The mapping and characterization of a novel binding site on HIV-1 gp120 for the

broadly neutralizing monoclonal antibody IgG1 b12

by

Jillian Waruk

A Thesis submitted to the Faculty of Graduate Studies of

The University of Manitoba

in partial fulfilment of the requirements of the degree of

DOCTOR OF PHILOSOPHY

Department of Medical Microbiology

University of Manitoba

Winnipeg

Copyright © 2011 by Jillian Waruk

Abstract

HIV infects target cells via fusion events following surface envelope glycoprotein binding to the CD4 receptor and a chemokine co-receptor. Despite the high sequence variability of envelope across and within HIV-1 subtypes, this process requires conserved sequences and structures on gp120, which also represent good targets for HIV-1 neutralizing antibodies. Few examples of HIV-1 broadly neutralizing antibodies exist, but these antibodies may hold the key to a protective HIV-1 vaccine. One such antibody,

IgG1 b12 (b12), binds the CD4 binding site on the HIV-1 envelope glycoprotein gp120.

To date, no vaccine preparations have been able to elicit a b12-like response. A complete understanding of the mechanism of b12 binding to gp120 is essential to successful design of an b12-like immune response.

Until now, strategies to map the b12 binding site on gp120 have utilized indirect techniques and/or core gp120 and have shown that b12 binds to a site on gp120 that overlaps the CD4 binding site. To more directly map the b12 epitope on intact gp120, epitope excision mass spectrometry mapping was carried out in the MALDI QqTOF platform. The putative epitope sequence was confirmed by tandem mass spectrometry sequencing. Epitope mapping revealed a novel binding site for IgG1 b12 at the gp120 amino terminus called Nterm. b12 bound a synthesized peptide of the epitope and the nature of the epitope was explored by ELISA. Although the Nterm epitope is involved in b12-gp120 interactions, ELISAs also show that the epitope does not make up the entire binding site on gp120. Rabbits immunized with a peptide version of the Nterm epitope

II do express antibodies that bind monomeric gp120, but these antibody responses do not neutralize HIV-1 in vitro.

These data indicate that the b12 binding site on gp120 is much more complex than previously thought. The b12 binds the Nterm sequence of gp120, perhaps in conjunction with the CD4 binding site. It has been shown that another HIV-1-neutralizing antibody,

4E10, also binds this novel Nterm epitope, and this may indicate a similar mechanism of action utilized by these two different antibodies. Though not able to elicit neutralizing antibodies on its own, this epitope may be an important element of the neutralizing b12 epitope and an important component of HIV-1 neutralizing antibody responses.

III Dedication

This thesis is dedicated to all the teachers in my life who have inspired me. I admire your passion for education and respect for those whom you teach.

Cette thèse est dédiée à tous les enseignants dans ma vie qui m'ont inspirée. J'admire votre passion pour l'éducation et votre respect pour vos étudiants.

Monsieur Gauvin, your devotion to promoting sciences to youth, especially females, was truly the encouragement I needed to get me here. Merci.

Monsieur Gamache, your humour engaged me, and your end devastated me. Your life provided me with focus. Je serai toujours un de tes petits bouts de choux.

Katie Waruk, sister, there are not enough words. I have gained so much from our friendship. Your support and dedication to me is inextricably tied to all my successes in life. Thank you.

IV Acknowledgements

I’d like to thank way too many people than I can fit into a <2 page long acknowledgements section. This is by no means an exhaustive list of the people I would like to acknowledge and thank for their support over the years, so please excuse me if your name does not appear herein, I do thank you all from the bottom of my heart.

I wish to first thank my advisor, Dr. Frank Plummer. His sometimes insane capacity for knowledge coupled with his drive to combat infectious disease here in Canada and in the Developing World make him a pleasure and an inspiration to work with. I consider myself lucky to have had Frank as my advisor.

On a day-to-day basis, I was managed by Dr. Blake Ball. Thank you, Blake, for your supervision and friendship. You taught me to be a worthy and thoughtful researcher.

My advisory committee provided both support and invaluable scientific advice throughout this process. Dr. Keith Fowke, your leadership in the collective HIV group has been invaluable. I will always think of you as my third advisor and almost as good a hockey player as me. Thank you Dr. Jody Berry for your guidance, especially with the inception of this project. Dr. Kevin Coombs, your adherence to the scientific process always steered me in the right direction. Dr. John Wilkins, you and your lab staff provided much needed help when I ran in to technical difficulties. Thanks also to Robbin Shattock for providing feedback on my thesis.

I would not have been able to complete the research study detailed herein without the help of out amazing technical staff, including John, Leslie, Sue, and Steve. They all have displayed large amounts of patience with me and their scientific input was and is valued. I performed the MS studies alongside Keding Cheng and would like to acknowledge the contribution of the other staff at the Manitoba Centre for Proteomics. Dr. David Montefiori and staff at the Laboratory of Immune Measurements performed the pseudovirus assays and were always available for discussions. I’d like to thank Ma, Binhua, and Ralph for invaluable discussions and Trent Bjorndahl and Gary for continued collaborations. Without the generous donations of reagents by Drs. Dennis Burton and Carlos Barbas III, this project never would have began. I’d like to thank Nicole and staff at Animal Care at the NML for taking good care of the animals that were sacrificed in the name of HIV vaccine research. Thanks to Jen for stats and thesis discussions.

Thank you to the Department of Medical Microbiology, in particular, Joanne Embree, Angela, Carol, Jude, Lynn, Eva, and Sharon. Without you I would not have graduated or been paid or even had a chair to sit on, and I’m not exaggerating.

I have seen many other students come and go from both the Plummer and Fowke labs, most who have made both major and minor contributions to my work and overall happiness. I was extremely lucky to end up with a bunch of students who made the lab a fun and congenial place to work. In moderately chronological order, I’d like to thank my

V friends Lyle, Paul, Sandy, Tammy, Shehzad, Hezhao, Françoise, Lindsay, Mariel, Allison, Crystal, Catherine, Meika, Caitlin (my antibuddy), JJ, Nadine, and Melissa, Aida, Derek, Lindsay, Winnie, and Were. I’m really fortunate I got to work with you all and look forward to our future collaborations.

I’d like to acknowledge and thank the colleages and staff at the University of Nairobi. Though my work did not involve the clinics in Kenya, their dedication to the eradication of HIV is praiseworthy and without their tireless efforts, my project would not have been funded. I would be remiss if I did not also mention the women of the Pumwani cohort. Their continued cooperation and contribution to this project will never be forgotten, not by any of us.

Thanks to all my friends outside the lab, your encouragement has meant a lot to me. Thanks to Leanne for allowing me to vent long-distance and for the trip last year. Thank you Lindsay for the lunches, thanks Laura for always being there. Jill#2, thanks for insisting that I be Jill#1 and, with Andy, for your willingness to go for beers. Thank you to Dale and the Wiebes. I will always remember how much you supported me. Thanks to my sports team families who always provided me with an outlet. Thanks to my health care team. Thank you, my chosen family, Jaime, Kerri, Judy, Marsha, Natalie, Caitlin, and JJ.

Last, but certainly not least, I’d like to thank each and every one of my family. They grounded me by never letting a chance to make fun of me go by and lifted me up with their interest in the value of my work and attempts to understand the jillitope. Thanks Aunts, Uncles, Cousins. Thank you Ivy. You just missed meeting the 1st Dr. Waruk. Thank you Shayla. Thanks Dad. Thank you Katie. Thanks Mom, keeper of my cats. My last thank you is reserved for my Alana. Everyday you remind me of the value of life and the science of compassion.

VI

Table of Contents

Abstract II

Dedication IV

Acknowledgements V

Table of Contents VII

List of Tables XII

List of Figures XIV

List of Copyrited Materials for which Permission was Obtained XVI

Section 1.0 Introduction 1 1.1 Human Immunodeficiency Virus 1 1.1.1 History of HIV/AIDS and HIV epidemiology 1 1.1.2 Transmission and prevention 3 1.1.3 HIV classification, infection, and protection 5 1.1.4 The HIV-1 envelope glycoprotein and mechanism of HIV-1 entry 9 1.1.4.1 The envelope trimer 9 1.1.4.2 Envelope 10 1.1.4.3 Entry 12 1.1.5 HIV infection kinetics, clinical manifectations, and treatment 14

1.2 Immune system – general 17 1.2.1 Innate immunity 17 1.2.2 Adaptive immunity 18 1.2.2.1 T cells 18 1.2.2.2 B cells and antibodies 20

1.3 Immune system – HIV-specific immunity 25 1.3.1 T cell responses 25 1.3.2 B cells and antibodies 26 1.3.2.1 B cells in infection 26 1.3.2.2 HIV-specific antibody responses 27 1.3.2.3 HIV-specific monoclonal antibodies 32 1.3.2.3.1 IgG1 b12 and CD4 binding site monoclonal 33 antibodies 1.3.2.3.1.i IgG1 b12 activity 33 1.3.2.3.1.ii IgG1 b12 antigen interface 34 1.3.2.3.1.iii Other CD4BS mAbs 36 1.3.2.3.2 HIV-1 monoclonal antibodies of other specificities 37

VII 1.3.2.4 HIV-1 evasion of antibody responses 42

1.4 HIV vaccines – trials and tribulations 43 1.4.1 HIV vaccine design 43 1.4.2 Antibody-based vaccine design and epitope mapping 45

Section 2.0 Hypotheses 50

Section 3.0 Specific Objectives 52

Section 4.0 Materials and Methods 53 4.1 General materials 53 4.1.1 Solutions 53 4.1.1.1 Mass spectrometry 53 4.1.1.2 Gel electrophoresis 55 4.1.1.3 Enzyme-Linked Immunosorbant Assay (ELISA) 56 4.1.1.4 Western blot 57 4.1.1.5 Serum antibody purification 57 4.1.1.6 Cell separations/infections 58 4.1.2 Antibodies and antigens 59 4.1.3 Animals 61 4.1.4 Human samples 62 4.1.5 Pseudovirus neutralization assay reagents 62 4.1.6 Other consumables 64 4.2 General methods 64 4.2.1 SDS Page gel electrophoresis 64 4.2.2 Coomasie stain 65 4.2.3 Silver stain 65 4.2.4 Mass spectrometry column preparation 65 4.2.5 Matrix-assisted laser desorption/ionization mass spectrometry 67 4.2.5.1 Epitope excision 67 4.2.5.2 Tandem mass spectrometry (MS/MS) 68 4.2.6 Peptide concentration calculations 69 4.2.7 ELISA 69 4.2.7.1 Antibody capture of peptide antigen 69 4.2.7.2 Sandwich ELISA for truncated gp120 binding 70 4.2.7.3 Competition assay 71 4.2.7.4 gp120-binding antibody detection in animal sera 71 4.2.7.5 p24 detection 72 4.2.8 Animal immunizations 73 4.2.8.1 Ethics 73 4.2.8.2 Antigen preparation 73 4.2.8.3 Immunization protocol 74 4.2.8.4 Sample collection 75 4.2.8.5 Serum inactivation 76 4.2.8.6 Serum IgG purification 76

VIII 4.2.8.7 Serum adsorption 77 4.2.9 Pseudovirus infection/neutralization protocol 77 4.2.9.1 Cell and pseudovirus preparation 77 4.2.9.2 Pseudovirus neutralization assay 78 4.2.10 PBMC-based neutralization assay 80 4.2.10.1 Ficol separation of PBMC from whole blood 80 4.2.10.2 Phytohemagglutinin (PHA) stimulation of PBMCs 80 4.2.10.3 Generation of viral stocks for PBMC neutralization assay 81 4.2.10.4 Tissue culture infectious dose (TCID50) assay 82 4.2.10.5 Virus calculations 83 4.2.10.6 PBMC infection/neutralization assay 84 4.2.10.6.1 Cell viability with treatment 84 4.2.10.6.2 Infections and neutralizations 85 4.3 Data analyses 86

Section 5.0 Results 89 5.1 Identification of a binding site for IgG1 b12 on HIV-1 gp120 by MALDI 89 QqTOF mass spectrometry epitope excision mapping 5.1.1 Rationale 89 5.1.2 Hypotheses 89 5.1.3 Objectives 90 5.1.4 Study Outline 90 5.1.5 Epitope excision mass spectrometry mapping of IgG1 b12identifies 90 specific binding sequences of gp120 5.1.6 Putative amino acid identity of the IgG1 b12 gp120 epitope identified 94 by MS 5.1.7 Tandem mass spectrometry (MS/MS) identification of the gp120 98 peptides involved in b12 interactions 5.1.8 Mass spectrometry epitope mapping of IgG1 b6 106 5.1.9 Summary 109 5.2. Confirmation of the antigenic specificity of IgG1 b12 for the Nterm sequence 111 of HIV-1 gp120 5.2.1 Rationale 111 5.2.2 Hypotheses 111 5.2.3 Objectives 112 5.2.4 Study Outline 112 5.2.5 IgG1 b12 binds to the synthetic Nterm peptide 112 5.2.6 Confirmation of a role of the Nterm sequence in IgG1 b12-gp120 117 interactions 5.2.7 The IgG1 b6 monoclonal Ab binding to the Nterm peptide 124 5.2.8 Summary 126 5.3 Analysis of the Nterm sequence – variation and vaccine suitability 128 5.3.1 Rationale 128 5.3.2 Hypotheses 129 5.3.3 Objectives 129 5.3.4 Study Outline 129

IX 5.3.5 Correlation of the amino acid variation at the Nterm epitope sequence 130 with the neutralization titre of IgG1 b12 5.3.6 Correlation of the amino acid variation at the CD4BS with the 138 neutralization titre of IgG1 b12 5.3.7 Characterization of the Nterm amino acid sequence and variation in 140 IgG1 b12 neutralization-tested virus 5.3.8 Amino acid sequence conservation at the Nterm in available HIV-1 147 sequences 5.3.9 Summary 153 5.4 Immunogenicity of the identified Nterm peptide in animal models and 154 neutralization inhibition 5.4.1 Rationale 154 5.4.2 Hypotheses 154 5.4.3 Objectives 154 5.4.4 Study Outline 155 5.4.5 Immunized animal sera antigen binding 155 5.4.6 IgG purification of rabbit sera and antigenicity tests 159 5.4.7 Pseudovirus neutralization assays 162 5.4.8 PBMC-based HIV-1 infection neutralization assays 168 5.4.9 Summary 177 5.5 General Results Summary 179

Section 6.0 Discussion 180 6.1 Identification of a binding site for IgG1 b12 on HIV-1 gp120 by 181 MALDI QqTOF mass spectrometry epitope excision mapping! 6.1.1 MS mapping and putative sequence of the IgG1 b12 epitope 181 6.1.2 MS/MS sequence identification confirms the putative b12 182 epitope 6.1.3 MS mapping of the CD4BS mAb IgG1 b6 185 6.2 Confirmation of the antigenic specificity of IgG1 b12 for the Nterm 186 sequence on HIV-1 gp120 6.2.1 b12 specifically binds the Nterm peptide 186 6.2.2 The role for Nterm in b12 binding to gp120 187 6.2.3 IgG1 b6 binds Nterm peptide weakly 190 6.3 Analyses of the Nterm sequence – variation and vaccine suitability 191 6.3.1 Nterm sequence variation correlates with b12 neutralization 191 sensitivity 6.3.2 The gp120 CD4BS variation correlates with b12 sensitivity 192 6.3.3 Sequence and chemical characteristics of Nterm and its 193 variants 6.3.4 The role of gp120 amino terminal region in HIV-1 infection 195 6.3.5 Nterm epitope cross-reactivity 196 6.4 Nterm peptide elicitation of antibody responses and their neutralization 197 profiles 6.4.1 Nterm peptide immunogenicity in animals 197 6.4.2 Animal serum and IgG pseudovirus-based neutralizations 198

X 6.4.3 Rabbit serum and IgG PBMC-based neutralizations 200 6.5 Experimental limitations 202 6.5.1 Mass spectrometry epitope mapping, epitope identification 202 6.5.2 ELISA confirmation of Nterm epitope antigenicity 202 6.5.3 Nterm sequence analysis 204 6.5.4 Animal studies 204 6.6 General discussion 205 6.6.1 The gp120 amino terminus as an antigen 205 6.6.2 Antibody-based vaccine design 205 6.6.3 IgG1 b12-like antibody elicitation 207 6.7 Model of IgG1 b12 binding to the Nterm epitope 209 6.8 Future Directions 211 6.9 Summary 214 7.0 References 216 8.0 Appendices 269 Appendix A List of Abbreviations 269 Appendix B Amino Acids 273 Appendix C Nterm amino acid sequences for neutralization-tested viruses 274 Appendix D Animal serum and IgG neutralization data 275

XI List of Tables Page Table 1 Prominent HIV-1 subtypes and their global distribution 3

Table 2 Antibody types and their cognate Fc receptor and function 23

Table 3 Animal characteristics 62

Table 4 Pseudoviruses for neutralizations 63

Table 5 Consummable labware used in experiments 64

Table 6 Animal immunization timeline for all three species 75

Table 7 A breakdown of MS tests performed and their corresponding figures 92

Table 8 Putative gp120 sequence results of the peaks identified by MS using 97 theoretical digests

Table 9 Sonar MS/MS sequence matches for the 1357 MS/MS spectrum 105

Table 10 Sonar MS/MS sequence matches for the 1609 MS/MS spectrum 107

Table 11 b12 neutralization concentration and expanded Nterm sequences of 133 all Clade B viruses tested against b12 for neutralization

Table 12 Amino acid characteristics 136

Table 13 Amino acid sequences implicated in b12-gp120 interactions of 139 Subtype B viral strains tested for b12 neutralization sensitivity

Table 14 Blast search results of the Nterm peptide against human proteins 143

Table 15 Types of amino acid variation in the published Nterm sequences that 145 have been tested for neutralization against IgG1 b12 (N = 93; includes all subtypes)

Table 16 Frequency of and amino acid changes that occur at more than 1% in 151 all of the available Nterm sequences in the Los Alamos HIV Sequence Database

Table 17 Sequence variation of Nterm among HIV-1 subtypes in the Los 152 Alamos Sequence Database

Table 18 Animal serum naming system 155

Table 19 Guinea pig sera neutralizations of pseudotyped pSG3!Env grown in 164

XII TZM-bl cells

Table 20 Rabbit sera neutralizations of pseudotyped pSG3!Env grown in 166 TZM-bl cells

Table 21 Rabbit IgG and sera pseudovirus neutralizations of pseudotyped 167 pSG3!Env grown in TZM-bl

Table 22 Data for TCID50 calculations for 4 viruses tested against donor D074 170

Table 23 Data for TCID50 calculations for 4 viruses tested against donor D039 171

Table 24 Summary of the PBMC-based neutralization assays 178

XIII List of Figures Page Figure 1 Cartoons of the viral protein and genetic arrangements of HIV-1 6

Figure 2 Crystal structures of gp120 12

Figure 3 Structure of the IgG antibody 21

Figure 4 IgG1 b12 and KZ52 MALDI mass spectrometry epitope mapping 93 spectra

Figure 5 mAb digestion control spectra 95

Figure 6 Endopeptidase autodigestion background spectra 96

Figure 7 The Nterm epitope sequence within the gp120 sequence 99

Figure 8 Fragmentations that can occur along a protein or peptide backbone 100 during ionizing MS/MS sequencing experiments

Figure 9 Spectra of monomeric gp120 digestion 102

Figure 10 MS/MS sequencing spectra of the tryptic peak 1356 103

Figure 11 MS/MS sequencing spectra of tryptic peaks 1608 104

Figure 12 Amino acid alignment of IgG1 b6 and b12 light and heavy chain 108 variable regions (Fvs)

Figure 13 MALDI QqTOF MS epitope excision mapping of IgG1 b6 110

Figure 14 Binding confirmation of the Nterm peptide 114

Figure 15 Potential Nterm secondary structure based on PSIPRED prediction 116 and crystal structure studies

Figure 16 Other HIV-specific mAbs do not bind Nterm peptide compared to 118 IgG1 b12

Figure 17 Competition ELISA between gp120 and Nterm peptide for binding 120 by b12

Figure 18 IgG1 b12 binds poorly to gp120 lacking its amino terminus 122

Figure 19 IgG1 b6 binds poorly to the Nterm epitope 125

Figure 20 Binding of IgG1 b6 to gp120 lacking the amino terminus 127

XIV

Figure 21 Linear regression correlation of IgG1 b12 neutralization with amino 132 acid variation at the Nterm sequence for the tested strains across multiple clades

Figure 22 Linear regression correlation of IgG1 b12 neutralization 135 concentration to clade B HIV strains with the number of amino acid variations of the expanded Nterm sequence of that same viral strain

Figure 23 Linear regression correlation of IgG1 b12 neutralization 137 concentration to clade B HIV strains with the number of amino acid variations and their score of the expanded Nterm sequence of that same viral strain

Figure 24 Linear regression correlation of IgG1 b12 neutralization 141 concentration to clade B HIV strains with the number of amino acid variations b12 binding site sequence of that same viral strain

Figure 25 Biochemical properties of the Clade B Nterm sequence and 146 sequence variants that have been tested for neutralization against IgG1 b12

Figure 26 Conservation of amino acid sequences in gp120 Nterm and the b12 149 binding site/CD4BS

Figure 27 Immunized animal sera binding to IIIB gp120 by ELISA 157

Figure 28 Immunized animal sera binding to streptavidin by ELISA 158

Figure 29 Purified IgG from immunized rabbits 160

Figure 30 Binding of rabbit sera, IgG-purified fraction, IgG-depleted sera, and 161 IgG add-back sera to IIIB gp120 in ELISA

Figure 31 Cell viability assays for donor D074 and D039 with IIIB virus 172

Figure 32 AZT inhibition of each viral strain IIIB and Bal 174

Figure 33 Viral growth for Day 7 harvests of IIIB-infected D074 PBMC 175

Figure 34 Nterm sequence and the functions of individual amino acids based 195 on alanine-scanning mutagenesis and binding studies

XV List of Copyrighted Materials for which Permission was Obtained

Figure 2 The crystallized structures of gp120-b12 fab interaction and gp120 16

XVI Section 1.0 Introduction

1.1 Human Immunodeficiency Virus

1.1.1 History of HIV/AIDS and HIV epidemiology

The Human Immunodeficiency Virus (HIV) is the causative agent of Acquired

Immunodeficiency Syndrome (AIDS). Worldwide, there are approximately 33.3 million people living with HIV/AIDS, 7.5% (2.6 million) of whom were infected in 2009 alone

(1). Since the Joint United Nations on HIV/AIDS (UNAIDS) World Health Organization

(WHO) began publishing Global HIV/AIDS reports in 2001 there have been approximately 2 million deaths due to HIV infection each year (1-9). Most effected by the HIV epidemic are the world’s poorest populations, notably those in Sub-Saharan

Africa, where two thirds of all infected people reside and where one half of AIDS-related deaths occur (1, 2, 9). While the Canadian statistics are not as bleak as those from many

African countries, the prevalence of HIV infections in Canada has risen from 50,000 infections in 2002 to 65,000 in 2008 (10). There are currently 1637 diagnosed HIV- infected people in Manitoba where there is an overrepresentation of aboriginals who are

HIV+ and where women are becoming infected at higher rates than ever before (11).

In the early 1980s, a conspicuous number of cases of pneumocystis pneumonia (PCP, causative agent Pneumocystis jirovecii – previously termed P. carinii) and Kaposi’s

Sarcoma were identified in previously healthy young adults males (12). Both illnesses typically afflict immunocompromised individuals (13, 14). Epidemiologic risk factors included being a bisexual or gay male, a Haitian immigrant, or a haemophiliac (15). The

1 causative agent of the immunodeficiencies was described and confirmed independently by two labs as the virus now known as HIV-1 (16-19).

There are two major types of HIV: HIV-type-1 and HIV-type-2 (HIV-1 and HIV-2, respectively). HIV-1 is responsible for the majority of the HIV pandemic. The progression to AIDS in HIV-2 infection is slower than in HIV-1 and is sometimes absent

(reviewed in (20)). Based on genetic sequencing and phylogenetic analyses there are three groups of HIV-1 – M, N, and O (21-23). Genetic analysis of simian immunodeficiency virus (SIV) and HIV suggests that all three HIV groups arose as a result of individual viral zoonotic transmission events from chimpanzees to humans in west equatorial Africa (24-26), although there is some evidence that group O may have been transmitted from gorillas (27). M (“Main”) HIV is responsible for 90% of the pandemic and is thought to have been transmitted to humans in the early 20th century in southern Cameroon (28). HIV N and O each arose from their own separate zoonotic transmission events; N (Non-M, Non-O) originated from chimpanzees in southern

Cameroon, while the origin of O (Outlier) is still undetermined (25). HIV-1 M is further broken down into subtypes, or clades, based on phylogenetic clustering. While HIV is a global pandemic, HIV subtypes display distinct geographic distributions (Table 1). The most common subtype in North America, Europe, and Australia is clade B. Clade C predominates in India and southern Africa, but many more subtypes are found in these regions and the rest of Africa and Asia (Table 1, IAVI, 2003). HIV is capable of genetic recombination on superinfection of a single target cell, and many of these recombinants are established circulating recombinant forms (CRF; 01_AE and 02_AG in Table 1).

2 Table 1. Prominent HIV-1 subtypes and their global distribution Main Clades Main geographic locations A Kenya, Rwanda, Burundi, Eastern Europe, Russia A1 Eastern Europe, Russia AB recombinant North America, South America, Europe, Russia, China, B Australia, South East Asia BF recombinant Eastern South America BC recombinant China China, India, Eastern and Southern African countries except C Kenya D Uganda, Sudan F G H Angola, Congo, D.R. Congo, Gabon J K 01_AE Angola, Congo, D.R. Congo, Gabon, South East Asia 02_AG Western, Central African countries A1D Kenya, Tanzania, Uganda, Europe

1.1.2 Transmission and prevention

HIV transmission occurs via the blood or mucosal secretions, primarily through penile- vaginal and penile-anal sex. The risk of acquiring HIV through heterosexual sex is low

(0.182% per sex act, although almost twice as frequent from male to female as female to male (29-31)), but sexual transmission accounts for over 3/4 of new HIV infections (32,

33). The risk of transmission fluctuates with contraception use (34), antiretroviral (ARV) use (35), viral load (30, 35-37), concurrence of sexually transmitted infections (STI) (32,

38-45), and male circumcision (46). Socially, condom education and distribution have been successful in promoting behavioral changes (47). The drop in HIV infection in

Uganda from 15% in 1991 to 5% 2001 has been linked to a controversial national prevention program known as the ABCs (Abstinence, Be faithful, use Condoms) (48-50).

3 Biomedical approaches to risk reduction have had mixed results, but are gaining favour

(46, 51-54). Although treatment of STIs has produced mixed results in terms of reduction in HIV incidence (55-59), clinical trials have clearly demonstrated that male circumcision provides a 60% reduction in incidence of HIV. For women, heterosexual transmission occurs when HIV crosses the vaginal epithelium and infects underlying target cells

(reviewed in (60)). Vaginal microbicides are a potentially powerful tool for HIV prevention as they give women the ability to protect themselves without their partner’s input (61-63). Early microbicide trials were unsuccessful, as exemplified by nonoxynol-

9, a spermicide ingredient that showed in vitro anti-HIV activity (64, 65), but resulted in increased ulcers and HIV incidence in clinical trials (66-69). Recent studies are more encouraging, however, as a gel containing Tenofovir, an anti-HIV-1 drug, reduced infections by 54% in adherent subjects when applied to the vaginal tract in a South

African population (55).

Vertical transmission of HIV from mother to child can occur in utero, during delivery, or through breast milk and, like sexual transmission, correlates with transmitter viral load

(70-74). A clinical trial of treatment of both mother and child with zidovudine showed protection of newborns (75, 76) and a single dose of nevirapine during delivery reduces the likelihood of vertical transmission (77). Combination antiretroviral therapy (ART) treatment of infected mothers in developed countries has decreased the chance of mother- to-child transmission to <2% (78, 79). Unfortunately, such prophylaxis is not available to all women, especially those in developing countries and breast milk replacement formula is often unavailable, expensive, and unsuitable in some rural areas (74, 80-82).

4

Blood-borne transmission of HIV occurs primarily through intravenous (IV) drug use and receipt of contaminated blood products. Needle exchange programs and blood bank screening work to reduce transmission, although the former is highly controversial and its efficacy is difficult to prove. Rarely occurring routes of transmission include oral sex, dental surgery and occupational exposure such as needle stick injuries (reviewed in (83)).

The development of ARVs that act against HIV is no doubt the most significant HIV- related discovery to date. In developed countries, monitoring of disease progression and use of ARVs have vastly reduced the burden of HIV infection (35). Unfortunately, the majority of the world’s new HIV infections occur in the poorest areas, which have inadequate access to health care or in high risk-taking, stigmatized populations. Often these people are also the hardest to access and have challenges complying with daily anti-

HIV medication. The creation of an effective vaccine that requires few contacts with health care and could be implemented on a global scale would have a profound effect on infection rates and would help the most susceptible people.

1.1.3 HIV classification, infection, and protection

HIV belongs to the family Retroviridae in the genus Lentiviridae. It is a single-stranded positive sense RNA virus and is a retrovirus owing to its ability to reverse transcribe its

RNA genome into DNA for integration into the host genome. The HIV genome is 9.2 kb in size and encodes three structural proteins (Gag p24, p17 and p55), two envelope

5 glycoproteins (gp120 and gp41), three enzymes (protease, reverse transcriptase, and integrase), and six accessory proteins (Vif, Vpr, Vpu, Rev, Tat, and Nef) (Figure 1).

Figure 1. Cartoons of the viral protein and genetic arrangements of HIV-1 (reviewed in (84, 85)).

HIV entry into target cells is mediated by a trimer of gp120-gp41 dimers (86, 87), discussed in greater detail below. Briefly, the early phase of infection begins when a gp120 engages its primary receptor, cluster of differentiation 4 (CD4), on the target cell, which are primarily macrophage and T cells (88-92). Other cell types that can become

HIV-infected include dendritic cells (DC), Langerhans cells and epithelial cells (93-97).

Conformational changes in envelope allow gp120 to interact with the co-receptor C-C chemokine receptor 5 (CCR5) on macrophage or T cells, or C-X-C chemokine receptor type 4 (CXCR4) on T cells (98-103). Further conformational changes mediate fusion

6 between the HIV membrane gp41 and the cell membrane (104, 105). As the virus enters the cell, it is uncoated. Reverse transcription is initiated in the cytosol by viral reverse transcriptase (RT) with the help of other viral and cellular factors (106). The wide genetic diversity in HIV is due to the notoriously low RT fidelity. The error rate of HIV

RT is estimated to result in 1 error/viral genome per replication cycle (107) and exhibits positive selection for non-synonymous mutations (108). The newly transcribed DNA provirus is shuttled to the cell nucleus in a pre-integration complex where HIV integrase integrates its genome into the host genome. The virus can remain latent as an HIV reservoir for the life of the cell.

The late phase of cellular infection begins when the integrated viral DNA is transcribed into mRNA. The unspliced mRNA is transported to the cytosol for Gag/Pol polyprotein synthesis (which are later cleaved by viral protease) and spliced mRNA is utilized for envelope and accessory protein translation (85). Envelope is expressed and modified in the endoplasmic reticulum (ER) and the Golgi apparatus.

Viral assembly occurs in the cytoplasm. The envelope trimer is transported to the cell membrane where, along with Gag, Pol, two copies of the viral genome, and accessory proteins, budding occurs (reviewed in (109, 110)). HIV can also be transferred between cells via syncytium formation (111, 112). HIV counteracts the ability of cell surface CD4 to capture budded virus through nef-induced internalization and degradation of surface- expressed CD4 (113, 114). Viral budding can also be blocked by the host cell protein tetherin (115, 116), which is inhibited by viral Vpu and Nef (117).

7 Initial infection is typically via CCR5-tropic virus, as CXCR4-tropic viruses have been demonstrated to be largely inadequate at establishing infection, regardless of mode of transmission (118, 119). By assessing the viral quasispecies of acute seroconverters and their partners, it was shown that a very small, homogeneous population of HIV caused new infections (120). In fact, studies of mother-to-child perinatal transmission show that only a single or very few variants bypass host defenses to establish infection (121-124).

Defenses range from target cell innate factors to retrovirus restriction mechanisms including apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3

(APOBEC3) and tripartite interaction motif 5 ! (TRIM5!) (125). APOBEC3G deaminates cytidine, leading to CG to AT hypermutation, often causing premature stop codon insertion (126). HIV Vif binds and inactivates APOBEC3G. TRIM5! prevents capsid formation in non-human primates, but does not affect HIV capsidation in humans

(127), although its function could be mirrored in humans through the design of drugs that mimic its action. Since the natural ligands of CCR5 and CXCR4, RANTES (Regulated upon Activation, Normal T-cell Expressed, and Secreted) and macrophage inflammatory protein (MIP)-1a block HIV entry into target cells (128-130), chemokines could be exploited for the development of novel treatment strategies.

It has also been discovered that some people exhibit a genetic basis for reduced susceptibility to infection. Some TRIM5! mutations have been correlated with susceptibility to HIV infection (131), and polymorphisms within the IRF-1 gene have been linked to reduced susceptibility to HIV-1 infection in a cohort of HIV-exposed seronegative (HESN) sex workers in Nairobi, Kenya (132). The CCR5"32 mutation is

8 common in Caucasians and encodes a mutant form of CCR5 that cannot be used by HIV as a co-receptor (133-136). There are human leukocyte antigen (HLA) genes that are associated with susceptibility to infection (137-139), reduced progression to AIDS (140-

142), and rapid progression to AIDS (142-145). Since HLA are a main component of the adaptive immune system, these data suggest that the immune system plays an important role in infection and progression to AIDS.

1.1.4 The HIV-1 envelope glycoprotein and mechanism of HIV-1 entry

1.1.4.1 The envelope trimer

The envelope glycoprotein spike expressed on the surface of HIV is composed of three molecules of gp120 that interact non-covalently with three molecules of gp41, which serve to anchor the entire structure into the HIV membrane (146, 147). gp120 and gp41 are initially expressed as a gp160 polyprotein and are cleaved in the cell by host furin before being shuttled to the membrane (86, 87, 148, 149). Interestingly, CD4 is also expressed in the ER and can bind to gp120 at the ER, which inhibits envelope translocation to the cell membrane (150-152). HIV overcomes this with Vpu, which binds to CD4 and signals it for degradation (153, 154). Both gp120 and gp41 are glycosylated, with >50% of the molecular mass of gp120 owing to its outer glycosylation coating (155, 156). The inner domain of gp120 lacks glycosylation, likely to facilitate interaction with gp41 (156). Approximately 14 (ranging from 4 to 35) spikes are expressed on the surface of an HIV virion (157) compared to influenza, which is a similar size virus and expresses approximately 450 spikes per virus particle (158). Antibody

(Ab) binding studies suggest that the positioning of the envelope trimer within the

9 membrane appears to be fairly rigid (159). Although the molecules are generally arranged in trimers (157), gp120 shedding does occur, resulting in monomeric gp120 in the circulation and viral spikes consisting of 0, 1, or 2 molecules of gp120 (160).

1.1.4.2 Envelope

The HIV gp120 molecule consists of 5 variable (V) regions and 5 constant (C) regions

(147, 161). Gp120 has been crystallized in conjunction with soluble CD4 and the 17b monoclonal Ab (mAb) fragment antigen binding domain (fab) (156, 162), with the immunoglobulin (Ig)G1 b12 fab (163), and ligated to F105 and b13 monoclonal Ab fabs

(164). The SIV gp120 has been crystallized in an unliganded state (165, 166) (Figure 2).

In each case, small variants of a “core structure” gp120 was crystallized, which consists of approximately 58% of monomeric gp120. Thus, gp120 is drastically altered to facilitate crystallization; it is missing the 82 amino-terminal amino acids, the V1/V2 loops (aa 121-203), the V3 loop (aa 300-328), and the carboxy-terminus (aa 493-511).

Amino acid substitutions were introduced to restrict core movement and fix its conformation (163, 167). More recently, core HIV has been crystallized with V3 (168) and the N and C termini added back (169). HIV (and SIV) gp120 is comprised of 2 domains - an inner and an outer domain - that are hypothesized to be relatively flexible with respect to one another while gp120 is monomeric, but are somewhat constrained in the trimer (170). The inner domain is located internal to the envelope trimer, is highly structurally diverse (171, 172), mediates gp120-gp41 interactions (156, 173-175), and as it is buried in the trimer, it is invisible to the immune system (174). The amino- and carboxy-termini of gp120 interact with one another (169) and were shown to be involved

10 in gp120-gp41 interactions (169, 176-178). The gp120 outer domain is accessible to the immune system and is thus heavily glycosylated and composed of the gp120 variable regions, which mutate at high rates (179). The variable regions are flanked by cysteines that interact and from which the variable loops outcrop (180, 181).

HIV-1 and HIV-2 share 60% sequence similarity, and their gp120 proteins are approximately 40% similar (20). Within a single individual diversity is most evident in the gp120 sequence (182-184). HIV-1 intraclade differences are 20% at the amino acid level, with >30% divergence in envelope (185). HIV envelope sequence diversity is greatest at the variable regions (161). Variable loops act as immunodominant, evolving shields for gp120. This is emphasized in studies showing that the removal of V1 and/or

V2 loops results in a virus that is still infective but highly susceptible to neutralizing Abs

(186, 187). Conversely, along with residues within the bridging sheet, V3 is involved in chemokine-receptor binding in the entry process, which means that it is subject to some variation constraint (188-190).

Gp41 is anchored to the envelope membrane by its C terminal transmembrane region and has an intracellular cytoplasmic tail of >100 amino acids. The cytoplasmic tail is involved in interactions with the HIV matrix and with host proteins (191). The amino terminus of gp41, termed the fusion peptide, is responsible for fusion between HIV and the target membrane. Gp41 also contains two hydrophobic heptad repeats that interact with one another to bring the target membrane close in order for fusion to occur (192,

193). The extracellular domain of gp41 has been resolved in the chemokine receptor-

11 bound state, but little is known about its structure on the surface of HIV in interaction with gp120 (193, 194).

Figure 2. Crystal structures of gp120. Top: gp120 core is co-crystallized with the b12 fab. Bottom row: structure of unliganded SIV gp120, b12-bound, and CD4/17b-bound gp120. The inner domains are in light grey, the outer domains in red, and the bridging sheets in blue. Figure is reprinted by permission from Macmillan Publishers Ltd: Nature, advanced online publication 15 February 2007 (doi:10.1038/sj.nature05580) (163).

1.1.4.3 Entry

HIV infection of target T cells and macrophage is a multi-step process. Gp120 binds

CD4 through its CD4 binding site (CD4BS). Initial studies suggested that the CD4BS residues were located in C1, C3, C4, and C5 (147, 195-199). Point mutagenesis studies

12 identified the amino acids on gp120 involved in CD4 binding, namely T257 (of C2),

D368, K370 (C3), W427, and D457 (C4) (200). CD4 and gp120 have been co- crystallized (Figure 2) (147, 156, 197, 200-203). Crystal structure data indicate that the

CD4BS is at the interface between the gp120 outer and inner domains and the bridging sheet of gp120, in a pocket-like structure. They also identified that residues of CD4 involved in binding lie between amino acids 25-64 with the side chain of phenylalanine at position 43 sitting well within the CD4BS pocket of gp120 (F43 accounts for 23% of the

CD4 interactions with gp120). F43 and R59 of CD4 interact with E368, G370, and W427 of gp120. These three amino acids in the CD4BS are conserved among primate immunodeficiency viruses and the region 365-371 and 425-430 account for 57% of the defined gp120-CD4 interface (200). The overall structure of the second domain of CD4

(D2) has been shown to be essential to gp120-CD4 interactions as well (204-206).

The gp120-CD4 interaction is characterized by favourable enthalpy and a large, unfavourable change in entropy, resulting in a modest affinity (207). The entropy change corresponds to conformational changes in gp120 that promote the secondary interaction of gp120 with the seven-transmembrane C-C or C-X-C chemokine receptor (CCR5 or

CXCR4), which allows membrane fusion to occur (98-103, 189, 208-210). On engagement of the co-receptor, the HIV envelope undergoes further conformational changes whereby gp120 and gp41 amino termini form a pre-hairpin intermediate (192,

211). Further conformational change in the gp41 hairpin pulls the HIV and the target cell membrane together. The gp41 amino terminus inserts its fusion peptide domain into the target membrane, allowing for fusion between HIV and the target cell membranes (193,

13 212-217). At this point, the gp120 amino terminus may interact with the target cell membrane as an anchor for virus to the target cell and aid in fusion (218).

HIV envelope interacts with other cell-surface molecules, such as heparin sulphate (219), which has been hypothesized to enable CXCR4-type infections (171, 220). DC-SIGN, a c-type lectin, allows the virus to attach to DC via gp120 mannans (221-223). Attachment can lead to viral uptake, degradation, and antigen presentation (224) or transport of HIV to the lymph nodes, where DC can facilitate the infection of CD4+ T cells in trans (219,

225, 226). Galectin-1, another c-type lectin expressed on numerous cell types including

DC and activated T and B cells (227), enhances infection by promoting binding of HIV envelope to CD4 (228). Conversely, it has been shown that the c-type lectin langerin on

Langerhans cells binds gp120 and leads to HIV degradation (229, 230). The gut homing receptor !4#7 binds HIV gp120 at the V2 region (231). The !4#7 receptor is expressed on mucosal CCR5+ CD4+ T cells, and may aid in the selection of R5-tropic viruses early in infection (232). It is thought that this interaction enhances infection and therefore may soon become the target of therapeutic design (233).

1.1.5 HIV infection kinetics, clinical manifestations, and treatment

Infection most often occurs sexually. The cervix and seminal plasma and cells are main sources of HIV at the genital tract (44, 234). Once HIV encounters the genital mucosa, it must cross the epithelial barrier. This can be achieved through breaks in the mucosa owing to trauma or STI and can be enhanced by c-type lectins such as DC-SIGN (219,

14 225, 226, 235, 236). Direct transfer across the epithelium via epithelial transcytosis can also bring HIV in proximity to target cells in the systemic compartment (237).

In the first few weeks post infection, a febrile illness termed acute seroconversion illness occurs. These symptoms coincide with a sharp rise in detectable HIV viral load (virus copies per ml) in the blood and a dip in the CD4 counts (CD4+ T cells per ml of blood).

No HIV-specific neutralizing Abs are seen at this stage of infection (238) and transmissibility is high (239, 240). The primary stage of HIV and SIV infection is characterized by a massive depletion of CD4+ T cells in the gut mucosa (241-243). This alteration of the gastrointestinal immune system has been proposed to cause an irreplaceable void that sets the stage for later systemic depletion of CD4+ T cells and immunodeficiency as HIV progresses (244, 245).

The viral load subsequently drops and reaches a set point that can vary widely between people (>1000 fold) and can be a predictor of progression (246, 247). This reduction in viral load corresponds to the induction of a CD8+ T cell response, which likely contributes to the drop (248-250). The Ab response, although ineffective at this point, is initiated (251, 252). This is the beginning of the clinically latent phase of infection, which still includes a high level of HIV replication; as many as 109 cells are newly infected daily (253). The majority of nascent virions are non-infectious owing to the large number of point mutations that occur in the reverse transcription process (253), while the rest can show a large diversity in their amino acid composition (254, 255). The duration of this stage varies widely (often depending on the set point viremia). An HIV infected

15 subject can remain positive without progression to AIDS and without ART therapy

(ART) for 2-30 years. Subjects who develop AIDS within the first few years of infection are referred to as rapid progressors (RP). Rare “protective” courses of infection have been defined: long-term non-progressors (LTNP), elite controllers (ECs), and viremic controllers (VCs). LTNPs are capable of maintaining low viral loads (usually defined by high CD4 count) for more than 7-10 years. ECs and VCs spontaneously achieve undetectable and low viral loads for greater than 1 year follow up, respectively (256).

Over time viral loads slowly rise while CD4 counts decline until a threshold is reached and AIDS occurs. The CDC and WHO defines AIDS illness as occurring at CD4 count below 200 and/or the development of opportunistic infections. This eventually leads to the death of the infected individual. Before a patient’s CD4 count drops below 350 cells/ml, they should be placed on ARV therapy, although in developing countries where resources are limited, this is not always achieved (257). There are currently 5 main classes of HIV ART licensed for use in North America: nucleoside reverse transcriptase inhibitors (NRTI; first approved for use in HIV+ patients in 1987) (258, 259), nonnucleoside reverse transcriptase inhibitors (NNRTI), protease inhibitors (260), integrase inhibitors (261), and entry/fusion inhibitors (192, 262). Due to the low fidelity of the viral replication, drug resistance inevitably develops to ARVs, requiring cocktails of drugs to be prescribed together (263). Unfortunately, although ARV treatment can lead to a rebound in CD4+ T cell levels, they rarely return to normal levels (264).

16 Ab tests are typically performed to diagnose HIV-1 infection. Unfortunately, as mentioned above, serum Ab levels are not detectable until the clinically latent stage of infection. Therefore, highly infectious early acute infections can give negative test results. Medical practitioners will often require patients to return at months 3 and 6 to be re-tested. Research into detection systems that can diagnose HIV infection at these early stages, especially in resource-poor settings where HIV infection diagnosis may be lacking, is ongoing (265).

1.2 Immune system – general

1.2.1 Innate immunity

There are two main arms of the human immune system that a pathogen encounters; these are termed innate and adaptive immunity. The hallmarks of innate immunity are that it is a quickly mounted response (within hours of exposure), it recognizes basic microbial molecular patterns, and it does not generate or maintain memory. Innate immunity involves mechanisms that are germ line-encoded and therefore the diversity of responses is limited (266).

Antigen presenting cells (APCs) are key players in the innate immune system and can secrete cytokines and express co-stimulatory molecules that provide the necessary second signal for the activation of adaptive immune cells. Antigen surveillance and presentation can be carried out by DC, monocytes, macrophage, and B cells. Innate immune cells are stimulated through germline-encoded pathogen recognition receptors (PRR) and bind certain common molecular motifs on pathogens, pattern-associated molecular patterns

17 (PAMP). There are several families of PRR, including the toll-like receptors (TLR)

(267), retinoic acid-inducible gene (RIG)-1-like receptors (RLR), nucleotide-binding oligomerization domain (NOD)-like receptor family (NLR), and the C-type lectin receptors (CLR) (266, 268, 269). Innate immune cells can also express Ab-binding receptors. These are termed Fragment Crystallizable Receptor (FcR; Table 1) and on antibody binding, signal for phagocytosis or degranulation. The complement cascade is initiated when antigen-Ab complexes are recognized and opsonized (270-272). HIV, however, has hijacked this system. It binds complement in either Ab-dependant or - independent fashions and uses the interaction with surface receptors to infect target cells in trans in the lymph node (270, 273).

1.2.2 Adaptive immunity

Adaptive immunity is defined as being a highly specific adaptable response that develops memory so that on re-exposure, the secondary response is swift and effective. Two main cells types mediate adaptive immunity: T and B cells.

1.2.2.1 T cells

T cells originate in the bone marrow from the same lymphocyte progenitor as B cells and natural killer (NK) cells. T cells mature in the thymus into either CD4+ or CD8+ T cells.

CD4+ T cells, or T helper (Th) cells are the major regulators of the adaptive immune system. Once activated, they provide activation signals to both B and CD8+ T cells. The

T cell receptor (TCR) binds to the presented peptide antigen in conjunction with HLA-

18 Class II on APCs (274). With co-stimulation from CD28 binding to CD80 or CD86,

CD40 ligand (CD40L)-CD40 interactions, and a cytokine signal, the CD4+ T cell is activated (275, 276). Activated CD4+ T cells clonally expand, express IL-2, and develop into memory or effector cells depending on whether they reside in the lymphoid tissue or migrate from the lymph node to non-lymphoid tissues, respectively (277).

There are several subsets of Th cells (278). Initially there were thought to be 2 main lineages of Th cells – Th1 and Th2. T helper 1 (Th1) type responses are generated towards intracellular pathogens, such as viruses, and are characterized by cell-mediated responses, the production of IL-2 and IFN-$, and IgG2 Ab response. T helper 2 (Th2) responses are directed at extracellular pathogens and defining characteristic are Ab production (IgG1, IgE, IgA) and the secretion of cytokines IL-4, IL-5, IL-10, and IL-13

(279). Other subsets of Th cells include: follicular Th that provide IL-4, IL-21, and

CD40L co-stimulation for germinal centre B cells (280-282), T regulatory cells (Tregs) that act to depress, or regulate, the immune response by production of IL-10 or TGF-# cytokines (283), and Th 17 cells. Th17 are pro-inflammatory cells defined by their ability to express IL-17 and play a role in maintaining the integrity of the mucosal barrier.

Th17 aid in protection from microbial pathogens at mucosal surfaces such as the gut by stimulating the secretion of chemokines by epithelial cells, which in turn recruit neutrophils, monocytes, and other lymphocytes (284-286).

CD8+ T cells, or cytotoxic T lymphocytes (CTL), like CD4+ T cells, require antigen recognition for activation. The TCR on CTL bind to antigen and HLA Class I, and if they

19 receive co-stimulation from DC or neighbouring CD4+ T cells, they become activated.

Activated CTL clonally expand and differentiate to effector and memory phenotypes.

Effector CTL migrate to the site of infection and carry out their cytolytic function

(through cytotoxic granule release of perforin and granzymes) and secrete IFN-$, lymphotoxin and TNF to activate inflammation and phagocytosis.

1.2.2.2 B cells and antibodies

B cells are lymphocytes responsible for the Ab-based humoral immune response. B cells largely reside in lymph nodes, secondary lymphoid tissue (such as the spleen), and in the bone marrow (287, 288). The B cell receptor (BCR) consists of membrane-anchored immunoglobulin and the Ig! or Ig# immunoglobulin co-receptors. Initially, mature, naïve

B cells express either membrane-bound IgM or IgD (289, 290). In the lymph node, antigens reach B cells by diffusion and BCR-mediated endocytosis or by display on macrophage, DC, or follicular DC (287, 291). Co-stimulation is provided by the antigen presenting cell or Th cells. B cells then form a germinal centre where they replicate and which serves as the site for Ab affinity maturation and memory B cell formation (280,

292). Activated B cells differentiate to memory cells or plasmablasts, then to plasma cells (289, 293). Plasma cells are terminally differentiated and secrete Abs into the bloodstream. They can traffic to the bone marrow to become long-lived plasma cells

(294-296). Memory B cells are long-lived cells that differentiate into Ab-secreting plasma cells on antigen re-exposure (289, 297).

20 Antibodies (Abs) (the IgG Ab is shown in Figure 3) are proteins of the humoral immune system produced by B cells that recognize antigen on invading pathogens independently or in conjunction with immune cells or other immune molecules. The site where Abs bind toxins and extracellular foreign molecules on pathogens is called an antigenic determinant, or epitope. Abs are capable of binding many types of antigens including proteins, carbohydrates and lipids. Antigenicity can be measured by ex vivo binding assays such as the enzyme-linked immunosorbant assay (ELISA). However, not all antigens are immunogenic. Immunogenicity is the ability of an antigen to elicit adaptive immune responses, for example B cell activation and Ab synthesis.

Figure 3. Structure of the IgG Antibody. The two antigen binding arms are highlighted in green. The Fc receptor/complement binding regions carry out Ab biological functions. Heavy and light chains are linked by disulfide bonds.

21 The immunogenicity of an epitope is determined by immunization or vaccination and then detection of de novo antigen binding by serum Abs.

Early in their development, B cells undergo a germ-line encoded, antigen-independent mechanism known as VDJ recombination, which preliminarily determines the class of Ab that is expressed on the surface of the naïve B cell (IgM) and is responsible for some Ab diversity (298). Upon antigen stimulation, B cells undergo class switch recombination

(CSR), which allows the integrity of the antigen binding site to remain intact while swapping out the immunoglobulin heavy chain constant region (299) and conferring differing biological functions on the resulting Ab. While Figure 3 shows the general structure of IgG, there are 5 classes of immunoglobulin that are based on the isotype of the heavy chain: IgG ($ chain), IgM (µ chain), IgD (% chain), IgA (! chain), and IgE (& chain), each with specific functions (Table 2). For example, IgG is preferentially expressed to combat smaller pathogens such as viruses and many bacteria by targeting them for phagocytosis, while IgA is relatively resistant to acidic environments, making it the ideal Ab to act at acidic mucosal surfaces such as the gut or vaginal lumen (300). The

Ab variable domain (Fv) contains the antigen-binding domain and consists of both the light and heavy chain with 3 hypervariable loops, or complementarity determining regions (CDRs), on each chain. CDR3, especially the CDR3 of the heavy chain, is often primarily responsible for antigen binding (301-303). The hallmarks of antigen recognition by Abs are specificity, diversity, and affinity maturation. Abs are able to distinguish between single amino acid variations within epitopes (specificity). Antibody repertoire diversity is illustrated by the estimation that a person may express up to 109

22 distinct Abs, although this is based on a cross-sectional survey and may not represent the

unfathomable potential diversity over a lifetime (302, 304, 305).

Table 2. Antibody types and their cognate Fc receptor and function. Data for this table are taken from reviews (306, 307). Ig Secreted Function Subtype FcR expressers Class form Naïve BCR Pentamer (monomer), IgM with J - - complement chain, C 4 H activation Membrane IgD Naïve BCR - - only 1 only - Fc$R1 all 4 subtypes (CD64) on Fc$RIIa/b/c 1 macrophage, (CD32) on Opsinization, neutrophils, monocytes(abc), complement eosinophils, DCs macrophage(abc), activation, 2 neutrophils(ac), ADCC, 3 only - Fc$R1 platelets(a), IgG Monomer neonatal (CD64) on Langerhans immunity, 3 macrophage, cells(a), B cells(c) negative B neutrophils, Fc$RIIIa/b (CD16) cell feedback eosinophils, DCs on macrophage(a), (RIIb) NK cells(a), T cells(a), 4 neutrophils(b), eosinophils(b) Monomer 1 (serum), Mucosal FcR!Ia (CD89) on neutrophils, Dimer protection, IgA monocytes, macrophage, eosinophils, with J complement 2 Kupffer cells, DCs chain activation (mucosa) Mast cell Fc&RI on mast cells, basophils, degranulation Langerhans cells, monocytes Monomer, in helminth IgE - Fc&RIIa/b (CD23) on B cells(ab), T CH4 response, allergy cells(b), monocytes(b), eosinophils(b), response macrophage(b)

Affinity maturation is a process by which additional mutations are increased via somatic

hypermutation and selection such that higher affinity Abs evolve at the V region in the

23 CDRs (308). Both CSR and affinity maturation processes require the expression of activation-induced cytidine deaminase and allow the evolution of higher affinity Abs that become the dominant Ab response (309, 310). Contributing to the ability of an Ab to interact with its cognate antigen is avidity, which is the measurement of the binding intensity provided by multiple contact points between binding pairs. Abs are bivalent, and therefore their avidities are greater if both arms bind antigen on the same virus.

Similarly, dimeric and pentameric Abs (IgA and IgM, respectively) have even greater avidity potential. Abs carry out their function by interacting with antigen either at linear sequences (linear epitope) or at multiple points in close proximity due to conformational folding of the antigen (conformational epitope). Abs interact with their cognate antigen through a variety of mechanisms, including electrostatic interactions, hydrogen bonds, van der Waals forces, and hydrophobic interactions (303). Typically, the affinity of an

Ab for its epitope is 10-7 to 10-11 M (the nature and specificity of Ab-antigen interactions are reviewed in (302, 303, 311, 312)). Together, the affinity and avidity of Ab for its particular antigen contribute to its overall binding capacity (313-317).

Abs can block or limit infection by steric neutralization and Ab-dependant cell-mediated viral inhibition (ADCVI). Neutralization blocks infection, sometimes by sterically blocking attachment or entry into a target cell. This direct mechanism of pathogen neutralization has been proven only rarely, as a firm quantitative relationship between Ab binding and loss of infectivity must be established (318). Additionally, Abs are also responsible for Ab-dependant cell-mediated cytotoxicity (ADCC). ADCC (a form of

ADCVI) is a process initiated when infected cells expressing foreign antigen on their

24 surface are bound by circulating Abs. The FcR on the surface of cytotoxic cells are cross-linked by binding the Fc regions and the infected cell is killed by release of cytotoxic granules (319-321). Other Ab antiviral mechanisms include opsinization for phagocytosis and complement activation. Abs secreted into mucosal surfaces (typically sIgA) can inhibit transcytosis or the ability of an invading organism to pass through the mucosal layer (322-324).

1.3 Immune system – HIV-specific immunity

1.3.1 T cell responses

CD4+ T cells are the main target cell of HIV infection. Their depletion throughout the course of HIV disease is a hallmark of progression, and therefore CD4 counts are an indicator of the health status of an infected patient (88-91). There is some evidence that

CD4+ T cell responses play a role in protection from disease progression. IFN-$ and IL-2 responses are associated with slower progression (325, 326). HIV-specific T cells can be detected despite the infection of not only activated T cells, but HIV-specific activated cells (327, 328). HIV can lead to the depletion of Tregs, which may account for some of the rampant and chronic immune activation seen in HIV infection (329).

Although not exclusively, the initial decline in HIV viremia is associated with an increase in HIV-reactive CTL (249, 330). It has been shown that CTL play a crucial role in determining the viral set point and viral loads negatively correlate with HIV-reactive

CTL (331). In a macaque model, depletion of CTL lead to a sharp rise in viral titre

(332). The current CTL literature focuses on determining what constitutes an effective

25 CTL response. Epitope mapping studies and the characterization of polyfunctionality in

CTL are providing further evidence that these responses play an intrinsic role in determining HIV progression (333). A CTL-based vaccine that protects from progression, even though it may not provide sterilizing immunity, is an important pursuit (334).

1.3.2 B cells and antibodies

1.3.2.1 B cells in infection

Even before the identification of HIV as the causative agent of AIDS (17), aberrant B cell activation leading to non-specific Ab production and hypergammaglobulinaemia alongside reduced T cell-dependant B cell activation were identified as hallmarks of

AIDS (325, 335). These are characterized by increased levels of Abs and autoreactive

Abs in serum (336) and unchecked B cell proliferation (337-340), coinciding with an increased expression of terminal-differentiation plasmablast markers and lowered expression of CD20 and CD21 (341-343). Despite high activation, the number of total B cells is reduced (341, 344-347). On initiation of ART, B cell dysregulation is mostly reversed, although the B cell memory compartment remains difficult to detect (348-350).

The memory pool of B cells likely contains valuable information on the progression of the Ab response that may provide information on HIV viral evolution. Using flow cytometry-based single memory B cell sorting and culture (351), the memory B cell repertoires were detailed in the blood of HIV+ donors (352). It was shown that the memory B cells specific for HIV envelope had higher somatic mutation rates than non-

HIV-specific memory B cells. This is perhaps indicative of a chronic immune response

26 that is constantly evolving to ‘keep up’ with the mutating envelope of the virus (353,

354). This study described Ab specificities throughout infection and has indicated a need for more archeological Ab studies.

1.3.2.2 HIV-specific antibody responses

The binding and conformational changes required for entry translate to several constraints on gp120 and gp41 sequence and structure, theoretically allowing for the development of a neutralizing Ab response. Despite this, the development of neutralizing

Abs in infection, especially autologously neutralizing Abs, arises later in infection and occurs only in approximately one quarter of infected individuals (355-361). Perhaps the most overwhelming questions currently facing the field concern the definition and value of an HIV-1 cross-subtype neutralizing response before and during infection.

Role of Abs in progression: Early dysfunction in B cell responses is due to initial destruction of infected Th cells that would normally stimulate an appropriate B cell response and/or due to some other as yet undefined mechanism (349). Several studies have shown a protective role for neutralizing Abs in progression to AIDS, although most evidence is circumstantial. Whether neutralization is provided by a mAb or a polyclonal subset of cross-reactive Abs is unknown, although a growing body of evidence suggests the latter (352, 362). A study of plasma transfusion from healthy HIV+ patients to symptomatic HIV+ patients suggested that Abs may play a role in slowing disease progression without preventing it (363). Long-term survivors with undetectable viral load and stable CD4 counts for over 12 years were shown to have both robust CTL and

27 neutralizing Ab responses (364). While Ab responses, particularly neutralizing ones, take a long time to develop in acute infection (range from 4 days to 45 weeks), the sera of some LTNP contain autologous neutralizing Abs (238, 365). Some of the most convincing data regarding the role of neutralizing Abs in slower disease progression came from a comparison of LTNPs and RP over 8 years. Near seroconversion, both patient groups exhibited a rise in neutralizing Abs followed by a decline. In LTNP, however, 1-4 years after the initial neutralizing Ab titres, neutralizing Abs resurfaced while the levels in RPs remained low or undetectable. The neutralizing Ab response in

LTNP was associated with elevated CD4 counts and proliferation, but there was no indication whether this was a cause or effect of viral suppression (366). In a subset of individuals who were taken off ART, viral rebound preceded a reduction in viremia that coincided with an increase in serum neutralizing Abs (367).

The positive association of neutralizing Abs with infection status is not always found.

Some studies have suggested that Abs play no role in control of viremia in EC populations (341, 355, 361, 368, 369). One such study is a very comprehensive cross- sectional study analyzing the neutralization breadth of sera from 103 infected individuals at varying stages of progression (361). Neutralizing Abs were able to develop in individuals with higher viral load due to prolonged exposure to antigen. Ab responses shape infection, and protective or not, place immune pressure on the infecting virus, as some data suggest that Ab responses lead to escape mutations in envelope (182, 370-

379). HIV-1 is able to quickly mutate and escape neutralization (182, 370-379). In some studies, escape from Ab responses is associated with an increase in the length of the

28 gp120 V regions and in the number of glycosylations sites (376, 380-384). Both these mechanisms likely act to further shield important functional or structural aspects of the gp120 (such as the CD4 binding site) from the humoral immune response seemingly without affecting viral fitness (383-385).

Neutralizing, and perhaps more importantly, cross-protective neutralizing Abs, are rarely measurable or exist at very low titres in the sera of infected individuals, but this does not undercut the importance and value of these Abs to instructing vaccine design (356-363,

379, 386-389). The Ab synthesis and repertoire of these individuals deserve study and are a model for a protective Ab-based vaccine.

Protection from pre-existing Abs: Despite the ambiguous evidence for the role of neutralizing Abs in HIV-1 progression, it has been shown that transmitted or founder virus and virus from acute infection are more sensitive to neutralizing Abs, indicating that pre-existing antibody responses could play a crucial role in protection from infection

(121). In some individuals who display natural resistance to infection by HIV (HIV exposed seronegative, or HESN), there is controversial evidence of HIV-specific neutralizing, transcytosis-inhibiting IgA at the genital mucosa (322, 323, 390-394).

However, whether or not all exposed, uninfected people express neutralizing Abs may not be the right focus of this debate, but rather what the specificity and quality are of Abs from those who do. Although it is still debated (324, 395-399), it has largely been rejected that Abs alone are sufficient to protect from vertical transmission (400-404). It was shown that the viruses involved in vertical transmission are resistant to neutralization

29 by the mother’s serum Abs while non-transmitting mothers were more likely to have autologously-neutralizing serum Abs (122-124, 405, 406). This suggests that the maternal Ab repertoire may impose limitations on viral growth and exert pressure on the antigenic sequence of perinatally-transmitted virus.

Vaccine and therapeutic potential: There is a considerable body of evidence that HIV-1- specific neutralizing Abs can protect from infection (407-409). In vivo animal studies have been useful in showing that sterilizing immunity with passive immunization (PI) of neutralizing mAb is achievable, as shown in humanized severe combined immunodeficient (SCID) mice with HIV-1 neutralizing mAb IgG1 b12 protection of the infused, challenged mice (407, 408) (more discussion on neutralizing mAbs is in section

1.3.2.3). Non-human primates are protected from infection by PI of neutralizing mAbs

(410-419) and lowered viral set point was seen in breakthrough infections (420). Perhaps the most biologically relevant studies of neutralizing mAb PI involve vaginal protection from infection, which represent (a) a natural route of transmission and (b) the potential to block transmission by vaccine-induced neutralizing Abs or microbicides containing neutralizing Abs (411, 415, 416).

Some studies contradict the protective effect of PI, especially in established HIV-1 (or

SIV) infection. A study of humanized SCID mice with an established HIV infection showed short term or no viral load reduction when neutralizing monoclonal Abs were administered, likely due to the developed escape mutants (421). Rhesus macaques progressing to disease were infused with purified Ig from chronically infected macaques,

30 resulting in a three-fold reduction in cell-associated viral load via ADCC, which was ultimately not protective for these animals (422). Perhaps a shortcoming of many experiments is the assumption that normal progression in macaques is related solely to neutralizing Abs. A more reasonable hypothesis is that neutralizing Abs are involved in viral suppression but they do not act alone, or by a single mechanism. In two recent human trials, infected individuals received PI of 4E10, 2F5, and 2G12, or 2F5 and 2G12 neutralizing mAbs. The 2F5/2G12 trial was performed in asymptomatic individuals and showed transient reduction in viral load concurrent with a rise in CD4 count and a burst of complement activity (423). The trial with the three mAb regimen required patients to halt ART while receiving the mAbs (424). Mirroring the previous trial, there was a delay in viral load rebound after ART cessation, suggesting a short-term reduction in viral replication. PI, in these instances, was mostly ineffective and highlights the need for more research into PI and the ensuing immune response, the Abs that are suited to this method, and mechanisms of escape.

There is a growing body of evidence that non-neutralizing Abs can also contribute to protective immunity. Despite an overall lack of protection, the VAX004 recombinant gp120 B/B vaccine (425) elicited ADCC responses in a subset of individuals, which correlated with a reduction in infections (321). Similarly, analysis of sera of individuals immunized in the ALVAC/AIDSVAX B/E combination trial suggests that Abs raised to the vaccine were neutralizing and capable of ADCC (426, 427). This is consistent with two studies of HIV+ subjects where more than half had detectable ADCC responses (428,

429). The study of protective functions of HIV-specific Abs (including ADCC,

31 phagocytosis, and complement activation) is an emerging field that will add important and previously overlooked information of Ab function both in illness and vaccination.

Whether or not non-neutralizing Abs play a role in enhancement of HIV infection is controversial. It has been suggested that Abs and molecules (such as soluble CD4) at low concentrations bind the HIV envelope trimer and expose the coreceptor binding site and increase infectivity of some HIV strains (430). The failed gp120 vaccine trials showed no overall enhancement of infection (431). In light of this, there is little evidence that

Ab-based vaccine design and therapeutics will lead to enhancement of HIV infectivity.

1.3.2.3. HIV-specific monoclonal antibodies

HIV entry can be blocked at various stages of entry by several mAbs targeting various epitopes of gp120 or gp41. The isolation and measurement of the ability of these mAbs to neutralize viral infection in vitro and in vivo in animal models has been described extensively ((423, 424), reviewed in (387)). Neutralizing mAbs are also able to block syncytium-based viral spread, a mechanism previously thought to hide virus from the Ab response (432, 433). It is generally accepted that neutralizing mAbs are able to bind to the oligomeric trimer on HIV, while non-neutralizing mAbs only bind to gp120 monomers or defective trimers (434-436). A recent study suggests that some powerful

HIV-1 neutralizing mAbs can achieve irreversible neutralization in vitro by inducing shedding of gp120 from the viral spike (437). This renders the spike incapable of fusion with the target cell membrane and inactivates the virion. Infection-neutralizing mAbs can bind the glycosylation on gp120 (438), the variable regions on gp120 (107), the CD4-

32 binding site (CD4BS) on gp120, intermediate conformations of the envelope after binding of gp120 to CD4 (439), a membrane-proximal region on gp41 (440-443), or can be quaternary structure dependant and bind only the intact trimer (444). There is potential for mAbs to be potent tools for immune therapies themselves while insight into their antigenic specificities and biological functions educate Ab-based vaccine design.

1.3.2.3.1 IgG1 b12 and CD4 binding site monoclonal antibodies

1.3.2.3.1.i IgG1 b12 activity

IgG1 b12, or b12, is one of the most studied monoclonal Abs to date and is the focus of this thesis. The Fab heavy and light chain fragments of b12 were initially cloned from the Ab repertoire of a long term HIV+ asymptomatic American male by phage display and selected by its ability to bind gp120 (445-447). b12 neutralizes HIV infection by binding gp120 at a conserved site overlapping the CD4BS (200) and so it is unsurprising that b12 is able to broadly neutralize viral strains across clades in vitro (448) (407, 415,

416, 448-459). Although it neutralizes HIV-1 subtype B strains most effectively, there is evidence that b12 also neutralizes primary viral isolates from subtypes A, C, D, E, F, AE, and BG (448, 460). b12 has been shown to block HIV-1 infection in vivo. PI of IgG1 b12 into SCID mice transplanted with human lymphocytes were protected from infection by

HIV-1 even when b12 was administered six to eight hours after inoculation (407, 453).

In two macaque studies, most animals administered b12 intravenously or vaginally were protected from infection on vaginal challenge with SHIV (415, 416). b12 also contributes to protection from infection in studies whereby animals are administered cocktails of neutralizing mAbs (449, 461).

33

It has been suggested that b12-induced irreversible shedding of gp120 contributes to its neutralizing potency (437). Both IgG1 b12 and a human IgA backbone carrying the b12 antigen-binding regions are able to block transcytosis of HIV-1 across epithelial barrier and subsequent infection of underlying cells in vitro (462). A study of several anti-gp120 and gp41 mAbs suggests that b12 is the only mAb able to block gp120 interaction with

DC-SIGN, which suggests an ability to block trans infection of CD4+ T cells via DC- captured virus (463). There is some evidence that b12 is able to bind bivalently to the surface of HIV by engaging two spikes at the same time, increasing its avidity for HIV-1 and further contributing to its remarkable neutralization capacity (159).

1.3.2.3.1.ii IgG1 b12 antigen interface

The epitope recognized by this potent neutralizing mAb has been an important focus for the design of an Ab-based vaccine. Initially, soluble CD4 (sCD4) was shown to block b12-gp120 interactions in an ELISA (445, 452) and so b12 was defined as a CD4BS mAb. b12 binds gp120 with a high affinity (1.1x108 M-1)(452) and induces very little change in the overall gp120 structure (163, 170). Methods used to describe the b12 epitope include competition assays (445, 446), mutational studies (421, 452, 464-468), co-crystallization studies, and a computational docking model (467, 468). Competition assays between b12 and CD4 and b12 and other CD4BS-specific mAbs corroborate the

CD4BS specificity (436, 445, 452, 469, 470). While non-neutralizing CD4BS mAbs do inhibit b12 binding to monomeric gp120, they are unable to block b12 neutralization of

HIV infection in vitro, suggesting different abilities to bind the HIV-1 trimer (469).

34 Shortly after being identified, b12 was tested for binding to gp120 versus gp120 point mutants in ELISA (452), which further confirm that the b12 epitope lies at and near amino acids previously shown to be involved in gp120-CD4 interactions (200).

Sequencing and mutational studies of b12 neutralization escaped virus identified amino acid P369 (part of the CD4BS) and D182 (in the V2 loop) as amino acids crucial to viral neutralization by b12 (464). With the crystallization of b12 came the computer docking studies whereby the structures of b12 and gp120 were aligned and potential interacting residues were identified (468). These putative amino acid binding points were validated and greatly expanded the size and complexity of the b12 epitope while again suggesting that the b12 interacts with the CD4BS. The V1 was also implicated in the b12 epitope.

Alanine-scanning mutagenesis of gp120 identified more amino acids important for b12- gp120 interactions (466). A sub-study of some of the b12-bound gp120 variants suggested that only just under two thirds of the variants had corresponding changes in b12 neutralization sensitivity. This suggests that some binding points may be unavailable on gp120 within the intact trimer. Again in this study, both of the V1/V2 and V3 deletion mutant showed reduced b12 binding. The V1/V2 and V3 loops are proximal to the

CD4BS and undergo important conformational changes upon CD4 ligation (471-475).

Mutation of N-linked glycosylation sites on gp120 also altered binding. Fluctuations in the overall structure of gp120 alters b12 access to its epitope. Together, these studies demonstrate that b12 binds the CD4BS (199, 476, 477) at a conformational epitope on

HIV-1 gp120 (452, 458, 466, 468, 478-483). The gp120-b12 fab co-crystallization studies also suggest that b12 binds only to the outer domain of the gp120 structure and only through its heavy chains (Figure 1) (163). Conversely, alanine-scanning mutagenesis of

35 b12 suggests that the L1 segment of the b12 mAb is involved in interactions, as well as amino acids within the CDR3 of the heavy chain (484). The epitope binding site (or paratope) of IgG1 b12 is also unique as it has an extended CDR3 region that allows it to insert into the F43 of gp120 (484), and an asymmetrical structure that may allow it to contact gp120 from two separate trimers at once (159, 485).

Despite all of the b12 epitope information gathered thus far, crystal studies utilized a highly modified gp120 construct and mutational studies could not distinguish between epitope sites and structurally important sites that are not part of the epitope. Therefore, epitope mapping studies using native gp120 are needed to gain more information on the b12 binding site on gp120.

1.3.2.3.1.iii Other CD4BS mAbs

B cells isolated from HIV-1 infected individuals allowed the discovery of a new CD4BS mAb, HJ16. The amino acid determinants of the HJ16 epitope appear to be different from b12 (486) and it has a broad but unique HIV-1 neutralization profile (487). VRC01 is a newly discovered broadly neutralizing mAb purified from an HIV+ individual (488).

VCR01 binds to gp120 at the CD4BS in a configuration reminiscent of CD4. It binds with comparable affinity to b12, but contacts gp120 with both heavy and lights chains, unlike b12. This might be a reflection of the fact that VRC01 represents a naturally- occurring Ab purified from an infected individual, whereas b12 was created by random assembly of a heavy and light chain pair that bound gp120 (445-447). VRC01 competes

36 with b12 for binding to gp120 but is able to neutralize many more primary isolates

(neutralization of 90% of a panel of circulating viruses versus 40%, respectively) (470).

Other CD4BS-specific Abs were generated at the same time as b12. Among them, IgG1 b6 (b6) has often been assayed alongside b12 as a poorly HIV-1-neutralizing CD4BS mAb control, in that it only neutralizes a small range of subtype B viruses that are also neutralized by b12 (445, 448, 452, 489). There is evidence that b12 binds the HIV-1 envelope trimer while b6 does not (490, 491), which may explain why, despite competing with one another for gp120 binding, b6 does not inhibit b12 neutralization (492).

Interestingly, b6 does induce partial shedding of gp120 from the HIV-1 envelope trimer, indicating that some weakly neutralizing Abs may also participate in this irreversible

HIV-inactivating phenomenon (437). This also suggests some shared b12 and b6 abilities despite the divergence in neutralization profiles. Although many do, not all amino acid changes in gp120 that affect b12 also affect b6 binding (466, 488, 489). This suggests that despite the epitope overlap between b12 and b6, there are differences in their specificity for gp120 and/or the HIV-1 trimer that may account for their differences in HIV-1 neutralization.

Anti-CD4 binding site Abs have been found in the serum of many individuals. A study of gp120 antigen-depleted sera from ART naïve HIV+ individuals showed that the two most broadly neutralizing sera were defined by high levels of CD4BS-specific Abs (493).

1.3.2.3.2 HIV-1 monoclonal antibodies of other specificities

37 IgG1 2G12 is one of the more potently neutralizing mAbs identified to date. It recognizes mannose-dependant glycosylations of the gp120 C2, C3, C4 and V4 regions

(438, 494-496). In phase 1 clinical trials, PI of 2G12 into HIV+ subjects was well tolerated (497), and in 2005 the 2G12, 2F5, and 4E10 mAbs were administered to HIV+ patients undergoing structured treatment interruption. mAb PI delayed but did not prevent viral rebound. The rebounding virus had mutated and was no longer susceptible to 2G12 neutralization, suggesting that 2G12 was responsible for the delay and that the virus was capable of 2G12 escape (424). Attempts to elicit 2G12-like Abs in animals have not yielded 2G12-like Ab responses (498, 499). The structure of 2G12 is unlike any described Abs to date in that its variable heavy (VH) domains are swapped between antigen binding arms. This renders the antigen binding sites rigid with respect to one another and allows the mAb to recognize the large glycans with greater avidity. This structure is unique to 2G12 and may therefore be essential for its specificity and powerful neutralizing function (500, 501).

gp41-specific neutralizing mAbs 2F5 (440-442, 502) and 4E10 (443) have been studied extensively for their mechanism of action and epitope specificity. Both Abs induce irreversible shedding of gp120 from the viral spike, which likely plays a large role in their neutralization potency (437). 2F5, when pre-infused into chimpanzees, was not protective of subsequent intravenous HIV-1 challenge, but this neutralizing mAb, despite not providing infection protection, affected positively the onset of acute infection and subsequent viremia (418). HIV+ patients infused with 2F5, 4E10, and 2G12 showed delayed viral rebound on antiviral cessation, but this was likely owing to the 2G12, since

38 after rebound, the circulating viruses displayed continued neutralization sensitivity to 2F5 and 4E10, but escaped 2G12 (424). Both mAbs bind within the 30 amino acid-long gp41 membrane proximal external region (MPER) (503). It has been suggested that both mAbs bind phospholipid in order to gain access the MPER, and it is still debated whether binding to the HIV-1 membrane contributes to their neutralizing capacity (218, 504-509).

The 2F5 MPER epitope was confirmed by mass spectrometry (510) and 4E10 epitope mapping was performed using a similar mass spectrometry technique as was used in this study (218). Mass spectrometry mapping studies expanded the 4E10 epitope map to include sequences outside the MPER, which will be discussed in greater detail in the

Discussion section 6.2.2. 4E10 has been crystallized in conjunction with the MPER, although the epitope required some sequence/structure alterations to improve crystallization (511). A third moderately neutralizing gp41 mAb, Z13e1, was shown to bind a sequence overlapping the 2F5 and 4E10 epitopes (512). Although they share similar binding affinities, what moderates the differences in the three gp41 mAbs’ abilities to neutralize HIV-1 is unclear and may involve as yet undescribed epitope determinants or mAb structure differences. gp41 Abs have been difficult to detect in infected sera (513); it was not until recently that MPER-specific Abs were identified in sera of a small number of infected individuals (389, 514, 515). This difficulty may be related to controversial reports in the HIV mAb field with regards to autoreactivity. In addition to phospholipid, it has been suggested that 2F5 and 4E10 are unsuitable targets for vaccine design because they bind to cardiolipin (516, 517). It has been hypothesized that autoreactivity explains why Abs with certain specificities are largely absent from

39 infected sera (516), although it may be because the MPER is sterically inaccessible on the intact trimer (159, 424, 518).

CD4 induced co-receptor binding site (CD4i) Abs neutralize HIV-1 by binding the gp120 co-receptor binding site. A well-studied CD4i mAb, 17b, and a newer mAb, E51, were shown to neutralize various HIV-1 strains only to a moderate degree (439, 519). This site is occluded on the unliganded HIV-1 trimer, and mAb activity is therefore optimal after engagement of CD4 with gp120. The gp120-CD4 interaction is quite stable and theoretically the CD4i epitope could be exposed for enough time to mount an Ab response (101). However, when gp120 binds CD4 on the surface of a cell, steric hindrance does not allow full-sized Ab to interact with the CD4i epitope (520). This can be overcome by fragment antigen-binding (fab) or a single chain Ab (scFv) or by temporary dissociation of the gp120-CD4 connection (520-522). It is therefore hypothesized that the function of such Abs is to block the evolution of CD4-independant virus (523). In several studies, the majority of infected study subjects tested had CD4i- specific Abs in their sera (352, 362, 523), but another study that putatively identified

CD4i Abs from HIV+ sera found them to be non-neutralizing (513).

While the majority of broadly neutralizing mAbs identified to date bind both the trimer and the gp120 or gp41 monomer, recent advances have allowed the isolation of trimer- specific quaternary structure-dependant mAbs (444, 524). 2909 is a highly type specific mAb that neutralizes few HIV-1 isolates potently (524). Although it is an interesting case study, the lack of broad neutralization limits its use in vaccine design. A study of

40 HIV+ individuals with broadly cross-reactive neutralizing sera generated PG9 and PG16 mAbs (444). Both mAbs are trimer-specific, potently neutralize across HIV subtypes

(525), and provide further evidence that HIV+ individuals do express Abs to quaternary epitopes (526). The complexity of their epitopes makes quaternary structure binding sites a difficult target for vaccine design.

The gp120 V regions mutate at a high rate, most likely to escape the Ab response (171).

The V regions are also immunodominant, in that they elicit the majority of the strain- specific Ab responses. Therefore, although mAbs to the variable regions can neutralize individual strains of HIV, they generally do not neutralize HIV-1 across clades (527,

528). There is some evidence that V3-specific Abs can provide some measure of protection (529, 530), although it has been suggested that V3-specific Abs in infected individuals are less cross-reactive than, for example, CD4 binding site-specific Abs

(379). For instance, the mAb 447-52D targets a conserved sequence at the V3 and its neutralization is restricted to clade B strains (448, 529, 531). There are efforts to generate Ab responses to the V3 region as it has some sequence constraints to maintain its structural integrity for chemokine receptor binding (190, 531).

To date, there exist a number of broadly neutralizing mAbs specific for many regions of the HIV-1 envelope and that affect different steps of HIV entry. These Abs, their epitopes, and their mechanisms of action are an important source of clues in the search for a protective HIV-1 vaccine.

41 1.3.2.4 HIV-1 evasion of antibody responses

There are three main features of gp120 that make it a poor target for robust neutralizing

Ab responses: sequence diversity, glycosylation shielding, and gp120 conformational masking and shedding (107, 532). As discussed earlier, the HIV RT is error-prone and gp120 can accept a large number of mutations, especially in the V regions, while maintaining function (171). The V regions form loops and V1-V4 are exposed on the envelope surface (179), occluding the constant regions from the immune system, even on monomeric gp120 (171, 474). High mutation rates (>5% within an individual/year) occur especially along sequences of gp120 that are exposed to Ab binding (375, 533, 534).

Thus, Ab responses are constantly being evaded (354, 370-378). Even so, cross-reactive

Abs are a promising tool for Ab-based vaccine design.

The second main feature of gp120 is its heavy glycosylation. Glycans are added to the spike proteins gp120 and gp41 by the host cell machinery. They create a shield as the immune system generally sees glycans as self molecules (371). Ab responses to sugar moieties are often weak or absent, and these Ab responses are not maintained by immune memory. Glycosylation patterns can also alter intracellular epitope processing for antigen presentation to Th cells (535). Importantly, resistance to the glycosylation- binding mAb 2G12 arose quickly in PI HIV+ subjects, suggesting carbohydrate-based Ab responses are easily evaded by HIV-1 (424). More research is required to fully understand how the immune response can overcome the HIV-1 gp120 glycan shield.

42 The third Ab-evasive feature of gp120 is its conformational masking and shedding, which leads to an accumulation of free monomeric gp120. Gp120 is highly flexible and there are thought to be many conforms of gp120 in existence at one time, both within the trimer and shed from the HIV surface, resulting in a variability in the accessibility of antigens (170, 536, 537). Shedding leads to a decrease in functional trimeric spikes on the surface of HIV. Shed gp120 may bind neutralizing Abs and render them inactive against the virus (538) and monomeric gp120 displays to the immune system a surface that is normally buried within the HIV surface trimer, eliciting Ab responses to non- neutralizing, otherwise inaccessible epitopes (171, 474). Circulating monomeric gp120 may also contribute to the activation of bystander T cells by interacting with CD4 or by other mechanisms, creating an undesired milieu of immune activation and rendering local

CD4+ T cells more susceptible to productive infection (539). Conformational plasticity, shedding, high mutation rates of the HIV envelope, and the dense coverage of the trimeric surface by mannose oligosaccharides make the HIV envelope a poor, ever shifting, target for the development of a successful Ab response.

1.4 HIV vaccines – trials and tribulations

1.4.1 HIV vaccine design

While the mechanism of action of some vaccines is still undefined (pertussis, typhoid, and live-attenuated influenza), to date, many effective vaccines function by eliciting neutralizing Abs in the human host (polio, rabies, and inactivated influenza) (540, 541).

A study of the effect of the smallpox vaccine showed that while no doubt T cells play a role in protection, the neutralizing Ab response was crucial for protection (542). Studies

43 of the immune response generated by the yellow fever vaccine, the Human Papilloma

Virus (HPV) vaccine, and the Hepatitis B Virus (HBV) vaccine show that all rely on neutralizing Abs for protection (543-546). Ab-based vaccine design has until now been quite successful, and this focus should continue on to HIV-1 vaccine design.

The immune mechanisms of protection from HIV-1 are still being defined and debated.

The first ever HIV-1 vaccine phase III trial (AIDSVAX; VAX003 and VAX004) consisting of monomeric gp120 immunizations generated neutralizing Ab responses

(547), but overall the vaccine proved to be ineffective with a 6% protective effect (425,

548-550). Vaccinees with low levels of gp120-specific responses had a moderately higher relative risk of infection compared to placebo recipients. There may therefore be a threshold level of protection that must be reached by a vaccine below which susceptibility to infection is increased. It was shown recently that Abs to gp160 persisted in more than 80% of vaccinees 8 years later (551). This injects hope that a vaccine can induce long lasting neutralizing Abs. A phase I trial (DP6-001) showed that a gp120

DNA prime, multivalent gp120 protein boost generated stronger cross-clade neutralizing

Ab responses than previous vaccine attempts (552, 553). The design of an HIV-1 vaccine capable of eliciting neutralizing Abs able to block infection will require a more in depth study of the mechanisms of Ab-virus interactions and more trials with different types of

HIV-1 envelope immunogens.

For many years much effort was put into developing vaccines to induce CTL responses to

HIV-1. The recent T cell-based phase IIB trial in humans tested the efficacy of a

44 Adenovirus 5 vector expressing Gag, Pol, and Nef (MRKAd5 HIV-1 gag/pol/nef) was halted prematurely, as the vaccinated group were not protected from HIV-1 infection, and in fact may have been at higher risk than the placebo group (554).

One of the most effective vaccines to date, the yellow fever vaccine, has recently been shown to induce a multi-parameter-type immune response (555-557). The most recent

HIV-1 vaccine phase III trial data is from the RV144 trial. The trial was the first of its kind – it was intended to elicit both T and B cell responses. The vaccine combined the

ALVAC canarypox vector consisting of HIV-1 subtype AE gp41 and B gp120, Gag, and protease prime and an AIDSVAX B/E gp120 boost. The vaccine provided just over 31% protective effect (558). Many hailed this trial as a major advance as it is the most effective vaccine to date and indicates that the field is inching nearer to a fully protective vaccine and that a vaccine that elicits protective CTL and Th responses in conjunction with Ab responses is a realistic goal.

1.4.2 Antibody-based vaccine design and epitope mapping

With the failure of Ab-based vaccines to date, a more rational design of Ab-inducing vaccines is required (548). Attempts to design better gp120, gp41, and trimer antigen constructs have provided a wealth of information about the nature of HIV Ab responses.

Trimeric envelope antigens are being designed and tested for their ability to elicit neutralizing Abs that can bind the intact trimer (559, 560). gp120 engineering has attempted to alter the structure of monomeric gp120 in order to elicit certain neutralizing

Ab specificities. Immunofocusing may be a useful tool for eliciting responses that echo

45 the broadly neutralizing mAbs that have been described to date. A molecule is thus engineered such that only the ideal target Ab recognizes it. This new form of the antigen can be tested for immunogenicity in order to see if it induces an Ab response with a similar potency as the mAb of interest. Many attempts have been made to engineer gp120 to bind solely to the potent neutralizing mAb b12 (478, 479, 481, 561), but to date, none of these antigens have elicited b12-like responses in animals (562).

Initial epitope mapping can performed by competition assays as was the case with b12, gp120 and CD4. CD4 inhibited b12 binding to gp120, and so b12 was defined as a

CD4BS mAb. Studies on HIV-specific monoclonal panels that were cross-competed for binding have yielded much information about antigen specificity of both neutralizing and non-neutralizing Abs (436, 448). Deletions or mutations of portions of the antigen and subsequent binding assays can also lend information on the epitope (466). A method has been described for general Ab characterization of a mixed pool of Abs (such as in serum) whereby carrier beads are coated with antigen and the Ab-specificities of choice can be depleted/purified (388). Another method with similar tactics is the purification and description of HIV-specific memory B cells (351, 352). An advantage of these methods is that the subjects from whom samples are taken can be pre-screened for clinical parameters, such as disease progression, in an attempt to purify and describe clinically protective Ab responses. Epitope mapping is an ever-expanding field that allows for better rational design of Ab-based vaccines for HIV.

46 The gold standard for more specific epitope mapping is mutational studies. Antigen point mutations allow for identification of precise amino acid residues involved in Ab-epitope interactions. Alanine-scanning mutagenesis is an elegant form of this technique, and has been applied to b12 mapping (466). The shortcoming of these assays is that they identify amino acids that participate in the epitope as well as amino acids that can lie far outside the epitope that are important for the structural integrity of the epitope (489). Co- crystallization allows delineation of the Ab-antigen interface with each molecule in its tertiary structure. The pitfall of this method, particularly with the b12 fab-gp120 co- crystallization, is that the assay involved a highly altered antigen. The gp120 that is used for crystallization is a highly modified core structure, lacking portions of the variable and constant regions and containing extra disulfide bonds that modify the structure and lock it in a “CD4-bound” state and restrict movements between the inner and outer domains

(163, 563). Phage and yeast display of antigens (or cloned Abs) to pan out antigen- specific Abs have also successfully generated new insights into Ab repertoires (564-566).

Peptide epitope libraries can also be screened this way, or by more conventional binding formats such as ELISA, but binding assays involving overlapping peptides of the antigen may garner information about linear epitopes only (379, 567). For all the epitope mapping experiments that have been applied to b12, none have identified an epitope that elicits neutralizing Abs in animal models, suggesting that more information, perhaps using newer methods of epitope mapping of the b12 epitope, is required.

The use of mass spectrometry in Ab epitope mapping is a relatively recent concept (385,

568-572). Mass spectrometry has been useful in determining the precise amino acid

47 sequence recognized by the HIV-1 IIIB p26-specific murine mAb 13-102-100 (572),

4E10 (218), and 2F5 (510, 570). In fact, the paper by Parker et al. further refined the 2F5

MPER epitope NEQELLELDKWASLWN to its core epitope, ELDKWA (510). Unlike other epitope mapping techniques, mass spectrometry epitope mapping is the most direct method of defining Ab specificity on an intact antigen. Therefore, it is important to examine the b12 gp120-binding region to gain precise information about the epitope that may guide vaccine design. In this study, matrix-assisted laser desorption/ionization

(MALDI) quadrupole-time of flight (QqTOF) mass spectrometry (MS) epitope mapping was performed. A solid surface (plate) is coated with an immobilized Ab-antigen complex and is inserted into the mass spectrometer. A laser excites the sample; the small, non-covalently linked antigen is ejected from the plate and the Ab, is singly charged, and is directed into the machine. The time of flight of the charged epitope to the detector is directly proportional to its size and the readout is expressed as mass/charge ratio, where charge = 1. There are two forms of MS epitope mapping: epitope excision and epitope extraction. Epitope extraction works best when mapping linear epitopes; the antigen is pre-digested before incubation with the immobilized Ab and the resulting peptide fragments that bind the Ab encapsulate the epitope. Epitope excision is suited for conformational epitope identification, but can also identify linear epitopes (573). Epitope excision allows for the incubation of the native antigen with the Ab before digestion with proteolytic enzymes. The epitope is protected from digestion and can be detected by the mass spectrometer. The studies reported in this thesis employed epitope excision to map the epitope of the well-described mAb IgG1 b12 to gain information on its conformational CD4BS epitope on HIV-1 gp120.

48

Once identified, the epitope underwent confirmation studies and sequence analysis to determines its relevance to a globally relevant vaccine. The epitope was tested for its ability to elicit Abs in mammalian models, as is the standard for novel epitope discovery.

The induced Abs were tested for in vitro neutralization of HIV. If these steps lead to the discovery of an epitope that can elicit Abs that neutralize HIV-1, then that antigen can go on to studies in larger animal models, primates, and eventually humans for safety and efficacy trials.

49 Section 2.0 Hypotheses

With the failure of gp120 immunogen-based vaccine trials, there has been a shift in focus in Ab studies to identify specific antigens of potently neutralizing Abs. The IgG1 b12 epitope has been studied extensively, but no immunogen constructs have been able to elicit b12-like Abs in animal studies. This is likely in part due to the conformational structure of the b12 epitope, and in part due to the hidden nature of the CD4 binding site within the gp120 trimer structure. The majority of b12 epitope mapping studies identifying epitope-binding sites employed either the core gp120, which lacks major portions of the entire molecule, or were indirect mutational studies that only truly identified amino acids important to the structure and accessibility of the b12 epitope. It is for these reasons that the IgG1 b12 epitope is the focus of highly specific and sensitive mass spectrometry epitope mapping studies reported herein. The general hypotheses for this study are as follows:

i. Epitope mapping of IgG1 b12 will identify contacts points between b12 and

HIV-1 gp120.

ii. Antigenicity of the regions of HIV-1 gp120 identified as part of the b12

epitope will be confirmed using traditional immunologic assays.

iii. Sequence variation of the b12 epitope in viral strains will correlate with

sensitivity to b12 neutralization, supporting its role in b12 neutralization of

50 viruses. Since b12 neutralizes across HIV-1 clades, the epitope will be well-

conserved.

iv. Immunogenicity of the b12 epitope will be demonstrated in animal

immunizations. The epitope will elicit a b12-like neutralizing Ab response.

51 Section 3.0 Specific Objectives

With the above hypotheses in mind, we developed the following specific objectives:

i. map the epitope of IgG1 b12 by mass spectrometry epitope excision and

confirm with tandem mass spectrometry sequencing

ii. with a peptide of the putative epitope, confirm the mapping results in b12

ELISA binding assays to determine that the epitope is truly recognized by b12

iii. analyze the available sequence information for the epitope and compare the

variation within the described epitope with published neutralization data and

determine the suitability of the epitope for cross-clade immunogenic potential

iv. test the immunogenicity of the epitope in multiple animal models and measure

the ability of immunized animal sera to neutralize HIV-1 in vitro.

52 Section 4.0 Materials and Methods

4.1 General materials

4.1.1 Solutions

4.1.1.1 Mass spectrometry

Coupling Buffer

“Buffer A” 0.1 M NaHCO3, 0.5 M NaCl

33.616 g NaHCO3, 116.88 g NaCl, 4 L ddH2O

“Buffer B” 0.1 M Na2CO3, 0.5 M NaCl

10.599 g Na2CO3, 29.22 g NaCl, 1 L ddH2O

Use Buffer B to adjust the pH of Buffer A to between 8.32 and 8.45

HCl

HCl concentrated, 1 mM

43.1 µl of 11.6N HCl, 500ml ddH2O

Acetate Buffer

0.1 M NaOAc, 0.5 M NaCl, pH 4.0

6.8g NaOAc 3H2O, 14.61g NaCl, Add 400ml H2O, adjust pH with acetic acid to 4.0

(very slowly). Total volume to 500ml with water.

Ethanolamine

1 M Ethanolamine

53 To 70 ml water add 6.2344 ml stock ethanolamine (16.04 M). Adjust pH with concentrated HCl to 8.0. Final volume to 100 ml with water.

10% Sodium Azide Stock Solution

10 mg NaN3,100 µl ddH20

Phosphate-buffered saline (PBS)

48.5 g PBS powder (contains 137.93 mM NaCl, 2.67 mM KCl, 8.1 mM Na2HPO4, 1.47 mM KH2PO4) (Invitrogen, Burlington, Ontario) per 1 litre ddH2O

Tris Buffered Saline (incubation and wash buffer)

25 mM Tris, 150 mM NaCl, pH 7.5

1.25 ml 1 M Tris pH 7.5, 2.5 ml 3 M NaCl, 46.25 ml H2O to 50 ml final volume

Mass Spectrometry Digestion Buffer

100 mM NH4HCO3

0.8 g NH4HCO3, 100 ml ddH20

Mass Spectrometry Wash Buffer

100 mM NH4HCO3, 10 mM n-Octyl-beta-D-glucopyranoside (BOG) detergent

0.145 g BOG, 50 ml Digestion Buffer

Dihydroxy Benzoate (DHB) matrix solution

54 0.08 g DHB, 250 µl Acetonitrile, 250 µl ddH20

4.1.1.2 Gel electrophoresis

SDS PAGE Gel (7.5%)

Separating gel

2.5 ml 1.5 M Tris pH 8.0, 0.1 ml 10% SDS, 2.5 ml acrylamide, 0.005 ml TEMED, 0.5 ml

Ammonium persulphate, 4.4 ml ddH2O

Stacking Gel

1.25 ml 1.5 M Tris pH 8.0, 0.05 ml 10% SDS, 0.65 ml acrylamide, 0.005 ml Temed, 0.25 ml Ammonium persulphate, 3.05 ml ddH2O

Running buffer

30.3 g Tris base, 144 g Glycine, 10 g SDS, make to 1L with ddH2O

Loading buffer 3X stock

1M Tris pH 6.8 2.4 ml, 3 ml 20% SDS, 3 ml Glycerol (100%), 1.6 ml B- mercaptoethanol, 0.006g Bromophenol blue

Stains

Coomasie Blue Brilliant R-250 staining solution (Bio-Rad, Hercules, CA)

Destain - 5% Acetic Acid in ddH20

Bio-Rad Silver Stain Kit (Bio-Rad, Hercules, CA)

55 4.1.1.3 Enzyme-Linked Immonosorbant Assay (ELISA)

Coating buffer

1.59 g Na2CO3, 2.93 g NaHCO3, 1 L ddH2O, pH 9.6

P24 ELISAs only Coating buffer

5.3 g Na2CO3, 4.3 g NaHCO3, 1 L ddH2O, pH 9.6

Blocking buffer

PBS, 10% Bovine Serum Albumin

10 g BSA in 100 µl PBS

Glycine Buffer

1 M glycine buffer in ddH2O, pH 2.5 with HCl

P24 ELISAs only Blocking/Dilution buffer

PBS, 2% Goat Serum, 0.01% polyoxyethylene sorbitan monolaurate (Tween 20)

Add 20 ml goat serum and 100 µl Tween 20 to 1 litre PBS

Dilution buffer

PBS, 1% Bovine Serum Albumin, 0.05% Tween 20

10 g BSA and 500 µl Tween 20 in 1 L PBS

Wash Buffer (all ELISAs)

56 PBS, 0.05% Tween 20

500 µl Tween 20 in 1 L PBS

DEA (all ELISAs)

0.101 g MgCl2•6H2O, dissolve in ddH2O, 97 ml Diethanolamine (Fisher Scientific,

Ottawa, Ontario), up to 1 L ddH2O, pH 9.8 with HCl

1 Sigma 104 substrate Tablet (Sigma, Oakville, Ontario) per 5ml DEA buffer

ABTS (all ELISAs) (see 4.1.3)

ABTS buffer (Roche, Mississauga, Ontario)

Add 1 Tablet per 5 ml ABTS buffer (Roche Diagnostics, Canada, Laval, Quebec)

4.1.1.4 Western blot

PBS 0.05% T20 (PBST)

To 1 L PBS add 500µl tween 20

Blocking buffer

Dissolve 10% Skim milk powder (Sigma, Oakville, Ontario) in PBST

4.1.1.5 Serum antibody purification

Tris Glycine buffer

1M glycine in ddH2O pH 3.3 with HCl

57

Neutralizing buffer

1.5 M Tris in ddH20 pH 8.8 with

4.1.1.6 Cell separations/infections

PBS

PBS, 2% fetal calf serum (FCS; Invitrogen, Burlington, Ontario)

Media

Culture media - RPMI 1640 10% FCS 1% penicillin/streptomycin/fungizone (P/S/F)

Hybridoma media - RPMI 1640 2% depleted FCS 1% P/S/F

FCS is depleted by running over a Protein-G Sepharose column (Amersham Bioscience,

Piscataway, NJ).

Supplemented media – RPMI 1640, 10% FCS, 1% penicillin/streptomycin (P/S), 10 u/ml recombinant IL-2, and 2 µg/ml polybrene

Complete media – RPMI 1640, 10% FCS, 20 u/ml IL-2, 1% P/S/F

Complete Growth Media (GM) - D-MEM 10% FBS, 25 mM HEPES (Gibco, Carslbad,

USA) and 50 µg/ml gentamicin (Sigma, Oakville, Ontario)

(unless otherwise stated, all reagents from Invitrogen, Burlington, Ontario)

10% Triton-X

10 ml Triton-X (Invitrogen, Burlington, Ontario) was added to 90 ml PBS

58

Britelite Reagent

Lyophilized Britelite Substrate Solution (PerkinElmer Life Sciences, Massachusetts,

USA) was reconstituted in 250 ml of Britelite Substrate Buffer Solution and stored at

-70°C for up to 1 month. Aliquots were thawed in a water bath in the dark immediately prior to use. The solution was gently vortexed and used within 60 minutes of thawing.

4.1.2 Antibodies and antigens

IgG1 b12, IgG1 KZ52, and IgG1 b6

All three mAbs were kindly donated by C. Barbas III and D.R. Burton, and are extensively described elsewhere (445-447, 452, 574). The b12 and b6 mAb variable regions were cloned from the Ab repertoire of a long term HIV+ asymptomatic American male by phage display and selected for their ability to bind gp120 and compete with soluble CD4 for binding to gp120. b12 is a broadlyHIV-1-neutralizing mAb, while b6 only weakly neutralizes a small number of HIV-1 strains. IgG1 KZ52 is a neutralizing mAb specific to the Ebola spike glycoprotein consisting of the same IgG1 backbone as b12 (575, 576).

Other mAb and pAb reagents mAb reagents obtained from the National Institutes of Health AIDS Research and

Reference Reagent Program (ARRRP) are the following (catalogue number in parentheses): IgG1 39F anti-gp120 V3 region (11437), IgG 19b anti-gp120 V3 region

(11436), IgG1 670-30D anti-gp120 C terminus (7370), IgG1 Chessie 6 anti-gp120 C1

59 region (810), IgG1 697-30D anti-gp120 C1/C2 region (7371), IgG1' 17b anti-gp120

(4091), IgG1' F105 anti-gp120 CD4bs (857) (577), IgG1' 2G12 anti-gp120 glycosylations (1476), IgG1' 4E10 anti-gp41 (10091), IgG1' 2F5 anti-gp41 (1475),

IgG1 91-5 anti-p24 (1238),

P24 detection ELISA reagents were mouse IgG1 anti-p24 for coating ELISA plates 183-

H12-5C (NIH ARRRP; 1513), rabbit polyclonal anti-p24 (ABI, Carlsbad, USA), biotin anti-rabbit IgG (Sigma, Oakville, Ontario), Streptavidin-Alkphosphatase (Jackson

ImmunoResearch Laboratories, Inc. West Grove, PA.). Secondary mAbs for detection of human and animal Abs were sheep anti-human IgG peroxidase (The Binding Site, San

Diego, USA), goat anti-mouse HRP, goat anti-guinea pig HRP, goat anti-rabbit HRP

(Southern Biotech, Birmingham, USA).

Gp120

Recombinant HIV-1 gp120 IIIB and MN strains were purchased from

Immunodiagnostics, Inc. (Woburn, MA). "NmCHO, or DNmCHO, an engineered gp120 molecule that has been truncated up to E52 (481), was also donated by D.R. Burton.

Antigens

Peptides

Nterm peptide - dBiotin-LWVTVYYGVPVWKEATTTLFCASDAK-COOH, and

Scrambled peptide - dBiotin-VWCAPLVYWTSTGELAVDKFVTATYK-COOH (United

60 Biochemical Research, Inc., Seattle, WA.). Both were resuspended in 5% formic acid

(Sigma, Oakville, ON), then diluted in the described buffers for experimentation.

Avidin/Streptavidin

Avidin acted as a carrier molecule in immunizations and streptavidin was used to measure background Ab levels for animal immunization. Streptavidin was used to coat

ELISA plates for anti-streptavidin Ab testing (both from Zymed Laboratories, Inc., San

Francisco, CA)

Freund’s Adjuvant

Complete Freund’s adjuvant was administered on the 1st immunization, followed by incomplete Freund’s adjuvant on subsequent injections (Fisher Scientific, Ottawa, ON)

4.1.3 Animals

Specific pathogen-free animals were ordered from Charles River Canada in Quebec and were received by the Veterinary Technical Services technicians. Mice and guinea pigs were group housed in filtered cages with a 12 hour light/darkness cycle. Rabbits were housed singly. The animals were allowed to acclimatize a minimum of 5 days before being used in the project. Other housing conditions such as room and cage temperature, relative humidity, light hours and number of animals per cage occurred according to the

Animal Care facility guidelines at the National Microbiology Laboratory, Public Health

Agency of Canada.

61 Table 3. Animal characteristics Species Strain Age or Weight Sex Total number immunized mouse Balb/c 5-7 weeks Female 40 (10 in each of 4 groups) guinea pig Hartley 300-350g Female 20 (5 in each of 4 groups) New Zealand rabbit 2-2.5kg Female 8 (2 in each of 4 groups) White

All three animal species were chosen for their abilities to produce consistently good amounts of Ab on peptide or protein immunization (578-583).

4.1.4 Human Samples

Blood samples were obtained from Winnipeg donors. They were healthy adults of a variety of age ranges. Their samples were obtained expressly for use of the peripheral blood mononuclear cells (PBMC) in HIV infection assays. Human sera tested for gp120 and truncated gp120 binding was obtained from HIV+ women attending the Pumwani sex-worker clinic co-sponsored by the University of Manitoba and the University of

Nairobi in the Pumwani district of Nairobi, Kenya. All Subjects gave informed consent and ethics approval was provided by both the University of Manitoba and University of

Nairobi Ethics Boards.

4.1.5 Pseudovirus neutralization assay reagents

Cells

TZM-bl cells were infected (NIH ARRRP, Bethesda, MD). TZM-Bl cells are HeLa cells engineered to express CD4, CXCR4, CCR5, and a tat-responsive firefly luciferase reporter gene that is under control of the HIV LTR (long terminal repeat) (584-586).

62 Pseudoviruses

The pseudovirus pSG3"Env HIV-1 backbone vector consists of the HIV genome less the envelope gene. The envelope genes from the HIV strains that were tested were chosen based on the triage three tier virus panel system and were cloned into the backbone virus

(587, 588). This triage system was devised to standardize neutralization testing across serum screening for vaccine trials. Briefly, the tier 1 tests the homologous virus strain and heterologous strains that are broadly sensitive to neutralization. If serum shows neutralization, the tier 2 expands the testing to other pre-selected strains within the clade of interest and tier 3 examines neutralization across subtypes. In this study, a panel of clade B that spanned tiers was tested. Of note, b12 neutralizes SF162 and QH0692.42, but not PVO.4.

Table 4. Pseudovirus strains used for neutralizations Strain Clade Tested animal Tier (587) IIIB B Guinea pig and Rabbit 1 SF162.LS B Guinea pig and Rabbit 1 6535.3 B Rabbit 1b QH0692.42 B Rabbit 2 SC422661.9 B Rabbit 2 RHPA4259.7 B Rabbit 2 THRO4156.18 B Rabbit 2 PVO.4 B Rabbit 3 SVA-MLV n/a Rabbit control

PBMC neutralization viruses

HIV-1 IIIB and Bal isolates were obtained through the NIH ARRRP (Bethesda, MD).

63 4.1.6 Other consumables

Table 5. Consumable labware used in experiments Consumable Source Sterile Culture Ware 25 cm2 T flask Corning 75 cm2 T flask Corning 96 well U bottom plates with lids Costar 15 ml sterile conical tube BD Falcon 50 ml sterile conical tube BD Falcon Tips and Pipettes 10, 200, 1000 µl pipette tips Fisherbrand 5, 10, 25 ml pipettes BD Falcon Sterile Filters 0.45 nm bottle-top filters BD Falcon 0.22 nm syringe-top filters and 3c.c. syringes BD Falcon Chemicals Trypan Blue Sigma (Oakville, Ontario) PHA Sigma (Oakville, Ontario) Pen/Strep/fungizone Gibco Miscellaneous Disposables Bottletop Filters Nalgene Reservoirs Costar Transfer pipettes Samco

4.2 General methods

4.2.1 SDS Page gel electrophoresis

Protein gels were poured in a BioRad (Mississauga, Ontario) MiniGel apparatus. The

7.5% SDS-PAGE separating gel was poured, allowed to set with a layer of isopropanol

(Sigma, Oakville, Ontario) on top, rinsed with distilled tap water, and then the staking gel was poured on top with the 10-well comb insert.

Protein samples were boiled with equal volume loading buffer for 7 minutes. Samples were loaded into wells at 10 ul (1-2 µg protein) per well. One well of 5 ul protein ladder

(1kb, or kaleidescope for Western blot gels, BioRad, Mississauga, Ontario) was run per

64 gel for size determination. Samples were run on the gel for 1 hour at 200 Volts. Gels were removed from the apparatus and immediately placed in either staining solution

(Coomasie or Silver Stain Buffer 1) or transfer buffer for Western blot.

4.2.2 Coomasie stain

Gels were placed in 20 ml Coomassie Blue stain for 30 minutes on a rocker. After 30 minutes, gels were placed in destain solution for up to 1 hour, or until bands became distinguishable.

4.2.3 Silver Stain

As per the silver staining kit (Pierce Protein Research Products, Rockford, USA), the protein gel was removed from the running apparatus and placed in solution 1 for 1.5 hours. The gel was transferred to solution 2 (sodium carbonate buffer) for 1.5 hours, then was placed in the silver stain until colour developed. The gel was placed in 5% acetic acid stop solution and dried between sheets of acetate.

4.2.4 Mass spectrometry column preparation

Abs were dialyzed in coupling buffer at room temperature for 4 hours before they were coated on beads. Before dialysis, 5 µg Ab were removed to a separate tube and labeled

BD (before dialysis). Dialysis occurred in 1L coupling buffer 4 hours; buffer was changed every hour except in last 2 hours.

65 Cyanogen bromide-activated Sepharose 4B beads (Sigma-Aldrich, Oakville, Ontario) were used for coupling. One ml beads were coated per 1 mg mAb (25µl per sample). 285 mg of dried beads were weighed into Eppendorf tubes and 1ml HCl buffer was added and incubated at room temperature for 30 minutes.

Beads were microcentrifuged for 30 seconds at 420xg. They were allowed to settle for 1-

2 minutes. The HCl buffer was aspirated with extreme care using a 23 ga needle with gentle suction. Beads were washed 3 times with 2-3 times bead volume coupling buffer to activate them (with same spinning and supernatant removal conditions as above). A small amount of buffer was left above beads after the last wash.

Five µl of dialyzed Ab were removed from dialysis tube, transferred to new tube, labeled

AD (after dialysis) and stored at 4°C. The remaining dialyzed Ab was transferred to the beads and coupled to beads for 2-4 hours at room temperature on a rocker. The mixture was microcentrifuged for 5 minutes at 100xg. The supernatant was removed and transferred to a tube labeled AC (after coupling) and stored at 4°C. The beads were washed twice with coupling buffer. 2-3 times bead volume ethanolamine was added to beads. Beads were blocked with 2 times bead volume at 4°C overnight on rotator.

On day 2, the column was spun down (30 seconds, 420xg) and washed with 1 ml coupling buffer 3 times. Following the wash with coupling buffer, the column was washed 3 times with 1 ml of acetate buffer. This 3 times wash was repeated 2 times (18 washes total - 9 of each buffer). The column was washed 3 more times with coupling

66 buffer, then once with ice-cold PBS. After the PBS wash, PBS (2 times bead volume) and sodium azide (to 0.05%) were added to the beads to prevent bacterial growth.

Coupled beads were stored at 4°C. To check for proper dialysis and coupling, a 7.5%

SDS PAGE gel was run with the samples BD, AD, and AC. Gel was silver stained according to a kit (Pierce Protein Research Products Rockford, USA).

4.2.5 Matrix-assisted laser desorption/ionization mass spectrometry

4.2.5.1 Epitope excision

Ab-bound beads were gently resuspended with a pipette tip and 15 µl beads mixture was removed (approximately 7.5 µl solid beads) to new tubes. Beads were washed with 400

µl tris-buffered saline (TBS) to dilute out sodium azide. Washes were repeated twice for a total of 3 washes. Microcentrifuge spinning for washes were at room temperature, at

960xg for 3 minutes, or in a Tomy microcentrifuge for 20 seconds. Supernatant was removed carefully using a 200 µl pipette so as not to lose any beads. After the last wash approximately 15 µl supernatant was left on top of beads.

Twenty-five µg recombinant gp120 IIIB or MN (Immunodiagnostics, Inc. Woburn, MA) was added to each tube of Ab-coupled beads; control tube received 25 µl TBS. Ab- antigen mixtures were incubated on ice for 2 hours with gentle vortexing every 10 minutes. Beads were centrifuged and washed 2 times with 400 µl TBS. Tubes were washed with 400 µl Digestion buffer. Supernatant was removed from beads.

67 Trypsin (500 ng, lysine/arginine sites, Calbiochem-Novabiochem Corporation, San

Diego, CA) or Glu-C endopeptidase (500 ng, glutamate cut site, Roche Diagnostics

Canada. Laval, Quebec) were incubated with beads at 37°C for 1 hour. Tubes were vortexed gently and incubated at 37°C for another hour. Unbound fragments were washed away 3 times with TBS, and then beads were washed with MS Wash Buffer containing BOG detergent. The final mixture was at a beads:wash buffer ratio of 1:1.

Tubes were stored at 4°C until mass spectrometry run.

One µl of the bead mixture was pipetted onto a gold-plated target plate, allowed to air- dry, and then spotted again. One µl dihydroxy benzoate (DHB) matrix solution was spotted on top of dried bead spot. The samples were analyzed using the Manitoba/SCIEX prototype hybrid quadrupole-time of flight mass spectrometer (QqTOF) coupled to a matrix-assisted laser desorption/ionization (MALDI) source as previously described (589,

590). The charge transferred to the binding epitopes by MALDI MS is typically +1, the resulting mass of each peak is the m/z value, in Daltons (Da). The MALDI system is not quantitative. All MS experiments were repeated 2-4 times.

4.2.5.2 Tandem mass spectrometry (MS/MS)

Tandem mass spectrometry (MS/MS) sequencing was performed on the same instrument and data were analyzed using BioMultiview (MDS Sciex) software. 1ug of soluble MN gp120 was digested with 20 ng either trypsin or Glu-C. The mixture was analyzed on the

MALDI QqTOF. Peaks 1356 and 1608 of the trypsin-gp120 mixture and peaks 1806,

1866, and 2096 of the Glu-C-gp120 mixture were selected for MS/MS. These fragment

68 peaks generated from the parent ion were compared to a list of theoretical ions for each peak (a, b and y ions were analyzed).

4.2.6 Peptide concentration calculations

Lyophilized peptides obtained from United Biochemical Research, Inc. (Seattle, WA.) were reconstituted in 5% formic acid. Peptides were diluted in ddH2O and absorbance was measured on the Spectramax Plus (Molecular Devices, Sunnyvale, CA) at an optical density of 280 nm. The concentration of each peptide was calculated as follows:

Concentration of Peptide = Abs280 x dilution factor x molecular weight / extinction coefficient

Where the molecular weight of the Nterm and Scrambled peptides is 2949.4 g/mol and the molecular weight of biotin is 244.3 g/mol (therefore Nterm + Biotin = 3193.7 g/mol).

The extinction coefficient is 13980 as calculated by the ProtoParam software

(http://ca.expasy.org/tools/protparam.html).

4.2.7 ELISA

Unless otherwise stated, all ELISAs were performed at a volume of 100 µl/well and all samples were tested in triplicate. All incubations occurred in a wet box consisting of a plastic container lined with wetted paper towels. Statistical differences in binding were calculated by unpaired student’s t test.

4.2.7.1 Antibody capture of peptide antigen

69 Ninety-six-well microtiter plates (NUNC, Mississauga, ON) were coated overnight at

4°C with a 2.5 µg/ml dilution of Ab in carbonate buffer. The next day, plates were blocked with 110 µl/well blocking buffer at 37°C for 2 hours. Plates were washed twice with distilled water. Biotinylated peptides were serially diluted in dilution buffer. One hundred µl of antigen dilution were added to wells and incubated at 37°C for 1 hour.

Plates were washed 10 times on a Wellwash 384 washer (Thermo Labsystems, Franklin,

MA). Streptavidin alkaline phosphatase (Jackson ImmunoResearch Laboratories, Inc.

West Grove, PA.) in dilution buffer was added to each well. Plates were incubated at room temperature for 30 minutes, washed 10 times with 1X wash buffer, rinsed twice with distilled water, and developed for colour at room temperature using MgCl2 diethanolamine substrate buffer plus para-nitrophenylphosphate (pNPP). All ELISA plate readings were performed at times 15, 30, and 60 minutes at an optical density of 405 nm on a Spectramax Plus (Molecular Devices, Sunnyvale, CA) using Softmax Pro software

(Molecular Devices, Sunnyvale, CA).

4.2.7.2 Sandwich ELISA for truncated gp120 binding

Microtiter plates were coated with 2.5 µg/ml capture Ab D7342 (sheep anti-gp120; Aalto

BioReagents, Dublin, Ireland) or with gp120 DNmCHO or IIIB at 250 ng/well in Sodium

Carbonate Buffer. After blocking, plates were incubated with 5 µg/ml gp120 DNmCHO or IIIB in dilution buffer for 1 hour at 37°C. Plates were washed and serial dilutions of

IgG1 b12 or patient sera in dilution buffer (starting at 5 µg/ml and 1/100, respectively, doubling dilutions) were incubated on the plate for 1 hour at 37°C. Plates were washed and sheep anti-human IgG linked to horseradish peroxidase (HRP; The Binding Site, San

70 Diego, CA) was added to the plate at 1:2000 in dilution buffer. Plates were incubated 1 hour at 37°C, washed, and plates were incubated with ABTS (Roche Diagnostics Canada,

Laval, Quebec, Canada) at room temperature. Colour change was measured as above.

4.2.7.3 Competition assay

To test for competitive binding between gp120 and Nterm by IgG1 b12 Ab, microtiter plates were coated with 2.5 µg/ml D7342, blocked (as above), and incubated with 1

µg/ml IIIB gp120 for 1 hour at 37°C. In separate tubes, 0.25 µg/ml IgG1b12 and serial dilutions of Nterm or Scrambled peptides were incubated together for 1 hour at 37°C.

The microtiter plates were washed and tube contents were transferred to the plates for another 1 hour incubation at 37°C. Conjugated goat anti-human IgG-HRP was incubated on plates and binding was detected as above. This competition assay measured the ability of the Nterm peptide to block b12 binding to gp120.

Alternatively, 96 well plates were coated with 2.5 µg/ml IgG1 b12 Ab. After blocking, doubling gp120 IIIB dilutions starting at 24 µg/ml and a fixed peptide concentration of

0.625 µg/ml were added to wells at the same time. The antigens were incubated on the plate together for 1 hour at 37°C. Plates were detected for binding of biotinylated peptide on addition of varying amounts of gp120 to wells. This assay measured the ability of gp120 to block IgG1 b12 binding to Nterm.

4.2.7.4 gp120-binding antibody detection in animal sera

71 Microtiter plates were coated with 2.5 µg/ml HIV IIIB gp120, blocked, washed, and incubated with doubling serial dilutions of animal sera for 1 hour at 37°C. Plates were washed and incubated with HRP labeled goat anti-animal (mouse, guinea pig, or rabbit)

Ab for 1 hour at 37°C. After washing, the plates were developed with ABTS (Roche,

Laval, QC.

4.2.7.5 p24 detection

Anti-p24 for coating ELISA plate

The 183-H12-5C hybridoma cells were initially grown up in 7 ml of culture media in T-

25 cm flasks at 37°C, 50% humidity, 5% CO2. Five ml of media were removed from each flask and transferred to 15 ml conical tubes for centrifugation (10 minutes, 600xg).

Supernatants were removed and discarded and pellets were resuspended in 25 ml hybridoma media and transferred to T-75 flasks. Cells were cultured for 5 days and the supernatant was collected and filtered at 0.45u. The supernatant was neutralized with

NaOH. The Protein-G column was cleaned with 10 ml of 0.1 M glycine buffer and equilibrated with 25 ml PBS. The supernatant was passed through the column at 2 ml per minute, and then PBS was used to wash unbound protein, as monitored on the BIO-RAD

UV Monitor. The Ab was eluted (i.e. the peak was collected as indicated on the UV monitor) with the glycine pH 2.5 buffer. The eluted Ab was dialyzed against 2 changes of

4 L of PBS. The OD280 of the sample was taken and the concentration determined using the extinction coefficient of 1.2 OD units equals 1 mg/ml. Aliquots were stored at -70°C.

p24 ELISA

72 Microtiter plates were incubated overnight at 4°C with 100 µl of 2 µg/ml recombinant mouse IgG1 anti-p24. Coated plates were tested for efficiency of coating using the assay below and the standard recombinant p24. Anti-p24-coated ELISA plates were blocked for 2 hours at 37°C with 110 µl PBS 2% goat serum, 0.05% Tween20. After rinsing the plate twice with distilled tap water, 100 µl of Triton X-treated samples were thawed and added to the ELISA plates. A p24 standard curve was run on every plate. The plates were incubated at 4°C overnight.

The next day, the plates were washed and rabbit polyclonal anti-p24 was added at a final dilution of 1:10,000. The plates were incubated for 90 minutes at 37°C, washed, and a

1:10,000 dilution of biotinylated anti-rabbit mAb was incubated on the plate for 90 minutes at 37°C. Plates were washed and streptavidin alkaline phosphatase in dilution buffer was incubated on the plates at room temperature for 30 minutes. Plates were washed developed for colour at room temperature using DEA substrate buffer plus alkaline phosphatase yellow (pNPP).

4.2.8 Animal immunizations

4.2.8.1 Ethics

Ethics approval was obtained for animal immunizations from the Animal Care

Committee under the Canadian Council on Animal Care (CCAC) guidelines at the

National Microbiology Laboratory under the Public Health Agency of Canada.

4.2.8.2 Antigen preparation

73 Biotinylated peptides were linked to avidin carrier molecule so as to increase the size of the antigen that was injected into the animals. In order to maximize the amount of antigen an animal received, an excess of biotinylated peptide (both Nterm and Scrambled peptides) was incubated with avidin. Peptide and avidin were diluted in sterile PBS and mixed together at a 1:1 weight ratio. This mixture was incubated for 45 minutes at 37°C

(example: 5 µg peptide was incubated with 50 µg avidin). HIV-1 IIIB gp120 was diluted appropriately in sterile PBS. Mock immunized received sterile PBS.

The antigens were mixed with the Freund’s adjuvant with a 22 ga needle immediately prior to subcutaneous injection in animals. The initial immunizations contained sterile

Complete Freund’s adjuvant. The subsequent immunizations contained sterile

Incomplete Freund’s Adjuvant (containing no Mycobacterial cell wall components).

4.2.8.3 Immunization protocol

Immunizations and bleeds were carried out by the technical staff at Animal Care facility at the National Microbiology Laboratory, Public Health Agency of Canada. Each mouse received injections of 100 µl (50 µl antigen, 50 µl adjuvant) consisting of 10 µg of protein (gp120 or peptide plus avidin). Guinea pigs received 200 µl (100 µl antigen, 100

µl adjuvant) consisting of 20 µg of protein. Rabbits received 200 µl injections, consisting of 25 µg protein. The peptide immunizations were carried out for all 4 boosters, while the gp120 and PBS immunizations were halted after the 3rd booster, as anti-gp120 serum Ab levels were high enough to proceed.

74 Briefly, 4 groups of each animal species were immunized. The first group received mock immunizations of straight phosphate buffered saline and Freund’s adjuvant. The second group was immunized with whole recombinant gp120 (strain IIIB) in PBS plus adjuvant.

The third group of animals received immunizations of avidin-linked biotinylated Nterm peptide in PBS plus adjuvant. The fourth group received immunizations of avidin-linked biotinylated Scrambled peptide plus adjuvant. When immunized, animals were washed with disinfectant at the site of injection, swabbed with alcohol, and then injected subcutaneously. The immunization timeline was as shown in Table 6 below.

Table 6. Animal immunization timeline for all three species. Day Immunization Sample collection 0 Pre-bleed for baseline 1 1st immunization 21 1st booster 28 Trial bleed 35 2nd booster 42 Trial bleed 49 3rd booster 56 Trial bleed 63 4th booster 70 Exsanguinations

4.2.8.4 Sample collection

The Day 0 bleed consisted of the collection of a small volume of blood for baseline serum Ab levels. Trial bleed sera were collected in BD Microtainer Tubes( (gold top, containing clot activators and gel for serum separation, BD, Franklin Lakes, NJ) and Ab levels were measured by gp120 ELISAs to determine whether or not the subsequent immunizations were required. Area-numbing EMLA cream was applied to ear 20-30 minutes prior to bleeding via ear artery (rabbits). The mice and guinea pigs were bled via the saphenous vein (no anesthetics were required).

75

The terminal bleed took place at day 70 for peptide-immunized animals, and at day 58 for

PBS and gp120-immunized animals (as determined by Day 58 trial bleed gp120 ELISA results). The final sample-taking was an exsanguination by cardiac puncture while animals were unconscious under general anesthetic (Isofluorane 2.5-5% with oxygen as carrier gas at 0.3-0.5 L/ml).

Once collected, the sera were filtered through a 220 nm sized filter and analyzed via

ELISA to test Ab levels and gp120 binding (Section 4.2.6.iv) and in infection neutralization assays (Sections 4.2.14 and 4.2.15). Both pseudovirus neutralizations and

PBMC neutralizations were tested, as these two assays do not always give the same results (591).

4.2.8.5 Serum inactivation

Animal sera were heat inactivated by incubating at 56°C for 1 hour.

4.2.8.6 Serum IgG purification

The animal serum IgG was purified on a 1ml Pierce HyTrap protein G column. The column was washed with 10 times the column volume (10 ml) PBS. Three ml of sample was added to the column. The column was washed with 5 ml of PBS, and then sample was eluted from the column using Glycine buffer, pH 3.3. The samples were neutralized with 1.5 M Tris HCl and were immediately desalted on columns (Pierce, Rockford, USA) and quantified.

76

Measuring concentration of IgG

The IgG was quantified using a BCA Protein Assay Kit quantification kit (Novagen,

Germany), as directed.

4.2.8.7 Serum adsorption

The rabbit sera were adsorbed against the same PBMC that were used in the infection studies. The sera were added at a 500µl volume to 500,000 PBMC at a concentration of

2x106 cells/ml in complete media and the mixture was incubated for 45 minutes at 37°C and 5.0 CO2. Cells were spun down and the serum supernatant was recovered.

4.2.9 Pseudovirus infection/neutralization assays

All pseudovirus assays were carried out at the Laboratory of Immune Measurements at the Duke Human Vaccines Institute of Duke University in collaboration with and under the supervision of David C. Montefiori. The Laboratory of Immune Measurements is the premiere lab in the world for performing the pseudovirus neutralization assay from past and current clinical vaccine trials (588).

4.2.9.1 Cell and pseudovirus preparations

Cells

Infectivity with Env-pseudotyped viruses was measured by luciferase reporter gene expression in TZM-bl cells. TZM-bl are adherent cells, and were maintained in T-75 culture flasks in Complete Growth Media (GM). For cell-splitting and preparation for

77 infection, monolayers were disrupted using Trypsin-EDTA (Invitrogen, Burlington,

Ontario). Briefly, media was removed from cell layer, and monolayer was rinsed 3 times with sterile PBS. Each flask received 2.5 ml of 0.25% Trypsin EDTA and was incubated at room temperature for 30-45 seconds. The trypsin was decanted, and the flask was incubated for another 4 minutes at 37°C. Ten ml of GM was used to resuspend cells, which were counted by trypan blue exclusion. Cells were used to seed a new T-75 flask at 106 cells in 15 ml complete growth media. The flasks were incubated at 37°C in 5%

CO2/95% air. Splitting was performed every 3 days.

Viruses

Pseudoviruses were grown up in 293T/17 cells. Supernatants were obtained for stocks by low-speed centrifugation and filtration (0.45 micron). Stocks were stored at -80°C in GM with 20% FBS.

Pseudovirus TCID50

A 2.5-times background relative light unit (RLU) was employed when determining positive infection in TCID50 assays. The standard inoculum utilized in order to curtail any virus-induced cytopathicity was 200 TCID50. This inoculum still allowed for a detection of a 2 fold reduction in infectivity.

4.2.9.2 Pseudovirus neutralization assay

Unless otherwise noted, all incubations occurred at 37°C with 5% CO2. This assay measures neutralization Ab titers in the range of 1:20 to 1:43,740.

78

Infections were carried out on 96-well flat-bottom culture plates (Costar, Nepean, ON).

One column on each plate was used for cell-only control, one column for cells + virus only. The first dilution of inhibitor (11 µl of animal serum) was added in duplicate across row H on the plate to wells containing 140 µl of GM. Fifty µl was removed from row H and transferred to row G (a 3-fold dilution). The dilutions were carried out up to row A.

Pre-immune sera was used as a negative control and IgG1 b12 was used as a positive control. The virus was thawed in a waterbath at ambient room temperature then diluted in GM to a concentration of 4,000 TCID50/ml. Each well receives 50 µl virus. TZM-bl cells were trypsinized and resuspended in GM containing 37.5 µg/ml DEAE dextran

(Sigma, Oakville, Ontario) at a density of 1x105 cells/ml. Each well received 100 µl of cell suspension so that the final concentration of DEAE dextran is 15 µg/ml. Plates were covered and incubated for 2 days.

After 48 hours, 150 µl of culture medium was removed from each well, leaving 100 µl.

Britelite Reagent was added at 100 µl to each well. Plates were incubated at room temperature for 2 minutes to allow cell lysis and mixed by pipetting up and down twice and transfer 150 µl to a corresponding 96-well black plate (Costar, Nepean, Ontario).

Plates were read immediately in a Model Victor2 Luminometer (PerkinElmer Life

Sciences, Waltham, MS). Percent neutralization was determined by calculating the difference in average RLU between test wells (cells + animal serum sample + virus) and cell only control wells divided by the difference in average RLU between virus control

(cell + virus) and cells only control, subtracting from 1 and multiplying by 100.

79

1 – (test wells–cells only wells) / (cells plus virus wells–cells only wells) x 100 = % Neutralization

Neutralizing Ab titers were tabulated as the reciprocal of the serum dilution required to reduce RLU by 50%.

4.2.10 PBMC-based neutralization assay

4.2.10.1 Ficoll separation of PBMC from whole blood

Peripheral blood mononuclear cell infection assay samples were collected from donors in

Winnipeg. Blood was collected by venipuncture into vacutainers containing anti- coagulant. Tubes were centrifuged for 7 minutes at 514xg and serum layer was removed.

Blood was diluted with PBS to 2 times starting volume. In 50 ml tubes, 12 ml ficoll

(Invitrogen, Burlington, Ontario) was added and up to 25 ml diluted blood was carefully layered on top. Tubes were centrifuged with the brakes off at 400xg for 25 minutes to separate lymph cells. White cell layer was carefully collected with transfer pipettes into fresh 50 ml tube, and diluted to 40 ml with sterile PBS/2% FCS. Lymphocytes were centrifuged at 600xg for 10 minutes. Supernatant was poured off and cell pellet was resuspended and washed with 25 ml R-10 culture media. Cells were centrifuged at 400xg for 10 minutes and were resuspended in culture media to 5x106 cells/ml. Cell counts were done using a haemocytometer and trypan blue exclusion.

4.2.10.2 Phytohemagglutanin (PHA) stimulation of PBMC

PBMC were spun down (10 minutes, 400xg) and resuspended at 2x106 cells/ml in culture media with 5 µg/ml PHA (Sigma, Oakville, Ontario). Cells were cultured for 72 hours in

80 37°C incubator (50% humidity, 5% CO2) in vented T-75 or T-25 flasks (Costar, Nepean,

Ontario).

4.2.10.3 Generation of viral stocks for PBMC assay

PBMC were isolated from Winnipeg donors using the ficoll procedure. Cells were PHA stimulated in T-75 flask for 72 hours with 5 µg/ml PHA. PBMC were washed twice with culture media, counted and return to flask at 2x107 cells/ml in supplemented media containing 10u/ml recombinant IL-2 and polybrene. Cells were taken to the Level 3 laboratory.

In the Level 3 laboratory, day 14 viral stock preparations were used to give an MOI of at least 2 and cells plus virus were incubated for 2 hr at 37°C. Supplemented media was added to dilute the cells to 4x106 cells/ml. Flasks were incubated overnight at 37°C, then without disturbing cells, ! of the media was removed and replaced with fresh complete media to 2x106 cells/ml. A 1 ml aliquot was removed for p24 ELISAs. The culture was incubated for 3 days at 37°C.

On day 7 the supernatants were collected and centrifuged at 514xg for 10 minutes and separated into 1 ml aliquots. Aliquots of supernatant were frozen at –80°C.

Supplemented media was added back to cells and they were cultured for another 7 days.

Supernatants were collected and stored in 1ml aliquots for re-infection. Stocks were titered by p24 ELISA after they were frozen for 24 hours.

81 4.2.10.4 Tissue culture infectious dose (TCID50) assay

Level 2 Laboratory

Local donor PBMC were isolated and stimulated with 5 µg/ml PHA. At the end of three days, cells were spun down at 400xg for 10 minutes, washed once with 40 ml complete media (re-spin at 400xg, 10 minutes), and viable cells were enumerated by haemocytometer and trypan blue exclusion. Only cell cultures that maintained 80% viability from date of isolation were used for TCID50 infection assays. Cells were brought up to 2x106 cells/ml in complete media.

On a 96-well microtiter plate (12 columns, 8 rows), 150 µl complete media was added to all columns (less rows 1 and 2 – p24 standard) except for column 2 and virus only control wells. Column 2 received 135 µl complete media (less rows 1 and 2). Plates were transferred to the level 3 Laboratory for virus dilutions and addition of PBMC.

Level 3 Laboratory

Viral stocks were thawed and 65 µl virus was added to column 2 wells (less rows 1 and

2). Wells were mixed by pipetting and 50 µl was transferred to column 3 (a 1:4 dilution).

Dilutions were continued across the plates (1:4, 1:16, 1:64, 1:256 and so on to

1:419,430). Each dilution was therefore done in sextuplicates. Fifty µl of virus was added to virus alone control wells (1:4 dilution). Cells were added to the entire 96-well microtiter plate, minus the wells that were used as virus only control wells, the p24 standard curve wells, and the blank wells. Cells were added at 100,000 cells per well in

82 50 µl complete media. Only one viral stock and one donor PBMC were tested on a single plate. Plates were incubated at 37°C for 4 hours.

4 hours later, plates were wrapped in parafilm and centrifuged at 330xg for 5 minutes.

Supernatants were removed (150 µl), discarded, and replaced with complete media.

Plates were resealed and spun again at 330xg for 5 minutes. One hundred and fifty ul of supernatant was removed and replaced with complete media. Plates were incubated at

37°C for 3 days.

On day 3, 100 µl of supernatants were removed and transferred to non-sterile 96-well plates containing 10% Triton-X. One hundred µl complete media was added back and culture plates were incubated for another 3 days. The plates containing the supernatants plus Triton-X were sealed and frozen at -70°C for at least 2 hours, until removal for p24

ELISA testing.

On days 6, 10, and 14, the methods were repeated as on day 3, but on day 14 the cultures were discarded in 50% bleach.

4.2.10.5 Virus calculations

The amount of virus used for subsequent PBMC neutralizations was calculated by the

TCID50 experiments. The TCID50 was calculated by assessing the number of positive replicates at each dilution. Then “h” is calculated, where h is the interpolated log10 value

83 of a dilution step. The h value is added to the dilution value at the step above 50% growth

(by number of positive replicates), where

h = (% infected wells at dilution above 50% infected) – 50% (% infected wells at dilution above 50%)-(% infected wells at dilution below 50%)

The multiplicity of infection (MOI) for subsequent infections was calculated by multiplying the desired MOI by the TCID50 and dividing by the number of target cells per well. This gives the volume of virus required for each well.

4.2.10.6 PBMC infection/neutralization assay

4.2.10.6.1 Cell viability with treatment

Cell viability to infection and treatment was done to assess the toxicity of the rabbit serum against PBMC in conjunction with 12 day in vitro HIV infection. As below in

4.2.15.ii, PBMC to be infected were incubated with serially diluted (1:4 dilutions) heat- inactivated, sterile-filtered, pre-adsorbed, or IgG-purified serum at 37°C for 1 hour before addition of IIIB test virus. These assays were set up in triplicate. After 12 days of infection and three harvest points on Days 3, 7, and 12, the leftover cells for each test were combined and diluted 1:2 with trypan blue. The cells were applied to a haemocytometer and counted under 40X magnification. Dead blue cells were counted as well as viable clear cells in one entire 5x5 grid on the haemocytometer. Viability was measured as 100 – (dead cells) / (total cells in field) x 100 = % viability. All cell viability determination was performed in the level 3 laboratory.

84 4.2.10.6.2 Infections and neutralizations

Infection/neutralization assays were carried out in PHA-stimulated PBMC on 96-well culture plates in a level 3 laboratory at the University of Manitoba. Final volumes in each well were 100 µl virus, 50 µl PBMC, and 50 µl test material (200 µl total). Control wells contained virus alone, cells alone, or cells plus virus without inhibitor; the rest of the volume was made up with complete media. Stimulated cells were harvested from flasks and spun down at 400xg for 5 minutes. Supernatants were poured off and cells were reconstituted in complete media and counted. Donor cells were brought up to 2x106 cells/ml.

Test samples were added to appropriate wells of a 96-well dilution plate (BD Falcon,

Franklin Lakes, NJ). Animal serum concentrations tested were 1/200, 1/800 and 1/3200.

IgG1 b12 concentrations were 4, 1, and 0.25 µg/ml. Infections were performed in triplicate. Once inhibitors were diluted, the plates were taken into the Level 3 lab for addition of virus. Virus was added at 100 µl/well in complete media and the inhibitors plus virus were incubated for 30 minutes at 37°C. After 30 minutes, 100,000 cells were added per well and plates were incubated for 4 hours at 37°C.

The PBMC were then washed by spinning down the plates at 330xg for 5 minutes, pipetting out 150 µl of complete media, replacing it, spinning down the plates at 330xg for another 5 minutes, then removing 150 µl media and replacing it again.

85 Cultures were incubated for 3 days. New 96-well non-sterile plates were labeled and 15

µl of 10% Triton X-100 was added to each well. One hundred µl of supernatant was harvested from each plate into Triton-X and plates were frozen at -70°C before being removed to the level 2 laboratory for p24 ELISA.

Fresh complete media (100 µl) was added back to each well and plates were incubated at

37°C for another 3 days. Day 6 culture supernatants were collected and frozen in Triton-

X as above for p24 ELISAs. Supernatant harvesting was repeated at days 10 and 14, and at the latter cultures were discarded in 50% bleach. P24 assays to detect viral growth were performed as described.

4.3 Data analyses

Mass spectrometry

Visualization/analysis of Spectra

All spectra generated were viewed, labeled, and analyzed on m/z software (Genomic

Solutions, http://bioinformatics.genomicsolutions.com/MoverZDL.html).

Prediction of enzyme digestion patterns

Theoretical digestion patterns of gp120 MN and IIIB by trypsin and Glu-C endopeptidases were generated using PeptideCutter software at http://www.expasy.org/tools/peptidecutter/.

Sonar MS/MS

86 Sonar MS/MS was employed in order to match the ion spectra obtained from MS/MS to the NCBI protein database. The Sonar MS/MS search engine was utilized, as it matches individual peptides sequenced to full-length proteins (592).

ELISA analysis

All ELISA plates were read, and p24 standard curves and p24 values were calculated on

Softmax Pro software (Molecular Devices, Sunnyvale, CA). ELISA data were imported into Microsoft Excel for data manipulation and analysis, but most data analysis and figure generation occurred in GraphPad Prism (GraphPad Software, Inc., San Diego, CA).

Nterm sequence analysis

In order to determine the sequence and biochemical properties of the Nterm sequences, two website were utilized: The Los Alamos Sequence and Immunology database tools

(http://www.hiv.lanl.gov/content/index), and the ExPASy site (http://br.expasy.org/).

The biochemical properties of Nterm were determined using the ProtParam tool. The secondary structure of the Nterm peptide was generated using the Protein Structure

Prediction Server (PSIPRED, http://bioinf.cs.ucl.ac.uk/psipred/). The hydrophobicity plot of the Nterm variants were generated using the ProtScale

(http://br.expasy.org/tools/protscale.html) Kyte & Doolittle scale (593) and the polarity plot using the Grantham scale (594), both using a window size of 3 amino acids.

Where sequence data correlated to neutralization data in the literature, sequence data were obtained from the Los Alamos HIV Sequence database. The sequences were

87 selected from Binley et al, 2004 for neutralization by IgG1 b12 (448). The neutralization data were gathered entirely from the Binley et al. paper. All data were entered into Excel and analysis was performed on GraphPad Prism software.

Neutralization analyses

Both Pseudovirus Luminometer and p24 ELISA data were collected and manipulated in

Excel.

88 Section 5.0 Results

5.1 Identification of a binding site for IgG1 b12 on HIV-1 gp120 by MALDI QqTOF mass spectrometry epitope excision mapping

5.1.1 Rationale

IgG1 b12 is one of the best-studied mAbs. The b12 epitope has been delineated previously by several means. Originally, b12 was characterized as a CD4-binding site- specific mAb through ELISA blocking experiments (452). Alanine-scanning mutagenesis follow-up studies further suggested that amino acids surrounding/overlapping those involved in CD4-gp120 interactions were involved in b12- gp120 interactions. Utilizing the x-ray crystallography-generated structures of both the

IgG1 b12 and gp120 core molecules, a computer 3D protein modeling program, further mutagenesis studies (468), and co-crystallizaion of b12 with gp120 confirmed earlier results (163). While all methods previously employed to define the b12 epitope are valid, the epitope excision method of epitope mapping is a more direct method of characterizing binding interfaces. Thus, epitope excision was performed on b12, KZ52 (an IgG1 control mAb), and b6, a CD4 binding site-specific non-neutralizing mAb cloned from the same individual as b12, in an attempt to further our understanding of the composition of the neutralizing b12 epitope.

5.1.2 Hypothesis

Direct epitope mapping of IgG1 b12 to gp120 will reveal a binding site at the CD4- binding site and may reveal new specificities proximal to the CD4BS.

89 5.1.3 Objectives i. To determine the epitope for b12 on HIV-1 B clade recombinant gp120 using a relatively new technique, epitope excision mass spectrometry. ii. To confirm the discovered epitope by tandem mass spectrometry sequencing. iii. To compare the epitope mapping results of b12 to the non-neutralizing mAb b6, which has similar origins but different biological functions from IgG1 b12.

5.1.4 Study Outline

For initial mapping experiments, IgG1 b12, b6, and KZ52 mAbs were covalently linked to carrier beads and incubated with whole, recombinant IIIB and/or MN gp120.

Endopeptidases digested the unprotected (i.e. unbound) sites on gp120, which were removed via washing. The remaining epitope/Ab-bound beads were subjected to MALDI mass spectrometry. Putative epitope sequences were identified from the resulting peaks.

Sequencing was performed by incubating monomeric gp120 with trypsin and selecting the previously identified peak sizes for MS/MS sequencing.

5.1.5 Epitope excision mass spectrometry of IgG1 b12 identifies specific binding sequences of gp120

It has been previously demonstrated that IgG1 b12 binds to the CD4 binding-site on HIV-

1 gp120. The goal was to confirm previous findings via mass spectrometry (MS) epitope mapping, and perhaps identify new binding determinants. MS had not yet been used to epitope map IgG1 b12. The technique used was epitope excision, or a protection assay, as the fragments bound to Abs are protected from digestion and subsequent washing

90 steps. Being highly glycosylated, the mAbs are relatively resistant to endopeptidases and remain intact and linked to the Sepharose beads during the digests. An entire list of the

MS experiments is represented in Table 7. All x axes terminate near 2400 m/z, as there were no usable data generated beyond that point, and to facilitate data visulalization.

The b12 epitope excision assay using soluble IIIB gp120 and Glu-C endopeptidase identified three specific peptic fragment peaks that represent peptides bound by b12 at mass-to-charge (m/z) ratios of 1806, 1866 and 2096 (Figure 4a; peak m/z are rounded off for results and discussion). The same three peaks were observed when the experiment was repeated with b12 and MN gp120 (Figure 4c; 1806, 1866, and 2096). As expected, the negative control mAb IgG1 KZ52 experiment, when incubated with MN gp120 and digested with Glu-C, generated no peaks at all (Figure 4e). These data show that b12 bound 3 peptide fragments of 1806, 1866, and 2096 Da.

When b12 was incubated with IIIB gp120 and digested with trypsin endopeptidase, a peak appeared at 1608 Da (Figure 4b). When incubated with MN gp120, b12 pulled out two fragments represented by peaks at 1356 Da and 1608 Da (Figure 4d). Figure 4f shows the results of KZ52 incubation with MN followed by trypsin digest. No peaks were observed in the KZ52 experiment.

91 Table 7. MS tests performed, their results, and corresponding figures. Gp120 Ab IgG1 Enzyme Test Resulting peaks Figure antigen Glu-C 1806, 1866, 2096 4a IIIB Trypsin 1608 4b Glu-C 1806, 1866, 2096 4c b12 MN Trypsin 1356, 1608 4d Glu-C No specific peaks 5a - Trypsin No specific peaks 5b Glu-C 1806, 1866, 2096 13a MN MS Trypsin 1356, 1608 13b b6 Glu-C No specific peaks 13c - Trypsin No specific peaks 13d Glu-C No specific peaks 4e MN Trypsin No specific peaks 4f KZ52 Glu-C No specific peaks 5c - Trypsin No specific peaks 5d a/b/y ion spectra of 1806, 1866, Trypsin 9 and 2096 IIIB MS/MS a/b/y ion spectra of 1356 and - Glu-C 9, 10, 11 1608 Glu-C Autodigest peaks (background) 6a - MS Trypsin Autodigest peaks (background) 6b

92

Figure 4. IgG1 b12 and KZ52 MALDI mass spectrometry epitope mapping spectra. b12 and KZ52 control were linked to sepharose beads and gp120 antigen was incubated with Ab-bound beads. Mixtures were digested and unbound fragments were washed away. Spectra a and b represent the gp120 IIIB antigen plus b12, spectra c and d represent gp120 MN with b12 and spectra e and f represent the mapping of gp120 MN to KZ52. The spectra on the left (a, c, and e) represent mixtures digested with Glu-C endopeptidase and the spectra on the right (b, d, and f) represent mixtures digested with trypsin. Specific peaks are labelled in Daltons. m/z is the mass-to-charge ratio and the y axes indicate the signal intensity of detectable ions. These data are single analyses represenatative of 3 independent experiments.

93 To show that the mAb alone was not responsible for the peaks observed by antigen+Ab mixture, antigen-free bead-linked b12 and KZ52 were digested with endopeptidases in the absence of antigen. No peaks matching the gp120 epitope data were observed when b12 was digested with Glu-C (Figure 5a) or trypsin (Figure 5b). KZ52 digestion also gave no gp120 specific peaks (Glu-C Figure 5c; trypsin Figure 5d).

Endopeptidase autodigestion peaks were also measured. The enzyme was incubated at

37°C in incubation buffer and spotted directly on the MALDI platform. Auto-digested

Glu-C spectra had no peaks matching the epitope mapping experiments (Figure 6a). The trypsin auto-digest also gave no peaks corresponding to those generated in the epitope mapping experiments (Figure 6b).

The IgG1 b12 epitope was mapped by mass spectrometry in both MN and IIIB gp120 and using both trypsin and Glu-C endopeptidases. Each mapping experiment generated the same sized gp120 epitope fragments regardless of the gp120 strain that was tested. The identified fragments subsequently underwent analysis to be identified.

5.1.6 Putative amino acid identity of the IgG1 b12 gp120 epitopes identified by MS

In order to initially determine the identity of mass spectrometry-generated peaks,

PeptideCutter software was employed to generate theoretical digests of each gp120 IIIB and MN with each Glu-C and trypsin. Based on the in silico digests, the putative epitope sequences are shown in Table 8. Notably, all of the sequences overlap two regions on gp120.

94

Figure 5. mAb digestion control spectra. Ab-bound Sepharose beads were incubated with endopeptidase without antigen in order to determine background spectral peaks. Figures a and b represent experiments with b12 and Figures c and d represent experiments with KZ52. Spectra on the left (a and c) represent the experiments with Glu- C endopeptidase; spectra on the right (b and d) are those performed with trypsin. m/z is the mass-to-charge ratio and the y axes indicate the signal intensity of detectable ions. These data are single analyses represenatative of 2 independent experiments.

95

Figure 6. Endopeptidase autodigestion background spectra. Figure a shows the self- digestion spectrum of Glu-C. Figure b shows the self-digestion spectrum of trypsin. Peak labels are expressed in Daltons. m/z is the mass-to-charge ratio and the y axes indicate the signal intensity of detectable ions. These data are single analyses represenatative of 2 independent experiments.

96 Table 8. Putative gp120 sequence identity of the peaks identified by MS using theoretical digests. Matching light and dark grey colours indicate overlapping sequences. Ab/Ag endopeptidase peaks sequence 1806 ATTTLFCASDAKAYDTE Glu-C 1866 KLWVTVYYGVPVWKE b12/IIIB 2096 TEKLWVTVYYGVPVWKE Trp 1608 LWVTVYYGVPVWK 1806 ATTTLFCASDAKAYDTE Glu-C 1866 KLWVTVYYGVPVWKE b12/MN 2096 TEKLWVTVYYGVPVWKE 1356 EATTTLFACSDAK Trp 1608 LWVTVYYGVPVWK

97 The putative epitope is located at the gp120 amino terminus (Figure 7a). The first 29 amino acids shown in the gp120 IIIB sequence (Figure 7a; and the first 30 amino acids in

MN, not shown) are a leader signal sequence, are removed during processing, and are not in the final gp120 protein product. Amino acids previously shown to be involved in b12- gp120 interactions are highlighted in colour and/or italics (421, 452, 464, 466, 468). The entire putative IgG1 b12 epitope sequence with its enzyme cut sites,

TEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTE, is shown in Figure 7b. The minimal putative epitope, whereby each segment of the epitope was able to maintain interaction with b12 upon digestion, consists of the 26 amino acid-long sequence

LWVTVYYGVPVWKEATTTLFCASDAK, and henceforth is referred to as “Nterm”.

5.1.7 Tandem mass spectrometry (MS/MS) identification of the gp120 peptides involved in b12 interactions

The same recombinant gp120 IIIB antigen was digested and examined using the MALDI

QqTOF platform and the peaks that were previously identified by epitope mapping were selected for sequencing by tandem mass spectrometry (MS/MS). We believe this to be ideal because (1) there was insufficient material from initial digests to sequence, and (2) other gp120 digest-generated differ from our peaks of interest by at least 8 Da. In

MS/MS sequencing, the parent ion amino acid backbone can be split into different ion fragment types, including a, b, and y, which are utilized for sequence identification with the Sonar MS/MS search engine (Figure 8). The a fragment is generated when a sequence is severed between the two carbons of the peptide backbone. The b fragment is generated when the carbon amino terminal to the nitrogen is severed. The y fragment is

98

Figure 7. The Nterm epitope sequence within the gp120 sequence. Figure a demonstrates the maximal (single underline) and minimal (double underline) epitope sequence as identified by MS in the IIIB gp120 sequence (NCBI accession # AAK49977). The coloured and/or italicized amino acids are previously identified binding residues for b12 (421, 452, 464, 466, 467). Figure b delineates the epitope with the theoretical endopeptidase cut sites.

99

Figure 8. Fragmentations that can occur along a protein or peptide backbone during ionizing MS/MS sequencing experiments. Fragmentation generates a, b, c, x, y, and z ions.

100 generated when the carbon proximal to the carboxy terminus from the nitrogen is severed. Therefore, for each individual amino acid, there can be gleened three data points of three slightly different sizes.

The spectrum of whole gp120 IIIB digestion with trypsin is shown in Figure 9a. The Glu-

C digestion of gp120 did not give as many detectable peptide fragments, nor were they as abundant (Figure 9b). The trypic peaks at m/z 1356 and 1608 were selected for MS/MS sequencing. Figure 10a shows sequence-matched a, b, and y fragments of the 1356 parent ion, which are plotted on the 1356 MS/MS spectrum (Figure 10b). Figure 11a shows the ion spectrum and sequence-matched a, b, and y fragments of the 1608 parent ion, which are plotted on the 1608 MS/MS spectrum (Figure 11b). Sequencing of the gp120 confirmed the tryptic Nterm epitope fragments 1356 and 1608 to the sequence

LWVTVYYGVPVWKEATTTLFCASDAK.

The MS/MS spectral peak lists and intensities from M/Z were inputed to the Sonar

MS/MS tool and matching sequences were identified by searching the National Center for Biotechnology Information (NCBI) virus-specific database. The Sonar MS/MS search engine matches both parent ions and digested amino acid sequences to published protein sequences to identify the protein used for sequencing and the sequence of the query. The 1356 parent ion most significantly matched to the amino acid sequence

EATTTLFCASDAK, which confirms the sequence predicted by the theoretical digests

(Table 9). The top match represents 1214 different HIV-1 gp120 sequences in the NCBI database, all of which contain the putative epitope sequence. The other top 4 full

101

Figure 9. Spectra of monomeric gp120 digestion. IIIB gp120 was incubated with the trypsin (a) or Glu-C (b) endopeptidase. The final mixture was run on the MALDI QqTOF mass spectrometer and resultant peaks were autolabelled using the m/z software. These digests were subsequently used for MS/MS tests. Peak labels are expressed in Daltons. These data are single analyses represenatative of 2 independent experiments.

102

Figure 10. MS/MS sequencing spectra of the tryptic peak 1356. Monomeric gp120 was incubated with trypsin, spotted on the gold plate with DHB, and run on the mass spectrometer. Peak 1356 sequenced and the Sonar MS/MS tool identified the gp120 sequence based on matched a, b, and y ions (in boxes)(a). The matching a, b, and y ions are identified on the 1356 MS/MS sequencing spectrum.

103

Figure 11. MS/MS sequencing spectra of tryptic peak 1608. Monomeric gp120 was incubated with trypsin, spotted on the gold plate with DHB, and run on the mass spectrometer. Peak 1608 sequenced and the Sonar MS/MS tool identified the gp120 sequence based on matched a, b, and y ions (in boxes)(a). The matching a, b, and y ions are identified on the 1608 MS/MS sequencing spectrum.

104 Table 9. Sonar MS/MS sequence matches for the 1356 MS/MS spectrum

1. The significance value refers to the expect value. This describes the number of chance sequences matches one can expect in the current search. All values below 1 are considered significant matches, closer to zero is more significant. 2. The identity of each protein gives the accession numbers, as well as the name.

105 protein identities were to HIV-1 gp120 sequence variants. The MS/MS sequencing analysis of the 1608 parent ion with the Sonar MS/MS tool identified the gp120 amino acid sequence LWVTVYYGVPVWK (Table 10), matching the in silico-identified sequence. Unlike the 1356 parent ion, other non-HIV sequences in the NCBI virus database were matched to the 1608 parent ion by Sonar MS/MS, although their significance scores were much closer to zero (less likely to occur by chance) than that of the gp120 sequence (1.7x10-7 vs. >0.19). These secondary matches belong to proteins not tested in this system (Yosoke Virus, Middleburg Virus, etc.). This, in conjunction with the low match scores, suggests that they are not the sequence of interest.

5.1.8 Mass spectrometry epitope mapping of IgG1 b6

When the IgG1 b12 antigen-binding domain (Fab) was originally purified, other gp120- binding Ab Fabs, including b6, were cloned from the same individual (445, 447). b6 binds the CD4BS on gp120 with comparable affinity to b12, but is weakly neutralizing

(445, 446, 574). To investigate if the b6 and b12 antigen binding regions have sequence identity, the heavy and light chain sequence alignments of b6 and b12 variable regions

(Fvs) were determined (Figure 12). The Kalign software alignment shows high similarity between the light chains (approximately 78.2% identity) and more diversity at the heavy chains (approximately 38.8% identity). Within the antigen-binding complementarity determining regions (CDR), the light chain sequences match at 16/28 amino acids

(57.1%) while the heavy chains match at only 3/45 amino acids (6.7%). Therefore, while the lights chains show some homology, the heavy chains are very different, which may account for epitope specificity or neutralization differences. One would therefore expect

106 Table 10. Sonar MS/MS sequence matches for the 1608 MS/MS spectrum

1. The significance value refers to the expect value. This describes the number of chance sequences matches one can expect in the current search. All values below 1 are considered significant matches, closer to zero is more significant. 2. The identity of each protein gives the accession numbers, as well as the name.

107 Kalign (2.0) alignment in ClustalW format

Heavy Chains b6 1 2 ---LEESGGGLVKPGGSLRLSCVGSGFTFSSAWMAWVRQAPGRGLEWVGLIKSKADGETT b12 QVQLVQSGAEVKKPGASVKVSCQASGYRFSNFVIHWVRQAPGQRFEWMGWINPYNGNK--

b6 3 DYATPVKGRFSISRNNLEDTVYLQMDSLRADDTAVYYCATQKPRYFD-LLSGQYRRVAGA b12 EFSAKFQDRVTFTADTSANTAYMELRSLRSADTAVYYCARVGPYSWDDSPQDNYY-----

b6 FDVWGHGTTVTVSP b12 MDVWGKGTTVIVSS

Kalign (2.0) alignment in ClustalW format Light Chains b6 1 2 E--LTQSPGTLSLSPGERATLSCRAGQSISSNYLAWYQQKPGQAPRLLIYGASNRATGIP b12 EIVLTQSPGTLSLSPGERATFSCRSSHSIRSRRVAWYQHKPGQAPRLVIHGVSNRASGIS

b6 3 DRFSGSGSGTDFTLSISRLEPEDFAVYYCQQYGTSPYTFGQGTQLDIKRT b12 DRFSGSGSGTDFTLTITRVEPEDFALYYCQVYGASSYTFGQGTKLERKRT

Figure 12. Amino acid alignment of IgG1 b6 and b12 light and heavy chain variable regions (Fvs). Alignments were performed with the multiple alignment freeware Kalign on the EMBL-EBI website (http://www.ebi.ac.uk/ ; freeware http://www.ebi.ac.uk/Tools/kalign/index.html). The bolded, numbered sequences are the 3 antigen-binding complementarity determining regions (CDR) and the unbolded sequences are the framework regions (FR) according to (595).

108 that the Nterm sequence would not be recognized by the b6 mAb.

The IgG1 b6 mAb epitope was determined by mass spectrometry epitope mapping. b6 was incubated with gp120 MN and digested with either Glu-C or trypsin endopeptidases.

MS Analysis of the digestion of IgG1 b6-bound gp120 identified the same peaks at m/z

1806, 1866, and 2096 (Figure 13a) as with the b12 Glu- C assays (Figure 4c). The trypsin digest identified peaks at m/z 1356 and 1608 (Figure 13b), as did the b12 spectra with trypsin (Figure 4d). The epitope mapping of IgG1 b6 by MALDI QqTOF mass spectrometry epitope excision demonstrated that b6 binds to the same site on gp120 as

IgG1 b12, at the amino terminus.

Sequence analysis of the two mAbs shows the differences that exist in their antigen- binding domains, which may in turn explain their different neutralization capabilities.

The mass spectrometry results suggest that IgG1 b12 and b6 are both able to bind the

Nterm sequence of gp120. This may mean that the Nterm sequence is not involved in b12 neutralization of HIV-1. Further inspection of the binding of both these mAbs to the

Nterm sequence by ELISA should lend more information to the role that Nterm may play in neutralization of HIV-1 by b12.

5.1.9 Summary

The application of mass spectrometry in epitope mapping is an extremely sensitive and useful tool in biologic research. We employed epitope excision mapping to determine aspects of the conformational epitope for IgG1 b12 on HIV-1 gp120. The results

109

Figure 13. MALDI QqTOF MS epitope excision mapping of IgG1 b6. Figure a shows the spectrum from IgG1 b6 incubated with MN gp120 and digested with Glu-C. Figure b shows the spectra of the Ab/antigen complex digested with trypsin. IgG1 b6 plus Glu-C (c) and trypsin (d) without antigen are also represented. Specific peaks are labelled in Daltons. These data represents 2 independent experiments.

110 obtained using 2 strains of HIV-1 gp120 and 2 different endopeptidases confirmed that the b12 epitope sequence is LWVTVYYGVPVWKEATTTLFCASDAK at the amino terminus of HIV-1 gp120. The sequence was confirmed by MS/MS. A novel IgG1 b12 epitope determinant, Nterm, has been identified.

5.2 Confirmation of the antigenic specificity of IgG1 b12 for the Nterm sequence of

HIV-1 gp120.

5.2.1 Rationale

Mass spectrometry epitope mapping has revealed a binding site for IgG1 b12 at the gp120 amino terminus. This novel finding warrants confirmation by other methods. One caveat to mass spectrometry epitope mapping is that identified epitope sequences cannot necessarily be confirmed in other systems, as was the case of the 4E10 MS epitope mapping, whereby a synthetic peptide of the identified epitope did not bind 4E10 in

ELISA (218). b12 binding to Nterm was confirmed by measuring direct binding to a synthetic Nterm peptide by ELISA. This determined that b12 bound the epitope sequence in a peptide format. Other binding assays were performed to ascertain if the b12 binding site on gp120 involves the amino terminus of gp120. The ability of b6 to bind to the Nterm peptide was also determine the specificity of b6 for the same region on gp120 as b12.

5.2.2 Hypotheses

Confirmatory binding assays will confirm the novel amino-terminal binding site for b12 on gp120. In other words, that the Nterm epitope peptide based on the MS studies is

111 antigenic. If the Nterm is involved in b12 neutralization of HIV-1, deletion of the Nterm from gp120 should eliminate b12 binding. B6 will bind Nterm peptide, reflecting its weak ability to neutralize HIV-1.

5.2.3 Objectives

The objective of these assays is to confirm the binding of the Nterm peptide by IgG1 b12.

ELISA binding assays will also determine the ability of IgG1 KZ52, b6, and other HIV- specific mAb binding to Nterm. Competition ELISAs and binding assays with mutant gp120 lacking its amino terminus will elucidate the role of the Nterm binding site in b12- gp120 interactions.

5.2.4 Study Outline

In order to confirm that the Nterm sequence is a part of the b12 epitope, the 26 amino acid-long sequence and a Scrambled control peptide were tested in ELISA binding experiments. Additional ELISA experiments were conducted to examine the binding of

Nterm peptide by b6 and other described HIV-1-specific mAbs. b12 was tested for its ability to bind HIV-1 gp120 lacking its amino terminus as well and competition ELISAs were done to measured the role of Nterm in b12 binding to gp120.

5.2.5. IgG1 b12 binds to the synthetic Nterm peptide

The Nterm peptide, of the sequence LWVTVYYGVPVWKEATTTLFCASDAK, was synthesized with a biotin molecule attached to its amino terminus. A biotinylated amino acid-scrambled version (termed “Scrambled” peptide) with the sequence

112 VWCAPLVYWTSTGELAVDKFVTATYK, was tested in tandem. Attempts to coat the

ELISA plates with peptide, then detect for b12 binding, were negative (data not shown), so the biotinylated peptides were assayed in a capture ELISA. Ninety-six-well microtiter

ELISA plates were coated with b12 or KZ52 and incubated with titrated biotinylated peptides. Figure 14 is representative of two separate experiments performed in triplicate. mAb binding to peptide was detected by measuring the biotinylated peptides remaining bound to b12 in each well after washing. b12 bound to the synthetic Nterm peptide

(Figure 14a). Peak binding of b12 to Nterm was seen at 1 µg/ml peptide and this binding titrated out to 0.031 µg/ml. The immobilized KZ52 mAb did not bind the Nterm peptide in a titratable manner. These data confirm that the Nterm peptide specifically binds to the antigen binding domain on b12. When b12 binding to Scrambled peptide was tested in this same system, no titratable binding was observed (Figure 14b). Notably, b12 only interacted with Nterm peptide at high concentrations of the peptide which may reflect low affinity to the peptide epitope. This is likely reflective of the fact that the Nterm sequence is not the only binding region of b12 on gp120; the overall affinity for the intact epitope is greater than the affinity for one of its parts. The negative control mAb did not bind the Nterm peptide and b12 did not bind the negative control peptide appreciably compared to the Nterm peptide. This is the first description of b12 binding to the gp120

Nterm sequence.

To further delineate the manner in which IgG1 b12 interacts with the Nterm sequence, the 2 biotinylated peptide halves were constructed. The halves reflect the two different peptides halves identified by MS epitope mapping. The first half peptide is the sequence

113

Figure 14. Binding confirmation of the Nterm peptide. A synthetic peptide version of the Nterm epitope binds to IgG1 b12 and not to KZ52 by indirect ELISA (a). IgG1 b12 does not bind a Scrambled version of the peptide (b) and interacts most strongly with the carboxy terminal end of the peptide, when the Nterm epitope peptide expressed as two halves (c). Error bars indicated variance about the mean and asterisks identify significant binding differences at each dilution of peptide (unpaired T test). Each experiment was performed in triplicate and figures represent 2 independent experiments.

114 Biotin-EKLWVTVYYGVPVWKE. Glutamic acid and lysine were added at the beginning of the sequence. It occurs naturally at that site, and was added to the sequence to increase the overall solubility of the peptide to facilitate dissolution. Nonetheless, the

E-E peptide was still hydrophobic (Grand average of hydropathicity = -0.081, according to the Kyte and Doolittle Scale; calculation available at http://www.expasy.ch/tools/protparam.html) and both peptides were dissolved in the same 5% formic acid solution. The second half of the epitope is the sequence dBiotin-

EATTTLFCASDAK. Both halves were tested for binding by b12 in the same ELISA protocol as the whole biotinylated peptides. b12 bound the second half of the epitope sequence much better, and in a dose-dependant manner, compared to the amino-terminal half (Figure 14c). The first half of the sequence, in fact, did not bind b12 at all.

Interestingly, these results are not in accordance with the initial mass spectrometry results where both halves of the gp120 Nterm sequence are bound by IgG1 b12.

To assess if the Nterm peptide structure may affect b12 binding, the Protein Structure

Prediction Server (PSIPRED, http://bioinf.cs.ucl.ac.uk/psipred/) was used to predict the structure of the Nterm peptide and was compared to the Nterm structure determined by previous crystallographic studies (169) (Figure 15). When expressed as a peptide, the secondary structure that make up the strands are predicted to be different from the crystal structure.

Other gp120-, gp41-specific mAbs were tested for binding to the Nterm peptide. Again, titrated peptide were added to ELISA wells coated with mAbs, and the ability of the mAb

115

Figure 15. Potential Nterm secondary structure based on PSIPRED prediction and crystal structure studies (169). Both structures ascribe coil and strand structures to the Nterm sequence.

116 to capture the Nterm peptide compared to b12 capture was determined. Due to mAb availability, Figure 16 represents an experiment performed in triplicate. The mAb 17b, a

CD4i Ab that binds and neutralizes HIV-1 upon gp120 ligation to CD4 (439), was unable to bind Nterm peptide (Figure 16a). 697-30D, a gp120 V2 loop-binding mAb (529), was unable to capture Nterm (Figure 16b). Compared to binding by b12, 670-30D, an IgG mAb specific for the C terminus of gp120 (182, 183, 596), was unable to bind Nterm peptide (Figure 16c). The IgG 4E10 mAb is a gp41 neutralizing mAb that binds to the membrane-proximal external region of gp41 (218, 597) and was previously shown to bind a sequence overlapping Nterm by MS mapping, but did not bind a peptide similar to the one tested here (218). 4E10 also showed no appreciable Nterm peptide binding in this system (Figure 16d). The 19b mAb, a gp120 V3-specific IgG mAb (598) also failed to bind to Nterm peptide, although it did show higher background binding at the highest concentration of peptide compared to the other tested mAbs (2 µg/ml; Figure 16e).

While some mAbs had higher background binding to Nterm peptide compared to others, none bound to Nterm as well and in a dose-dependant manner as did b12, indicating other mAbs are non-specific or interact with Nterm peptide only weakly. There is therefore a specific interaction between b12 and the Nterm peptide.

5.2.6 Confirmation of a role of the Nterm sequence in IgG1 b12–gp120 interactions

ELISAs were performed to test competitive binding between gp120 and Nterm peptide by b12. ELISA plates were coated with IIIB gp120 and b12 plus Nterm or Scrambled peptide were added to the wells. b12 and gp120 coating concentrations were constant

117

Figure 16. Other HIV-specific mAbs do not bind Nterm peptide compared to IgG1 b12. Triplicate ELISA binding assays show mAbs 17b (a), 697-30D (b), 670-30D (c), 4E10 (d), and 19b (e) binding to Nterm peptide (black lines) compared to b12 binding (grey lines). Error bars indicated variance about the mean and asterisks identify significant binding differences at each dilution of peptide (unpaired T test). Each experiment was performed in triplicate.

118 and the amount of peptide was titrated out from 1 µg/ml. The Nterm peptide did not notably reduce b12 binding to gp120 (Figure 17a). There was a small decrease in the b12 binding to gp120 at the higher concentrations of Nterm (1 µg/ml peptide) but this did not dilute out as the peptide was diluted. The Scrambled peptide demonstrated some non- specific inhibition at the highest dilutions (1 and 0.5 µg/ml peptide). Both adding the peptide plus mAb at the same time and pre-incubating the mAb/peptide together gave the same results. These experiments were performed in triplicate, twice. This experiment shows that b12 binding to Nterm does not inhibit gp120 binding, which either indicates that the Nterm is not part of the b12 epitope on gp120, or, more likely, that the binding of b12 to its intact conformational epitope gp120 out-competes its interaction with the

Nterm portion of its epitope alone.

To test the ability of gp120 to block Nterm peptide binding to b12, ELISA plates were coated with b12, and constant concentrations of biotinylated Nterm peptide (0.625 µg/ml) and titrated gp120 starting at 48 µg/ml were added to the wells at the same time. Figure

17b shows that the whole, monomeric gp120 blocks b12 binding to Nterm peptide in a dose-dependant manner starting at 48 µg/ml. This concentration of gp120 reduces Nterm binding by b12 to levels (from 0.6 OD to 0.2 OD units) almost as low Scrambled peptide background. At 48 µg/ml, the molecular ratio of gp120:Nterm is over 1:1 (1:1 is at 23.4

µg/ml gp120). As gp120 is diluted out, just under 50% reduction in b12 binding to

119

Figure 17. Competition ELISA between gp120 and Nterm peptide for binding by b12. In Figure a, the wells were coated with a gp120. Peptide and IgG1 b12 were added to wells together and IgG binding to gp120 was measured. The no peptide bar indicated baseline gp120 binding (0.25µg/ml gp120). In Figure b, wells were coated with IgG1 b12 and biotinylated peptide was added with gp120. The first set of bars indicates the baseline peptide binding levels. Biotinylated peptide binding was measured. In both assays, the lighter coloured bars indicate Nterm peptide and the darker bars indicate Scrambled peptide assays. Error bars indicated variance about the mean and asterisks indicate significant inhibition; figures represent results from 2 independent experiments.

120 Nterm is still seen at a 0.1:1 ratio (at 2.34 µg/ml gp120). The complete inability of gp120 to inhibit Scrambled peptide binding to b12 suggests that Scrambled peptide background binding to b12 is truly non-specific for the b12 antigen-binding domain. These data indicate that gp120 and Nterm bind to the same location on IgG1 b12, in other words, they have overlapping paratopes, such that gp120 binding occludes the Nterm binding site on b12. Together, these competition assays show that b12 can interact with gp120 in the presence of the Nterm peptide. It suggests that the binding site on b12 for Nterm is blocked by gp120 binding, either through specific or non-specific steric hindrance.

To further elucidate the importance of the Nterm region in b12-gp120 interactions, b12 binding to gp120 lacking the amino terminus was tested. DNmCHO gp120 (also referred to as "NmCHO, or DeltaNmCHO), a construct of JR-FL gp120 with the amino terminal

52 amino acids deleted (481), was used to confirm that the gp120 amino terminus is involved in the b12 epitope. This was performed with ELISA plates directly coated with

DNMmCHO or IIIB and with plates coated with anti-gp120 Ab D7342 then the

DNmCHO or IIIB was captured on the plate. b12 or KZ52 was titrated on the plate and

IgG1 binding was detected. Figure 18a shows that b12 had a reduced ability to bind to

DNmCHO as compared to IIIB. While the OD of b12 binding to IIIB reached above 2.0 units, the binding of DNmCHO by b12 did not reach higher than 0.3 OD. There was a small amount of measurable b12 binding to DNmCHO that was not reflected with the

KZ52 negative control Ab (0.25 compared to 0.08), but this did not dilute out. The KZ52 bound neither gp120. These data indicate that an intact amino terminus on gp120 is important for b12 binding to gp120.

121

Figure 18. IgG1 b12 binds poorly to gp120 lacking its amino terminus. ELISA plates were coated with DNmCHO or IIIB gp120 and binding by b12 compared to the negative control mAb KZ52 was detected (a). b12 binding to DNmCHO and IIIB gp120 was also compared to binding by positive control HIV+ human sera to DNmCHO (b). In b, the 2 µg/ml IgG1 b12 is equivalent to a 1:100 dilution of HIV+ sera (doubling dilutions were performed). Error bars are based on experiments done in triplicate; figures represent results from 2 independent experiments.

122 To demonstrate that HIV-1 envelope-specific Abs other than b12 were able to bind the

DNmCHO gp120, despite lacking its amino terminus, DNmCHO was captured on ELISA plates by sheep anti-gp120 D7342. Both lots of DNmCHO are included in the figure (04 and 05). Pooled HIV+ sera were incubated on the plates and binding was detected by labeled anti-human Ab. Figure 18b shows that HIV+ sera bound DNmCHO gp120 at levels comparable to b12 binding to IIIB. The lowered ability of b12 to bind DNmCHO therefore supports the notion that the amino terminal sequence of gp120 is involved in

IgG1 b12 recognition of gp120.

The competition assays suggest that b12 binds to Nterm peptide through a region of its antigen-binding domain that overlaps its binding surface for whole gp120. b12 interaction with the Nterm peptide, however, is not sufficient enough to block b12 binding to intact gp120. b12 does display a great reduction in binding to a gp120 construct that lacks its amino terminal 52 amino acids. This indicates that there may be a role for this region of gp120 in b12-gp120 interactions. A caveat to these studies is that no information is gleaned about the contribution of the Nterm sequence to b12 neutralization of HIV by binding on the intact envelope trimer.

The data in this section show that b12 is able to bind to a synthetic Nterm peptide compared to a Scrambled version of the epitope. Negative control mAbs did not bind

Nterm. Whole, monomeric gp120 blocks Nterm peptide binding by b12, although Nterm peptide does not block b12 binding to gp120. Finally, b12 binds poorly to a gp120 construct lacking the amino terminus. Taken together, the data in this section support the

123 hypothesis that the gp120 Nterm sequence is, at least partly, involved in IgG1 b12-gp120 interactions. Much of the data of the previous 2 sections of Results are reported in the patent CA 2562385.

5.2.7 The IgG1 b6 monoclonal Ab binding to the Nterm peptide

The result of the IgG1 b6 mass spectrometry epitope mapping experiments showed that b6 bound the same epitope as b12, therefore b6 was also tested for binding to biotinylated

Nterm peptide. The ELISAs were performed twice in triplicate as in section 5.2.6.

Briefly, plates were coated with b6 and biotinylated peptides were incubated on the plate and detected for capture by b6. The b6 results can be compared to the b12 and KZ52 results from Figure 14, as these experiments were performed at the same time, on the same ELISA plate. The results are in Figure 19; the grey b12 and KZ52 lines in the graph are the same data represented in Figure 14a. Although b6 bound Nterm peptide appreciably better than KZ52, and binding was dose-dependant, the b6 mAb bound

Nterm peptide at a much lower level compared to b12. b6 binding to Nterm reached a maximum of OD 0.74 units at 1 µg/ml Nterm, compared to the maximum b12 OD >1.75 units at the same Nterm concentration. Similar to Figure 14b, b6 was tested for binding to the Scrambled peptide (Figure 19, open squares). b6 does not bind to Scrambled peptide compared to the albeit low-level b6 binding to Nterm peptide (0.2 versus 0.74 maximum OD units, respectively). ELISA b6-Nterm peptide binding studies suggest that

IgG1 b6 displays reduced binding to the Nterm peptide when compared to IgG1 b12.

124

Figure 19. IgG1 b6 binds poorly to the Nterm epitope. The binding of IgG1 b6 to biotinylated Nterm peptide (closed squares) was measured compared to IgG1 b12 (grey triangles) and KZ52 (black Xs) by ELISA. IgG1 b6 binding to Nterm peptide was also compared to its binding to the Scrambled peptide (open squares). In both graphs the b12 and KZ52 results that were displayed earlier in Figure 14 are represented with grey lines. All wells were coated with Ab at the same concentration. Error bars indicated variance about the mean; this figure represents results from 2 independent experiments.

125 ELISAs were employed to test b6 binding to the amino terminally-truncated gp120

DNmCHO as described, whereby the DNmCHO was captured in ELISA wells by D7342

Ab, b6 was added, and binding was measured. b6 had reduced binding to gp120 lacking the amino terminus compared to intact IIIB gp120 (Figure 20a). As in the case of b12 binding to DNmCHO (Figure 18a), the binding curve did not dilute out well across the mAb dilutions. This reduction in binding of b6 to DNmCHO compared to IIIB was not as drastic as the reduction in b12 binding to DNmCHO, but the binding differences between b6 and b12 on DNmCHO, despite being significant, were only 0.1 OD at most dilutions (Figure 20b). These data, together with the data shown in Figure 19, suggest at most low level binding of IgG1 b6 to the amino terminus of gp120.

5.2.8. Summary

The mapping experiments revealed the gp120 amino terminus Nterm sequence is involved in b12-gp120 interactions. To confirm this, b12 binding to biotinylated Nterm peptide was tested by ELISA. b12 bound Nterm peptide compared to a negative control mAbs and did not bind a Scrambled peptide, and, when expressed as 2 separated peptides, interacted with the carboxy half of Nterm. Competitive ELISAs showed that the Nterm peptide did not interfere with the b12-gp120 binding, but gp120 inhibited

Nterm binding, indicating a shared binding site on b12. Further supporting the role for

Nterm in b12 binding to gp120, b12 bound poorly to truncated gp120 lacking its amino terminus.

126

Figure 20. Binding of IgG1 b6 to gp120 lacking the amino terminus. IgG1 b6 binding to gp120 DNmCHO, which lacks the amino terminal 52 amino acids of the C1 region. b6 binding to IIIB and DNmCHO gp120 are plotted with IgG1 KZ52 binding (a). The binding of IgG1 b12, b6, and KZ52 to truncated gp120 are compared with an expanded x-axis to appreciate the differences in binding to DNmCHO (b). Asterisks indicate significant binding differences between b6 and b12 based on paired t tests. Error bars are based on experiments done in triplicate; figures represent results from 3 independent experiments.

127 Nterm was also identified by mass spectrometry as the b6 epitope. Both b6 and b12 are

CD4BS-specific mAbs, but b6 only weakly neutralizes HIV-1 in vitro (448). b6 bound

Nterm peptide weakly compared to b12, suggesting a potential affinity difference between the two mAbs for the Nterm peptide. The two mAbs share some homology in their light chain CDR, which may account for some of the specificity overlap. Like b12, but to a lesser degree, b6 had a reduced ability to bind to DNmCHO gp120. Therefore, the Nterm binding site on gp120 may play a role in b6 interactions with gp120, but role that the Nterm sequence has in their dissimilar neutralization profiles has yet to be determined.

This section confirms that IgG1 b12 binds to the Nterm sequence of gp120, thus proving its antigenicity. As b12 is one of the most broadly neutralizing HIV-1 Abs described to date, it is important to determine whether or not this Nterm amino acid sequence or peptide could make a suitable vaccine candidate by comparing existing data on HIV-1 gp120 sequence variations within the Nterm sequence in the context of b12 neutralization profiles.

5.3 Analysis of the Nterm sequence – variation and vaccine suitability.

5.3.1 Rationale

Through mass spectrometry and ELISA studies a 26 amino acid-long sequence at the amino terminus of gp120 has been identified as being involved in IgG1 b12 binding. If the Nterm sequence is truly involved in b12 neutralization of HIV, it follows that the neutralization titres required by b12 to inhibit in vitro infection will correlate with sequence variation of Nterm of that tested HIV strain. For a vaccine candidate to be

128 worthy of study, not only must the epitope be immunogenic (see immunogenicity studies in section 5.4), but the sequence should elicit an Ab response that will act against evolving gp120 sequences across multiple subtypes and strains found all over the world.

Therefore, the epitope should be well conserved with respect to its sequence and the biochemistry of variants. With a large source of envelope sequence information available in the Los Alamos HIV sequence database and a wealth of in vitro b12 neutralization studies, these types of analyses are feasible.

5.3.2 Hypotheses

The in vitro neutralization of various HIV strains by b12 will correlate with the sequence variation within the Nterm epitope. Since b12 is a broadly neutralizing mAb, the biochemical properties of Nterm sequence variants will be relatively conserved.

5.3.3 Objectives i. To demonstrate through analysis of existing HIV-1 gp120 sequence data and IgG1 b12 neutralization data that variation in the Nterm sequence correlates with the resistance of that HIV-1 strain to b12 neutralization. ii. To define the Nterm sequence conservation and variation and subsequent biochemical changes that are affected by Nterm variation. iii. To investigate if the Nterm sequence would be a suitable candidate for a cross-clade protective vaccine (i.e. is well conserved across HIV-1 subtypes).

5.3.4 Study Outline

129 To determine if the HIV gp120 Nterm sequence variation correlates with b12 neutralization capacity and the nature and number of Nterm sequence variants that exist, available Nterm sequences and b12 neutralization data were co-analyzed. The HIV-1 sequences were obtained from the Los Alamos HIV Sequence Database or the NCBI protein database. Viruses for which b12 neutralization capacity was tested in the J.

Binley et al. paper published in the Journal of Virology in December of 2004 were selected for the analysis in sections 5.3.5, 5.4.6 and 5.3.7, (448). This paper detailed the neutralization titres of several neutralizing and non-neutralizing mAbs, including b12, against multiple subtypes and strains of HIV-1. This is the largest, most comprehensive study of mAb neutralizations to date (in some experiments, >90 HIV strains were tested).

Analysis was conducted with all clades and was also performed against the subset of clade B viruses. The biochemical properties of the Nterm amino acid sequence and its variants were analyzed to determine their chemical nature. Both the sequence variation at

Nterm and previously identified b12 binding specificities were studied. For section 5.3.8, the analysis was conducted for the entire HIV-1 envelope protein database.

5.3.5 Correlation of the amino acid variation at the Nterm epitope sequence with the neutralization titre of IgG1 b12

The IgG1 b12 mAb has been tested for neutralization of in vitro infection by various methods and in various labs. It has been tested in both PBMC and in cell lines infections, in lab-adapted HIV-1 strains as well as primary isolates ((318, 445, 448, 460, 464, 574), to list a few). In order to analyze the wealth of neutralization data for b12 and compare them to available sequence data, it is most advantageous to use neutralization data

130 generated by a single laboratory in a single study. Such a study exists. In 2004, the

Burton laboratory (Scripps Research Institute, La Jolla, California) published a paper in

Journal of Virology whereby many (>90) strains of HIV from multiple clades and from various origins were tested in a pseudovirus assay (448). This study represents the most comprehensive data set to date. I therefore compared the Burton sets of analyses to determine if sequence variations at the Nterm sequence correlate with neutralization sensitivity of a given virus. Both the b12 titre required to neutralize 50% and 90% of viral growth were examined and compared with the number of amino acid variations from the Nterm consensus. Taking into account b12 neutralization titre, the variation at the Nterm sequence from the Los Alamos HIV-1 sequence database consensus correlates with the 50% neutralization titre (rs=0.04437, p=0.01842; Figure 21a) and with 90% neutralization titre (rs=0.3534, p=0.000211; Figure 21b). This suggests that, especially at more stringent neutralization titres, the variation at the Nterm, regardless of subtype, may affect the ability of b12 to neutralize that HIV-1 strain.

While b12 does neutralize HIV-1 across clades, it preferentially neutralizes clade B virus.

Inter-clade amino acid variability is greater than intra-clade amino acid variation (599).

Taking these two concepts into account, it seems only natural that, across all subtypes, variation away from the consensus Nterm sequence, which matches the clade B sequence, should correlate with a reduced sensitivity to b12 neutralization. For this reason, the same analysis as above was done for the clade B sequences only. Table 11 shows the

50% and 90% neutralization titres in Binley et al. and the sequences of the tested clade B viruses. The table is arranged with the most sensitive viruses at the top. Only viruses

131

Figure 21. Correlation of IgG1 b12 neutralization with amino acid variation at the Nterm sequence for the tested strains across multiple clades (448). Figure a represents the 50% neutralization titres (N=41), figure b represents the 90% neutralization data (N=31) versus numbers of amino acid variations in Nterm from the consensus. All sequences were gathered from the Los Alamos Sequence Database. The rs and p values are calculated via Spearman’s rank correlation.

132 Table 11. b12 neutralization concentration and expanded Nterm sequences of all Clade B viruses tested against b12 for neutralization in (448). Amino acid variations in the Nterm sequence are highlighted in red. Seq. 50% 90% Amino acid sequence name conc.1 conc. 1 MN 0.003 0.02 TEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTE SF162 0.03 0.08 VEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTE JRFL 0.09 0.17 VEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTE BaL 0.19 0.25 TEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTE NL43 0.06 0.28 TEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTE IIIB 0.04 0.35 TEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTE HT92594 0.17 0.35 VEQLWVTVYYGVPVWKEATTTLFCASDAKAYDRE TTQZ458 0.08 0.68 TEQLWVTVYYGVPVWKEATTTLFCASDAKAYDTE USJRCSF 0.16 1.17 VEKLWVTVYYGVPVWKETTTTLFCASDAKAYDTE TT692 0.24 1.56 AENLWVTVYYGVPVWKEATTTLFCASDAKAYETE HT92593 0.23 2.81 AENLWVTVYYGVPVWKEATTTLFCASDAKAYETE US93073 0.5 5.71 AEKMWVTVYYGVPVWKEATTTLFCASDAKATDTE US91056 0.49 6.24 AEQSWVTVYYGVPVWREATTTLFCASDAKAYDTE TT515 0.69 6.54 TEQLWVTVYYGVPVWKEATTTLFCASDAKAYDVE US44 1.34 7.81 VEQTWVTVYYGVPVWKEANTTLFCASDAKAYNTE US5768 1.3 10 ADKLWVTVYYGVPVWKETTTTLFCASDAKAYDTE TH93305 1.23 10.75 VEDLWVTVYYGVPVWKAANTTLFCASDAKAYDTE FRBX08 0.35 19.47 ADQLWVTVYYGVPVWKDATTTLFCASDAKAYDKE BR92021 2.18 21.41 EDKLWVTVYYGVPVWKEATTTLFCASDAKAYDTE US1196 2.69 24.13 EDNLWVTVYYGVPVWKEATTTLFCASDAKAYDTE US92712 9.22 37.2 AEESWVTVYYGVPVWKEATTTLFCASDAKAYDTE BRVLGC2 6.75 50 TENLWVTVYYGVPVWKEATTTLFCASDAKAYDTE BR92020 27.5 50 KDNLWVTVYYGVPVWKEATTTLFCASDAKAYKAE 1. Neutralization concentration (µg/ml) of b12 to achieve 50% or 90% neutralization of the tested pseudovirus

133 with complete Nterm sequence data and measurable titres (i.e. <50µg/ml neutralization titre) are included. The number of amino acid variations were analyzed for their correlation to b12 neutralization titre (Figure 22). Both the 50% (rs=0.5334, p=0.005289;

Figure 22a) and 90% neutralization titres (rs=0.2208, p=0.0316; Figure 22b) significantly correlated with amino acid variation within the Nterm sequence.

Amino acid variations can impose different modification changes depending on the amino acid change. To help determine the consequences of amino acid variation analysis, amino acid physical properties were tabulated (Table 12). The properties were obtained from the ExPASy website (www.expasy.ch/). The residues are referred to by their individual letter abbreviations in column 2. Size, charge, hydrophobicity, and polarity of a protein or peptide can all be affected by amino acid variation. A score based on the charge, polarity, hydrophobicity, and size was attributed to amino acid variations in order to give weight to the biochemical change associated with amino acid variation.

The difference between the consensus amino acid and the new amino acid was given a score based on a shift in these properties. Each property was arbitrarily assigned a score of 1, meaning a change from a polar amino acid to a non-polar amino acid was given the same score as a shift of a small side chain to a bulky one. Conversely, the change of a side chain from one size to an only slightly smaller size was a given score of zero. Each variation was assessed a score. When the variation score was correlated with the 50% and 90% neutralization titres, there was significant correlation in both cases (Figure 23a; r2=0.1734 and p=0.0481 for 50% neutralization, and 22b r2=0.2250 and p=0.0298 for

134

Figure 22. Correlation of IgG1 b12 neutralization concentration to clade B HIV strains with the number of amino acid variations of the Nterm sequence of that same viral strain. Fifty percent neutralization titres are correlated in (a) and ninety percent neutralization titre correlations are shown in (b). The data points shown here correspond to the sequences represented in Table 11. Only sequences with complete Nterm data and measurable neutralization titres (i.e. <50ug/ml) are included. The rs and p values are calculated via Spearman’s rank correlation.

135 Table 12. Amino acid characteristics. The information tabulated here was used to calculate the nature of amino acid changes along the studied sequences Amino Group Charge Polarity Hydrophobicity1 Size acid/abbrev Glycine G Aliphatic n/a Nonpolar +/- - Alanine A Aliphatic n/a Nonpolar ++ + Cysteine C Sulfur-containing n/a Nonpolar +++ + Isoleucine I Aliphatic n/a Nonpolar ++++ ++ Leucine L Aliphatic n/a Nonpolar ++++ ++ Valine V Aliphatic n/a Nonpolar ++++ ++ Proline P Cyclic Bend Nonpolar -- +++ Methionine M Sulfur-containing n/a Nonpolar ++ +++ Phenylalanine F Aromatic n/a Nonpolar +++ ++++ Tryptophan W Aromatic n/a Nonpolar -- +++++ Tyrosine Y Aromatic/hydroxyl n/a Non/Polar - ++++ Serine S Hydroxyl n/a Polar - + Threonine T Hydroxyl n/a Polar - + Aspartic acid D Acid - Polar --- ++ Arginine R Basic + Polar ---- ++ Lysine K Basic + Polar ---- ++ Glutamic acid E Acid - Polar --- +++ Histidine H Basic n/a Polar --- +++ Asparagine N Amide n/a Polar --- +++ Glutamine Q Amide n/a Polar --- +++ 1. hydrophobicity was determined from the Kyte and Doolittle scale (593)

136

Figure 23. Correlation of IgG1 b12 neutralization concentration to clade B HIV strains with the number of amino acid variations and their score of the Nterm sequence of that same viral strain. 50% neutralization titres are correlated in (a) and 90% neutralization titre correlations are shown in (b). Only sequences with complete Nterm data and measurable neutralization titres (i.e. <50ug/ml) are included. The rs and p values are calculated via Spearman rank correlation.

137 90% neutralization). These data show that amino acid varitaion and the type of variation away from the Nterm consensus sequence correlates with the capacity of b12 to neutralize that strain. This indirectly supports the suggestion that the Nterm sequence might be involved in the neutralizing epitope of IgG1 b12.

5.3.6 Correlation of amino acid variation at the CD4BS with the neutralization titre of IgG1 b12

In previous epitope mapping studies, the b12 binding site on gp120 was mapped to residues overlapping the CD4BS (163, 447, 466). The sequence variation in the b12 epitope at the CD4BS, if it is indeed involved in HIV neutralization should, like Nterm, correlate with b12 neutralization capacity. Similar to section 5.3.5., the sensitivity of viral strains to neutralization by b12 was correlated to variation at previously identified b12 binding sites on gp120 (163, 199, 200, 466). Unlike the Nterm sequence, the previously identified b12 epitope is conformational, therefore two sequence fragments with three binding sites total were analyzed for sequence variation (first region – amino acids 363-375, the second at amino acids 461-474, according to HxB2 alignment). Their location in the gp120 sequence can be seen in Figure 7. The subtype B strains, sequence fragments, and variants are shown in Table 13. The bolded yellow amino acids were previously shown to be involved in b12 recognition of gp120 (163, 199, 200, 466). The flanking amino acids to each previously identified amino acid were included in the analysis. The total number of amino acid changes both within the b12 binding sites and flanking regions are shown along with the neutralization sensitivity of each viral strain.

138 Table 13. Amino acid sequences implicated in b12-gp120 interactions of subtype B viral strains tested for b12 neutralization sensitivity. The amino acids implicated in b12-gp120 interactions are bolded in yellow. Underlined amino acids are involved in the CD4BS. The variations away from the subtype B consensus are indicated in red. Sequence vs. Sequence vs. Strain 50%1 90%1 consensus2 consensus2 QSSGGDPEIVMHS3 DTNDTEIFRPGGGD3 MN 0.003 0.02 QSSGGDPEIVMHS DTNDTEIFRPGGGD SF162 0.03 0.08 QSSGGDPEIVMHS ISNTTEIFRPGGGD JRFL 0.09 0.17 HSSGGDPEIVMHS NENGTEIFRPGGGD BaL 0.19 0.25 HSSGGDPEIVTHS EDNKTEVFRPGGGD NL43 0.06 0.28 QSSGGDPEIVTHS NNNGSEIFRPGGGD IIIB 0.04 0.35 QSSGGDPEIVTHS NNNGSEIFRPGGGD HT92594 0.17 0.35 QPSGGDPEIVLHS ENNETETFRPGGGD TTQZ458 0.08 0.68 QSSGGDPEIVMHS GXNGTEIFRPGGGD USJRCSF 0.16 1.17 HSSGGDPEIVMHS NESEIEIFRPGGGD TT692 0.24 1.56 QSSGGDPEIVMHS VNGTRETFRPGGGD HT92593 0.23 2.81 PSSGGDPEIVMHS XTNGTEIFRPGGGD US93073 0.5 5.71 HSSGGDPEIVMHS NDNDTEIFRPGGGD US91056 0.49 6.24 QSSGGDPEIVMHS NTNGTEIFRPGGGD TT515 0.69 6.54 QSSGGDVEIVMHS GTNETETFRPGGGN US44 1.34 7.81 NSSGGDPEIVMHT NDNQTEIFRPVGGD US5768 1.3 10 QSSGGDPEIEMHS SNTSEEVFRPGGGN TH93305 1.23 10.75 QSSGGDPEIVMHS NGTANETFRPGGGD FRBX08 0.35 19.47 QSSGGDPEIVMHS SSSGKEIFRPGGGD BR92021 2.18 21.41 HSSGGDPEIVMHS NNSTXETFRPGGGD US1196 2.69 24.13 RSSGGDPEIVTHS XXTXTEIFRPGGGD US92712 9.22 37.2 QSSGGDPEIMTLM SENETEIFRPGGGD BR92020 27.5 50 PPSGGDPEIVFHS EMNTTEIFRPGGGD 1. Neutralization concentration (µg/ml) of b12 from Binley, et al (448) 2. Consensus for subtype B according to the Los Alamos HIV Sequence Database 3. Underlined amino acids in CD4 binding (156)

139 Statistical analysis shows that the 90% (but not the 50%) titres correlate with amino acid variation at the previously described b12 binding site. The SGGDPEIVM sequence shows a high level of conservation, while RPGGGD has only a single amino acid change in one variant – a glycine to a valine in the strain US44. Proline 369, which was mapped to the b12 binding site in multiple studies also varied in one strain only (TT515). This proline to valine change represents a change from an amino acid that can introduce a structural bend to a smaller, highly hydrophobic molecule. Neither of these single changes result in the strain being particularly more or less sensitive to b12 neutralization, according to their neutralization titres and placement in the table. The centre epitope sequence, 462TND, has many possible variants across all analyzed sequences, and therefore this region may not play as strong a role in neutralization as other binding points.

5.3.7 Characterization of the Nterm amino acid sequence and variation in IgG1 b12 neutralization-tested virus

To assess if the Nterm sequence or peptide may make a suitable vaccine, analysis of the sequence with respect to its crossreactivity to human proteins and physical characteristics if variants on a global scale were analyzed.

It has been suggested that so few broadly neutralizing Abs can be found in infected patients because they are cross-reactive to self proteins and are eliminated from the antibody repertoire. The cross-reactivity of some HIV-1-neutralizing mAbs has also been proposed (516, 517, 600, 601). Therefore, the Nterm sequence was examined for identity

140

Figure 24. Liner regression correlation of concentration for IgG1 b12 neutralization of clade B HIV strains with the number of amino acid variations at the b12 binding site sequence. 50% neutralization titres (a; N=21) and 90% neutralization titre (b; N=22) correlations were calculated by Spearman’s rank correlation. Only sequences with complete b12 binding site sequence data and measurable neutralization titres (i.e. <50ug/ml) are included.

141 to human protein sequences by a set of Blast searches through ExPASy (Table 14). In order to analyze such a short sequence against the human protein databases Swiss-prot,

UniProtKB, and TrEMBL, the Expected Value Threshold was raised from the baseline 10 to 100 (allowing more random matches to occur in the search). The sequence

LWVTVYYGVPVWKEATTTLFCASDAK matched only at the sequence 46KEATTTL to an intracellular nucleus membrane protein. Next, the sequence was broken down into pentamer motifs, which is the shortest amino acid motif that has been observed while maintaining antigenicity, and hence Ab interactions (602). Because the sequences tested are only five amino acids in length, any matches are considered “low similarity” matches

(603). Of the twenty-two 5-mers overlapping by one amino acid, six matched human protein sequences. The E values for all the matches were high (>1 is high), but these magnitudes reflect a poor match in combination with the short length of the queried sequence. When the analysis was repeated with adjustments for short sequence queries

(Expect threshold 20,000, word size 2, PAM30 matrix), the Nterm sequence showed only low level identity to the highly mutable human Ab variable regions (data not shown). The consensus Nterm sequence has no noteworthy cross-reactivity to human proteins.

To assess the global types of amino acid variations seen at the Nterm sequence across all

HIV-1 clades, Nterm sequences were gathered from the Los Alamos website. Though there are nearly 1000 Nterm sequences in the Los Alamos HIV Sequence Database, a subset of 93 sequences (from the Binley et al. 2005 publication) were chosen to facilitate analysis. The types of change were determined by the four criteria in Table 12. The substitutions seen in the analyzed Nterm sequences were conservative in nature: 48.8% of

142 Table 14. Blast search results of the Nterm peptide against human proteins.

Epitope/epitope fragment sequence Top hit E Value1 and Blast match LWVTVYYGVPVWKEATTTLFCASDAK 26, RRP12-like protein; Weakly expressed nucleus membrane protein LWVTV No hits found WVTVY No hits found VTVYY No hits found TVYYG No hits found VYYGV No hits found YYGVP 89, Pumilio homolog 2; RNA-binding protein, regulates translation and mRNA stability by binding the 3'-UTR of mRNA targets YGVPV No hits found GVPVW 76, cDNA FLJ55508; nuclear envelope protein VPVWK 62, WDR59; interacts with ligase complexes PVWKE 64, Rab11 family-interacting protein 2; acting in the regulation of the transport of vesicles from the endosomal recycling compartment to the plasma membrane VWKEA No hits found WKEAT No hits found KEATT No hits found EATTT No hits found ATTTL No hits found TTTLF No hits found TTLFC 82, Tumor necrosis factor ligand superfamily member 22; Cytokine that binds to TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR TLFCA No hits found LFCAS No hits found FCASD No hits found CASDA No hits found ASDAK No hits found 1Expect Value Threshold of 100 2The sequence match is located within the signal peptide and is not found in the mature protein

143 changes were given a score of 0; 41.9% were given a score of 1 compared to 1.2% with a score of 3 and 8.1% with a score of 2 (Table 15). Therefore, variation in the Nterm sequence tends toward conservation of the biochemical properties of the individual amino acids.

The biochemical properties over the length of the Nterm peptide sequences were determined using software from the Expasy website (www.expasy.ch; http://www.expasy.ch/tools/protscale.html). Subtype B only was graphed for ease of analysis. Hydrophobicity was determined using the Kyte & Doolittle scale (Figure 25a)

(593). A window of 3 amino acids was used to calculate the hydrophobicity at each amino acid to assess the hydrophobicity trends along the epitope, which also means that the amino and carboxy termini hydrophobicity values take into account neighbouring amino acids outside the minimal expitope, but still within the larger sequence originally identified by MS. Each coloured line indicates a different amino acid sequence. The consensus sequence is in grey in the forefront of all other data lines. Except for the very amino terminus of the sequence, the sequences are hydrophobic in the first third of the sequence, then the second third is relatively hydrophilic, and the final third is hydrophobic but for the carboxy terminus. Most of the sequences fall under or near the same plotted line as the consensus, especially around the end of the first third and at the last third of the sequence. The variation occurs mainly in the amino terminus and in the centre of the sequence.

144 Table 15. Types of amino acid variations in the published Nterm sequences that have been tested for neutralization against IgG1 b12 (N = 93; includes all subtypes). aa # of times change type of change3 Score4 total changes1 occurs2 E-D 26 conserved (slightly smaller) 0 K-R 14 conserved 0 42 T-S 1 conserved 0 V-I 1 conserved 0 T-D 13 conserved (neg to OH) 1 T-K 10 conserved (smaller, OH to +) 1 T-E 7 conserved (larger, to -) 1 36 T-N 3 conserved (larger) 1 L-M 2 conserved (slightly larger) 1 K-T 1 conserved (neg to OH) 1 L-S 3 nonp-pol, hydrophob-phil 2 A-T 2 nonp-pol, hydrophob-phil 2 E-A 1 pol-nonp, hydrophil-phob, 2 7 smaller L-T 1 nonp-pol, hydrophob-phil 2 L-K 1 Neut-+, nonp-pol, hydrophil-phob 3 1 1. aa subs = amino acid changes that occur within the Nterm sequences available at the Los Alamos 2. number of times each amino acid change occurs within the available sequences 3. the amino acid changes can be referred back to Table 11 earlier in this section: neg = negative, OH = hydroxy group, nonp = nonpolar, pol = polar, hydrophob and phob = hydrophobic, hydrophil and phil = hydrophilic, neut = neutral. + = positively charged, - = negatively charged 4. the score is based upon the number of amino acid characteristics that are altered upon the amino acid change, based on Table 12

145

Figure 25. Biochemical properties of the Clade B Nterm sequence and sequence variants that have been tested for neutralization against IgG1 b12. Plotted are the hydrophobicity (a), polarity (b), and charge (c) of the amino acids along the Nterm sequence. (a) and (b) were analysed using a window of 3 amino acids. All tests were performed at the ExPASy website. Where necessary, the flanking amino acids were included in the analysis. The consensus sequence is in grey.

146 The polarity along the Nterm Clade B sequences was plotted using the Zimmerman scale, and the sliding 3 amino acid window (Figure 25b) (604). Like the hydrophobicity plot, the majority of sequences follow at or near the polarity pattern of the consensus sequence. Not surprising, since non-polar molecules tend to be hydrophobic (Table 12), the shape of the polarity graph is nearly a mirror image of the hydrophobicity plot. As with the hydrophobicity plot, only a small amount of variation exists at the amino terminus and the middle of the Nterm sequence.

The least variant of the three biochemical properties along the subtype B sequences is the charge (Figure 25c). The charge is invariant but for the sequence TH93305. TH93305 has a glutamic acid to alanine substitution at the center of the sequence (the sequence can be seen in Table 11). This substitution also accounts for the hydrophobicity and polarity skewing of that sequence from the consensus visible in Figures 25a and b. Thus, the subtype B Nterm sequence is well conserved, both by amino acid sequence, and also by the biochemical properties of the variants.

5.3.8 Amino acid sequence conservation at the Nterm in available HIV-1 sequences

Our hypothesis is that the Nterm region is involved in IgG1 b12 binding to gp120 and may contribute to the potent cross-clade neutralization exhibited by b12. If this region is to be considered as a globally relevant vaccine candidate, the Nterm epitope sequence should be represented across all the major HIV-1 subtypes with very few amino acid variations. Thus, to examine this, analysis of all available gp120 Nterm sequences at the

147 Los Alamos HIV Sequence Database (http://hiv-web.lanl.gov/content/index) was performed.

Variation at each amino acid position along the Nterm epitope sequence of all Nterm sequences at Los Alamos shows the static nature of most of the sequence (Figure 26a).

Compared to the entire C1 at approximately 84%, the amino acid conservation at Nterm was 92.4%. Across the 26 amino acids examined, only 5 residues occured at less than

90% frequency in the 977 sequences tested. The three residues with the lowest conservation were K46 (68.7%), E47 (65.0%) and T49 (36.0%). Overall, the variation in the Nterm was 8%, which is drastically lower than the average intraclade gp120 variation of 30% (185). The 26 amino acids surrounding a sequence of amino acids in the gp120

C3 region shown to be involved in both CD4 binding and b12 binding (Q368 to Q393,

Table 13 and Figure 7) were more variant (18.1%; Figure 26b). Twelve of the 26 CD4BS epitope amino acids were less than 90% conserved (versus 5/26 Nterm). The overall conservation of this CD4BS region was 81.9%. While still higher than the average gp120 conservation of 70% across HIV-1 clades, the Nterm sequence showed a higher level of conservation.

To target an antigenic region of a variable protein across multiple clades may require the inclusion of peptides reflecting the sequences of more than one clade. Therefore, the most common Nterm sequence amino acids variants across clades were determined. The fewer the variants, the better coverage of HIV-1 sequences would be acheived. The

Nterm sequence, while 92% conserved across clades, does have some amino acids that

148

Figure 26. Conservation of amino acid sequences in gp120 Nterm (amino acid positions 34-62) and the b12 binding site/CD4BS (amino acid positions 368-93). (a) Amino acid variation at each position in the Nterm sequence away from the consensus sequence. (b) Amino acid variation along a sequence overlapping CD4BS contact residues. Sequence data were obtained from the Los Alamos Sequence Database (http://www.hiv.lanl.gov/content/sequence/HIV/mainpage.html/). N = 977 for Nterm, N = 1765 for CD4BS.

149 are highly variable. The most common single amino acid variation is E to D at position

47. In more than 30% of sequences, the second most common amino acid at this point is aspartic acid (Table 16). This substitution is conservative, although a vaccine containing both variants would cover nearly 100% of known sequences at that point. Similarly, the lysine of K46 is replaced very often with arginine. The T49 amino acid shows the lowest conservation, at 36%. Three more amino acids can be toggled into that position (lysine, glutamic acid, aspartic acid) to cover most of the rest of sequences at that point.

Therefore, good sequence coverage of the Nterm epitope sequence can be achieved by few variants.

It is also well documented that HIV clades exist in geographically distinct regions, and it is important to understand what the sequences of the most common circulating strains by region are. Table 17 shows the most common sequences and the consensus at the Nterm sequence for the most common HIV-1 subtypes, including two circulating recombinant forms. The number of sequences used to determine the overall consensus (1765) exceeds the summed numbers for the tabulated subtypes (1344) because the subtypes that were not well represented in the database (incomplete data or fewer than 20 sequences) were omitted. Except for these two subtypes, the sequences from the Nterm regions in the other clades are, for the majority, made up of the consensus plus one or two amino acid substitutions. The Nterm sequence identified by mass spectrometry with gp120 is also the most common sequence in clades B and D (52% and 57.4% frequency, respectively).

The same intact Nterm epitope sequence is also seen at low levels in clade C and A1D sequences (1.1% and 15.4%, respectively). Perhaps more interesting is that 71% over all

150 Table 16. Frequency of and amino acid changes that occur at more than 1% in all of the available Nterm sequences in the Los Alamos HIV Sequence Database. N = 977. Amino % acid >30% >20% >10% 5-10% 5-1% conserved frequency L M S 90.3 W Y 94.0 V A 93.8 T 99.6 V I 98.0 Y 99.1 Y 98.1 G 100 V 99.7 P 100 V A 98.4 W 99.4 K R E 68.7 E D 65.0 A T 98.8 T K E D N A E S 39.0 T P A 88.7 T V I P 90.6 L 99.7 F 99.7 C 99.5 A 99.5 S 99.6 D N 96.5 A V 97.4 K R N 91.2

151 Table 17. Most represented and variations from consensus of Nterm sequences among HIV-1 subtypes in the Los Alamos Sequence Database. The bottom row represents data from the entire set of sequences. Percent Percent Percent 0 Clade (total 0-2 most variant number of Most represented Nterm sequence variant represented amino sequences) amino sequence acids1 acids1 A (38) LWVTVYYGVPVWKDAETTLFCASDAK 52.6 0 71.1 A1 (59) LWVTVYYGVPVWKDAETTLFCASDAK 39.6 0 52.8 B (529) LWVTVYYGVPVWKEATTTLFCASDAK 52 52 95.2 C (469) LWVTVYYGVPVWKEAKTTLFCASDAK 34.5 1.1 73.2 D (61) LWVTVYYGVPVWKEATTTLFCASDAK 57.4 57.4 93.7 G (30) LWVTVYYGVPVWEDADTPLFCASDAK 20 0 0 01_AE (71) LWVTVYYGVPVWRDADTTLFCASDAK 52.1 0 19.7 02_AG (61) LWVTVYYGVPVWRDAETTLFCASDAK 32.8 0 34.2 A1D (26) LWVTVYYGVPVWKDAETTLFCASDAK 26.9 15.4 72.9 Consensus LWVTVYYGVPVWKEATTTLFCASDAK 22.6 22.6 71.0 (1765) 1. variation from the consensus sequence

152 sequences is composed of the Nterm sequence with 2 or fewer variations. Taken together, the amino acid sequence analyses suggest that the Nterm sequence and/or few variants thereof would be suitable cross-clade immunogens.

5.3.9 Summary

The main analysis in this section examines the sequences of HIV-1 strains that have been previously tested for neutralization sensitivity in a single comprehensive study (448).

The amino acid variation within the Nterm sequence is expected to correlate with b12 neutralization sensitivity. Variation does correlate significantly, especially at 90% reduction in infectivity. The Nterm sequence lies within the C1 region of gp120, and so it would be expected that amino acid changes in this region would be conserved. This is the case based on the biochemical properties of each amino acid change of variants. The potential for cross-clade coverage by vaccination with Nterm was investigated. The

Nterm sequence is much more conserved than the gp120 overall, more conserved than gp120 C regions, and more conserved than another part of the b12 epitope. The main sequence variations in this region are few and predictable and with little amino acid toggling, many HIV-1 sequences could be encompassed using Nterm as a cross-clade vaccinogen.

153 5.4 Immunogenicity of the Nterm epitope peptide in animal models and neutralization inhibition.

5.4.1 Rationale

Epitope mapping and ELISA binding experiments confirmed that the Nterm sequence of gp120 is involved in IgG1 b12-gp120 interactions. Sequence analysis of the Nterm region showed that variation in this region in HIV-1 correlates with b12 neutralization sensitivity and that the Nterm region is highly conserved across many clades and thus may be a desirable target for vaccine design. The 26-mer was assessed for its ability to elicit Abs in animal models. The sera of immunized animals were then tested for HIV-1 infection neutralization in two in vitro systems. From these studies, it will be determined if the Nterm peptide has promise for inclusion in a human-targeted vaccine.

5.4.2 Hypotheses

The Nterm epitope peptide is immunogenic in animals. Abs from immunized animals will bind gp120. Neutralization assays will show that the Nterm-specific antibodies are neutralizing across multiple HIV-1 strains.

5.4.3 Objectives i. To determine whether or not the Nterm peptide is immunogenic in mice, guinea pigs, and rabbits by immunization with peptide linked to an avidin carrier. ii. To determine whether or not the Nterm peptide elicits a neutralizing Ab response in immunized animals.

154 5.4.4 Study Outline

Groups of mice (4 per group), guinea pigs (5 per group), and rabbits (2 per group) (Table

18) were immunized with avidin-linked Nterm peptide, avidin-linked Scrambled peptide, gp120, or PBS only over 56 days. Animals were exsanguinated and the sera and IgG- purified samples were tested for binding to IIIB gp120 by ELISA. The guinea pig sera, rabbit sera, and the rabbit IgG samples were tested for neutralization of many HIV strains in pseudovirus neutralization assays. The rabbit sera and IgG-purified samples were tested for PBMC infection neutralization in an in-house assay using two donor PBMC and two viral strains.

Table 18. Animal serum naming system. Animals and their serum samples were labeled based on the immunogen and animal number. Animal Immunization group PBS gp120 Nterm Scrambled Mouse MuPBS Mu120 MuNt MuScr (pooled) Guinea pig GpPBS #1-5 Gp120 #6-101 GpNt #11-15 GpScr #16-20 Rabbit RPBS #1, 2 R120 #3, 4 RNt #5, 6 RScr #7, 8 1. The guinea pig 10 (Gp120 10) fell ill and had to be euthanized and so is omitted from analysis.

5.4.5 Immunized animal sera antigen binding

Having established the antigenicity of Nterm for IgG1 b12, the sera of animals immunized with Nterm were tested for binding to the native gp120. This would confirm that the epitope is immunogenic.

The exsanguinated animal sera were heat-inactivated, filtered, and tested for gp120 binding by ELISA. Microtitre plates were coated with gp120 and bound animal IgG was

155 measured using an animal-specific secondary Ab. The gp120-immunized rabbit sera bound gp120 with the greatest intensity, with maximal binding stretching beyond the dilutions of this experiment (Figure 27c, red line). The gp120-immunized mouse and guinea pig sera also bound to gp120 (Figure 27a and b, respectively, red lines). Thus, gp120 was immunogenic in all three animal species. Both the mouse and guinea pig animal sera were pooled for each group to conserve samples. The Nterm peptide- immunized mouse and guinea pig sera did not bind to gp120 above background (Figure

27 a and b, respectively, green lines versus blue and black lines). This indicates that the

Nterm peptide is likely not immunogenic in these two animal species, or if it is, the resulting antibodies do not bind the Nterm sequence on gp120. The Nterm peptide- immunized rabbit RNt #5 serum did bind gp120 moderately over the background (Figure

27c, dark green line). The Nterm peptide was thus immunogenic in this rabbit.

To demonstrate that the animals were capable of producing Ab responses to injected antigen, the animal sera binding to streptavidin was measured. Despite only 33% sequence identity, there is significant chemical and structural homology between avidin and streptavidin (605), which may explain the cross-reactivity. Avidin was used as a carrier, linking four peptides (Nterm or Scrambled) together in order to increase the immunogenicity of the peptide. The animals that were immunized with peptide (Nterm or

Scrambled) developed Abs that cross-reacted with streptavidin. The pooled mouse sera from the Nterm and Scrambled peptide-immunized animals bound to streptavidin compared to the gp120- and PBS-immunized pools (Figure 28a). The same was seen for the peptide-immunized guinea pigs and rabbits (Figure 28b and c, respectively).

156

Figure 27. Immunized animal sera binding to IIIB gp120 by ELISA. Mice (a), guinea pigs (b), and rabbits (c) were immunized with sterilized PBS (black lines), HIV-1 gp120 IIIB in PBS (red lines), Nterm peptide linked to avidin in PBS (green lines), or Scrambled peptide linked to avidin in PBS (blue lines). Assays were performed in triplicate (error bars are included) and these data are representative of 2 experiments.

157

Figure 28. Immunized animal sera binding to streptavidin by ELISA. Figure a shows the murine specificity; Figure b shows the guinea pig results; Figure c shows the rabbit binding. Animals immunized without avidin carriers (PBS and gp120-immunized animals – GpPBS #4, RPBS #1 and Gp120 #7, R120 #3, respectively) are shown with grey lines. The animals that were immunized with Nterm linked to avidin (GpNt #1-5 and RNt #5,6) are shown in yellow and black, and the animals immunized with Scrambled peptide linked to avidin are shown in colour (GpScr #16-19, RScr #7,8). Samples were run in triplicates and error bars indication the variation about the mean are included.

158 5.4.6 IgG purification of rabbit sera and antigenicity tests

Purified IgG was tested to ensure that no serum components were inhibiting the Ab responses in the ELISA and neutralization assays. The sera of immunized rabbits were

IgG purified for side-by-side comparisons of the gp120 binding and neutralization profile of whole sera versus the IgG fraction. IgG was purified from all rabbits at a concentration ranging 1.12 mg/ml to 3.42 mg/ml (Figure 29).

Purified rabbit IgG was tested for gp120 specificity to ensure the Nterm peptide- immunized rabbit Ab responses were not inhibited by other serum components. The

ELISA gp120-binding assay compared native serum, serum depleted of IgG, and IgG-add back serum from all four immunized groups (Figure 30). As expected, none of the IgG samples from PBS-immunized RPBS #1 and RPBS #2 and Scrambled peptide- immunized RScr #7 and RScr #8 bound to gp120 (Figures 30a and b panels and g and h panels, respectively, red lines). Purified IgG from both gp120-immunized R120 #3 and

R120 #4, bound gp120 (Figures 30c and d, red lines). This suggests that the IgG fraction contained the gp120-binding Abs. IgG from both Nterm peptide-immunized rabbits RNt

#5 and RNt #6 bound to gp120 (Figures 30e and f, red lines). In the gp120 and Nterm peptide-immunized serum, the IgG binding to gp120 closely followed the intact serum binding (red and black lines, respectively). This shows that the negative immunogenicity profiles of these animals are not due to inhibitory factors in their serum. The depleted sera (blue lines), in all gp120 and Nterm peptide-immunized animals, were unable to bind gp120, indicating that the IgG fractionation efficiently extracted all detectable gp120- binding Ab responses.

159

Figure 29. Purified IgG from immunized rabbits. Rabbit serum was run over a Protein G column, neutralized and desalted. IgG concentration was measured using a BCA kit.

160

Figure 30. Binding of rabbit sera (black), IgG-purified fraction (red), IgG-depleted sera (blue), and IgG add-back sera (purple) to IIIB gp120 in ELISA. The title indicates the rabbit. The x-axis shows the serum dilutions. This figure includes error bars and represents the experiment performed in triplicate.

161 When IgG was added back to the depleted sera, the gp120 binding was restored in the gp120 and Nterm peptide-immunized samples (purple lines).

IgG was purified from all eight rabbits. The IgG fractions from both the gp120- immunized and Nterm peptide-immunized animals recognized gp120 in an ELISA format. This binding reflected the binding by intact sera, indicating that there were no factors in the sera that affected gp120 binding by ELISA. The IgG fraction of rabbit serum is responsible for the gp120 binding as was shown with depletion studies. In rabbits, the Nterm antigen successfully elicited an IgG Ab to gp120.

5.4.7 Pseudovirus neutralization assays

Having established gp120-specificity of the animal sera, and since not all Abs that bind

HIV envelope can neutralize virus (182, 435, 469, 491), the samples were tested for HIV-

1 neutralization in vitro. The tested viruses were all subtype B (Table 4), but not all shared the same Nterm sequence (Appendix C). Pseudovirus assays were performed in collaboration with The Collaboration for AIDS Vaccine Discovery Network by technical staff at the Immune Monitoring Core Laboratory at Duke University. Guinea pig and rabbit sera and rabbit IgG were tested using their pseudovirus system. Infections were performed in TZM-bl cell lines and the pseudovirus consisted of a pSG3"Env HIV-1 backbone. TZM-bl contained a luciferase reporter gene under the control of the HIV

LTR that allowed for detection of successful infection (606). 50% inhibitory dose (ID50) indicates the amount of sample required to reduce by half measurable luciferase activity

162 infection cultures. Wherever possible, both pre-bleed and exsanguination samples were tested.

Due to limited sample quantities, the guinea pig sera of 2 animals in each group were tested against two HIV-1 strains: IIIB and SF162 (Table 19). Sera from both gp120- immunized guinea pigs Gp120 #7 and Gp120 #10 neutralized the IIIB pseudovirus (ID50

2115 and 1936, respectively) and less so the SF162 virus (ID50 133 and 29, respectively). Serum from the Nterm peptide-immunized GpNT #2 exsanguination sample inhibited IIIB (ID50 pre-bleed 45, exsanguination 67) and SF162 (ID50 pre-bleed

53, exsanguination 127) only slightly compared to the pre-bleed samples. This small difference in neutralization post-immunization was not seen in the GpNt #3 serum (IIIB pre- bleed 75 ID50, exanguination 24 ID50; SF162 pre-bleed 102 ID50, exsanguination

<20 ID50, indicating that there is no inhibition of viral growth above background by either guinea pig. Scrambled peptide-immunized sera ID50s were at background levels

(ID50 ranging from <20 to 39), as were the PBS-immunized serum ID50s (ranging from

<20 to 28 ID50). The control sera of the Scrambled peptide and PBS-immunized animals were non-neutralizing (<20 ID50). These data suggest that the sera from gp120- immunized guinea pigs display some protection from infection at a 50% neutralization titre, but that the Nterm peptide-immunized guinea pig sera do not. Therefore, the Nterm peptide did not induce the production of HIV-neutralizing Abs in guinea pigs.

Nterm peptide-immunized rabbits were the only animals whose serum and Abs bound to gp120 (Figures 28 and 30). Therefore, we were hopeful that these serum Abs would have

163 Table 19. Guinea pig sera neutralizations of pseudotyped pSG3"Env grown in TZM-bl cells. Viruses were pseudotyped with IIIB and SF162. Infections were performed in triplicate. Pseudovirus assays were performed by technical staff at Laboratory of Immune Measurements at the Duke Human Vaccines Institute of Duke University. ID501 Animal Sample IIIB SF162 Pre-bleed 45 53 GpNt #12 Exsanguination 67 127 Pre-bleed 75 102 GpNt #13 Exsanguination 24 <20 Pre-bleed 29 21 GpScr #16 Exsanguination 39 27 Pre-bleed <20 <20 GpScr #19 Exsanguination 32 35 GpPBS #2 Exsanguination 28 <20 GpPBS #4 Exsanguination 28 <20 Gp120 #7 Exsanguination 2115 133 Gp120 #10 Exsanguination 1936 29 positive control --- 443 346 negative control --- <20 <20 1. 50% Inhibitory dose values are the reciprocal dilution at which the relative luminescence units (RLU) were reduced by 50% compared to untreated control wells.

164 some neutralizing properties. Unfortunately, the rabbit serum pseudovirus neutralization assays gave similar results to the guinea pig sera (Table 20). The gp120-immunized

R120 #3 serum neutralized infection of TZM-bl cells by IIIB virus (1921 ID50 compared to pre-bleed <20 ID50) and inhibited SF162 at a low-level (66 ID50 compared to pre- bleed <20 ID50). The gp120-immunized R120 #4 serum neutralized IIIB virus infection only (43740 ID50 compared to pre-bleed <20 ID50). The Nterm peptide-immunized samples RNt #5 and #6 both did not inhibit IIIB and SF162 (all <20 ID50) and so neutralization testing was not extented to the entire panel of viruses. RPBS #1 and #2 and RScr #7 and #8 all gave <20 ID50. While the gp120 antigen was able to elicit a narrow neutralizing Ab response, Nterm was unable to elicit HIV-1 neutralizing Abs. To determine if animal serum components could be blocking Ab function in this system, purified rabbit IgG was tested for infection neutralization. The purified IgG and the depleted sera were also tested for neutralization (Table 21). The results showed that the

IgG-purified samples of both the gp120-immunized animals neutralized viral infection, mirroring the native serum neutralizations. R120 #3 serum was again able to neutralize

IIIB (3205 ID50), as was the R120 #3 purified IgG (507 ID50, compared to depleted serum <20 ID50). To a lesser extent R120 #3 also neutralized SF162 (ID50 51), as in

Table 19, but the purified IgG fraction did not. The lack of neutralization by purified IgG may be because some Ab is lost in the purifiction process. R120 #4 serum and the IgG fraction inhibited IIIB only (ID50 >43,740 and 33,722, respectively). Neither Nterm peptide-immunized sera, nor their purified IgG neutralized the tested pseudoviruses (all

ID50 <20).

165 Table 20. Rabbit sera neutralizations of pseudotyped pSG3"Env grown in TZM-bl cells. Pre-bleeds and exansguination samples were tested. All samples but R120 #3 and #4 were tested against IIIB and SF162 pseudotypes only. #3 and #4 were tested for ID50 against an expanded clade B panel. Infections were performed in triplicate. Pseudovirus assays were performed by technical staff at Laboratory of Immune Measurements at the Duke Human Vaccines Institute of Duke University. ID501 THRO4156.18 RHPA4259.7 SC422661.9 SVA Animal Sample QH0692 6535.3 PVO.4 SF162 - MLV IIIB

Pre- RPBS <20 <20 nd nd nd nd nd nd Nd bleed #1 Exsang <20 <20 nd nd nd nd nd nd Nd Pre- RPBS <20 <20 nd nd nd nd nd nd Nd bleed #2 Exsang <20 <20 nd nd nd nd nd nd Nd Pre- R120 <20 <20 <20 <20 <20 <20 <20 <20 <20 bleed #3 Exsang 1921 66 25 <20 <20 <20 <20 <20 <20 Pre- R120 <20 <20 <20 <20 <20 <20 <20 <20 <20 bleed #4 Exsang 43740 <20 <20 <20 <20 <20 <20 <20 <20 Pre- RNt <20 <20 nd nd nd nd nd nd Nd bleed #5 Exsang <20 <20 nd nd nd nd nd nd Nd Pre- RNt <20 <20 nd nd nd nd nd nd Nd bleed #6 Exsang <20 <20 nd nd nd nd nd nd Nd Pre- RScr <20 <20 nd nd nd nd nd nd Nd bleed #7 Exsang <20 <20 nd nd nd nd nd nd Nd Pre- RScr <20 <20 nd nd nd nd nd nd Nd bleed #8 Exsang <20 <20 nd nd nd nd nd nd Nd 1. Values are the reciprocal dilution at which the relative luminescence units (RUL) were reduced by 50% compared to untreated control wells. 2. Scr = Scrambled peptide

166 Table 21. Rabbit IgG and sera neutralizations of pseudotyoped pSG3"Env grown in TZM-bl. All samples were tested against a panel of clade B viruses. Infections were performed in triplicate. Pseudovirus assays were performed by technical staff at Laboratory of Immune Measurements at the Duke Human Vaccines Institute of Duke University. ID501 THRO4156.18 RHPA4259.7 SC422661.9 SVA Animal Sample QH0692 6535.3 PVO.4 SF162 - MLV IIIB

IgG <20 <20 <20 <20 <20 <20 <20 <20 <20 RPBS Depleted <20 <20 <20 <20 <20 <20 <20 <20 <20 #1 Exsang <20 <20 <20 <20 <20 <20 <20 <20 <20 IgG <20 <20 <20 <20 <20 <20 <20 <20 <20 RPBS Depleted <20 <20 <20 <20 <20 <20 <20 <20 <20 #2 Exsang <20 <20 <20 <20 <20 <20 <20 <20 <20 IgG 507 <20 <20 <20 <20 <20 <20 <20 <20 R120 Depleted <20 <20 <20 <20 <20 <20 <20 <20 <20 #3 Exsang 3,205 51 <20 <20 <20 <20 <20 <20 <20 IgG 33,722 <20 <20 <20 <20 <20 <20 <20 <20 R120 Depleted <20 <20 <20 <20 <20 <20 <20 <20 <20 #4 Exsang >43,740 <20 <20 <20 <20 <20 <20 <20 <20 IgG <20 <20 <20 <20 <20 <20 <20 <20 <20 RNt Depleted <20 <20 <20 <20 <20 <20 <20 <20 <20 #5 Exsang <20 <20 <20 <20 <20 <20 <20 <20 <20 IgG <20 <20 <20 <20 <20 <20 <20 <20 <20 RNt Depleted <20 <20 <20 <20 <20 <20 <20 <20 <20 #6 Exsang <20 <20 <20 <20 <20 <20 <20 <20 <20 IgG <20 <20 <20 <20 <20 <20 <20 <20 <20 RScr Depleted <20 <20 <20 <20 <20 <20 <20 <20 <20 #7 Exsang <20 <20 <20 <20 <20 <20 <20 <20 <20 IgG <20 <20 <20 <20 <20 <20 <20 <20 <20 RScr Depleted <20 <20 <20 <20 <20 <20 <20 <20 <20 #8 Exsang <20 <20 <20 <20 <20 <20 <20 <20 <20 PBS <20 <20 <20 <20 <20 <20 <20 <20 <20 gp120 Pooled <20 <20 <20 21 <20 <20 <20 <20 <20 Nterm Pre-bleed 26 <20 <20 24 <20 <20 <20 <20 <20 Scra 28 <20 <20 35 24 24 <20 <20 <20

167 The RPBS #1 and #2 and the RScr #7 and #8 were non-neutralizating (all ID50<20) and the pre-bleeds in all cases were low-to-undetectable (ID50 from <20 to 35). Both the sera and IgG fraction of gp120-immunized animals were neutralizing to only one or two viral strains, and Nterm peptide-immunized animals do not express HIV-1 pseudotyped virus- neutralizing Abs.

The pseudovirus neutralization assays demonstrate that, while gp120-immunized animals raised a narrow HIV-neutralizing Abs, the Nterm peptide-immunized animals did not.

The pseudovirus assay is an efficient, sensitive and high-throughput means to test for neutralization, but the caveat to this system is its relative artificiality. Infections are performed on a uniform cell line by pseudoviruses. Thus, we also tested serum neutralizations in PBMC culture with intact virus – a system that arguably mimics natural infection slightly better than the pseudovirus system, especially as PBMC are a heterologous mixture of cell types found in the blood.

5.4.8. PBMC-based HIV-1 infection neutralization assays

The ability of animal sera to neutralize HIV-1 was tested in a PBMC-based infection system. While the pseudovirus-based system gave negative results for neutralization by

Nterm peptide-immunized animals, these two systems are not always in agreement and should both be employed (607).

Animal sera neutralizations were tested against two viral strains in two donor PBMC.

PBMC were separated from blood by density-gradient centrifugation and stimulated with

168 the mitogen PHA before the addition of virus and inhibitors (sera or IgG). After incubation, the virus and the inhibitor were washed from the cells and, unless neutralized, the virus that infected the target cells in the incubation period grows and spreads to other cells. HIV p24 production and secretion by infected cells was measured and quantified by p24 ELISA. Not all HIV grows at the same rate in all cells, so neutralizations were performed on PBMC from two human donors (D074 and D039). TCID50s for four viruses, IIIB, Bal, SF162, and THA were performed on both donors to determine the best viruses to use. The Bal and IIIB viruses grew better in both donors (Tables 22 and 23) compared to SF162 and THA viruses, thus they were chosen for these studies.

In order to determine if the rabbit sera, IgG, or any forms of sera inactivation treatments were more or less toxic to PBMC, the viability of PBMC after incubation with heat- inactivated serum, heat-inactivated and sterile-filtered serum, filtered and PMBC adsorbed serum, and filtered, purified IgG was measured. Both D074 (Figure 31a) and

D039 (Figure 31b) viability were similar across each treatment group compared to each other and to virus-infected controls. Thus, any effects of the animal samples on HIV replication are more likely due to the Ab present in the samples and not altered viability of the PBMC.

Infections were therefore carried out for both D074 and D039 with the viruses IIIB and

Bal at MOI 0.1 and using the heat-inactivated, filtered serum samples. Pre-bleeds were pooled due to limited sample volume. The viruses were pre-incubated with the animal sera for 1 hour before the entire mixture was added to the cells. Supernatants of infections

169 Table 22. Data for TCID50 calculations for 4 viruses tested against donor D074. Day 7 TCID50 were used to calculate subsequent MOIs. Infections were performed in sextuplicate. Number of wells1 with detectable p24 production at each dilution 1:1,048,576 1:4,194,304 1:262,144

Virus 1:16,384 1:65,536 Day strain 1:1024 1:4096 1:256 1:16 1:64 1:4

Bal 6 6 6 6 3 2 0 0 0 0 0 IIIB 6 6 6 0 0 0 0 0 0 0 0 3 SF162 6 6 0 0 0 0 0 0 0 0 0 THA 2 0 0 0 0 0 0 0 0 0 0 Bal 6 6 6 6 6 6 6 6 5 2 1 IIIB 6 6 6 6 6 6 6 3 1 1 2 7 SF162 6 6 6 6 5 2 0 0 0 0 0 THA 6 3 1 1 0 0 0 0 0 0 0 Bal 6 6 6 6 6 6 6 6 4 3 5 IIIB 6 6 6 6 6 6 6 6 3 2 3 12 SF162 6 6 6 6 6 4 0 2 1 0 0 THA 6 6 3 2 2 1 0 1 0 0 0 1. out of 6 wells total

170

Table 23. Data for TCID50 calculations for 4 viruses tested against donor D039. Day 7 TCID50 were used to calculate subsequent MOIs. Infections were performed in sextuplicate. From these data, Bal and IIIB were selected for future studies. Number of wells1 with growth at each dilution 1:1,048,576 1:4,194,3 1:262,144

Virus 1:16,384 1:65,536 Day strain 1:1024 1:4096 1:256 1:16 1:64 1:4 04

Bal 6 6 6 6 3 2 0 0 0 0 0 IIIB 6 6 0 0 0 0 0 0 0 0 0 3 SF162 0 0 0 0 0 0 0 0 0 0 0 THA 0 0 0 0 0 0 0 0 0 0 0 Bal 6 6 6 6 6 6 6 6 4 5 1 IIIB 6 6 6 6 6 6 4 2 1 3 0 7 SF162 6 6 5 1 0 1 0 0 0 0 0 THA 1 1 0 0 0 0 0 0 0 0 0 Bal 6 6 6 6 6 6 6 6 6 5 4 IIIB 6 6 6 6 6 6 6 5 3 4 0 12 SF162 6 6 6 4 0 5 0 0 1 0 0 THA 5 4 2 0 0 1 0 0 0 0 0 1. out of 6 wells total

171

Figure 31. Cell viability assays for donor D074 (a) and D039 (b) with IIIB virus. A direct comparison of the cell viability to determine the of effect different treatments of rabbit sera on cell viability with the addition of HIV IIIB. Viability was determined on day 12 of infection by trypan blue exclusion enumeration. The cells alone were uninfected PBMC; the virus only were PBMC infected without any addition of rabbit sera at the MOIs of 0.1 and 1 virus/cell. Sera were treated by heat inactivation, IgG purification, 220 nm pore filtration or pre-adsorption to autologous PBMC.

172 were harvested at days 3, 7, and 12 and were tested for p24 levels by ELISA. AZT was used as a positive control for inhibition of growth and was demonstrably able to inhibit

IIIB infection (Figure 32).

The panel of rabbit sera and purified IgG were tested for HIV IIIB neutralization in

Donor 074. ELISA OD405 of p24 concentration from harvestes of day 7 infection of

D074 by IIIB are shown in Figure 33. Data are grouped by sample and the shading of the bars corresponds to the sample dilution. The sera were diluted 1:4 as were IgG samples

(starting at 10 µg/ml). The dashed horizontal lines indicate untreated virus growth. The serum at 1:400 from the gp120-immunized animal R120 #3 inhibited viral growth (OD values drop below the dashed line, Figure 33b, blue shaded grouping), indicating modest inhibition, while serum from R120 #4 showed higher neutralization (greater than 50%) down 1:1600 dilution (Figure 33c, blue shaded grouping).

Purified IgG from R120 #3 showed variable HIV neutralization across the dilutions that may reflect variations in PBMC infections (Figure 33b, fourth grouping). Purified IgG from R120 #4, however, again neutralizated virus down to the 1:1600 dilution (Figure

33c, yellow shaded grouping). The pooled gp120 pre-bleed had widely varying effects on viral growth that do not follow the dilution pattern and therefore are not indicative of specific neutralization events. The gp120-immunized sera and IgG data confirm the results seen with the pseudovirus assay, where there was neutralization observed by both samples, which were stronger in R120 #4 than R120 #3, and the IgG neutralizations were weaker than the serum neutralizations.

173

Figure 32. AZT inhibition of each viral strain IIIB (a) and Bal (b) in PBMC from D074. The data are expressed as concentration of p24 calculated from a standard curve, were performed in triplcate, and represent 2 independent experiments.

174

Figure 33. Viral growth for Day 7 harvests of IIIB-infected D074 PBMC. Both rabbit sera and IgG purifications were tested for each animal, samples are listed on the x-axis. Five or six dilutions (depending on space and sample availability) of each test sample were tested. Dashed lines indicate viral growth without inhibitor. Blue and yellow shaded bars indicate neutralizations that echo the pseudovirus neutralizations. All infections were performed in triplicate. Error bars are shown.

175 The Nterm peptide-immunized RNt #5 serum inhibited viral growth at only lower concentrations, indication that these are likely artefacts and not real neutralizations

(Figure 33c, third grouping). This is re-enforced by the inability of RNt #6 serum to neutralize HIV IIIB (Figure 33d, second grouping). All the PBS-immunized samples did not neutralize HIV (Figure 33a and b). The Scrambled peptide group pre-bleed samples non-specifically neutralized HIV, while the Scrambled peptide-immunized samples did not neutralize although the RScr IgG #7 IgG, RScr #8 serum, and RSc #8 IgG did seem to enhance viral growth independant of sample dilution (Figure 33e).

Table 24 summarizes the results of all of the infection studies in the PBMC assay. A more comprehensive dataset is available in the Appendix D, Tables D1-D4. 50% neutralizing serum dilutions were tabulated as they corresponded to IgG concentration.

Superscript numbers indicate observed non-specific neutralization due to lack of association between the OD values and the calculated p24 values based on the p24 standard curve (data not shown) and to a lack of titratable neutralization. These neutralizing events also were not observed in the neutralizations in the other donor and/or the other virus, further suggesting that they are artefacts of the PBMC infections. Indeed, the PBMC assay was more variable than the pseudovirus assay, likely owing to the use of

PBMC instead of cell lines and viral strains instead of pseudotyped virus. Nonetheless, echoing Figure 33 and the pseudovirus assays, only gp120-immunized animals produced sera and IgG capable of neutralizing HIV-1 IIIB. The serum and IgG from R120 #4 gave a more consistent neutralization profile in both IIIB and Bal and in both D074 and D039.

In PBMC from both donors, IIIB virus was more readily neutralized. No other sera or

176 IgG fractions were able to appreciably neutralize by 50% either IIIB or Bal in either donor. Abs in RNt #5 or RNt #6 were not neutralizing, indicating again that the Nterm, in this antigenic state, does not elicit an HIV-1 neutralizing response.

The PBMC assays served to confirm the general conclusions that were taken from the pseudovirus neutralization assays. IIIB gp120 was able to elicit autologous IIIB HIV neutralization and milder heterologous neutralization of SF162 and Bal viruses. Purified

IgG neutralized IIIB, but serum samples tended to neutralize more potently. Although

Nterm was able to elicit Abs that recognize IIIB HIV gp120, this specificity did not translate to neutralization of Bal or IIIB viruses in the PBMC system.

5.4.9. Summary

Immunization of mice, guinea pigs and rabbits with Nterm gave rise to gp120-specific

Abs in the rabbits only, indicating that Nterm is immunogenic in rabbits. Each animal was capable of generating an Ab response to streptavidin, a form of the peptide carrier used for immunization, indicating that the peptide preparations were immunogenic and

Ab-production abilities were intact. The rabbit serum IgG was purified, and the resulting

IgG from the gp120- and Nterm peptide-immunized sera maintained gp120 binding and were the source of gp120 binding, as depleted sera did not bind gp120. Despite the ability to bind whole, soluble gp120, the sera and IgG of Nterm immunized animals were incapable of neutralizing HIV-1 in a pseudovirus assay. Gp120-immunized animal sera and IgG showed neutralization activity against few, but not all viral strains studied. Once having established that the animal sera and IgG were not toxic to PBMC, they were

177 Table 24. Summary of the PBMC-based neutralization assays. This table reflects the results from all of the p24 ELISA results for all three time points, D3, D7, D12, and all tested dilutions of sera and IgG. 50% neutralization (serum reciprocal Donor HIV strain rabbit dilution) PBS #1 no PBS #2 no gp120 #3 yes (400) gp120 #4 yes (1600) IIIB Nt #5 no Nt #6 no Scr #7 no Scr #8 no D074 PBS #1 no1 PBS #2 no gp120 #3 no gp120 #4 yes (100) Bal Nt #5 no2 Nt #6 no Scr #7 no Scr #8 no PBS #1 no PBS #2 no gp120 #3 yes (6400) gp120 #4 yes (100) IIIB Nt #5 no Nt #6 no Scr #7 no Scr #8 no D039 PBS #1 no PBS #2 no gp120 #3 no gp120 #4 no Bal Nt #5 no Nt #6 no2 Scr #7 no2 Scr #8 no 1. observed OD405 and p24 values do not agree and neutralizations do not titrate out 2. observed neutralization titres do not titrate out

178 tested for neutralizations in a PBMC infection assay. Nterm samples were negative for neutralization, while gp120 samples showed moderate protection to IIIB, but less so to

Bal virus. Together, these data suggest that the Nterm epitope, while immunogenic, does not elicit HIV-1-neutralizing Abs in rabbits.

5.5 General Results Summary

The data herein show that the HIV-neutralizing mAb IgG1 b12 binds to the amino terminus of HIV gp120, a previously undefined point of contact. b12 specifically bound a synthetic Nterm peptide, but this interaction did not block b12-gp120 interactions, indicating that the Nterm sequence may not be the sole b12 point of contact on gp120.

The Nterm peptide and gp120 share a binding site at the b12 antigen bindong domain. In tandem to these studies, the IgG1 b6 epitope on gp120 was mapped to the Nterm as well.

The weakly neutralizing CD4BS b6 mAb bound Nterm peptide poorly in ELISA, which supports that the Nterm may be a part of the b12 neutralizing epitope. The Nterm sequence variations in the Los Alamos HIV Sequence Database correlate with b12 neutralization and the highly conserved nature of the Nterm across subtypes make it an ideal candidate for vaccination. Animals immunized with the Nterm peptide express gp120-specific Abs that are unable to neutralize HIV. This suggests that Nterm, or at least the Nterm peptide construct on its own, does not represent an HIV-neutralization- inducing immunogen.

179 6.0 Discussion

HIV vaccine efforts need to better understand how to elicit neutralizing Ab responses, as epitope mapping and immunization studies based on neutralizing antibodies have thus far been unsuccessful. Efforts focused on harnessing the putative IgG1 b12 CD4BS epitope for a preventative vaccine have been fruitless, and a novel approach, such as the highly sensitive technique of mass spectrometry epitope mapping, is required (568).

The hypotheses addressed in this thesis are that epitope mapping of IgG1 b12 will identify contacts points between b12 and gp120, and that the antigenicity of the gp120 regions identified will be confirmed by ELISA. The sequence variation of the b12 epitope in viral strains will correlate with b12 sensitivity to neutralization and the epitope will be well conserved across clades. Finally, the immunogenicity of the b12 epitope will be demonstrated in animal immunizations and the epitope will elicit a b12-like neutralizing Ab response.

These studies are the first to identify a binding site for IgG1 b12 on gp120 by mass spectrometry. This is the first time the b12 epitope has been expanded to include a region in gp120 C1. The Nterm epitope was analyzed for sequence suitability for a vaccine and the results were favourable; Nterm is a highly conserved sequence of gp120 that would require few modifications to generate a polyvalent vaccine. The Nterm peptide elicited

Abs in rabbits, mainly IgG, that bound to gp120, and variation in gp120 binding between the animals highlights that not all species reacted to immunogens in the same way. These

Abs, however, did not neutralize HIV-1 in vitro. The Nterm sequence is a part of the b12

180 epitope but Abs to the Nterm peptide do not mirror the ability of b12 to potently neutralize HIV. The Nterm epitope is nonetheless a part of the intact b12 epitope on gp120 and should be considered in future vaccine design endeavors.

6.1. Identification of a binding site for IgG1 b12 on HIV-1 gp120 by MALDI

QqTOF mass spectrometry epitope excision mapping

6.1.1 MS mapping and putative sequence of the b12 epitope

This is the first report of epitope mapping of IgG1 b12 by epitope excision mass spectrometry, a very sensitive system that identifies the binding interface between two proteins and sequences amino acid residues. As the b12 epitope is conformation- dependant (445), epitope excision was chosen because this approach allows the detection of conformational epitopes using whole monomeric gp120 (573). Epitope excision should therefore identify amino acids involved in the binding of intact gp120 to IgG1 b12 by directly dissecting the Ab-antigen interface. MS was expected to identify binding sequences encompassing the CD4BS. Based on in silico digests, no CD4BS sequences were identified by MS epitope mapping. Instead, a putative amino-terminal epitope,

Nterm, was identified (Table 8, Figure 7). This binding site was confirmed in two gp120 strains and using two different endopeptidases.

The amino acids shown previously to be involved in b12-gp120 interactions were identified by mutational studies, neutralization escape studies, computational docking of the crystal structures, and co-crystallization studies (Figure 7) (421, 452, 464, 466, 468).

The putative sequence identity of each MS peak did not overlap amino acids previously

181 indicated to be involved in gp120-b12 interactions. The identified epitope is one linear sequence located in the 1st constant region (C1) at the amino terminus of the gp120 sequence – LWVTVYYGVPVWKEATTTLFCASDAK (“NTerm”). This is the first report of the involvement of C1, and more specifically, the amino terminus of gp120, in the IgG1 b12 epitope on HIV-1 gp120.

6.1.2 MS/MS sequence identification confirms the putative b12 epitope

Using monomeric IIIB gp120, MS/MS confirmed the putative sequence of the Nterm epitope. The gp120 tryptic peak at m/z 1356 was matched to the HIV-1 gp120 sequence

EATTTLFCASDAK (Figure 10). Notably, the other matched sequences in the Sonar

MS/MS analysis are variants of the same HIV gp120 sequence (Table 9). The Sonar

MS/MS tool matched the gp120 tryptic peak at m/z 1608 to the epitope sequence

LWVTVYYGVPVW (Figure 11, Table 10). The gp120 epitope of b12, as measured by

MALDI QqTOF MS and MS/MS, is LWVTVYYGVPVWKEATTTLFCASDAK.

The Glu-C endopeptidase-based resultant peaks intensities were lower than the trypsin endopeptidase and required longer incubation periods to sufficiently digest gp120. This resulted in lower intensities of available parent ions for sequencing. This may indicate a lower activity of the enzyme relative to trypsin. While Glu-C was acceptable as a reagent for epitope identification, it was not utilized for sequencing purposes.

The data generated in these experiments did not implicate the CD4BS in the b12 epitope.

The CD4BS was encompassed within the detectable peak size range for each strain and

182 peptidase (<2400m/z), so these regions theoretically could have been detected. The lack of CD4BS data could be explained in several ways including lack of protection from endopeptidase digestion, or weak interactions between amino acids that did not survive the washing step. Additionally, a consequence of the non-quantitative nature of the

MALDI QqTOF platform is the eclipsing of one spectral species by another, due to differences in signal intensity of the detectable ions. b12 may also have a high affinity for the collective CD4BS epitope, but on protein fragmentation b12 may not maintain individual contact points with as high apparent affinity as the Nterm sequence.

Therefore, the results of this study do not preclude other b12 contact points on gp120.

Since KZ52 did not bind any gp120 fragments, it is likely that Nterm is a part of the b12 epitope,.

The Nterm sequence is a newly defined epitope for b12. This is the first study to map the b12 epitope using intact gp120. In the crystallization-based mapping studies of b12 and gp120, heavily deglycosylated and truncated gp120 core was used (156). The core structure is modified to mimic the gp120 conformation upon sCD4 ligation, and its ability to bind sCD4 is preserved (156, 608). While the structure at the CD4BS itself is similar to unliganded gp120, the overall conformation is different from unliganded gp120 (163).

The use of this drastically altered core gp120 may, in part, explain why interaction of b12 with the C1 region, specifically at the Nterm, of gp120 was not identified (165, 166, 168).

The exact location of the gp120 amino terminus and the Nterm sequence in the natural gp120 and trimer structure is still unknown. A publication of the crystal structure of

183 gp120 core with the N and C termini added back partially solved the location of both termini on gp120 (169). The sequence from the Nterm epitope VWKE is a #-strand

(Figure 15) and interacts with other #-strands to form the conformationally invariant “7- stranded #-sandwich”, off of which emanates the structurally flexible gp120 inner domain in 3 “layers”, and that interacts with gp41. The carboxy terminal end of the Ntem epitope (ATTTLFCASDAK) sits within “layer 1”, but this layer 1 structure only exists in the CD4-bound conformation of gp120. The amino terminus of the Nterm sequence

(LWVTVYYGVP) interacted with the gp120 carboxy terminus, although their interaction with gp41 within the trimer did not fit with previous cryoelectron tomography studies of the shape of the HIV-1 trimer (209). Therefore, there is not enough information to model the Nterm structure on gp120 in conjunction with b12 binding and in the trimer at this time. The direct epitope mapping by mass spectrometry may therefore add information on the epitope of IgG1 b12 and to the structure of the Nterm on gp120.

The b12 epitope has been mapped by several other methods (385, 436, 445, 452, 466,

468, 478-481, 483, 609-612). Soluble CD4 (sCD4) binding and point mutation studies showed that amino acids previously implicated in gp120-sCD4 binding participate in b12 binding (200, 445, 452), and the b12 epitope was subsequently expanded by using a crystal structure docking model and alanine scanning mutagenesis studies (452, 466, 468)

(Figure 7a). In 2 assays, amino acids V36, Y40, and W45 (all located in the Nterm) were also tested by site-directed mutagenesis. Only the W45S mutation affected b12 binding by reducing binding by one third. The gp120 core protein crystallized in conjunction with b12 Fab demonstrated that the interaction occurs only through CDR3 of the b12

184 heavy chain (163). The authors suggest that the binding interface is expansive enough to be considered a complete Ab-antigen interface (613), confirming data describing the light chain promiscuity of this and other CD4BS Abs (447, 595). b12 mutational studies, however, suggest both its heavy and light chains are involved in gp120 interactions (484) and that there are undefined contact points between b12 and gp120. The data surrounding the nature of the b12 interaction with gp120 at the CD4BS are strong and inarguable. Therefore, it is not the goal of this discussion to dispute the established points of contact between b12 and gp120, but to suggest new ones and to question whether or not they are involved in HIV-1 neutralization.

6.1.3 MS mapping of the CD4BS mAb IgG1 b6

Another CD4BS Ab, IgG1 b6, underwent epitope mapping by mass spectrometry in the experiments described in this thesis. The mapping experiments showed that b6, like b12, binds to the Nterm sequence of gp120. This is perhaps not surprising, since b6 and b12 were cloned from the same individual and they share sequence homology at their light chain Fv, similar affinity for monomeric gp120, and similar binding footprints (163, 452,

464, 466, 468, 491, 614, 615). b12 is potently and broadly HIV neutralizing while b6 is only weakly neutralizing for clade B strains. This may reflect their different abilities to bind the intact HIV-1 envelope trimer or the existence of b6 antigenic determinants peripheral to those available only in intact trimer (448). b12 is able to bind to any form of the HIV-1 trimer, while b6 may only interact with inactivated, partially gp120-shed trimers on the surface of HIV-1 (490, 492). This observation is supported by studies in which b12 effectively inhibited b6 virus capture, while b6 only slightly inhibited b12

185 virus capture (490, 616). That both b6 and b12 bind to the Nterm epitope might also suggest that the Nterm sequence is not involved in the more potent neutralization activity of b12 versus b6, although this idea ignores the possibility that the mAbs may have different abilities to bind to the Nterm sequence. MALDI QqTOF MS is not quantitative and cannot address this issue, but ELISA binding assays can.

6.2. Confirmation of the antigenic specificity of IgG1 b12 for the Nterm sequence on

HIV-1 gp120.

6.2.1 b12 specifically binds the Nterm peptide

While MS epitope mapping suggested a new specificity of b12 on gp120, the antigenicity of that sequence needed to be confirmed, as MS mapping has not always been confirmed by ELISA (218). ELISA binding assays were performed with synthetic, biotinylated peptide representing Nterm and a negative control. A scrambled version of Nterm was used as a negative control (“Scrambled”). b12 bound to the Nterm peptide (Biotin-

LWVTVYYGVPVWKEATTTLFCASDAK). The negative control Ebola-specific KZ52

Ab did not (Figure 14a), nor do other anti-HIV mAbs (Figure 16), showing that b12 binding to the Nterm peptide is antigen-specific and not mediated by the heavy chain Fc. b12 did not bind the Scrambled peptide, demonstrating Nterm specificity (Figure 14b).

The small detectable background binding of b12 for the Scrambled peptide at the highest concentration likely represents background binding due to similar chemical characteristics such as the hydrophobic nature of the peptide. b12 binding to Nterm peptide occured only at high Nterm concentrations, suggesting that although Nterm

186 peptide can be bound independently of gp120, b12 has a reduced ability to bind the peptide than the epitope intact on monomeric gp120.

To further investigate the nature of the Nterm binding by b12, the epitope was expressed in two halves (determined by the two epitope halves identified by MS epitope mapping) and were tested for binding by b12 in ELISA. This experiment suggests that b12 bound the second half of the epitope (EATTTLFCASDAK) much better than the first half

(EKLWVTVYYGVPVWKE; Figure 14c). The presence of both sequence halves in MS mapping suggested that the Nterm sequence within gp120 may be presented differently.

It is not uncommon that epitope fragments have conformation-dependence in some binding determinants and not others (218), and the predicted secondary structure of the

Nterm peptide was somewhat different from the structure reported by Pancera, et al

(Figure 15), indicating removing Nterm from its setting within gp120 may have altered its structure. Together with the previous ELISA data, this study indicates that the structure of the Nterm peptide may not be identical to the structure of this sequence on gp120, which may affect immunogenic studies with this peptide immunogen. The b12

ELISA experiment set does, however, corroborate the MS sequencing result that b12 binds to gp120 Nterm in a specific manner.

6.2.2 The role for Nterm in b12 binding to gp120

To determine the role of the Nterm sequence in b12 binding to monomeric gp120, competition assays between gp120 and Nterm peptide were performed. Monomeric IIIB gp120 blocked b12 binding to the Nterm peptide but not Scrambled peptide (Figure 17b).

187 At a ratio of 10 molecules Nterm peptide to 1 molecule of gp120, the Nterm peptide binding was reduced by approximately 50%, suggesting the overall affinity of b12 for gp120 is higher than for the Nterm peptide. These data further suggest that gp120 blocks the paratope of Nterm on b12, either specifically or sterically. Conversely, Nterm peptide did not inhibit b12 binding to gp120 (Figure 17a). At the highest Scrambled and Nterm peptide concentrations, a steep drop in binding was observed, most likely reflecting the system being non-specifically overwhelmed with peptide. These results strongly suggest that the Nterm region is not the sole binding point on gp120 for b12 and raise the possibility that both the previously-identified CD4BS epitope and the Nterm are both involved in b12-gp120 interactions. Another explanation for the lack of competition of

Nterm peptide for gp120 binding is that gp120 sterically occludes the Nterm binding site on b12 without occupying it. The phenomenon of an Ab having multiple specificities is only recently being investigated in HIV infection (617). Unfortunately, the current understanding of the b12-gp120 contact points described by crystallization studies involve only the gp120 core and molecule and cannot resolve this point (163).

To delineate the role of the Nterm sequence and its surrounding region in the b12 epitope, we obtained a JR-FL strain-derived monomeric gp120 construct, "NmCHO, which lacks the amino terminal 52 amino acids (481). b12 bound "NmCHO poorly (Figure 18a) compared to wild-type IIIB gp120. While these gp120 constructs are from different HIV-

1 strains, b12 previously has been shown to bind wild type JR-FL gp120 at nearly identical levels as IIIB gp120 (538), and so IIIB serves well as a positive control. These

188 data suggest that the amino terminal 52 amino acids of gp120 are involved in successful binding by b12, owing in part or in full to the Nterm sequence.

The results of the b12 epitope mapping and ELISA experiments are striking when considered in context with a recent epitope mapping study of the neutralizing mAb 4E10

(218). 4E10 was already known to bind the gp41 MPER sequence NWFN/DIT (443,

618). Epitope excision and epitope extraction mass spectrometry mapping identified additional epitope determinants at the amino-terminal regions of both gp120 (amino acids

LWVTVYYGVPV) and gp41 as being involved in 4E10 binding. Further MS mapping showed that 4E10 bound the Nterm sequence even in the absence of gp41. Neither gp120, nor these regions have been previously shown to bind 4E10. This may help explain why some HIV strains with the NWFN/DIT MPER sequence are resistant to neutralization by 4E10 (448, 619, 620). It was suggested that the amino terminal gp41 epitope (in the “fusogenic” region) may not have been previously described because commercially available gp41 constructs generally lack that region. Notably, the 4E10 epitope at the gp120 amino terminus overlaps the Nterm epitope sequence described in this thesis. 4E10 did not bind a synthetic peptide of this sequence, nor several variants thereof, suggesting conformational requirement for 4E10 binding. Conversely, our data suggest that the Nterm peptide binding may not require overall conformational stability.

Interestingly, the first half of the Nterm peptide that did not bind b12 in ELISA is the identified 4E10 epitope that was not bound by 4E10 in ELISA. This further highlights the importance of the native antigen and structural context for Ab-epitope interactions.

189 The authors suggest that the highly conserved nature of this Nterm sequence may explain the potent cross-clade neutralization displayed by 4E10 (218).

6.2.3 IgG1 b6 binds Nterm peptide weakly

Why does this Nterm sequence appear to be involved in the binding of b12, b6, and

4E10? Each experiment used a mild detergent to reduce non-specific background results, so the results are not likely an artifact of the experimental system (621, 622). This begs the question: if the Nterm sequence is involved in b12 and 4E10 neutralization of HIV-1 across clades, what does it mean that the poorly neutralizing mAb b6 binds the Nterm sequence? If the Nterm sequence is involved in b12 neutralization, then the ability of b12 and b6 to bind Nterm peptide might be different. ELISAs showed that b6 binds to

Nterm at levels only slightly above the KZ52 background mAb and well below b12

(Figure 19). This suggests that Nterm could conceivably play a role in the b12 neutralizing epitope. b6 also exhibited slightly higher binding to "NmCHO gp120 than the b12 and KZ52 mAbs, although none of the binding levels drop off substantially enough to be considered dose-dependent binding. Given that "NmCHO was constructed in an attempt to induce an Ab response to the CD4BS, and that b12 was previously reported to bind to the construct (481), it is difficult to explain the lack of b6 and b12 binding to "NmCHO observed in this study. Although these two mAbs do not bind,

HIV+ sera bound "NmCHO and confirmed that it retains gp120 antigenic characteristics.

Therefore, the only conclusion that could be drawn from this experiment, in our hands, is that b12 and b6 showed no to extremely low level binding to "NmCHO. From these studies, it is apparent that, by ELISA, the Nterm peptide may represent only a weak

190 determinant of the entire conformational b6 epitope on HIV-1 gp120. These studies do not contradict the potential role for the Nterm in b12 binding and subsequent neutralization of HIV-1.

If Nterm plays a role in neutralization, perhaps amino acid variation in this sequence correlates with viral b12 neutralization sensitivity. In that case, this sequence may be suitable for a cross-clade neutralizing Ab-based HIV vaccine. This possibility can be addressed in two fashions: sequence analysis and animal immunizations.

6.3 Analyses of the Nterm sequence – variation and vaccine suitability.

6.3.1 Nterm sequence variation correlates with b12 neutralization sensitivity

Defining an HIV epitope that is able to elicit a broadly neutralizing antibody response has been elusive to date. HIV displays phenomenal sequence diversity globally and within a single infected individual, posing a unique problem for vaccine design. Therefore, a major focus of HIV vaccine design is to determine antigens that are both immunogenic, creating a neutralizing immune response in the host, and conservative, such that the targeted sequence is constrained and can vary little or very predictably over clades and strains. IgG1 b12 potently neutralizes HIV-1 across clades and therefore its epitope is likely well conserved.

The Nterm sequence variation among published sequences was correlated to the ability of b12 to neutralize that strain. A multitude of studies have measured b12 neutralization against many strains of HIV (inexhaustive list: (159, 318, 436, 445, 448, 452, 460, 464,

191 466, 468, 469, 481, 492, 562, 574, 595, 623, 624)) but this study focused on the most comprehensive data set from a single publication (448). The Los Alamos HIV Sequence

Database Nterm consensus sequence, which is identical to the IIIB and MN sequence identified by MS analysis, was used to assess amino acid variation. Nterm sequence variation correlated significantly with b12 neutralization titres (Figure 21). The 50% neutralization did not correlate as well with Nterm amino acid variation, but this may be because these data are less stringent than the 90% neutralization. These data support the possibility that the Nterm sequence may be involved in b12 neutralization. Given the potential bias for b12 to neutralize clade B viruses (445-447, 625), a similar analysis was performed with only subtype B sequences (Table 11). The clade B Nterm sequence variations correlated with neutralization sensitivity, although this analysis did not take into account the type of amino acid change. Therefore, charge, polarity, hydrophobicity, and size traits were used to attribute scores to amino acids variations within the studied gp120 sequences. For the purposes of this study, each characteristic was assigned equal value. In the variations + score data, both 50% and 90% graphs show significant correlation between sequence and neutralization titre (Figure 23). Overall, the Nterm sequence shows low levels of fairly conserved variation that is reflected in the chemical properties of the variants, and this variation correlates with b12 neutralization titres.

6.3.2 The gp120 CD4BS variation correlates with b12 sensitivity

Variation at three parts of the previously-described conformational CD4BS epitope was also compared to b12 neutralization sensitivity (156, 421, 464, 466, 468) (Table 13). The sequence 462TND is the most variable of the three epitope sequences studied, despite

192 the fact that mutations at the amino acids at 462 or 463 reduced b12 binding to monomeric gp120 by >50% (466, 468). The other two b12 binding regions appear to be much more conserved, which may also reflect their roles in CD4 binding. At 90% neutralization, there is a correlation between neutralization and sequence variation around the CD4BS epitope. These data reflect the Nterm sequence correlations. Therefore, these data do lend credence to the suggestion that the Nterm region may be involved with the b12 neutralizing epitope, perhaps in conjunction with the CD4BS, and highlight the conserved nature of the Nterm epitope.

6.3.3 Sequence and chemical characteristic of Nterm and its variants

Overall gp120 sequence conservation is approximately 70%, whereas variable region conservation is only about 20-30% (626). The Nterm sequence is much more conserved than C1 in general than the analysed CD4BS sequence (Figure 26). Using the score described in 6.3.1, the most common clade B Nterm sequence variations scored a zero, meaning that they are highly conserved. 90.7% of changes scored <2 and only 9.3% scored >2, suggesting that the amino acid changes that do occur in the Nterm sequence are conservative. The hydrophobicity pattern of each variant peptide was also similar

(Figure 25a). Similar to hydrophobicity, the polarity and charge of each residue shows consistent patterns among the analyzed sequences (Figure 25b and c). Overall, the middle of the Nterm sequence has the most variation, suggesting some chemical flexibility in this region may not affect the role of the Nterm sequence in infection

(Figure 26). Together, the chemical data show that the Nterm sequence is remarkably consistent, and that variants tend to exhibit nearly uniform biochemical properties with

193 the consensus sequence. This is likely because the Nterm sequence is important for gp120 function, and makes the Nterm sequence a stronger candidate as a vaccine antigen.

Both the Nterm and CD4BS are highly conserved regions indicating that, in the right vaccine preparation, they may elicit neutralizing Abs to functionally or structurally constrained sequences. Such Abs are the ultimate goal of vaccine design.

Nonetheless, taking into account HIV’s ability to vary its gp120 sequence, even Nterm sequence-based vaccination may require multiple variant epitopes where a small number of amino acids are toggled with two or more variant amino acids. To further determine variation in the amino acids comprising the available Nterm sequences, the frequencies of amino acids at each point along the sequence were compiled and tabulated (Table 17).

The majority of amino acid variations along the Nterm sequence exist in fewer than five percent of reported Nterm sequences. To assess the most common Nterm variants across

HIV subtypes, a consensus Nterm sequence for each subtype was generated (Table 18).

The overall consensus sequence matches the subtypes B and D consensuses, while consensuses of the other subtypes vary within the 46KEATT50 region. A vaccine consisting of the consensus sequences of the subtypes B, C, and D (2 variants) would cover 36.2% of the strains represented in the database. The top six variant consensus sequences cover 50.3% of the sequences database. This highlights how difficult it is to get total coverage for a sequence in HIV, even within a highly conserved region.

Therefore, it may be more sensible to create subtype-specific variant-containing vaccines and immunize individuals with cocktails based on the predominant subtypes of their geographical location. Polyvalent vaccines are an important area of research and have

194 been shown effective in other diseases, such as pneumococcus (627) and human papilloma virus (HPV) (628).

6.3.4 The role of gp120 amino terminal region in HIV-1 infection

Within the Nterm epitope, the amino acid regions 35W-45W and 54L-60A in C1 have been described previously as “nearly invariant sequences” that would be suitable HIV vaccine candidates (629). In separate studies, deletion of the amino terminal 32 amino acids of gp120, insertion of amino acids at the amino terminus, or mutation of the amino acids 36V, 40Y, 45W, V44, and F53 reduced associations between gp120 and gp41 (147,

176, 177). The slightly hydrophobic nature of the sequence corresponds with the hypothesized role of the Nterm involvement in gp41 interactions near the viral surface

(147, 176, 177, 630, 631). Mutagenesis showed that amino acids W35, V38, Y39, Y40,

G41, V42, and L52 are involved in normal envelope processing and P43 and W45 are important for gp120 processing and incorporation into the virion (630)(Figure 34).

Figure 34. Nterm sequence and the functions of individual amino acids based on alanine- scanning mutagenesis and binding studies (169, 177, 630).

The direct or indirect involvement of gp120 sequences at or near the C1 amino terminal region in binding to the CD4 molecule is controversial (198, 199, 632, 633). Some

195 evidence suggests that C1 lies in a conformation that allows for CD4 access to gp120, but when altered by mutagenesis, a structural change blocks CD4 contact (632, 634). Also supporting the importance of C1 in gp120-CD4 interactions, structural or otherwise, is the existence of C1-specific Abs that block gp120 binding to CD4 and HIV binding to

CD4+T cells (479, 635-637). The important role of the Nterm sequence in HIV-1 pathogenicity is evidenced by the biochemically conserved nature of its variants, therefore the prevalence of these variants across clades and the ability of this sequence to elicit cross-reactive neutralizing Ab responses deserves further investigation.

6.3.5 Nterm epitope cross-reactivity

Some research suggests that broadly neutralizing Abs to HIV are difficult to isolate because they are cross-reactive with human proteins, as in the case of 2F5 with cardiolipin (516). The B cells that express autoreactive Abs, should they arise, would therefore be removed during B cell development (517, 600, 601, 638). To determine if the Nterm sequence can be used as a non-self immunogen in humans, the Nterm sequence was subjected to a blast sequence alignment against known human proteins

(Table 15). Of relevance to Ab development, all but one matching protein are intracellular, and therefore unlikely to be exposed to B cell responses. The sequence

50TTLFC matched to tumour necrosis factor ligand factor 2, more commonly known as

TNF-! signal sequence that is intracellularly cleaved from the mature, secreted protein.

Thus, it is unlikely that any B cell Ab responses to this epitope will raise any self Abs or be deleted or rendered anergic due to similarities to host proteins.

196 6.4 Nterm peptide elicitation of antibody responses and their neutralization profiles

While some human HIV vaccine trials contained constructs including Nterm sequences, none of these trials have reported especially high levels of neutralizing Ab production, and none have released any detailed information of Ab specificities beyond binding to envelope monomers and trimers (547). There is therefore still much value to be gained in dissecting the immunogenicity of the Nterm sequence in mice, guinea pigs, and rabbits.

6.4.1 Nterm peptide immunogenicity in animals

Animal sera were used for all tests in section 5.5. Animal serum is preferred over plasma for neutralization assays since heparin sulphate and other anti-coagulants can affect viral growth (639). The animal sera were heat-inactivated to negate any anti-viral effects of complement, which may lead to viral lysis and inactivation.

The gp120-immunized animals showed varying abilities to produce gp120-specific Abs

(Figure 27). At all tested dilutions, the rabbit sera bound gp120 strongly. Guinea pig sera bound well with a titratable effect. The mouse sera binding was the lowest. Of all

Nterm peptide-immunized animals, only the rabbits developed Abs that bound gp120.

Therefore, species-specific differences exist with respect to the abilities of each animal to produce Abs to the Nterm peptide immunogen. The implications of this are important; if a putative epitope is poorly immunogenic in one species, this does not imply that the epitope is not immunogenic in another species, as demonstrated by HIV vaccines that are protective in one animal model and not in another (558, 640, 641).

197 In order to better define the IgG responses in rabbits, IgG was purified from sera (Figure

29). The depleted sera were negative for gp120 binding, indicating that serum IgG is mainly responsible for gp120 binding. IgG did not bind gp120 better than sera; it is thus unlikely that there are non-specific components in the serum that interfere with the IgG binding to gp120. It is therefore reasonable to use either IgG or animal sera for neutralization assays.

6.4.2 Animal serum and IgG pseudovirus-based neutralizations

The ability of animal sera to neutralize HIV was tested by a pseudovirus inhibition assay.

This assay is the current gold standard for testing vaccine responsiveness for several reasons including high reproducibility by use of clonal cell lines and a consistent HIV-1 pSG3"Env backbone virus. All tested viruses were subtype B, but they did not all share the same Nterm sequence as the consensus and the immunogen, allowing us to test animal sera against Nterm variants (Appendix C).

Murine sera were not tested for pseudovirus neutralizations since gp120 binding was absent in the Nterm peptide-immunized animals and because this system is not ideal for testing mouse sera (D. Montefiori, personal communication). The guinea pig sera samples were tested with two pseudotyped viruses, IIIB and SF162 (Table 21). Both strains are clade B tier 1 viruses and easily neutralized. The gp120-immunized guinea pigs sera easily neutralized the homologous IIIB and the heterologous SF162 to a lesser degree. Nterm immunized sera did not neutralize virus above the background levels from the pre-immunization samples, mirroring the ELISA gp120 binding assays. The lack of

198 neutralization in guinea pig samples indicates that, in this system, the Nterm epitope is not effective at eliciting neutralizing Abs.

The rabbit sera were also tested for neutralization against an extended panel of pseudoviruses (Table 22). Three viruses (6535.3, RHPA4259.7, and THRO4156.18) had variant amino acids in the Nterm sequence. The 6535.3 isolate is a tier 1B virus, while the other two are tier 2 viruses, indicating that they are slightly more resistant to neutralization. RHPA also has an amino acid substitution, T49N, which introduces an N- linked glycosylation site to the Nterm sequence. This may affect its susceptibility to neutralization. Much like the guinea pig neutralizations, the rabbit sera from the gp120 immunized animals inhibited IIIB, and at high concentrations, inhibited SF162 growth.

Virus 6535.3 was inhibited only barely above background, while the rest of the tested viruses were resistant to neutralization by the gp120-immunized sera. The Nterm peptide-immunized rabbit sera showed no neutralizing ability against the tier 1 viruses, so experiments were not extended to the other viruses. In these rabbit immunizations, the

Nterm epitope was immunogenic, but did not elicit Abs capable of neutralizing HIV-1.

The pseudovirus inhibition assay with rabbit sera was repeated with purified IgG (Table

23). The SF162 neutralization was nearly absent, which may indicate that either another

Ab isotype was responsible for that neutralization, or that the specific Abs were at low levels so as to be undetectable once the sample underwent the rigors of purification. The

Nterm peptide-immunized IgG fractions were unable to neutralize any tested viruses, indicating that the Nterm-specific rabbit IgG were not HIV-1 neutralizing.

199 6.4.3 Rabbit serum and IgG PBMC-based neutralizations

The pseudovirus-based neutralization system gave negative results for Nterm for both rabbits and guinea pigs, but while good for screening a large number of samples against a large number of viral strains, this system is somewhat artificial in its employment of cell lines and pseudoviruses and it is not always to be in accordance with the PBMC-based neutralization system (607). PBMC consist of immune cells of the blood and closer reflect in vivo HIV-1 infection than cell lines. Therefore, the animal samples were tested by PBMC neutralizations of IIIB and Bal. Only the gp120- but not the Nterm peptide- immunized rabbits were able to neutralize HIV IIIB. These results correspond to the earlier pseudovirus assays; gp120 can elicit neutralizing Abs in rabbits while the Nterm peptide cannot.

The overall results of all PBMC infections are tabulated in Table 24. The IIIB infections demonstrate that gp120-immunized animal sera and IgG neutralize IIIB virus. The Bal infections of D074 gave similar results but had higher background (Appendix D2). These experiments highlight a shortcoming of the PBMC assay versus the pseudovirus assay.

Although the pseudovirus system is more artificial than the PBMC studies, it is more controlled and is not affected by human-to-human and day-to-day variations in blood cells. Therefore, a valid approach to studying responses to a new antigen is to use both protocols in tandem (469, 492, 607). In this study there were some detectable Nterm peptide-immunized sample neutralizations, but they were not corroborated in other donor/viruses and the neutralization capacity of the pre-bleed samples were also somewhat high, indicating that these responses are not specifically neutralizing. Similar

200 results were obtained from donor 039.

The gp120-specific Abs may have been unable to neutralize as well as b12 partly due to the nature of the elicited paratope on the serum Abs. Although the overall structure of an

Ab antigen-binding site is convex, it has been suggested that the precise paratope structures correspond with their epitope. Peptide-specific paratopes are slightly grooved

(311), while protein-specific Abs tend to have a relatively flat paratope, and in the case of intact virus-linked epitopes, the length of the CDR H3 loop averages four amino acids longer than for any other antigen source (301). b12 has an unusually long, portruding

CDR H3 of eighteen amino acids, which may aid in its remarkable neutralization ability

(468). Therefore, a peptide immunogen may limit the structure of the elicited Ab paratope, which may alter function or access to the epitope within its native protein, and this should be taken into consideration in future mAb-based and b12-based vaccine design.

The Nterm peptide, although antigenic and immunogenic, was unable to elicit HIV-1 neutralizing Abs. It is, however, likely a part of a larger, more complex conformational epitope, and was not successful as a lone, linear epitope. The immunization system employed was the homologous prime-boost system. In this scheme, the animal immunogen preparations were the same at each time point, save for the adjuvant. There is some evidence, however, that heterologous boosting (example: monomeric gp120 booster) may enhance a vaccine immune response (642). The use of an immunogen in its native protein would theoretically instruct the maturing immune response to recognize

201 the peptide within its protein environment and structure and enhance the response to the

Nterm peptide.

6.5 Experimental limitations

6.5.1. Mass spectrometry epitope mapping, epitope identification

The mass spectrometry epitope mapping is a newer method that is highly sensitive, but it does have its drawbacks. In this system, the CD4BS regions that were previously identified as b12 binding sites were not identified, likely for reasons previously discussed. Notwithstanding, the identification of the Nterm epitope was a solid, repeatable outcome. The stringency of the washes was increased in attempt to lower the

Nterm abundance and detect the CD4BS, but the opposite experiments were not performed with decrease of stringency. These washes were optimized to reduce backgrounds, so reducing their stringency may not have been a suitable alternative.

Another alternative may have been to use a gp140 (gp120 + gp41 membrane external region) molecule or the gp120 core molecule to map the b12 epitope, but at the time this experiment was performed, gp120 core was unavailable and gp140 preparations were not standardized. Instead of using the gp120 epitope fragments from the b12 MS mapping experiments for sequencing, a separate experiment with digested gp120 was used for epitope mapping. Attempts to sequence b12-captured fragments were hindered by low yields of epitope fragments and so the specific tryptic peaks generated by gp120 digestion were used instead.

6.5.2. ELISA confirmation of Nterm epitope antigenicity

202 The Nterm epitope is hydrophobic, and therefore did not dissolve in water, which is the standard practice for epitope binding assays. Five percent formic acid successfully solubilized the peptides and allowed the ELISAs to be performed. A shortcoming of the

ELISA system itself was that the b12 was unable to bind Nterm peptide-coated wells.

This may have been because Nterm peptide was not coating the wells at a high enough concentration or because the Nterm peptide was not accessible or in the correct orientation for b12 binding. This drawback of being unable to control the conformation of the epitope recurs in the experimental limitations of the fourth section. We were therefore unable to measure dilutions of b12 binding to peptide, and unable to test competition by gp120 for b12 binding to Nterm by coating plates with Nterm, as was done with gp120. A final limitation of this section is that some high backgrounds were recorded with the Scrambled peptide. The Nterm binding was, however, significantly higher than the Scrambled peptide and reflected a real biochemical interaction.

The affinity of b12 for the Nterm peptide was not determined. Surface plasmon resonance experiments were performed on a Biacore 2000 machine, but there were high background affinities for the Scrambled peptide and the Nterm experiments were not reproducible (data not shown). In the absence of functioning surface plasmon resonance experiments, the apparent affinity of b12 for Nterm using an inhibitor could not be tested, as Nterm peptide did not inhibit b12-gp120 interactions. An alternative was direct competition between biotinylated Nterm and unbiotinylated Nterm. Unbiotinylated

Nterm was synthesized, but this reagent was either unable to remain in solution or

203 degraded quickly, as the concentration of the peptide dropped quickly over time (data not shown). It was therefore not utilized in experiments.

6.5.3. Nterm sequence analysis

The data in this section supported but did not prove the claim that the Nterm is involved in the neutralizing activity of b12. Another drawback in the analysis was subtype B bias in the Los Alamos HIV sequence database. Therefore, much of this section focused only on clade B sequences. This reduced the N but increased the assurance that the data were obtained in an unbiased fashion. These analyses also did not take into account the effect of mutations associated with CTL-mediated selection pressure, although the overall low rate of mutations in the Nterm epitope sequence suggests low pressure.

6.5.4. Animal studies

The animals that were sacrificed for this experiment were highly inbred, and therefore it would be expected that their responses to antigen would be the same within groups.

While the trends matched, purified IgG levels were not the same across all animals. The neutralization data was also inconsistent within groups. Some of the sera seemed to enhance the viral growth above background. Replicates and sample numbers were too low to determine if this is a true phenomenon or an artifact of human error. Infection enhancement is a concern for Ab-based vaccine design, although the enhancement seen here seems to be independent of the vaccinogen. Perhaps the most important note to make for all of the observed inconsistencies is the group number. There were only two

204 rabbits per immunization group, which magnifies any small differences in the care of the animals, the handling of the immunizations, and the handling of the samples.

6.6 General Discussion

6.6.1 The gp120 amino terminus as an antigen

The Nterm sequence was able to elicit Abs that bound IIIB gp120, though these Abs were unable to block IIIB infection. There is a possibility that the Nterm epitope is hidden within the envelope trimer, as it is hypothesized to be involved in C5/gp41 interactions

(169, 176). A seminal study performed in 1995 showed a disparity between monomeric gp120 binding and neutralization (538). The authors showed that the tested viruses did contain the epitopes in question, but concluded that these epitopes must exist on the immunologically silent face of the HIV trimer. When gp120 is shed, its underbelly is exposed to the immune response and Abs can be made to the biologically irrelevant, typically silent epitopes (160, 171, 643). This does not explain why b12 would have access to the Nterm region, unless the composition of the b12 epitope on gp120 is different from on the intact trimer, or the Nterm position in the trimer is more available to the immune system than previously thought. There are 3 mAbs that have been described that bind within the Nterm epitope region described here: 7E2/4, M85, and 4D4#85 (474,

636, 644). 7E2/4 and M85 are reported to bind within the amino acids 31-51 and both bind monomeric gp120. All three mAbs bind linear conformation-dependent epitopes at the amino terminus and 7E2/4 and M85 bind weakly to oligomeric gp120 (474).

6.6.2 Antibody-based vaccine design

205 Perhaps some of the most important HIV vaccine-related questions concern the definition of a neutralizing Ab response. Is a neutralizing response that acts against many different strains and subtypes of HIV possible? Will such broadly neutralizing responses require a monoclonal or a polyclonal-type response? Finally, specific to this thesis, what role could the Nterm epitope play in neutralizing Ab-based vaccine design?

Initially, the HBV-style of vaccination was attempted – injecting individuals with HIV-1 gp120 – in the AIDSVAX trial. Although a predictable failure due to the highly variant immunodominant regions and immune shielding glycosylation (645), this trial, in this author’s opinion, paved the way for current trials (646). There are no examples of studies of pre-exposure mAb prophylaxis in humans, but there has been some research into post- exposure passive immunization (PI). Although protective in rhesus macaques (647, 648), intravenously administered mAbs were unable to protect infected individuals from viral rebound on cessation of ART (424). Despite the failure of this human trial, the overwhelming success of prophylactic PI in animal studies strengthens the resolve that a vaccine based on broadly neutralizing mAbs such as IgG1 b12, 2G12, 4E10, and 2F5 may be protective.

Perhaps such ideas as insertion into scaffolding or immunofocusing will help to present the Nterm epitope structure properly or to enhance b12-inspired vaccine design. gp140 consists of gp120 expressed with gp41 and a stop codon inserted just upstream of the gp41 transmembrane region. It is capable of oligomerization, and has been developed for use as a trimer. gp140 alone could not elicit neutralizing or protective Abs in humans or

206 animals (649-652), but it was shown that the majority of neutralizing Abs generated to gp140 were V1-specific. Therefore, a new strategy was employed: insert V1 from diverse

HIV strains into the gp140 scaffold and generate cross clade responses (560). Another approach to HIV envelope-based research is gp120 engineering. The goal of this research is to introduce modifications such as glycosylations and deletions that will focus the immune response on certain areas of gp120 (478, 481, 653). As of yet, envelope engineering has failed to generate a product that induces neutralizing Abs. Attempts have also been made to turn the core gp120 molecule and other envelope constructs into more potent immunogens (654). HIV-1 primary isolates tend to be more resistant to neutralization than lab-adapted strains (434, 538, 655, 656) and may also represent a more appropriate antigen for eliciting in vivo neutralizing Ab responses (657).

It has been shown that some infected HIV-individuals have a CTL response to the Nterm epitope (658). Although the authors did not correlate such a response with infection progression, this may be a starting point for a vaccine that includes the Nterm sequence that elicits both Ab and CTL responses (659, 660). A vaccine that relies not only on the induction of a neutralizing Ab response, but also induces robust Th and CTL responses, is a much more promising focus for vaccine design. The RV144 vaccine is an example of such an endeavor, and also happens to be the only HIV vaccine to date that has shown moderate protection from infection in a phase III trial (558).

6.6.3 IgG1 b12-like antibody elicitation

207 There have been many attempts to generate the b12 epitope and elicit b12-like Abs in immunization. Immunofocusing has attempted to produce an antigen that displays only

CD4BS to the immune system. gp120 was mutated to be hyperglycosylated and lacking amino acids 473-476, a mutation that was shown to reduce non-neutralizing CD4BS mAb binding (such as b6) while b12 retained its ability to bind this gp120 construct. The immunofocussed gp120 was unable to elicit a neutralizing Ab response better than wild type gp120 (478, 562). The same alterations were made to trimeric gp140, but it too did not elicit a better neutralizing Abs response (661).

Technologically advanced studies, such as the use of MS in Ab epitope mapping, may be the key to new means of redefining and re-interpreting old mAbs and their specificities.

Several groups are also taking macromolecular approaches to design b12-like epitopes.

These methods are often used to design and alter drugs such as antibiotics and involve processes such as structural constraining and/or protein modification, designing miniproteins that duplicate the functional or epitope region of choice, and creation of small molecule mimetopes that structurally and chemically mimic the region of choice

(662). Mimetics are typically used to ameliorate drug specificities and functions and to overcome resistances and negative reactions. A mimetope binds the Ab at a site overlapping the paratope (antigenic sequence), but is not the same sequence as the epitope. A phage-display peptide library was tested and a mimetope for b12 was identified - B2.1 (484, 663). Unfortunately, the mimetope, when expressed as a peptide, showed much reduced affinity for b12, and the crystallization characterization of the mimetope-b12 interaction suggests the mimetope binds to b12 inappropriately at its

208 paratope (664). The design of a b12-based vaccine has been elusive, but the combination of new techniques with long-standing knowledge makes this a more attainable goal.

6.7 Model of IgG1 b12 binding to the Nterm Epitope

To develop a model of b12 binding to the Nterm sequence on gp120, it may be helpful to consider a similar scenario that has been described for a gp41 MPER epitope determinant for mAbs 2F5, 4E10, and Z13e1 (511, 512, 665). Interestingly, although these mAbs bind gp41 with comparable affinity, they do not neutralize with the same strength; the hierarchy of neutralization potency is 2F5>4E10>Z13e1. The basis for the different abilities is unclear. There may be other contact points on envelope that differ between the three mAbs. Alternately, the observed differences may be a result of the structure of the mAbs and their ability to engage the MPER while gp41 is in the viral envelope spike, akin to b6 versus b12 binding to the intact spike. The MPER, like its name suggests, lies close to the viral membrane at the base of the trimer and steric hindrance may partially or fully block access to these epitopes. Some of the obstacles to the complete understanding of the MPER mAb specificities for what may be a partially or fully hidden linear determinant may be applied to b12 binding to the Nterm sequence.

As mentioned in the Introduction, upon interaction of gp120 with the target cell CD4 and co-receptor molecules, the envelope pre-hairpin intermediate is formed. At this point, the hydrophobic gp120 amino terminus interacts with the hydrophobic gp41 amino terminus to form part of the fusion machinery. The gp41 sequence inserts into the target membrane, while the gp120 Nterm may interact with the target cell membrane (666-668).

209 This gp120 amino terminal conformational change emphasizes the flexible nature of

Nterm. This entry process invites the question: at what point in this process could b12 bind to Nterm and/or neutralize infection?

The gp120 amino terminus is thought to interact with C5 and gp41 at a point on the trimer that may be hidden from the immune response (436, 669, 670), which may explain why Abs to the Nterm are not neutralizing. The flexibility of gp120 that would be required for the interaction of b12 with the CD4BS and Nterm at a single Fab could be high. There is a study that has been largely overlooked that tests the kinetics of b12 neutralization (318). In this study, b12 neutralization occurred after HIV-1 attached to the target cell. More specifically, 80% of b12-coated virus was still able to bind to target cells, while 20% was neutralized before receptor engagement. This indicates that, although b12 binds the CD4BS, this binding either does not necessarily inhibit CD4 binding, or is out competed by CD4. Another directly conflicting paper published at nearly the same time showed that b12 neutralized mostly at pre-CD4 attachment (458).

Both studies used T cell line-adapted (TCLA) strains of HIV. A more recent study showed that b12 neutralized HIV-1 primary isolate JR-FL pre- and post-CD4 attachment, but was unable to block infection post-CD4/CCR5 attachment (513). TCLA virus is more sensitive to neutralization than primary isolates, and therefore the results from this third study may better reflect the in vivo b12 neutralization activity (434, 538, 655, 656,

671). This study confirms not only that b12 neutralizes post-CD4 attachment, but also that b12 can neutralize HIV at multiple steps in entry, perhaps by multiple mechanisms.

The Nterm sequence may be hidden on the trimer; perhaps conformational change after

210 CD4 ligation to trimeric gp120 exposes the amino terminus to b12. Therefore, the Nterm sequence may in truth be a contributor in the post-CD4 attachment b12 neutralization.

The identification of the Nterm epitope alone on mass spectrometry may be a result of the fact that b12 does not bind the Nterm epitope and the CD4BS at the same time when gp120 is in its CD4-unliganded state and off the spike. This explains why Abs that are specific only for the Nterm are not neutralizing and do not behave like b12. The ability of the b6 mAb to bind Nterm but not to neutralize viral infection may, in part, be a case of differing affinities for the Nterm, as suggested in the ELISA. A similar model has been suggested for 4E10 binding to Nterm, where the Nterm is engaged in the pre-hairpin intermediate following both CD4 and CCR5 engagement (211), resulting in a “molecular trap” where fusion is frozen. For b12, the mAb is brought into proximity of the fusion mechanism by interaction with the CD4BS. For 4E10, 2F5, and Z13e1, there is a dilemma with understanding how these mAbs can access the MPER and neutralize. One suggestion is that these Abs bind the phospholipid of the viral membrane to access the

Nterm and the MPER epitopes (507, 509). Together, the b12 and 4E10 datasets may present a new mechanism of post-receptor engagement Ab neutralization.

6.8 Future Directions

Several avenues can be explored to enhance or carry on the IgG1 b12 epitope mapping research presented here. Below are some examples of both. Currently, the Nterm sequence is being tested for binding to IgG1 b12 by nuclear magnetic resonance (NMR) spectroscopy. Initial studies support the data presented in this thesis; b12 binds Nterm.

Further mass spectrometry assays employing gp120 core and oligomeric envelope may

211 lend information as to when b12 binds the Nterm and not the CD4BS. Also, since large amounts of sCD4 are required to measurably reduce b12 binding to gp120 (672), it may be feasible to pre-incubate gp120 or gp140 oligomers with sCD4, then map b12 binding to gp120. Another future direction is to define the amino acids involved in b12 binding to Nterm by testing overlapping peptides by both MS and ELISA. b12 epitope mapping using purified HIV-1 would yield information on the b12 epitope on the intact trimer, which may identify new epitopes, confirm old ones, or substantiate the epitope described here. Another avenue would be to perform MS epitope mapping on CD4BS mAbs with different HIV-1 neutralization profiles from b12 and b6, such as b13 and VRC01. b13 was isolated alongside b12 and b6, is weakly neutralizing, and has also been co- crystallized with gp120 core (164, 445-447). The information from VRC01 may be more biologically relevant as it is a naturally-occurring Ab (488), whereas b12 was cloned and selected from heavy and light chain libraries.

Determining the exact differences in the specificities of b12 and b6 and how they affect the neutralization profiles of each mAb will provide the key to what makes a neutralizing

CD4BS Ab. In these studies, both b6 and b12 bind the Nterm sequence, but it appears that b12 binds better to the Nterm peptide, and confirmation of this by surface plasmon resonance or other methodologies will lend more credence to this observation. As the field of crystallization and co-crystallization advance, it will be interesting to see a comparison of the binding of two such mAbs with gp120 with the intact termini.

212 Determining the role of the Nterm in b12 binding on the trimer could be more problematic. Mutation at amino acids within the Nterm, or deletion of the Nterm from gp120 substantially reduces the ability of gp120 to interact with gp41 (177), which may reduce virus production and infectivity. One possible way of getting past this may be to use the SOS-Env pseudovirus. The SOS envelope contains a disulfide link between gp120 S505 and gp41 S605. This holds the gp120-gp41 structure together so that, once

CD4 and CCR5 bind gp120, conformational changes that allow fusion are blocked.

Without apparent detriment to the infection system, addition of dithiothreitol (DTT) reduces the gp120-gp41 bond and infection ensues (673, 674). This construct may be able to maintain the interactions between gp120 and gp41 for the examination of the effects of Nterm sequence mutation on b12 capture and neutralization.

As mentioned, a heterologous immunization strategy, whereby Nterm peptide is the priming immunogen and gp120 or gp140 with intact Nterm is the boost, may enhance responses to Nterm in its native environment. To determine the potential use of the

Nterm peptide as an immunogen, the animal sera could be further characterized for binding to HIV trimer with gp140 or cells expressing the HIV trimer. Also, the animal sera could be tested for non-neutralization functions such as ADCC, complement activation, and transcytosis inhibition. The immunogenicity of the Nterm peptide may be tested in other mammalian species. Also, alternative ways of expressing epitopes

(example: circularizing, embedding in a protein scaffold, or creating a multiple chain peptide (675, 676)) may constrain the peptide flexibility while maintaining antigenicity and perhaps elicit better Ab responses (511).

213

Screening the sera of infected individuals for Nterm sequence-specific Abs would indicate if this epitope has any clinical relevance. Also, testing other neutralizing Abs isolated from infected individuals and for Nterm epitope specificity would lend further support to the proposed role for Nterm sequences in neutralization by both b12 and 4E10.

On a more fundamental level, the entire binding interface between gp120 and b12 has yet to be described in one set of experiments. Additionally, once the epitope is defined, it must be translated into an immunogen that elicits neutralizing Abs in humans. Once these studies are achieved for b12 and/or other HIV-1 neutralizing Abs, the goal to curb the pandemic spread of HIV can become a reality.

6.9 Summary

The work detailed in this thesis has employed the MS epitope excision platform to define the epitope of a well-studied mAb, IgG1 b12. Previously, b12 has been shown to bind the HIV gp120 CD4 binding site. MS unveiled a new interaction site for b12 at the amino terminal sequence of gp120. In this experiment, the first hypothesis of this thesis, that MS will identify the CD4BS as the b12 epitope, was disproved. The b12 binding

Nterm epitope was validated in ELISA with the Nterm peptide, confirming the antigenicity of the epitope. The sequence variation of Nterm in the Los Alamos HIV

Sequence Database correlates with the potency of b12 neutralization. Supporting the third hypothesis, Nterm was determined to be a highly conserved sequence that should make a suitable global vaccine candidate. Animals were immunized with Nterm peptide

214 and although the rabbits expressed gp120-binding Abs, these Abs could not neutralize infection in vitro. This proves the first half of the final hypothesis, that the Nterm peptide is immunogenic, but disproves the second half of the hypothesis, that b12-like neutralizing Abs will be generated. Nevertheless, this is the first description of the Nterm as a binding site for b12 and one of the first studies to suggest that the gp120 Nterm sequence plays a role in cross-clade HIV-1 neutralizating Ab responses. In conjunction with the findings that the broadly neutralizing mAb 4E10 binds to a sequence within the b12 Nterm epitope, the data in this thesis emphasize the importance of characterizing broadly neutralizing antibody responses on their native antigen and the potential value of such findings to future Ab-based vaccine design.

215 Section 7.0 References

1. UNAIDS/WHO. 2009. AIDS Epidemic Update: December 2009. Geneva. 2. UNAIDS/WHO. 2007. AIDS Epidemic Update: December 2007. Geneva. 3. UNAIDS/WHO. 2006. AIDS Epidemic Update: December 2006. Geneva. 4. UNAIDS/WHO. 2005. AIDS Epidemic Update: December 2005. Geneva. 5. UNAIDS/WHO. 2004. AIDS Epidemic Update: December 2004. Geneva. 6. UNAIDS/WHO. 2003. AIDS Epidemic Update: December 2003. Geneva. 7. UNAIDS/WHO. 2002. AIDS Epidemic Update: December 2002. Geneva. 8. UNAIDS/WHO. 2001. AIDS Epidemic Update: December 2001. Geneva. 9. UNAIDS/WHO. 2008. AIDS Epidemic Update: December 2008. Geneva. 10. PHAC. 2010. HIV/AIDS Epi Updates. Centre for Infectious Disease Prevention and Control. 11. CDCU. 2007. Manitoba Health Statistical Update on HIV/AIDS: January 1985 - December 2006. Communicable Disease Control Branch. 12. 1981. Pneumocystis pneumonia--Los Angeles. MMWR Morb Mortal Wkly Rep 30:250-252. 13. 1981. Kaposi's sarcoma and Pneumocystis pneumonia among homosexual men-- New York City and California. MMWR Morb Mortal Wkly Rep 30:305-308. 14. 1981. Follow-up on Kaposi's sarcoma and Pneumocystis pneumonia. MMWR Morb Mortal Wkly Rep 30:409-410. 15. 1982. Update on acquired immune deficiency syndrome (AIDS)--United States. MMWR Morb Mortal Wkly Rep 31:507-508, 513-504. 16. Popovic, M., M. G. Sarngadharan, E. Read, and R. C. Gallo. 1984. Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science 224:497-500. 17. Barre-Sinoussi, F., J. C. Chermann, F. Rey, M. T. Nugeyre, S. Chamaret, J. Gruest, C. Dauguet, C. Axler-Blin, F. Vezinet-Brun, C. Rouzioux, W. Rozenbaum, and L. Montagnier. 1983. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 220:868-871. 18. Sarngadharan, M. G., M. Popovic, L. Bruch, J. Schupbach, and R. C. Gallo. 1984. Antibodies reactive with human T-lymphotropic retroviruses (HTLV-III) in the serum of patients with AIDS. Science 224:506-508. 19. Gallo, R. C., S. Z. Salahuddin, M. Popovic, G. M. Shearer, M. Kaplan, B. F. Haynes, T. J. Palker, R. Redfield, J. Oleske, B. Safai, and et al. 1984. Frequent detection and isolation of cytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS. Science 224:500-503. 20. de Silva, T. I., M. Cotten, and S. L. Rowland-Jones. 2008. HIV-2: the forgotten AIDS virus. Trends Microbiol 16:588-595. 21. Robertson, D. L., J. P. Anderson, J. A. Bradac, J. K. Carr, B. Foley, R. K. Funkhouser, F. Gao, B. H. Hahn, M. L. Kalish, C. Kuiken, G. H. Learn, T. Leitner, F. McCutchan, S. Osmanov, M. Peeters, D. Pieniazek, M. Salminen, P. M. Sharp, S. Wolinsky, and B. Korber. 2000. HIV-1 nomenclature proposal. Science 288:55-56.

216 22. Simon, F., P. Mauclere, P. Roques, I. Loussert-Ajaka, M. C. Muller-Trutwin, S. Saragosti, M. C. Georges-Courbot, F. Barre-Sinoussi, and F. Brun-Vezinet. 1998. Identification of a new human immunodeficiency virus type 1 distinct from group M and group O. Nat Med 4:1032-1037. 23. Gurtler, L. G., L. Zekeng, J. M. Tsague, A. van Brunn, E. Afane Ze, J. Eberle, and L. Kaptue. 1996. HIV-1 subtype O: epidemiology, pathogenesis, diagnosis, and perspectives of the evolution of HIV. Arch Virol Suppl 11:195-202. 24. Hahn, B. H., G. M. Shaw, K. M. De Cock, and P. M. Sharp. 2000. AIDS as a zoonosis: scientific and public health implications. Science 287:607-614. 25. Keele, B. F., F. Van Heuverswyn, Y. Li, E. Bailes, J. Takehisa, M. L. Santiago, F. Bibollet-Ruche, Y. Chen, L. V. Wain, F. Liegeois, S. Loul, E. M. Ngole, Y. Bienvenue, E. Delaporte, J. F. Brookfield, P. M. Sharp, G. M. Shaw, M. Peeters, and B. H. Hahn. 2006. Chimpanzee reservoirs of pandemic and nonpandemic HIV-1. Science 313:523-526. 26. Gao, F., E. Bailes, D. L. Robertson, Y. Chen, C. M. Rodenburg, S. F. Michael, L. B. Cummins, L. O. Arthur, M. Peeters, G. M. Shaw, P. M. Sharp, and B. H. Hahn. 1999. Origin of HIV-1 in the chimpanzee Pan troglodytes troglodytes. Nature 397:436-441. 27. Van Heuverswyn, F., Y. Li, C. Neel, E. Bailes, B. F. Keele, W. Liu, S. Loul, C. Butel, F. Liegeois, Y. Bienvenue, E. M. Ngolle, P. M. Sharp, G. M. Shaw, E. Delaporte, B. H. Hahn, and M. Peeters. 2006. Human immunodeficiency viruses: SIV infection in wild gorillas. Nature 444:164. 28. Korber, B., M. Muldoon, J. Theiler, F. Gao, R. Gupta, A. Lapedes, B. H. Hahn, S. Wolinsky, and T. Bhattacharya. 2000. Timing the ancestor of the HIV-1 pandemic strains. Science 288:1789-1796. 29. Kilmarx, P. H. 2009. Global epidemiology of HIV. Curr Opin HIV AIDS 4:240- 246. 30. Wilson, D. P., M. G. Law, A. E. Grulich, D. A. Cooper, and J. M. Kaldor. 2008. Relation between HIV viral load and infectiousness: a model-based analysis. Lancet 372:314-320. 31. Boily, M. C., R. F. Baggaley, L. Wang, B. Masse, R. G. White, R. J. Hayes, and M. Alary. 2009. Heterosexual risk of HIV-1 infection per sexual act: systematic review and meta-analysis of observational studies. Lancet Infect Dis 9:118-129. 32. Royce, R. A., A. Sena, W. Cates, Jr., and M. S. Cohen. 1997. Sexual transmission of HIV. N Engl J Med 336:1072-1078. 33. Cohen, M. S. 2007. Preventing sexual transmission of HIV. Clin Infect Dis 45 Suppl 4:S287-292. 34. Johnson, B. T., L. A. Scott-Sheldon, T. B. Huedo-Medina, and M. P. Carey. Interventions to reduce sexual risk for human immunodeficiency virus in adolescents: a meta-analysis of trials, 1985-2008. Arch Pediatr Adolesc Med 165:77-84. 35. Attia, S., M. Egger, M. Muller, M. Zwahlen, and N. Low. 2009. Sexual transmission of HIV according to viral load and antiretroviral therapy: systematic review and meta-analysis. Aids 23:1397-1404. 36. Baeten, J. M., E. Kahle, J. R. Lingappa, R. W. Coombs, S. Delany-Moretlwe, E. Nakku-Joloba, N. R. Mugo, A. Wald, L. Corey, D. Donnell, M. S. Campbell, J. I.

217 Mullins, and C. Celum. 2011. Genital HIV-1 RNA Predicts Risk of Heterosexual HIV-1 Transmission. Sci Transl Med 3:77ra29. 37. Quinn, T. C., M. J. Wawer, N. Sewankambo, D. Serwadda, C. Li, F. Wabwire- Mangen, M. O. Meehan, T. Lutalo, and R. H. Gray. 2000. Viral load and heterosexual transmission of human immunodeficiency virus type 1. Rakai Project Study Group. N Engl J Med 342:921-929. 38. Zhu, J., F. Hladik, A. Woodward, A. Klock, T. Peng, C. Johnston, M. Remington, A. Magaret, D. M. Koelle, A. Wald, and L. Corey. 2009. Persistence of HIV-1 receptor-positive cells after HSV-2 reactivation is a potential mechanism for increased HIV-1 acquisition. Nat Med 15:886-892. 39. Coombs, R. W., P. S. Reichelderfer, and A. L. Landay. 2003. Recent observations on HIV type-1 infection in the genital tract of men and women. Aids 17:455-480. 40. Reynolds, S. J., A. R. Risbud, M. E. Shepherd, J. M. Zenilman, R. S. Brookmeyer, R. S. Paranjape, A. D. Divekar, R. R. Gangakhedkar, M. V. Ghate, R. C. Bollinger, and S. M. Mehendale. 2003. Recent herpes simplex virus type 2 infection and the risk of human immunodeficiency virus type 1 acquisition in India. J Infect Dis 187:1513-1521. 41. Fleming, D. T., and J. N. Wasserheit. 1999. From epidemiological synergy to public health policy and practice: the contribution of other sexually transmitted diseases to sexual transmission of HIV infection. Sex Transm Infect 75:3-17. 42. Galvin, S. R., and M. S. Cohen. 2004. The role of sexually transmitted diseases in HIV transmission. Nat Rev Microbiol 2:33-42. 43. Henin, Y., L. Mandelbrot, R. Henrion, R. Pradinaud, J. P. Coulaud, and L. Montagnier. 1993. Virus excretion in the cervicovaginal secretions of pregnant and nonpregnant HIV-infected women. J Acquir Immune Defic Syndr 6:72-75. 44. Clemetson, D. B., G. B. Moss, D. M. Willerford, M. Hensel, W. Emonyi, K. K. Holmes, F. Plummer, J. Ndinya-Achola, P. L. Roberts, S. Hillier, and et al. 1993. Detection of HIV DNA in cervical and vaginal secretions. Prevalence and correlates among women in Nairobi, Kenya. Jama 269:2860-2864. 45. Cohen, M. S. 2006. Amplified transmission of HIV-1: missing link in the HIV pandemic. Trans Am Clin Climatol Assoc 117:213-224; discussion 225. 46. Bailey, R. C., S. Moses, C. B. Parker, K. Agot, I. Maclean, J. N. Krieger, C. F. Williams, R. T. Campbell, and J. O. Ndinya-Achola. 2007. Male circumcision for HIV prevention in young men in Kisumu, Kenya: a randomised controlled trial. Lancet 369:643-656. 47. Chan, D. J. 2005. Factors affecting sexual transmission of HIV-1: current evidence and implications for prevention. Curr HIV Res 3:223-241. 48. Collins, C., T. J. Coates, and J. Curran. 2008. Moving beyond the alphabet soup of HIV prevention. Aids 22 Suppl 2:S5-8. 49. Murphy, E. M., M. E. Greene, A. Mihailovic, and P. Olupot-Olupot. 2006. Was the "ABC" approach (abstinence, being faithful, using condoms) responsible for Uganda's decline in HIV? PLoS Med 3:e379. 50. Asamoah-Odei, E., J. M. Garcia Calleja, and J. T. Boerma. 2004. HIV prevalence and trends in sub-Saharan Africa: no decline and large subregional differences. Lancet 364:35-40.

218 51. Nagelkerke, N. J., S. Moses, S. J. de Vlas, and R. C. Bailey. 2007. Modelling the public health impact of male circumcision for HIV prevention in high prevalence areas in Africa. BMC Infect Dis 7:16. 52. Auvert, B., D. Taljaard, E. Lagarde, J. Sobngwi-Tambekou, R. Sitta, and A. Puren. 2005. Randomized, controlled intervention trial of male circumcision for reduction of HIV infection risk: the ANRS 1265 Trial. PLoS Med 2:e298. 53. Gray, R. H., X. Li, G. Kigozi, D. Serwadda, F. Nalugoda, S. Watya, S. J. Reynolds, and M. Wawer. 2007. The impact of male circumcision on HIV incidence and cost per infection prevented: a stochastic simulation model from Rakai, Uganda. Aids 21:845-850. 54. Gray, R. H., G. Kigozi, D. Serwadda, F. Makumbi, S. Watya, F. Nalugoda, N. Kiwanuka, L. H. Moulton, M. A. Chaudhary, M. Z. Chen, N. K. Sewankambo, F. Wabwire-Mangen, M. C. Bacon, C. F. Williams, P. Opendi, S. J. Reynolds, O. Laeyendecker, T. C. Quinn, and M. J. Wawer. 2007. Male circumcision for HIV prevention in men in Rakai, Uganda: a randomised trial. Lancet 369:657-666. 55. Abdool Karim, Q., S. S. Abdool Karim, J. A. Frohlich, A. C. Grobler, C. Baxter, L. E. Mansoor, A. B. M. Kharsany, S. Sibeko, K. Mlisana, Z. Omar, T. N. Gengiah, S. Maarschalk, N. Arulappan, M. Mlotshwa, L. Morris, D. Taylor, and C. T. Group. 2010. Effectiveness and Safety of Tenofovir Gel, an Antiretroviral Microbicide, for the Prevention of HIV Infection in Women. Science. 56. Paz-Bailey, G., M. Sternberg, A. J. Puren, L. E. Markowitz, R. Ballard, S. Delany, S. Hawkes, O. Nwanyanwu, C. Ryan, and D. A. Lewis. 2009. Improvement in Healing and Reduction in HIV Shedding with Episodic Acyclovir Therapy as Part of Syndromic Management among Men: A Randomized, Controlled Trial. J Infect Dis 200:1039-1049. 57. Kimani, J., R. Kaul, N. J. Nagelkerke, M. Luo, K. S. MacDonald, E. Ngugi, K. R. Fowke, B. T. Ball, A. Kariri, J. Ndinya-Achola, and F. A. Plummer. 2008. Reduced rates of HIV acquisition during unprotected sex by Kenyan female sex workers predating population declines in HIV prevalence. Aids 22:131-137. 58. Korenromp, E. L., R. G. White, K. K. Orroth, R. Bakker, A. Kamali, D. Serwadda, R. H. Gray, H. Grosskurth, J. D. Habbema, and R. J. Hayes. 2005. Determinants of the impact of sexually transmitted infection treatment on prevention of HIV infection: a synthesis of evidence from the Mwanza, Rakai, and Masaka intervention trials. J Infect Dis 191 Suppl 1:S168-178. 59. Watson-Jones, D., H. A. Weiss, M. Rusizoka, J. Changalucha, K. Baisley, K. Mugeye, C. Tanton, D. Ross, D. Everett, T. Clayton, R. Balira, L. Knight, I. Hambleton, J. Le Goff, L. Belec, and R. Hayes. 2008. Effect of herpes simplex suppression on incidence of HIV among women in Tanzania. N Engl J Med 358:1560-1571. 60. Miller, C. J., and R. J. Shattock. 2003. Target cells in vaginal HIV transmission. Microbes Infect 5:59-67. 61. Klasse, P. J., R. J. Shattock, and J. P. Moore. 2006. Which topical microbicides for blocking HIV-1 transmission will work in the real world? PLoS Med 3:e351. 62. Stein, Z. A. 1994. Vaginal microbicides and prevention of HIV infection. Lancet 343:362-363.

219 63. Stone, A. 2002. Microbicides: a new approach to preventing HIV and other sexually transmitted infections. Nat Rev Drug Discov 1:977-985. 64. Malkovsky, M., A. Newell, and A. G. Dalgleish. 1988. Inactivation of HIV by nonoxynol-9. Lancet 1:645. 65. Hicks, D. R., L. S. Martin, J. P. Getchell, J. L. Heath, D. P. Francis, J. S. McDougal, J. W. Curran, and B. Voeller. 1985. Inactivation of HTLV-III/LAV- infected cultures of normal human lymphocytes by nonoxynol-9 in vitro. Lancet 2:1422-1423. 66. Roddy, R. E., L. Zekeng, K. A. Ryan, U. Tamoufe, S. S. Weir, and E. L. Wong. 1998. A controlled trial of nonoxynol 9 film to reduce male-to-female transmission of sexually transmitted diseases. N Engl J Med 339:504-510. 67. Roddy, R. E., M. Cordero, K. A. Ryan, and J. Figueroa. 1998. A randomized controlled trial comparing nonoxynol-9 lubricated condoms with silicone lubricated condoms for prophylaxis. Sex Transm Infect 74:116-119. 68. Kreiss, J., E. Ngugi, K. Holmes, J. Ndinya-Achola, P. Waiyaki, P. L. Roberts, I. Ruminjo, R. Sajabi, J. Kimata, T. R. Fleming, and et al. 1992. Efficacy of nonoxynol 9 contraceptive sponge use in preventing heterosexual acquisition of HIV in Nairobi prostitutes. Jama 268:477-482. 69. Van Damme, L., G. Ramjee, M. Alary, B. Vuylsteke, V. Chandeying, H. Rees, P. Sirivongrangson, L. Mukenge-Tshibaka, V. Ettiegne-Traore, C. Uaheowitchai, S. S. Karim, B. Masse, J. Perriens, and M. Laga. 2002. Effectiveness of COL-1492, a nonoxynol-9 vaginal gel, on HIV-1 transmission in female sex workers: a randomised controlled trial. Lancet 360:971-977. 70. Newell, M. L. 1998. Mechanisms and timing of mother-to-child transmission of HIV-1. Aids 12:831-837. 71. Nduati, R., G. John, D. Mbori-Ngacha, B. Richardson, J. Overbaugh, A. Mwatha, J. Ndinya-Achola, J. Bwayo, F. E. Onyango, J. Hughes, and J. Kreiss. 2000. Effect of breastfeeding and formula feeding on transmission of HIV-1: a randomized clinical trial. Jama 283:1167-1174. 72. Luzuriaga, K., and J. L. Sullivan. 2000. Viral and immunopathogenesis of vertical HIV-1 infection. Pediatr Clin North Am 47:65-78. 73. Kourtis, A. P., M. Bulterys, S. R. Nesheim, and F. K. Lee. 2001. Understanding the timing of HIV transmission from mother to infant. Jama 285:709-712. 74. Bulterys, M., M. G. Fowler, K. K. Van Rompay, and A. P. Kourtis. 2004. Prevention of mother-to-child transmission of HIV-1 through breast-feeding: past, present, and future. J Infect Dis 189:2149-2153. 75. Dickover, R. E., E. M. Garratty, S. A. Herman, M. S. Sim, S. Plaeger, P. J. Boyer, M. Keller, A. Deveikis, E. R. Stiehm, and Y. J. Bryson. 1996. Identification of levels of maternal HIV-1 RNA associated with risk of perinatal transmission. Effect of maternal zidovudine treatment on viral load. Jama 275:599-605. 76. Connor, E. M., R. S. Sperling, R. Gelber, P. Kiselev, G. Scott, M. J. O'Sullivan, R. VanDyke, M. Bey, W. Shearer, R. L. Jacobson, and et al. 1994. Reduction of maternal-infant transmission of human immunodeficiency virus type 1 with zidovudine treatment. Pediatric AIDS Clinical Trials Group Protocol 076 Study Group. N Engl J Med 331:1173-1180.

220 77. Guay, L. A., P. Musoke, T. Fleming, D. Bagenda, M. Allen, C. Nakabiito, J. Sherman, P. Bakaki, C. Ducar, M. Deseyve, L. Emel, M. Mirochnick, M. G. Fowler, L. Mofenson, P. Miotti, K. Dransfield, D. Bray, F. Mmiro, and J. B. Jackson. 1999. Intrapartum and neonatal single-dose nevirapine compared with zidovudine for prevention of mother-to-child transmission of HIV-1 in Kampala, Uganda: HIVNET 012 randomised trial. Lancet 354:795-802. 78. 2005. Mother-to-child transmission of HIV infection in the era of highly active antiretroviral therapy. Clin Infect Dis 40:458-465. 79. Cooper, E. R., M. Charurat, L. Mofenson, I. C. Hanson, J. Pitt, C. Diaz, K. Hayani, E. Handelsman, V. Smeriglio, R. Hoff, and W. Blattner. 2002. Combination antiretroviral strategies for the treatment of pregnant HIV-1-infected women and prevention of perinatal HIV-1 transmission. J Acquir Immune Defic Syndr 29:484-494. 80. Tozzi, A. E., P. Pezzotti, and D. Greco. 1990. Does breast-feeding delay progression to AIDS in HIV-infected children? Aids 4:1293-1294. 81. Coutsoudis, A., F. Dabis, W. Fawzi, P. Gaillard, G. Haverkamp, D. R. Harris, J. B. Jackson, V. Leroy, N. Meda, P. Msellati, M. L. Newell, R. Nsuati, J. S. Read, and S. Wiktor. 2004. Late postnatal transmission of HIV-1 in breast-fed children: an individual patient data meta-analysis. J Infect Dis 189:2154-2166. 82. Luzuriaga, K. 2007. Mother-to-child Transmission of HIV: A Global Perspective. Curr Infect Dis Rep 9:511-517. 83. Levy, J. A. 2009. HIV pathogenesis: 25 years of progress and persistent challenges. Aids 23:147-160. 84. Swanson, C. M., and M. H. Malim. 2008. SnapShot: HIV-1 proteins. Cell 133:742, 742 e741. 85. Turner, B. G., and M. F. Summers. 1999. Structural biology of HIV. J Mol Biol 285:1-32. 86. Robey, W. G., B. Safai, S. Oroszlan, L. O. Arthur, M. A. Gonda, R. C. Gallo, and P. J. Fischinger. 1985. Characterization of envelope and core structural gene products of HTLV-III with sera from AIDS patients. Science 228:593-595. 87. Allan, J. S., J. E. Coligan, F. Barin, M. F. McLane, J. G. Sodroski, C. A. Rosen, W. A. Haseltine, T. H. Lee, and M. Essex. 1985. Major glycoprotein antigens that induce antibodies in AIDS patients are encoded by HTLV-III. Science 228:1091- 1094. 88. McDougal, J. S., M. S. Kennedy, J. M. Sligh, S. P. Cort, A. Mawle, and J. K. Nicholson. 1986. Binding of HTLV-III/LAV to T4+ T cells by a complex of the 110K viral protein and the T4 molecule. Science 231:382-385. 89. Klatzmann, D., E. Champagne, S. Chamaret, J. Gruest, D. Guetard, T. Hercend, J. C. Gluckman, and L. Montagnier. 1984. T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV. Nature 312:767-768. 90. Klatzmann, D., F. Barre-Sinoussi, M. T. Nugeyre, C. Danquet, E. Vilmer, C. Griscelli, F. Brun-Veziret, C. Rouzioux, J. C. Gluckman, J. C. Chermann, and et al. 1984. Selective tropism of lymphadenopathy associated virus (LAV) for helper-inducer T lymphocytes. Science 225:59-63.

221 91. Gartner, S., P. Markovits, D. M. Markovitz, M. H. Kaplan, R. C. Gallo, and M. Popovic. 1986. The role of mononuclear phagocytes in HTLV-III/LAV infection. Science 233:215-219. 92. Dalgleish, A. G., P. C. Beverley, P. R. Clapham, D. H. Crawford, M. F. Greaves, and R. A. Weiss. 1984. The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature 312:763-767. 93. Moore, J. S., F. Rahemtulla, L. W. Kent, S. D. Hall, M. R. Ikizler, P. F. Wright, H. H. Nguyen, and S. Jackson. 2003. Oral epithelial cells are susceptible to cell- free and cell-associated HIV-1 infection in vitro. Virology 313:343-353. 94. Moore, J. S., S. D. Hall, and S. Jackson. 2002. Cell-associated HIV-1 infection of salivary gland epithelial cell lines. Virology 297:89-97. 95. Miller, C. J., and J. Hu. 1999. T cell-tropic simian immunodeficiency virus (SIV) and simian-human immunodeficiency viruses are readily transmitted by vaginal inoculation of rhesus macaques, and Langerhans' cells of the female genital tract are infected with SIV. J Infect Dis 179 Suppl 3:S413-417. 96. Hladik, F., P. Sakchalathorn, L. Ballweber, G. Lentz, M. Fialkow, D. Eschenbach, and M. J. McElrath. 2007. Initial events in establishing vaginal entry and infection by human immunodeficiency virus type-1. Immunity 26:257-270. 97. Kawamura, T., F. O. Gulden, M. Sugaya, D. T. McNamara, D. L. Borris, M. M. Lederman, J. M. Orenstein, P. A. Zimmerman, and A. Blauvelt. 2003. R5 HIV productively infects Langerhans cells, and infection levels are regulated by compound CCR5 polymorphisms. Proc Natl Acad Sci U S A 100:8401-8406. 98. Deng, H., R. Liu, W. Ellmeier, S. Choe, D. Unutmaz, M. Burkhart, P. Di Marzio, S. Marmon, R. E. Sutton, C. M. Hill, C. B. Davis, S. C. Peiper, T. J. Schall, D. R. Littman, and N. R. Landau. 1996. Identification of a major co-receptor for primary isolates of HIV-1. Nature 381:661-666. 99. Doranz, B. J., J. Rucker, Y. Yi, R. J. Smyth, M. Samson, S. C. Peiper, M. Parmentier, R. G. Collman, and R. W. Doms. 1996. A dual-tropic primary HIV-1 isolate that uses fusin and the beta-chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors. Cell 85:1149-1158. 100. Dragic, T., V. Litwin, G. P. Allaway, S. R. Martin, Y. Huang, K. A. Nagashima, C. Cayanan, P. J. Maddon, R. A. Koup, J. P. Moore, and W. A. Paxton. 1996. HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5. Nature 381:667-673. 101. Doranz, B. J., S. S. Baik, and R. W. Doms. 1999. Use of a gp120 binding assay to dissect the requirements and kinetics of human immunodeficiency virus fusion events. J Virol 73:10346-10358. 102. Moore, J. P. 1997. Coreceptors: implications for HIV pathogenesis and therapy. Science 276:51-52. 103. Feng, Y., C. C. Broder, P. E. Kennedy, and E. A. Berger. 1996. HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science 272:872-877. 104. Helseth, E., U. Olshevsky, D. Gabuzda, B. Ardman, W. Haseltine, and J. Sodroski. 1990. Changes in the transmembrane region of the human immunodeficiency virus type 1 gp41 envelope glycoprotein affect membrane fusion. J Virol 64:6314-6318.

222 105. Stein, B. S., S. D. Gowda, J. D. Lifson, R. C. Penhallow, K. G. Bensch, and E. G. Engleman. 1987. pH-independent HIV entry into CD4-positive T cells via virus envelope fusion to the plasma membrane. Cell 49:659-668. 106. Sierra, S., B. Kupfer, and R. Kaiser. 2005. Basics of the virology of HIV-1 and its replication. J Clin Virol 34:233-244. 107. Johnson, W. E., and R. C. Desrosiers. 2002. Viral persistance: HIV's strategies of immune system evasion. Annu Rev Med 53:499-518. 108. Canducci, F., M. C. Marinozzi, M. Sampaolo, S. Berre, P. Bagnarelli, M. Degano, G. Gallotta, B. Mazzi, P. Lemey, R. Burioni, and M. Clementi. 2009. Dynamic features of the selective pressure on the human immunodeficiency virus type 1 (HIV-1) gp120 CD4-binding site in a group of long term non progressor (LTNP) subjects. Retrovirology 6:4. 109. Carter, C. A., and L. S. Ehrlich. 2008. Cell biology of HIV-1 infection of macrophages. Annu Rev Microbiol 62:425-443. 110. Ganser-Pornillos, B. K., M. Yeager, and W. I. Sundquist. 2008. The structural biology of HIV assembly. Curr Opin Struct Biol 18:203-217. 111. Johnson, D. C., and M. T. Huber. 2002. Directed egress of animal viruses promotes cell-to-cell spread. J Virol 76:1-8. 112. Gupta, P., R. Balachandran, M. Ho, A. Enrico, and C. Rinaldo. 1989. Cell-to-cell transmission of human immunodeficiency virus type 1 in the presence of azidothymidine and neutralizing antibody. J Virol 63:2361-2365. 113. Rhee, S. S., and J. W. Marsh. 1994. Human immunodeficiency virus type 1 Nef- induced down-modulation of CD4 is due to rapid internalization and degradation of surface CD4. J Virol 68:5156-5163. 114. Piguet, V., Y. L. Chen, A. Mangasarian, M. Foti, J. L. Carpentier, and D. Trono. 1998. Mechanism of Nef-induced CD4 endocytosis: Nef connects CD4 with the mu chain of adaptor complexes. Embo J 17:2472-2481. 115. Neil, S. J., T. Zang, and P. D. Bieniasz. 2008. Tetherin inhibits retrovirus release and is antagonized by HIV-1 Vpu. Nature 451:425-430. 116. Van Damme, N., D. Goff, C. Katsura, R. L. Jorgenson, R. Mitchell, M. C. Johnson, E. B. Stephens, and J. Guatelli. 2008. The interferon-induced protein BST-2 restricts HIV-1 release and is downregulated from the cell surface by the viral Vpu protein. Cell Host Microbe 3:245-252. 117. Neil, S. J., V. Sandrin, W. I. Sundquist, and P. D. Bieniasz. 2007. An interferon- alpha-induced tethering mechanism inhibits HIV-1 and Ebola virus particle release but is counteracted by the HIV-1 Vpu protein. Cell Host Microbe 2:193- 203. 118. Mariani, S. A., E. Vicenzi, and G. Poli. 2010. Asymmetric HIV-1 co-receptor use and replication in CD4(+) T lymphocytes. J Transl Med 9 Suppl 1:S8. 119. Margolis, L., and R. Shattock. 2006. Selective transmission of CCR5-utilizing HIV-1: the 'gatekeeper' problem resolved? Nat Rev Microbiol 4:312-317. 120. Zhu, T., N. Wang, A. Carr, D. S. Nam, R. Moor-Jankowski, D. A. Cooper, and D. D. Ho. 1996. Genetic characterization of human immunodeficiency virus type 1 in blood and genital secretions: evidence for viral compartmentalization and selection during sexual transmission. J Virol 70:3098-3107.

223 121. Keele, B. F., E. E. Giorgi, J. F. Salazar-Gonzalez, J. M. Decker, K. T. Pham, M. G. Salazar, C. Sun, T. Grayson, S. Wang, H. Li, X. Wei, C. Jiang, J. L. Kirchherr, F. Gao, J. A. Anderson, L. H. Ping, R. Swanstrom, G. D. Tomaras, W. A. Blattner, P. A. Goepfert, J. M. Kilby, M. S. Saag, E. L. Delwart, M. P. Busch, M. S. Cohen, D. C. Montefiori, B. F. Haynes, B. Gaschen, G. S. Athreya, H. Y. Lee, N. Wood, C. Seoighe, A. S. Perelson, T. Bhattacharya, B. T. Korber, B. H. Hahn, and G. M. Shaw. 2008. Identification and characterization of transmitted and early founder virus envelopes in primary HIV-1 infection. Proc Natl Acad Sci U S A 105:7552-7557. 122. Scarlatti, G., T. Leitner, V. Hodara, E. Halapi, P. Rossi, J. Albert, and E. M. Fenyo. 1993. Neutralizing antibodies and viral characteristics in mother-to-child transmission of HIV-1. Aids 7 Suppl 2:S45-48. 123. Scarlatti, G., V. Hodara, P. Rossi, L. Muggiasca, A. Bucceri, J. Albert, and E. M. Fenyo. 1993. Transmission of human immunodeficiency virus type 1 (HIV-1) from mother to child correlates with viral phenotype. Virology 197:624-629. 124. Wolinsky, S. M., C. M. Wike, B. T. Korber, C. Hutto, W. P. Parks, L. L. Rosenblum, K. J. Kunstman, M. R. Furtado, and J. L. Munoz. 1992. Selective transmission of human immunodeficiency virus type-1 variants from mothers to infants. Science 255:1134-1137. 125. Heeney, J. L., A. G. Dalgleish, and R. A. Weiss. 2006. Origins of HIV and the evolution of resistance to AIDS. Science 313:462-466. 126. Sheehy, A. M., N. C. Gaddis, J. D. Choi, and M. H. Malim. 2002. Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 418:646-650. 127. Stremlau, M., C. M. Owens, M. J. Perron, M. Kiessling, P. Autissier, and J. Sodroski. 2004. The cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old World monkeys. Nature 427:848-853. 128. Promadej-Lanier, N., D. L. Hanson, P. Srinivasan, W. Luo, D. R. Adams, P. C. Guenthner, S. Butera, R. A. Otten, and E. N. Kersh. Resistance to Simian HIV infection is associated with high plasma interleukin-8, RANTES and Eotaxin in a macaque model of repeated virus challenges. J Acquir Immune Defic Syndr 53:574-581. 129. Vangelista, L., M. Secchi, and P. Lusso. 2008. Rational design of novel HIV-1 entry inhibitors by RANTES engineering. Vaccine 26:3008-3015. 130. Cocchi, F., A. L. DeVico, A. Garzino-Demo, S. K. Arya, R. C. Gallo, and P. Lusso. 1995. Identification of RANTES, MIP-1 alpha, and MIP-1 beta as the major HIV-suppressive factors produced by CD8+ T cells. Science 270:1811- 1815. 131. Nakajima, T., E. E. Nakayama, G. Kaur, H. Terunuma, J. I. Mimaya, H. Ohtani, N. Mehra, T. Shioda, and A. Kimura. 2009. Impact of novel TRIM5alpha variants, Gly110Arg and G176del, on the anti-HIV-1 activity and the susceptibility to HIV-1 infection. Aids. 132. Ball, T. B., H. Ji, J. Kimani, P. McLaren, C. Marlin, A. V. Hill, and F. A. Plummer. 2007. Polymorphisms in IRF-1 associated with resistance to HIV-1 infection in highly exposed uninfected Kenyan sex workers. Aids 21:1091-1101.

224 133. Liu, R., W. A. Paxton, S. Choe, D. Ceradini, S. R. Martin, R. Horuk, M. E. MacDonald, H. Stuhlmann, R. A. Koup, and N. R. Landau. 1996. Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell 86:367-377. 134. Tang, J., C. Rivers, E. Karita, C. Costello, S. Allen, P. N. Fultz, E. E. Schoenbaum, and R. A. Kaslow. 1999. Allelic variants of human beta-chemokine receptor 5 (CCR5) promoter: evolutionary relationships and predictable associations with HIV-1 disease progression. Genes Immun 1:20-27. 135. Huang, Y., W. A. Paxton, S. M. Wolinsky, A. U. Neumann, L. Zhang, T. He, S. Kang, D. Ceradini, Z. Jin, K. Yazdanbakhsh, K. Kunstman, D. Erickson, E. Dragon, N. R. Landau, J. Phair, D. D. Ho, and R. A. Koup. 1996. The role of a mutant CCR5 allele in HIV-1 transmission and disease progression. Nat Med 2:1240-1243. 136. Carrington, M., M. Dean, M. P. Martin, and S. J. O'Brien. 1999. Genetics of HIV- 1 infection: chemokine receptor CCR5 polymorphism and its consequences. Hum Mol Genet 8:1939-1945. 137. MacDonald, K. S., J. E. Embree, N. J. Nagelkerke, J. Castillo, S. Ramhadin, S. Njenga, J. Oyug, J. Ndinya-Achola, B. H. Barber, J. J. Bwayo, and F. A. Plummer. 2001. The HLA A2/6802 supertype is associated with reduced risk of perinatal human immunodeficiency virus type 1 transmission. J Infect Dis 183:503-506. 138. Hardie, R. A., M. Luo, B. Bruneau, E. Knight, N. J. Nagelkerke, J. Kimani, C. Wachihi, E. N. Ngugi, and F. A. Plummer. 2008. Human leukocyte antigen-DQ alleles and haplotypes and their associations with resistance and susceptibility to HIV-1 infection. Aids 22:807-816. 139. Lacap, P. A., J. D. Huntington, M. Luo, N. J. Nagelkerke, T. Bielawny, J. Kimani, C. Wachihi, E. N. Ngugi, and F. A. Plummer. 2008. Associations of human leukocyte antigen DRB with resistance or susceptibility to HIV-1 infection in the Pumwani Sex Worker Cohort. Aids 22:1029-1038. 140. Martin, M. P., and M. Carrington. 2005. Immunogenetics of viral infections. Curr Opin Immunol 17:510-516. 141. Carrington, M., G. W. Nelson, M. P. Martin, T. Kissner, D. Vlahov, J. J. Goedert, R. Kaslow, S. Buchbinder, K. Hoots, and S. J. O'Brien. 1999. HLA and HIV-1: heterozygote advantage and B*35-Cw*04 disadvantage. Science 283:1748-1752. 142. Magierowska, M., I. Theodorou, P. Debre, F. Sanson, B. Autran, Y. Riviere, D. Charron, and D. Costagliola. 1999. Combined genotypes of CCR5, CCR2, SDF1, and HLA genes can predict the long-term nonprogressor status in human immunodeficiency virus-1-infected individuals. Blood 93:936-941. 143. Altfeld, M., E. T. Kalife, Y. Qi, H. Streeck, M. Lichterfeld, M. N. Johnston, N. Burgett, M. E. Swartz, A. Yang, G. Alter, X. G. Yu, A. Meier, J. K. Rockstroh, T. M. Allen, H. Jessen, E. S. Rosenberg, M. Carrington, and B. D. Walker. 2006. HLA Alleles Associated with Delayed Progression to AIDS Contribute Strongly to the Initial CD8(+) T Cell Response against HIV-1. PLoS Med 3:e403. 144. Ngumbela, K. C., C. L. Day, Z. Mncube, K. Nair, D. Ramduth, C. Thobakgale, E. Moodley, S. Reddy, C. de Pierres, N. Mkhwanazi, K. Bishop, M. van der Stok, N. Ismail, I. Honeyborne, H. Crawford, D. G. Kavanagh, C. Rousseau, D. Nickle, J.

225 Mullins, D. Heckerman, B. Korber, H. Coovadia, P. Kiepiela, P. J. Goulder, and B. D. Walker. 2008. Targeting of a CD8 T cell env epitope presented by HLA- B*5802 is associated with markers of HIV disease progression and lack of selection pressure. AIDS Res Hum Retroviruses 24:72-82. 145. MacDonald, K. S., K. R. Fowke, J. Kimani, V. A. Dunand, N. J. Nagelkerke, T. B. Ball, J. Oyugi, E. Njagi, L. K. Gaur, R. C. Brunham, J. Wade, M. A. Luscher, P. Krausa, S. Rowland-Jones, E. Ngugi, J. J. Bwayo, and F. A. Plummer. 2000. Influence of HLA supertypes on susceptibility and resistance to human immunodeficiency virus type 1 infection. J Infect Dis 181:1581-1589. 146. Lu, M., S. C. Blacklow, and P. S. Kim. 1995. A trimeric structural domain of the HIV-1 transmembrane glycoprotein. Nat Struct Biol 2:1075-1082. 147. Kowalski, M., J. Potz, L. Basiripour, T. Dorfman, W. C. Goh, E. Terwilliger, A. Dayton, C. Rosen, W. Haseltine, and J. Sodroski. 1987. Functional regions of the envelope glycoprotein of human immunodeficiency virus type 1. Science 237:1351-1355. 148. Hallenberger, S., M. Moulard, M. Sordel, H. D. Klenk, and W. Garten. 1997. The role of eukaryotic subtilisin-like endoproteases for the activation of human immunodeficiency virus glycoproteins in natural host cells. J Virol 71:1036-1045. 149. Bosch, V., and M. Pawlita. 1990. Mutational analysis of the human immunodeficiency virus type 1 env gene product proteolytic cleavage site. J Virol 64:2337-2344. 150. Lama, J., A. Mangasarian, and D. Trono. 1999. Cell-surface expression of CD4 reduces HIV-1 infectivity by blocking Env incorporation in a Nef- and Vpu- inhibitable manner. Curr Biol 9:622-631. 151. Crise, B., L. Buonocore, and J. K. Rose. 1990. CD4 is retained in the endoplasmic reticulum by the human immunodeficiency virus type 1 glycoprotein precursor. J Virol 64:5585-5593. 152. Hoxie, J. A., J. D. Alpers, J. L. Rackowski, K. Huebner, B. S. Haggarty, A. J. Cedarbaum, and J. C. Reed. 1986. Alterations in T4 (CD4) protein and mRNA synthesis in cells infected with HIV. Science 234:1123-1127. 153. Schubert, U., L. C. Anton, I. Bacik, J. H. Cox, S. Bour, J. R. Bennink, M. Orlowski, K. Strebel, and J. W. Yewdell. 1998. CD4 glycoprotein degradation induced by human immunodeficiency virus type 1 Vpu protein requires the function of proteasomes and the ubiquitin-conjugating pathway. J Virol 72:2280- 2288. 154. Margottin, F., S. P. Bour, H. Durand, L. Selig, S. Benichou, V. Richard, D. Thomas, K. Strebel, and R. Benarous. 1998. A novel human WD protein, h-beta TrCp, that interacts with HIV-1 Vpu connects CD4 to the ER degradation pathway through an F-box motif. Mol Cell 1:565-574. 155. Burton, D. R. 2006. Structural biology: images from the surface of HIV. Nature 441:817-818. 156. Kwong, P. D., R. Wyatt, J. Robinson, R. W. Sweet, J. Sodroski, and W. A. Hendrickson. 1998. Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393:648-659.

226 157. Zhu, P., J. Liu, J. Bess, Jr., E. Chertova, J. D. Lifson, H. Grise, G. A. Ofek, K. A. Taylor, and K. H. Roux. 2006. Distribution and three-dimensional structure of AIDS virus envelope spikes. Nature 441:847-852. 158. Yamaguchi, M., R. Danev, K. Nishiyama, K. Sugawara, and K. Nagayama. 2008. Zernike phase contrast electron microscopy of ice-embedded influenza A virus. J Struct Biol 162:271-276. 159. Klein, J. S., P. N. Gnanapragasam, R. P. Galimidi, C. P. Foglesong, A. P. West, Jr., and P. J. Bjorkman. 2009. Examination of the contributions of size and avidity to the neutralization mechanisms of the anti-HIV antibodies b12 and 4E10. Proc Natl Acad Sci U S A 106:7385-7390. 160. Thali, M., C. Furman, E. Helseth, H. Repke, and J. Sodroski. 1992. Lack of correlation between soluble CD4-induced shedding of the human immunodeficiency virus type 1 exterior envelope glycoprotein and subsequent membrane fusion events. J Virol 66:5516-5524. 161. Starcich, B. R., B. H. Hahn, G. M. Shaw, P. D. McNeely, S. Modrow, H. Wolf, E. S. Parks, W. P. Parks, S. F. Josephs, R. C. Gallo, and et al. 1986. Identification and characterization of conserved and variable regions in the envelope gene of HTLV-III/LAV, the retrovirus of AIDS. Cell 45:637-648. 162. Kwong, P. D., R. Wyatt, S. Majeed, J. Robinson, R. W. Sweet, J. Sodroski, and W. A. Hendrickson. 2000. Structures of HIV-1 gp120 envelope glycoproteins from laboratory-adapted and primary isolates. Structure 8:1329-1339. 163. Zhou, T., L. Xu, B. Dey, A. J. Hessell, D. Van Ryk, S. H. Xiang, X. Yang, M. Y. Zhang, M. B. Zwick, J. Arthos, D. R. Burton, D. S. Dimitrov, J. Sodroski, R. Wyatt, G. J. Nabel, and P. D. Kwong. 2007. Structural definition of a conserved neutralization epitope on HIV-1 gp120. Nature 445:732-737. 164. Chen, L., Y. D. Kwon, T. Zhou, X. Wu, S. O'Dell, L. Cavacini, A. J. Hessell, M. Pancera, M. Tang, L. Xu, Z. Y. Yang, M. Y. Zhang, J. Arthos, D. R. Burton, D. S. Dimitrov, G. J. Nabel, M. R. Posner, J. Sodroski, R. Wyatt, J. R. Mascola, and P. D. Kwong. 2009. Structural basis of immune evasion at the site of CD4 attachment on HIV-1 gp120. Science 326:1123-1127. 165. Chen, B., E. M. Vogan, H. Gong, J. J. Skehel, D. C. Wiley, and S. C. Harrison. 2005. Determining the structure of an unliganded and fully glycosylated SIV gp120 envelope glycoprotein. Structure 13:197-211. 166. Chen, B., E. M. Vogan, H. Gong, J. J. Skehel, D. C. Wiley, and S. C. Harrison. 2005. Structure of an unliganded simian immunodeficiency virus gp120 core. Nature 433:834-841. 167. Dey, B., M. Pancera, K. Svehla, Y. Shu, S. H. Xiang, J. Vainshtein, Y. Li, J. Sodroski, P. D. Kwong, J. R. Mascola, and R. Wyatt. 2007. Characterization of human immunodeficiency virus type 1 monomeric and trimeric gp120 glycoproteins stabilized in the CD4-bound state: antigenicity, biophysics, and immunogenicity. J Virol 81:5579-5593. 168. Huang, C. C., M. Tang, M. Y. Zhang, S. Majeed, E. Montabana, R. L. Stanfield, D. S. Dimitrov, B. Korber, J. Sodroski, I. A. Wilson, R. Wyatt, and P. D. Kwong. 2005. Structure of a V3-containing HIV-1 gp120 core. Science 310:1025-1028. 169. Pancera, M., S. Majeed, Y. E. Ban, L. Chen, C. C. Huang, L. Kong, Y. D. Kwon, J. Stuckey, T. Zhou, J. E. Robinson, W. R. Schief, J. Sodroski, R. Wyatt, and P.

227 D. Kwong. 2010. Structure of HIV-1 gp120 with gp41-interactive region reveals layered envelope architecture and basis of conformational mobility. Proc Natl Acad Sci U S A 107:1166-1171. 170. Kwong, P. D., M. L. Doyle, D. J. Casper, C. Cicala, S. A. Leavitt, S. Majeed, T. D. Steenbeke, M. Venturi, I. Chaiken, M. Fung, H. Katinger, P. W. Parren, J. Robinson, D. Van Ryk, L. Wang, D. R. Burton, E. Freire, R. Wyatt, J. Sodroski, W. A. Hendrickson, and J. Arthos. 2002. HIV-1 evades antibody-mediated neutralization through conformational masking of receptor-binding sites. Nature 420:678-682. 171. Poignard, P., E. O. Saphire, P. W. Parren, and D. R. Burton. 2001. gp120: Biologic aspects of structural features. Annu Rev Immunol 19:253-274. 172. Kong, L., C. C. Huang, S. J. Coales, K. S. Molnar, J. Skinner, Y. Hamuro, and P. D. Kwong. 2010. Local conformational stability of HIV-1 gp120 in unliganded and CD4-bound states as defined by amide hydrogen/deuterium exchange. J Virol 84:10311-10321. 173. Leavitt, M., E. J. Park, I. A. Sidorov, D. S. Dimitrov, and G. V. Quinnan, Jr. 2003. Concordant modulation of neutralization resistance and high infectivity of the primary human immunodeficiency virus type 1 MN strain and definition of a potential gp41 binding site in gp120. J Virol 77:560-570. 174. Yang, X., E. Mahony, G. H. Holm, A. Kassa, and J. Sodroski. 2003. Role of the gp120 inner domain beta-sandwich in the interaction between the human immunodeficiency virus envelope glycoprotein subunits. Virology 313:117-125. 175. Wyatt, R., P. D. Kwong, E. Desjardins, R. W. Sweet, J. Robinson, W. A. Hendrickson, and J. G. Sodroski. 1998. The antigenic structure of the HIV gp120 envelope glycoprotein. Nature 393:705-711. 176. Ivey-Hoyle, M., R. K. Clark, and M. Rosenberg. 1991. The N-terminal 31 amino acids of human immunodeficiency virus type 1 envelope protein gp120 contain a potential gp41 contact site. J Virol 65:2682-2685. 177. Helseth, E., U. Olshevsky, C. Furman, and J. Sodroski. 1991. Human immunodeficiency virus type 1 gp120 envelope glycoprotein regions important for association with the gp41 transmembrane glycoprotein. J Virol 65:2119-2123. 178. Guilhaudis, L., A. Jacobs, and M. Caffrey. 2002. Solution structure of the HIV gp120 C5 domain. Eur J Biochem 269:4860-4867. 179. Leonard, C. K., M. W. Spellman, L. Riddle, R. J. Harris, J. N. Thomas, and T. J. Gregory. 1990. Assignment of intrachain disulfide bonds and characterization of potential glycosylation sites of the type 1 recombinant human immunodeficiency virus envelope glycoprotein (gp120) expressed in Chinese hamster ovary cells. J Biol Chem 265:10373-10382. 180. Hoxie, J. A. 1991. Hypothetical assignment of intrachain disulfide bonds for HIV- 2 and SIV envelope glycoproteins. AIDS Res Hum Retroviruses 7:495-499. 181. Gregory, T., J. Hoxie, C. Watanabe, and M. Spellman. 1991. Structure and function in recombinant HIV-1 gp120 and speculation about the disulfide bonding in the gp120 homologs of HIV-2 and SIV. Adv Exp Med Biol 303:1-14. 182. Nyambi, P. N., H. A. Mbah, S. Burda, C. Williams, M. K. Gorny, A. Nadas, and S. Zolla-Pazner. 2000. Conserved and exposed epitopes on intact, native, primary human immunodeficiency virus type 1 virions of group M. J Virol 74:7096-7107.

228 183. Zolla-Pazner, S., J. O'Leary, S. Burda, M. K. Gorny, M. Kim, J. Mascola, and F. McCutchan. 1995. Serotyping of primary human immunodeficiency virus type 1 isolates from diverse geographic locations by flow cytometry. J Virol 69:3807- 3815. 184. Gao, F., D. L. Robertson, C. D. Carruthers, S. G. Morrison, B. Jian, Y. Chen, F. Barre-Sinoussi, M. Girard, A. Srinivasan, A. G. Abimiku, G. M. Shaw, P. M. Sharp, and B. H. Hahn. 1998. A comprehensive panel of near-full-length clones and reference sequences for non-subtype B isolates of human immunodeficiency virus type 1. J Virol 72:5680-5698. 185. McMichael, A. J. 2006. HIV vaccines. Annu Rev Immunol 24:227-255. 186. Stamatatos, L., and C. Cheng-Mayer. 1998. An envelope modification that renders a primary, neutralization-resistant clade B human immunodeficiency virus type 1 isolate highly susceptible to neutralization by sera from other clades. J Virol 72:7840-7845. 187. Cao, J., N. Sullivan, E. Desjardin, C. Parolin, J. Robinson, R. Wyatt, and J. Sodroski. 1997. Replication and neutralization of human immunodeficiency virus type 1 lacking the V1 and V2 variable loops of the gp120 envelope glycoprotein. J Virol 71:9808-9812. 188. Rizzuto, C., and J. Sodroski. 2000. Fine definition of a conserved CCR5-binding region on the human immunodeficiency virus type 1 glycoprotein 120. AIDS Res Hum Retroviruses 16:741-749. 189. Rizzuto, C. D., R. Wyatt, N. Hernandez-Ramos, Y. Sun, P. D. Kwong, W. A. Hendrickson, and J. Sodroski. 1998. A conserved HIV gp120 glycoprotein structure involved in chemokine receptor binding. Science 280:1949-1953. 190. Wu, L., N. P. Gerard, R. Wyatt, H. Choe, C. Parolin, N. Ruffing, A. Borsetti, A. A. Cardoso, E. Desjardin, W. Newman, C. Gerard, and J. Sodroski. 1996. CD4- induced interaction of primary HIV-1 gp120 glycoproteins with the chemokine receptor CCR-5. Nature 384:179-183. 191. Emerson, V., D. Holtkotte, T. Pfeiffer, I. H. Wang, M. Schnolzer, T. Kempf, and V. Bosch. Identification of the cellular prohibitin 1/prohibitin 2 heterodimer as an interaction partner of the C-terminal cytoplasmic domain of the HIV-1 glycoprotein. J Virol 84:1355-1365. 192. Chan, D. C., and P. S. Kim. 1998. HIV entry and its inhibition. Cell 93:681-684. 193. Chan, D. C., D. Fass, J. M. Berger, and P. S. Kim. 1997. Core structure of gp41 from the HIV envelope glycoprotein. Cell 89:263-273. 194. Zwick, M. B., E. O. Saphire, and D. R. Burton. 2004. gp41: HIV's shy protein. Nat Med 10:133-134. 195. Linsley, P. S., J. A. Ledbetter, E. Kinney-Thomas, and S. L. Hu. 1988. Effects of anti-gp120 monoclonal antibodies on CD4 receptor binding by the env protein of human immunodeficiency virus type 1. J Virol 62:3695-3702. 196. Cordonnier, A., Y. Riviere, L. Montagnier, and M. Emerman. 1989. Effects of mutations in hyperconserved regions of the extracellular glycoprotein of human immunodeficiency virus type 1 on receptor binding. J Virol 63:4464-4468. 197. Cordonnier, A., L. Montagnier, and M. Emerman. 1989. Single amino-acid changes in HIV envelope affect viral tropism and receptor binding. Nature 340:571-574.

229 198. Syu, W. J., J. H. Huang, M. Essex, and T. H. Lee. 1990. The N-terminal region of the human immunodeficiency virus envelope glycoprotein gp120 contains potential binding sites for CD4. Proc Natl Acad Sci U S A 87:3695-3699. 199. Lasky, L. A., G. Nakamura, D. H. Smith, C. Fennie, C. Shimasaki, E. Patzer, P. Berman, T. Gregory, and D. J. Capon. 1987. Delineation of a region of the human immunodeficiency virus type 1 gp120 glycoprotein critical for interaction with the CD4 receptor. Cell 50:975-985. 200. Olshevsky, U., E. Helseth, C. Furman, J. Li, W. Haseltine, and J. Sodroski. 1990. Identification of individual human immunodeficiency virus type 1 gp120 amino acids important for CD4 receptor binding. J Virol 64:5701-5707. 201. Wu, H., P. D. Kwong, and W. A. Hendrickson. 1997. Dimeric association and segmental variability in the structure of human CD4. Nature 387:527-530. 202. Wang, J. H., Y. W. Yan, T. P. Garrett, J. H. Liu, D. W. Rodgers, R. L. Garlick, G. E. Tarr, Y. Husain, E. L. Reinherz, and S. C. Harrison. 1990. Atomic structure of a fragment of human CD4 containing two immunoglobulin-like domains. Nature 348:411-418. 203. Ryu, S. E., P. D. Kwong, A. Truneh, T. G. Porter, J. Arthos, M. Rosenberg, X. P. Dai, N. H. Xuong, R. Axel, R. W. Sweet, and et al. 1990. Crystal structure of an HIV-binding recombinant fragment of human CD4. Nature 348:419-426. 204. Moebius, U., L. K. Clayton, S. Abraham, S. C. Harrison, and E. L. Reinherz. 1992. The human immunodeficiency virus gp120 binding site on CD4: delineation by quantitative equilibrium and kinetic binding studies of mutants in conjunction with a high-resolution CD4 atomic structure. J Exp Med 176:507- 517. 205. Moebius, U., L. K. Clayton, S. Abraham, A. Diener, J. J. Yunis, S. C. Harrison, and E. L. Reinherz. 1992. Human immunodeficiency virus gp120 binding C'C" ridge of CD4 domain 1 is also involved in interaction with class II major histocompatibility complex molecules. Proc Natl Acad Sci U S A 89:12008- 12012. 206. Sweet, R. W., A. Truneh, and W. A. Hendrickson. 1991. CD4: its structure, role in immune function and AIDS pathogenesis, and potential as a pharmacological target. Curr Opin Biotechnol 2:622-633. 207. Myszka, D. G., R. W. Sweet, P. Hensley, M. Brigham-Burke, P. D. Kwong, W. A. Hendrickson, R. Wyatt, J. Sodroski, and M. L. Doyle. 2000. Energetics of the HIV gp120-CD4 binding reaction. Proc Natl Acad Sci U S A 97:9026-9031. 208. Platt, E. J., D. M. Shea, P. P. Rose, and D. Kabat. 2005. Variants of human immunodeficiency virus type 1 that efficiently use CCR5 lacking the tyrosine- sulfated amino terminus have adaptive mutations in gp120, including loss of a functional N-glycan. J Virol 79:4357-4368. 209. Liu, J., A. Bartesaghi, M. J. Borgnia, G. Sapiro, and S. Subramaniam. 2008. Molecular architecture of native HIV-1 gp120 trimers. Nature 455:109-113. 210. Duenas-Decamp, M. J., P. J. Peters, D. Burton, and P. R. Clapham. 2009. Determinants flanking the CD4 binding loop modulate macrophage tropism of human immunodeficiency virus type 1 R5 envelopes. J Virol 83:2575-2583. 211. Gustchina, E., C. A. Bewley, and G. M. Clore. 2008. Sequestering of the prehairpin intermediate of gp41 by peptide N36Mut(e,g) potentiates the human

230 immunodeficiency virus type 1 neutralizing activity of monoclonal antibodies directed against the N-terminal helical repeat of gp41. J Virol 82:10032-10041. 212. Melikyan, G. B., R. M. Markosyan, H. Hemmati, M. K. Delmedico, D. M. Lambert, and F. S. Cohen. 2000. Evidence that the transition of HIV-1 gp41 into a six-helix bundle, not the bundle configuration, induces membrane fusion. J Cell Biol 151:413-423. 213. Freed, E. O., D. J. Myers, and R. Risser. 1990. Characterization of the fusion domain of the human immunodeficiency virus type 1 envelope glycoprotein gp41. Proc Natl Acad Sci U S A 87:4650-4654. 214. Doms, R. W., and J. P. Moore. 2000. HIV-1 membrane fusion: targets of opportunity. J Cell Biol 151:F9-14. 215. Qiang, W., Y. Sun, and D. P. Weliky. 2009. A strong correlation between fusogenicity and membrane insertion depth of the HIV fusion peptide. Proc Natl Acad Sci U S A 106:15314-15319. 216. White, J. M., S. E. Delos, M. Brecher, and K. Schornberg. 2008. Structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme. Crit Rev Biochem Mol Biol 43:189-219. 217. Weissenhorn, W., A. Dessen, S. C. Harrison, J. J. Skehel, and D. C. Wiley. 1997. Atomic structure of the ectodomain from HIV-1 gp41. Nature 387:426-430. 218. Hager-Braun, C., H. Katinger, and K. B. Tomer. 2006. The HIV-neutralizing monoclonal antibody 4E10 recognizes N-terminal sequences on the native antigen. J Immunol 176:7471-7481. 219. Geijtenbeek, T. B., D. S. Kwon, R. Torensma, S. J. van Vliet, G. C. van Duijnhoven, J. Middel, I. L. Cornelissen, H. S. Nottet, V. N. KewalRamani, D. R. Littman, C. G. Figdor, and Y. van Kooyk. 2000. DC-SIGN, a dendritic cell- specific HIV-1-binding protein that enhances trans-infection of T cells. Cell 100:587-597. 220. Mondor, I., M. Moulard, S. Ugolini, P. J. Klasse, J. Hoxie, A. Amara, T. Delaunay, R. Wyatt, J. Sodroski, and Q. J. Sattentau. 1998. Interactions among HIV gp120, CD4, and CXCR4: dependence on CD4 expression level, gp120 viral origin, conservation of the gp120 COOH- and NH2-termini and V1/V2 and V3 loops, and sensitivity to neutralizing antibodies. Virology 248:394-405. 221. Mitchell, D. A., A. J. Fadden, and K. Drickamer. 2001. A novel mechanism of carbohydrate recognition by the C-type lectins DC-SIGN and DC-SIGNR. Subunit organization and binding to multivalent ligands. J Biol Chem 276:28939- 28945. 222. Feinberg, H., D. A. Mitchell, K. Drickamer, and W. I. Weis. 2001. Structural basis for selective recognition of oligosaccharides by DC-SIGN and DC-SIGNR. Science 294:2163-2166. 223. Su, S. V., P. Hong, S. Baik, O. A. Negrete, K. B. Gurney, and B. Lee. 2004. DC- SIGN binds to HIV-1 glycoprotein 120 in a distinct but overlapping fashion compared with ICAM-2 and ICAM-3. J Biol Chem 279:19122-19132. 224. Muratori, C., R. Bona, E. Ruggiero, G. D'Ettorre, V. Vullo, M. Andreotti, and M. Federico. 2009. DC contact with HIV-1-infected cells leads to high levels of Env- mediated virion endocytosis coupled with enhanced HIV-1 Ag presentation. Eur J Immunol 39:404-416.

231 225. Pope, M., M. G. Betjes, N. Romani, H. Hirmand, P. U. Cameron, L. Hoffman, S. Gezelter, G. Schuler, and R. M. Steinman. 1994. Conjugates of dendritic cells and memory T lymphocytes from skin facilitate productive infection with HIV-1. Cell 78:389-398. 226. Kwon, D. S., G. Gregorio, N. Bitton, W. A. Hendrickson, and D. R. Littman. 2002. DC-SIGN-mediated internalization of HIV is required for trans- enhancement of T cell infection. Immunity 16:135-144. 227. Rabinovich, G. A., L. G. Baum, N. Tinari, R. Paganelli, C. Natoli, F. T. Liu, and S. Iacobelli. 2002. Galectins and their ligands: amplifiers, silencers or tuners of the inflammatory response? Trends Immunol 23:313-320. 228. Ouellet, M., S. Mercier, I. Pelletier, S. Bounou, J. Roy, J. Hirabayashi, S. Sato, and M. J. Tremblay. 2005. Galectin-1 acts as a soluble host factor that promotes HIV-1 infectivity through stabilization of virus attachment to host cells. J Immunol 174:4120-4126. 229. de Witte, L., A. Nabatov, M. Pion, D. Fluitsma, M. A. de Jong, T. de Gruijl, V. Piguet, Y. van Kooyk, and T. B. Geijtenbeek. 2007. Langerin is a natural barrier to HIV-1 transmission by Langerhans cells. Nat Med 13:367-371. 230. de Jong, M. A., and T. B. Geijtenbeek. 2010. Langerhans cells in innate defense against pathogens. Trends Immunol 31:452-459. 231. Arthos, J., C. Cicala, E. Martinelli, K. Macleod, D. Van Ryk, D. Wei, Z. Xiao, T. D. Veenstra, T. P. Conrad, R. A. Lempicki, S. McLaughlin, M. Pascuccio, R. Gopaul, J. McNally, C. C. Cruz, N. Censoplano, E. Chung, K. N. Reitano, S. Kottilil, D. J. Goode, and A. S. Fauci. 2008. HIV-1 envelope protein binds to and signals through integrin alpha4beta7, the gut mucosal homing receptor for peripheral T cells. Nat Immunol 9:301-309. 232. Grivel, J. C., R. J. Shattock, and L. B. Margolis. 2010. Selective transmission of R5 HIV-1 variants: where is the gatekeeper? J Transl Med 9 Suppl 1:S6. 233. O'Rourke, S. M., B. Schweighardt, P. Phung, D. P. Fonseca, K. Terry, T. Wrin, F. Sinangil, and P. W. Berman. Mutation at a single position in the V2 domain of the HIV-1 envelope protein confers neutralization sensitivity to a highly neutralization-resistant virus. J Virol 84:11200-11209. 234. Bagasra, O., H. Farzadegan, T. Seshamma, J. W. Oakes, A. Saah, and R. J. Pomerantz. 1994. Detection of HIV-1 proviral DNA in sperm from HIV-1- infected men. Aids 8:1669-1674. 235. Gurney, K. B., J. Elliott, H. Nassanian, C. Song, E. Soilleux, I. McGowan, P. A. Anton, and B. Lee. 2005. Binding and transfer of human immunodeficiency virus by DC-SIGN+ cells in human rectal mucosa. J Virol 79:5762-5773. 236. Spira, A. I., P. A. Marx, B. K. Patterson, J. Mahoney, R. A. Koup, S. M. Wolinsky, and D. D. Ho. 1996. Cellular targets of infection and route of viral dissemination after an intravaginal inoculation of simian immunodeficiency virus into rhesus macaques. J Exp Med 183:215-225. 237. Hocini, H., and M. Bomsel. 1999. Infectious human immunodeficiency virus can rapidly penetrate a tight human epithelial barrier by transcytosis in a process impaired by mucosal immunoglobulins. J Infect Dis 179 Suppl 3:S448-453. 238. Pilgrim, A. K., G. Pantaleo, O. J. Cohen, L. M. Fink, J. Y. Zhou, J. T. Zhou, D. P. Bolognesi, A. S. Fauci, and D. C. Montefiori. 1997. Neutralizing antibody

232 responses to human immunodeficiency virus type 1 in primary infection and long- term-nonprogressive infection. J Infect Dis 176:924-932. 239. Hollingsworth, T. D., R. M. Anderson, and C. Fraser. 2008. HIV-1 transmission, by stage of infection. J Infect Dis 198:687-693. 240. Ahlgren, D. J., M. K. Gorny, and A. C. Stein. 1990. Model-based optimization of infectivity parameters: a study of the early epidemic in San Francisco. J Acquir Immune Defic Syndr 3:631-643. 241. Mehandru, S., M. A. Poles, K. Tenner-Racz, A. Horowitz, A. Hurley, C. Hogan, D. Boden, P. Racz, and M. Markowitz. 2004. Primary HIV-1 infection is associated with preferential depletion of CD4+ T lymphocytes from effector sites in the gastrointestinal tract. J Exp Med 200:761-770. 242. Veazey, R. S., M. DeMaria, L. V. Chalifoux, D. E. Shvetz, D. R. Pauley, H. L. Knight, M. Rosenzweig, R. P. Johnson, R. C. Desrosiers, and A. A. Lackner. 1998. Gastrointestinal tract as a major site of CD4+ T cell depletion and viral replication in SIV infection. Science 280:427-431. 243. Brenchley, J. M., T. W. Schacker, L. E. Ruff, D. A. Price, J. H. Taylor, G. J. Beilman, P. L. Nguyen, A. Khoruts, M. Larson, A. T. Haase, and D. C. Douek. 2004. CD4+ T cell depletion during all stages of HIV disease occurs predominantly in the gastrointestinal tract. J Exp Med 200:749-759. 244. Brenchley, J. M., D. A. Price, T. W. Schacker, T. E. Asher, G. Silvestri, S. Rao, Z. Kazzaz, E. Bornstein, O. Lambotte, D. Altmann, B. R. Blazar, B. Rodriguez, L. Teixeira-Johnson, A. Landay, J. N. Martin, F. M. Hecht, L. J. Picker, M. M. Lederman, S. G. Deeks, and D. C. Douek. 2006. Microbial translocation is a cause of systemic immune activation in chronic HIV infection. Nat Med 12:1365- 1371. 245. Brenchley, J. M., D. A. Price, and D. C. Douek. 2006. HIV disease: fallout from a mucosal catastrophe? Nat Immunol 7:235-239. 246. Mellors, J. W., C. R. Rinaldo, Jr., P. Gupta, R. M. White, J. A. Todd, and L. A. Kingsley. 1996. Prognosis in HIV-1 infection predicted by the quantity of virus in plasma. Science 272:1167-1170. 247. Bonhoeffer, S., G. A. Funk, H. F. Gunthard, M. Fischer, and V. Muller. 2003. Glancing behind virus load variation in HIV-1 infection. Trends Microbiol 11:499-504. 248. Wilson, J. D., G. S. Ogg, R. L. Allen, C. Davis, S. Shaunak, J. Downie, W. Dyer, C. Workman, S. Sullivan, A. J. McMichael, and S. L. Rowland-Jones. 2000. Direct visualization of HIV-1-specific cytotoxic T lymphocytes during primary infection. Aids 14:225-233. 249. Koup, R. A., J. T. Safrit, Y. Cao, C. A. Andrews, G. McLeod, W. Borkowsky, C. Farthing, and D. D. Ho. 1994. Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome. J Virol 68:4650-4655. 250. Borrow, P., H. Lewicki, B. H. Hahn, G. M. Shaw, and M. B. Oldstone. 1994. Virus-specific CD8+ cytotoxic T-lymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection. J Virol 68:6103-6110.

233 251. Tomaras, G. D., and B. F. Haynes. 2009. HIV-1-specific antibody responses during acute and chronic HIV-1 infection. Curr Opin HIV AIDS 4:373-379. 252. Tomaras, G. D., N. L. Yates, P. Liu, L. Qin, G. G. Fouda, L. L. Chavez, A. C. Decamp, R. J. Parks, V. C. Ashley, J. T. Lucas, M. Cohen, J. Eron, C. B. Hicks, H. X. Liao, S. G. Self, G. Landucci, D. N. Forthal, K. J. Weinhold, B. F. Keele, B. H. Hahn, M. L. Greenberg, L. Morris, S. S. Karim, W. A. Blattner, D. C. Montefiori, G. M. Shaw, A. S. Perelson, and B. F. Haynes. 2008. Initial B-cell responses to transmitted human immunodeficiency virus type 1: virion-binding immunoglobulin M (IgM) and IgG antibodies followed by plasma anti-gp41 antibodies with ineffective control of initial viremia. J Virol 82:12449-12463. 253. Coffin, J. M. 1995. HIV population dynamics in vivo: implications for genetic variation, pathogenesis, and therapy. Science 267:483-489. 254. Preston, B. D., B. J. Poiesz, and L. A. Loeb. 1988. Fidelity of HIV-1 reverse transcriptase. Science 242:1168-1171. 255. Roberts, J. D., K. Bebenek, and T. A. Kunkel. 1988. The accuracy of reverse transcriptase from HIV-1. Science 242:1171-1173. 256. Okulicz, J. F., V. C. Marconi, M. L. Landrum, S. Wegner, A. Weintrob, A. Ganesan, B. Hale, N. Crum-Cianflone, J. Delmar, V. Barthel, G. Quinnan, B. K. Agan, and M. J. Dolan. 2009. Clinical outcomes of elite controllers, viremic controllers, and long-term nonprogressors in the US Department of Defense HIV natural history study. J Infect Dis 200:1714-1723. 257. Jain, V., and S. G. Deeks. When to start antiretroviral therapy. Curr HIV/AIDS Rep 7:60-68. 258. Mitsuya, H., K. J. Weinhold, P. A. Furman, M. H. St Clair, S. N. Lehrman, R. C. Gallo, D. Bolognesi, D. W. Barry, and S. Broder. 1985. 3'-Azido-3'- deoxythymidine (BW A509U): an antiviral agent that inhibits the infectivity and cytopathic effect of human T-lymphotropic virus type III/lymphadenopathy- associated virus in vitro. Proc Natl Acad Sci U S A 82:7096-7100. 259. Broder, S. 1990. Clinical applications of 3'-azido-2',3'-dideoxythymidine (AZT) and related dideoxynucleosides. Med Res Rev 10:419-439. 260. Korant, B. D., and C. J. Rizzo. 1997. The HIV protease and therapies for AIDS. Adv Exp Med Biol 421:279-284. 261. Rowley, M. 2008. The discovery of raltegravir, an integrase inhibitor for the treatment of HIV infection. Prog Med Chem 46:1-28. 262. Dorr, P., M. Westby, S. Dobbs, P. Griffin, B. Irvine, M. Macartney, J. Mori, G. Rickett, C. Smith-Burchnell, C. Napier, R. Webster, D. Armour, D. Price, B. Stammen, A. Wood, and M. Perros. 2005. Maraviroc (UK-427,857), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor CCR5 with broad-spectrum anti-human immunodeficiency virus type 1 activity. Antimicrob Agents Chemother 49:4721-4732. 263. Simon, V., D. D. Ho, and Q. Abdool Karim. 2006. HIV/AIDS epidemiology, pathogenesis, prevention, and treatment. Lancet 368:489-504. 264. Chun, T. W., D. C. Nickle, J. S. Justement, J. H. Meyers, G. Roby, C. W. Hallahan, S. Kottilil, S. Moir, J. M. Mican, J. I. Mullins, D. J. Ward, J. A. Kovacs, P. J. Mannon, and A. S. Fauci. 2008. Persistence of HIV in gut-associated

234 lymphoid tissue despite long-term antiretroviral therapy. J Infect Dis 197:714- 720. 265. Pilcher, C. D., K. A. Christopoulos, and M. Golden. 2010. Public health rationale for rapid nucleic acid or p24 antigen tests for HIV. J Infect Dis 201 Suppl 1:S7- 15. 266. Abbas, A. K., A. H. Lichtman, and S. Pillai. 2007. Cellular and Molecular Immunology. Saunders Elsevier, Philadelphia. 267. Coffman, R. L., A. Sher, and R. A. Seder. 2010. Vaccine adjuvants: putting innate immunity to work. Immunity 33:492-503. 268. Iwasaki, A., and R. Medzhitov. 2004. Toll-like receptor control of the adaptive immune responses. Nat Immunol 5:987-995. 269. Higgins, S. C., and K. H. Mills. 2010. TLR, NLR Agonists, and Other Immune Modulators as Infectious Disease Vaccine Adjuvants. Curr Infect Dis Rep 12:4- 12. 270. Banki, Z., H. Stoiber, and M. P. Dierich. 2005. HIV and human complement: inefficient virolysis and effective adherence. Immunol Lett 97:209-214. 271. Porter, R. R., and K. B. Reid. 1979. Activation of the complement system by antibody-antigen complexes: the classical pathway. Adv Protein Chem 33:1-71. 272. Fearon, D. T., and R. M. Locksley. 1996. The instructive role of innate immunity in the acquired immune response. Science 272:50-53. 273. Malaspina, A., S. Moir, D. C. Nickle, E. T. Donoghue, K. M. Ogwaro, L. A. Ehler, S. Liu, J. A. Mican, M. Dybul, T. W. Chun, J. I. Mullins, and A. S. Fauci. 2002. Human immunodeficiency virus type 1 bound to B cells: relationship to virus replicating in CD4+ T cells and circulating in plasma. J Virol 76:8855-8863. 274. Lamarre, D., A. Ashkenazi, S. Fleury, D. H. Smith, R. P. Sekaly, and D. J. Capon. 1989. The MHC-binding and gp120-binding functions of CD4 are separable. Science 245:743-746. 275. Ben-Sasson, S. Z., J. Hu-Li, J. Quiel, S. Cauchetaux, M. Ratner, I. Shapira, C. A. Dinarello, and W. E. Paul. 2009. IL-1 acts directly on CD4 T cells to enhance their antigen-driven expansion and differentiation. Proc Natl Acad Sci U S A 106:7119-7124. 276. Tseng, S. Y., and M. L. Dustin. 2002. T-cell activation: a multidimensional signaling network. Curr Opin Cell Biol 14:575-580. 277. Chaplin, D. D. Overview of the immune response. J Allergy Clin Immunol 125:S3-23. 278. O'Shea, J. J., and W. E. Paul. 2010. Mechanisms underlying lineage commitment and plasticity of helper CD4+ T cells. Science 327:1098-1102. 279. Muller, I., J. A. Garcia-Sanz, R. Titus, R. Behin, and J. Louis. 1989. Analysis of the cellular parameters of the immune responses contributing to resistance and susceptibility of mice to infection with the intracellular parasite, Leishmania major. Immunol Rev 112:95-113. 280. Vinuesa, C. G., M. A. Linterman, C. C. Goodnow, and K. L. Randall. 2010. T cells and follicular dendritic cells in germinal center B-cell formation and selection. Immunol Rev 237:72-89.

235 281. Zaretsky, A. G., J. J. Taylor, I. L. King, F. A. Marshall, M. Mohrs, and E. J. Pearce. 2009. T follicular helper cells differentiate from Th2 cells in response to helminth antigens. J Exp Med 206:991-999. 282. King, I. L., and M. Mohrs. 2009. IL-4-producing CD4+ T cells in reactive lymph nodes during helminth infection are T follicular helper cells. J Exp Med 206:1001-1007. 283. Sakaguchi, S., M. Miyara, C. M. Costantino, and D. A. Hafler. 2010. FOXP3+ regulatory T cells in the human immune system. Nat Rev Immunol 10:490-500. 284. Korn, T., E. Bettelli, M. Oukka, and V. K. Kuchroo. 2009. IL-17 and Th17 Cells. Annu Rev Immunol 27:485-517. 285. Dubin, P. J., and J. K. Kolls. 2008. Th17 cytokines and mucosal immunity. Immunol Rev 226:160-171. 286. Dandekar, S., M. D. George, and A. J. Baumler. Th17 cells, HIV and the gut mucosal barrier. Curr Opin HIV AIDS 5:173-178. 287. Batista, F. D., and N. E. Harwood. 2009. The who, how and where of antigen presentation to B cells. Nat Rev Immunol 9:15-27. 288. Hoek, K. L., L. E. Gordy, P. L. Collins, V. V. Parekh, T. M. Aune, S. Joyce, J. W. Thomas, L. Van Kaer, and E. Sebzda. Follicular B cell trafficking within the spleen actively restricts humoral immune responses. Immunity 33:254-265. 289. Radbruch, A., G. Muehlinghaus, E. O. Luger, A. Inamine, K. G. Smith, T. Dorner, and F. Hiepe. 2006. Competence and competition: the challenge of becoming a long-lived plasma cell. Nat Rev Immunol 6:741-750. 290. Carsetti, R., M. M. Rosado, and H. Wardmann. 2004. Peripheral development of B cells in mouse and man. Immunol Rev 197:179-191. 291. Batista, F. D., D. Iber, and M. S. Neuberger. 2001. B cells acquire antigen from target cells after synapse formation. Nature 411:489-494. 292. Jacob, J., and G. Kelsoe. 1992. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. II. A common clonal origin for periarteriolar lymphoid sheath-associated foci and germinal centers. J Exp Med 176:679-687. 293. Schittek, B., and K. Rajewsky. 1990. Maintenance of B-cell memory by long- lived cells generated from proliferating precursors. Nature 346:749-751. 294. Manz, R. A., S. Arce, G. Cassese, A. E. Hauser, F. Hiepe, and A. Radbruch. 2002. Humoral immunity and long-lived plasma cells. Curr Opin Immunol 14:517-521. 295. Benner, R., W. Hijmans, and J. J. Haaijman. 1981. The bone marrow: the major source of serum immunoglobulins, but still a neglected site of antibody formation. Clin Exp Immunol 46:1-8. 296. McMillan, R., R. L. Longmire, R. Yelenosky, J. E. Lang, V. Heath, and C. G. Craddock. 1972. Immunoglobulin synthesis by human lymphoid tissues: normal bone marrow as a major site of IgG production. J Immunol 109:1386-1394. 297. Dogan, I., B. Bertocci, V. Vilmont, F. Delbos, J. Megret, S. Storck, C. A. Reynaud, and J. C. Weill. 2009. Multiple layers of B cell memory with different effector functions. Nat Immunol 10:1292-1299. 298. Jung, D., and F. W. Alt. 2004. Unraveling V(D)J recombination; insights into gene regulation. Cell 116:299-311. 299. Manis, J. P., M. Tian, and F. W. Alt. 2002. Mechanism and control of class- switch recombination. Trends Immunol 23:31-39.

236 300. Macpherson, A. J., K. D. McCoy, F. E. Johansen, and P. Brandtzaeg. 2008. The immune geography of IgA induction and function. Mucosal Immunol 1:11-22. 301. Collis, A. V., A. P. Brouwer, and A. C. Martin. 2003. Analysis of the antigen combining site: correlations between length and sequence composition of the hypervariable loops and the nature of the antigen. J Mol Biol 325:337-354. 302. Mian, I. S., A. R. Bradwell, and A. J. Olson. 1991. Structure, function and properties of antibody binding sites. J Mol Biol 217:133-151. 303. Braden, B. C., and R. J. Poljak. 1995. Structural features of the reactions between antibodies and protein antigens. Faseb J 9:9-16. 304. Holmberg, D., A. A. Freitas, D. Portnoi, F. Jacquemart, S. Avrameas, and A. Coutinho. 1986. Antibody repertoires of normal BALB/c mice: B lymphocyte populations defined by state of activation. Immunol Rev 93:147-169. 305. Berek, C., G. M. Griffiths, and C. Milstein. 1985. Molecular events during maturation of the immune response to oxazolone. Nature 316:412-418. 306. Nimmerjahn, F., and J. V. Ravetch. 2008. Fcgamma receptors as regulators of immune responses. Nat Rev Immunol 8:34-47. 307. Woof, J. M., and D. R. Burton. 2004. Human antibody-Fc receptor interactions illuminated by crystal structures. Nat Rev Immunol 4:89-99. 308. Papavasiliou, F. N., and D. G. Schatz. 2002. Somatic hypermutation of immunoglobulin genes: merging mechanisms for genetic diversity. Cell 109 Suppl:S35-44. 309. Muramatsu, M., K. Kinoshita, S. Fagarasan, S. Yamada, Y. Shinkai, and T. Honjo. 2000. Class switch recombination and hypermutation require activation- induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102:553-563. 310. Honjo, T., K. Kinoshita, and M. Muramatsu. 2002. Molecular mechanism of class switch recombination: linkage with somatic hypermutation. Annu Rev Immunol 20:165-196. 311. MacCallum, R. M., A. C. Martin, and J. M. Thornton. 1996. Antibody-antigen interactions: contact analysis and binding site topography. J Mol Biol 262:732- 745. 312. Greenspan, N. S. 2010. Cohen's Conjecture, Howard's Hypothesis, and Ptashne's Ptruth: an exploration of the relationship between affinity and specificity. Trends Immunol 31:138-143. 313. Zhu, Z., A. S. Dimitrov, K. N. Bossart, G. Crameri, K. A. Bishop, V. Choudhry, B. A. Mungall, Y. R. Feng, A. Choudhary, M. Y. Zhang, Y. Feng, L. F. Wang, X. Xiao, B. T. Eaton, C. C. Broder, and D. S. Dimitrov. 2006. Potent neutralization of Hendra and Nipah viruses by human monoclonal antibodies. J Virol 80:891- 899. 314. Wu, H., D. S. Pfarr, Y. Tang, L. L. An, N. K. Patel, J. D. Watkins, W. D. Huse, P. A. Kiener, and J. F. Young. 2005. Ultra-potent antibodies against respiratory syncytial virus: effects of binding kinetics and binding valence on viral neutralization. J Mol Biol 350:126-144. 315. Kausmally, L., K. Waalen, I. Lobersli, E. Hvattum, G. Berntsen, T. E. Michaelsen, and O. H. Brekke. 2004. Neutralizing human antibodies to varicella-

237 zoster virus (VZV) derived from a VZV patient recombinant antibody library. J Gen Virol 85:3493-3500. 316. Icenogle, J., H. Shiwen, G. Duke, S. Gilbert, R. Rueckert, and J. Anderegg. 1983. Neutralization of poliovirus by a monoclonal antibody: kinetics and stoichiometry. Virology 127:412-425. 317. Cavacini, L. A., C. L. Emes, J. Power, M. Duval, and M. R. Posner. 1994. Effect of antibody valency on interaction with cell-surface expressed HIV-1 and viral neutralization. J Immunol 152:2538-2545. 318. McInerney, T. L., L. McLain, S. J. Armstrong, and N. J. Dimmock. 1997. A human IgG1 (b12) specific for the CD4 binding site of HIV-1 neutralizes by inhibiting the virus fusion entry process, but b12 Fab neutralizes by inhibiting a postfusion event. Virology 233:313-326. 319. Forthal, D. N., G. Landucci, K. S. Cole, M. Marthas, J. C. Becerra, and K. Van Rompay. 2006. Rhesus macaque polyclonal and monoclonal antibodies inhibit simian immunodeficiency virus in the presence of human or autologous rhesus effector cells. J Virol 80:9217-9225. 320. Xiao, P., J. Zhao, L. J. Patterson, E. Brocca-Cofano, D. Venzon, P. A. Kozlowski, R. Hidajat, T. Demberg, and M. Robert-Guroff. 2010. Multiple vaccine-elicited nonneutralizing antienvelope antibody activities contribute to protective efficacy by reducing both acute and chronic viremia following simian/human immunodeficiency virus SHIV89.6P challenge in rhesus macaques. J Virol 84:7161-7173. 321. Forthal, D. N., P. B. Gilbert, G. Landucci, and T. Phan. 2007. Recombinant gp120 vaccine-induced antibodies inhibit clinical strains of HIV-1 in the presence of Fc receptor-bearing effector cells and correlate inversely with HIV infection rate. J Immunol 178:6596-6603. 322. Devito, C., J. Hinkula, R. Kaul, L. Lopalco, J. J. Bwayo, F. Plummer, M. Clerici, and K. Broliden. 2000. Mucosal and plasma IgA from HIV-exposed seronegative individuals neutralize a primary HIV-1 isolate. Aids 14:1917-1920. 323. Devito, C., J. Hinkula, R. Kaul, J. Kimani, P. Kiama, L. Lopalco, C. Barass, S. Piconi, D. Trabattoni, J. J. Bwayo, F. Plummer, M. Clerici, and K. Broliden. 2002. Cross-clade HIV-1-specific neutralizing IgA in mucosal and systemic compartments of HIV-1-exposed, persistently seronegative subjects. J Acquir Immune Defic Syndr 30:413-420. 324. Becquart, P., H. Hocini, B. Garin, A. Sepou, M. D. Kazatchkine, and L. Belec. 1999. Compartmentalization of the IgG immune response to HIV-1 in breast milk. Aids 13:1323-1331. 325. Rosenberg, E. S., J. M. Billingsley, A. M. Caliendo, S. L. Boswell, P. E. Sax, S. A. Kalams, and B. D. Walker. 1997. Vigorous HIV-1-specific CD4+ T cell responses associated with control of viremia. Science 278:1447-1450. 326. Younes, S. A., B. Yassine-Diab, A. R. Dumont, M. R. Boulassel, Z. Grossman, J. P. Routy, and R. P. Sekaly. 2003. HIV-1 viremia prevents the establishment of interleukin 2-producing HIV-specific memory CD4+ T cells endowed with proliferative capacity. J Exp Med 198:1909-1922. 327. Dai, J., L. M. Agosto, C. Baytop, J. J. Yu, M. J. Pace, M. K. Liszewski, and U. O'Doherty. 2009. Human immunodeficiency virus integrates directly into naive

238 resting CD4+ T cells but enters naive cells less efficiently than memory cells. J Virol 83:4528-4537. 328. Douek, D. C., J. M. Brenchley, M. R. Betts, D. R. Ambrozak, B. J. Hill, Y. Okamoto, J. P. Casazza, J. Kuruppu, K. Kunstman, S. Wolinsky, Z. Grossman, M. Dybul, A. Oxenius, D. A. Price, M. Connors, and R. A. Koup. 2002. HIV preferentially infects HIV-specific CD4+ T cells. Nature 417:95-98. 329. Cadogan, M., and A. G. Dalgleish. 2008. HIV immunopathogenesis and strategies for intervention. Lancet Infect Dis 8:675-684. 330. Betts, M. R., D. R. Ambrozak, D. C. Douek, S. Bonhoeffer, J. M. Brenchley, J. P. Casazza, R. A. Koup, and L. J. Picker. 2001. Analysis of total human immunodeficiency virus (HIV)-specific CD4(+) and CD8(+) T-cell responses: relationship to viral load in untreated HIV infection. J Virol 75:11983-11991. 331. Musey, L., J. Hughes, T. Schacker, T. Shea, L. Corey, and M. J. McElrath. 1997. Cytotoxic-T-cell responses, viral load, and disease progression in early human immunodeficiency virus type 1 infection. N Engl J Med 337:1267-1274. 332. Jin, X., D. E. Bauer, S. E. Tuttleton, S. Lewin, A. Gettie, J. Blanchard, C. E. Irwin, J. T. Safrit, J. Mittler, L. Weinberger, L. G. Kostrikis, L. Zhang, A. S. Perelson, and D. D. Ho. 1999. Dramatic rise in plasma viremia after CD8(+) T cell depletion in simian immunodeficiency virus-infected macaques. J Exp Med 189:991-998. 333. Almeida, J. R., D. Sauce, D. A. Price, L. Papagno, S. Y. Shin, A. Moris, M. Larsen, G. Pancino, D. C. Douek, B. Autran, A. Saez-Cirion, and V. Appay. 2009. Antigen sensitivity is a major determinant of CD8+ T-cell polyfunctionality and HIV suppressive activity. Blood. 334. Appay, V., D. C. Douek, and D. A. Price. 2008. CD8+ T cell efficacy in vaccination and disease. Nat Med 14:623-628. 335. Lane, H. C., H. Masur, L. C. Edgar, G. Whalen, A. H. Rook, and A. S. Fauci. 1983. Abnormalities of B-cell activation and immunoregulation in patients with the acquired immunodeficiency syndrome. N Engl J Med 309:453-458. 336. Ng, V. L. 1996. B-lymphocytes and autoantibody profiles in HIV disease. Clin Rev Allergy Immunol 14:367-384. 337. Racz, P., K. Tenner-Racz, F. van Vloten, H. Schmidt, M. Dietrich, J. C. Gluckman, N. L. Letvin, and G. Janossy. 1990. Lymphatic tissue changes in AIDS and other retrovirus infections: tools and insights. Lymphology 23:85-91. 338. Pantaleo, G., C. Graziosi, J. F. Demarest, O. J. Cohen, M. Vaccarezza, K. Gantt, C. Muro-Cacho, and A. S. Fauci. 1994. Role of lymphoid organs in the pathogenesis of human immunodeficiency virus (HIV) infection. Immunol Rev 140:105-130. 339. He, B., X. Qiao, P. J. Klasse, A. Chiu, A. Chadburn, D. M. Knowles, J. P. Moore, and A. Cerutti. 2006. HIV-1 envelope triggers polyclonal Ig class switch recombination through a CD40-independent mechanism involving BAFF and C- type lectin receptors. J Immunol 176:3931-3941. 340. Cuss, A. K., D. T. Avery, J. L. Cannons, L. J. Yu, K. E. Nichols, P. J. Shaw, and S. G. Tangye. 2006. Expansion of functionally immature transitional B cells is associated with human-immunodeficient states characterized by impaired humoral immunity. J Immunol 176:1506-1516.

239 341. Doria-Rose, N. A., R. M. Klein, M. M. Manion, S. O'Dell, A. Phogat, B. Chakrabarti, C. W. Hallahan, S. A. Migueles, J. Wrammert, R. Ahmed, M. Nason, R. T. Wyatt, J. R. Mascola, and M. Connors. 2009. Frequency and phenotype of human immunodeficiency virus envelope-specific B cells from patients with broadly cross-neutralizing antibodies. J Virol 83:188-199. 342. Peruchon, S., N. Chaoul, C. Burelout, B. Delache, P. Brochard, P. Laurent, F. Cognasse, S. Prevot, O. Garraud, R. Le Grand, and Y. Richard. 2009. Tissue- specific B-cell dysfunction and generalized memory B-cell loss during acute SIV infection. PLoS One 4:e5966. 343. Avery, D. T., J. I. Ellyard, F. Mackay, L. M. Corcoran, P. D. Hodgkin, and S. G. Tangye. 2005. Increased expression of CD27 on activated human memory B cells correlates with their commitment to the plasma cell lineage. J Immunol 174:4034- 4042. 344. Nagase, H., K. Agematsu, K. Kitano, M. Takamoto, Y. Okubo, A. Komiyama, and K. Sugane. 2001. Mechanism of hypergammaglobulinemia by HIV infection: circulating memory B-cell reduction with plasmacytosis. Clin Immunol 100:250- 259. 345. Moir, S., A. Malaspina, Y. Li, T. W. Chun, T. Lowe, J. Adelsberger, M. Baseler, L. A. Ehler, S. Liu, R. T. Davey, Jr., J. A. Mican, and A. S. Fauci. 2000. B cells of HIV-1-infected patients bind virions through CD21-complement interactions and transmit infectious virus to activated T cells. J Exp Med 192:637-646. 346. Moir, S., and A. S. Fauci. 2009. B cells in HIV infection and disease. Nat Rev Immunol 9:235-245. 347. Anderson, S. M., M. M. Tomayko, and M. J. Shlomchik. 2006. Intrinsic properties of human and murine memory B cells. Immunol Rev 211:280-294. 348. Nilssen, D. E., O. Oktedalen, and P. Brandtzaeg. 2004. Intestinal B cell hyperactivity in AIDS is controlled by highly active antiretroviral therapy. Gut 53:487-493. 349. Morris, L., J. M. Binley, B. A. Clas, S. Bonhoeffer, T. P. Astill, R. Kost, A. Hurley, Y. Cao, M. Markowitz, D. D. Ho, and J. P. Moore. 1998. HIV-1 antigen- specific and -nonspecific B cell responses are sensitive to combination antiretroviral therapy. J Exp Med 188:233-245. 350. De Milito, A. 2004. B lymphocyte dysfunctions in HIV infection. Curr HIV Res 2:11-21. 351. Scheid, J. F., H. Mouquet, N. Feldhahn, B. D. Walker, F. Pereyra, E. Cutrell, M. S. Seaman, J. R. Mascola, R. T. Wyatt, H. Wardemann, and M. C. Nussenzweig. 2009. A method for identification of HIV gp140 binding memory B cells in human blood. J Immunol Methods 343:65-67. 352. Scheid, J. F., H. Mouquet, N. Feldhahn, M. S. Seaman, K. Velinzon, J. Pietzsch, R. G. Ott, R. M. Anthony, H. Zebroski, A. Hurley, A. Phogat, B. Chakrabarti, Y. Li, M. Connors, F. Pereyra, B. D. Walker, H. Wardemann, D. Ho, R. T. Wyatt, J. R. Mascola, J. V. Ravetch, and M. C. Nussenzweig. 2009. Broad diversity of neutralizing antibodies isolated from memory B cells in HIV-infected individuals. Nature 458:636-640. 353. Burton, D. R., and P. Poignard. 2009. HIV: Immune memory downloaded. Nature 458:584-585.

240 354. Arendrup, M., C. Nielsen, J. E. Hansen, C. Pedersen, L. Mathiesen, and J. O. Nielsen. 1992. Autologous HIV-1 neutralizing antibodies: emergence of neutralization-resistant escape virus and subsequent development of escape virus neutralizing antibodies. J Acquir Immune Defic Syndr 5:303-307. 355. van Gils, M. J., Z. Euler, B. Schweighardt, T. Wrin, and H. Schuitemaker. 2009. Prevalence of cross-reactive HIV-1-neutralizing activity in HIV-1-infected patients with rapid or slow disease progression. Aids 23:2405-2414. 356. Euler, Z., M. J. van Gils, E. M. Bunnik, P. Phung, B. Schweighardt, T. Wrin, and H. Schuitemaker. 2010. Cross-reactive neutralizing humoral immunity does not protect from HIV type 1 disease progression. J Infect Dis 201:1045-1053. 357. Pantophlet, R., and D. R. Burton. 2006. GP120: target for neutralizing HIV-1 antibodies. Annu Rev Immunol 24:739-769. 358. Montefiori, D. C., L. Morris, G. Ferrari, and J. R. Mascola. 2007. Neutralizing and other antiviral antibodies in HIV-1 infection and vaccination. Curr Opin HIV AIDS 2:169-176. 359. Simek, M. D., W. Rida, F. H. Priddy, P. Pung, E. Carrow, D. S. Laufer, J. K. Lehrman, M. Boaz, T. Tarragona-Fiol, G. Miiro, J. Birungi, A. Pozniak, D. A. McPhee, O. Manigart, E. Karita, A. Inwoley, W. Jaoko, J. Dehovitz, L. G. Bekker, P. Pitisuttithum, R. Paris, L. M. Walker, P. Poignard, T. Wrin, P. E. Fast, D. R. Burton, and W. C. Koff. 2009. Human immunodeficiency virus type 1 elite neutralizers: individuals with broad and potent neutralizing activity identified by using a high-throughput neutralization assay together with an analytical selection algorithm. J Virol 83:7337-7348. 360. Sather, D. N., J. Armann, L. K. Ching, A. Mavrantoni, G. Sellhorn, Z. Caldwell, X. Yu, B. Wood, S. Self, S. Kalams, and L. Stamatatos. 2009. Factors associated with the development of cross-reactive neutralizing antibodies during human immunodeficiency virus type 1 infection. J Virol 83:757-769. 361. Doria-Rose, N. A., R. M. Klein, M. G. Daniels, S. O'Dell, M. Nason, A. Lapedes, T. Bhattacharya, S. A. Migueles, R. T. Wyatt, B. T. Korber, J. R. Mascola, and M. Connors. 2010. Breadth of human immunodeficiency virus-specific neutralizing activity in sera: clustering analysis and association with clinical variables. J Virol 84:1631-1636. 362. Binley, J. M., E. A. Lybarger, E. T. Crooks, M. S. Seaman, E. Gray, K. L. Davis, J. M. Decker, D. Wycuff, L. Harris, N. Hawkins, B. Wood, C. Nathe, D. Richman, G. D. Tomaras, F. Bibollet-Ruche, J. E. Robinson, L. Morris, G. M. Shaw, D. C. Montefiori, and J. R. Mascola. 2008. Profiling the specificity of neutralizing antibodies in a large panel of plasmas from patients chronically infected with human immunodeficiency virus type 1 subtypes B and C. J Virol 82:11651-11668. 363. Vittecoq, D., S. Chevret, L. Morand-Joubert, F. Heshmati, F. Audat, M. Bary, T. Dusautoir, A. Bismuth, J. P. Viard, F. Barre-Sinoussi, and et al. 1995. Passive immunotherapy in AIDS: a double-blind randomized study based on transfusions of plasma rich in anti-human immunodeficiency virus 1 antibodies vs. transfusions of seronegative plasma. Proc Natl Acad Sci U S A 92:1195-1199.

241 364. Cao, Y., L. Qin, L. Zhang, J. Safrit, and D. D. Ho. 1995. Virologic and immunologic characterization of long-term survivors of human immunodeficiency virus type 1 infection. N Engl J Med 332:201-208. 365. Moore, J. P., Y. Cao, D. D. Ho, and R. A. Koup. 1994. Development of the anti- gp120 antibody response during seroconversion to human immunodeficiency virus type 1. J Virol 68:5142-5155. 366. Carotenuto, P., D. Looij, L. Keldermans, F. de Wolf, and J. Goudsmit. 1998. Neutralizing antibodies are positively associated with CD4+ T-cell counts and T- cell function in long-term AIDS-free infection. Aids 12:1591-1600. 367. Montefiori, D. C., T. S. Hill, H. T. Vo, B. D. Walker, and E. S. Rosenberg. 2001. Neutralizing antibodies associated with viremia control in a subset of individuals after treatment of acute human immunodeficiency virus type 1 infection. J Virol 75:10200-10207. 368. Bailey, J. R., K. G. Lassen, H. C. Yang, T. C. Quinn, S. C. Ray, J. N. Blankson, and R. F. Siliciano. 2006. Neutralizing antibodies do not mediate suppression of human immunodeficiency virus type 1 in elite suppressors or selection of plasma virus variants in patients on highly active antiretroviral therapy. J Virol 80:4758- 4770. 369. Pereyra, F., M. M. Addo, D. E. Kaufmann, Y. Liu, T. Miura, A. Rathod, B. Baker, A. Trocha, R. Rosenberg, E. Mackey, P. Ueda, Z. Lu, D. Cohen, T. Wrin, C. J. Petropoulos, E. S. Rosenberg, and B. D. Walker. 2008. Genetic and immunologic heterogeneity among persons who control HIV infection in the absence of therapy. J Infect Dis 197:563-571. 370. Wrin, T., L. Crawford, L. Sawyer, P. Weber, H. W. Sheppard, and C. V. Hanson. 1994. Neutralizing antibody responses to autologous and heterologous isolates of human immunodeficiency virus. J Acquir Immune Defic Syndr 7:211-219. 371. Wei, X., J. M. Decker, S. Wang, H. Hui, J. C. Kappes, X. Wu, J. F. Salazar- Gonzalez, M. G. Salazar, J. M. Kilby, M. S. Saag, N. L. Komarova, M. A. Nowak, B. H. Hahn, P. D. Kwong, and G. M. Shaw. 2003. Antibody neutralization and escape by HIV-1. Nature 422:307-312. 372. Von Gegerfelt, A., J. Albert, L. Morfeldt-Manson, K. Broliden, and E. M. Fenyo. 1991. Isolate-specific neutralizing antibodies in patients with progressive HIV-1- related disease. Virology 185:162-168. 373. Tsang, M. L., L. A. Evans, P. McQueen, L. Hurren, C. Byrne, R. Penny, B. Tindall, and D. A. Cooper. 1994. Neutralizing antibodies against sequential autologous human immunodeficiency virus type 1 isolates after seroconversion. J Infect Dis 170:1141-1147. 374. Tremblay, M., and M. A. Wainberg. 1990. Neutralization of multiple HIV-1 isolates from a single subject by autologous sequential sera. J Infect Dis 162:735- 737. 375. Sagar, M., X. Wu, S. Lee, and J. Overbaugh. 2006. Human immunodeficiency virus type 1 V1-V2 envelope loop sequences expand and add glycosylation sites over the course of infection, and these modifications affect antibody neutralization sensitivity. J Virol 80:9586-9598.

242 376. Richman, D. D., T. Wrin, S. J. Little, and C. J. Petropoulos. 2003. Rapid evolution of the neutralizing antibody response to HIV type 1 infection. Proc Natl Acad Sci U S A 100:4144-4149. 377. Moog, C., H. J. Fleury, I. Pellegrin, A. Kirn, and A. M. Aubertin. 1997. Autologous and heterologous neutralizing antibody responses following initial seroconversion in human immunodeficiency virus type 1-infected individuals. J Virol 71:3734-3741. 378. Montefiori, D. C., I. Y. Zhou, B. Barnes, D. Lake, E. M. Hersh, Y. Masuho, and L. B. Lefkowitz, Jr. 1991. Homotypic antibody responses to fresh clinical isolates of human immunodeficiency virus. Virology 182:635-643. 379. Li, Y., K. Svehla, M. K. Louder, D. Wycuff, S. Phogat, M. Tang, S. A. Migueles, X. Wu, A. Phogat, G. M. Shaw, M. Connors, J. Hoxie, J. R. Mascola, and R. Wyatt. 2009. Analysis of neutralization specificities in polyclonal sera derived from human immunodeficiency virus type 1-infected individuals. J Virol 83:1045- 1059. 380. Rademeyer, C., P. L. Moore, N. Taylor, D. P. Martin, I. A. Choge, E. S. Gray, H. W. Sheppard, C. Gray, L. Morris, and C. Williamson. 2007. Genetic characteristics of HIV-1 subtype C envelopes inducing cross-neutralizing antibodies. Virology 368:172-181. 381. Moore, P. L., N. Ranchobe, B. E. Lambson, E. S. Gray, E. Cave, M. R. Abrahams, G. Bandawe, K. Mlisana, S. S. Abdool Karim, C. Williamson, and L. Morris. 2009. Limited neutralizing antibody specificities drive neutralization escape in early HIV-1 subtype C infection. PLoS Pathog 5:e1000598. 382. Gray, E. S., P. L. Moore, I. A. Choge, J. M. Decker, F. Bibollet-Ruche, H. Li, N. Leseka, F. Treurnicht, K. Mlisana, G. M. Shaw, S. S. Karim, C. Williamson, and L. Morris. 2007. Neutralizing antibody responses in acute human immunodeficiency virus type 1 subtype C infection. J Virol 81:6187-6196. 383. Bunnik, E. M., M. S. Lobbrecht, A. C. van Nuenen, and H. Schuitemaker. 2009. Escape from autologous humoral immunity of HIV-1 is not associated with a decrease in replicative capacity. Virology 397:224-230. 384. van Gils, M. J., E. M. Bunnik, J. A. Burger, Y. Jacob, B. Schweighardt, T. Wrin, and H. Schuitemaker. 2010. Rapid escape from preserved cross-reactive neutralizing humoral immunity without loss of viral fitness in HIV-1-infected progressors and long-term nonprogressors. J Virol 84:3576-3585. 385. Koch, M., M. Pancera, P. D. Kwong, P. Kolchinsky, C. Grundner, L. Wang, W. A. Hendrickson, J. Sodroski, and R. Wyatt. 2003. Structure-based, targeted deglycosylation of HIV-1 gp120 and effects on neutralization sensitivity and antibody recognition. Virology 313:387-400. 386. Burton, D. R. 2002. Antibodies, viruses and vaccines. Nat Rev Immunol 2:706- 713. 387. Mascola, J. R. 2003. Defining the protective antibody response for HIV-1. Curr Mol Med 3:209-216. 388. Dhillon, A. K., H. Donners, R. Pantophlet, W. E. Johnson, J. M. Decker, G. M. Shaw, F. H. Lee, D. D. Richman, R. W. Doms, G. Vanham, and D. R. Burton. 2007. Dissecting the neutralizing antibody specificities of broadly neutralizing

243 sera from human immunodeficiency virus type 1-infected donors. J Virol 81:6548-6562. 389. Shen, X., R. J. Parks, D. C. Montefiori, J. L. Kirchherr, B. F. Keele, J. M. Decker, W. A. Blattner, F. Gao, K. J. Weinhold, C. B. Hicks, M. L. Greenberg, B. H. Hahn, G. M. Shaw, B. F. Haynes, and G. D. Tomaras. 2009. In vivo gp41 antibodies targeting the 2F5 monoclonal antibody epitope mediate human immunodeficiency virus type 1 neutralization breadth. J Virol 83:3617-3625. 390. Kaul, R., D. Trabattoni, J. J. Bwayo, D. Arienti, A. Zagliani, F. M. Mwangi, C. Kariuki, E. N. Ngugi, K. S. MacDonald, T. B. Ball, M. Clerici, and F. A. Plummer. 1999. HIV-1-specific mucosal IgA in a cohort of HIV-1-resistant Kenyan sex workers. Aids 13:23-29. 391. Devito, C., K. Broliden, R. Kaul, L. Svensson, K. Johansen, P. Kiama, J. Kimani, L. Lopalco, S. Piconi, J. J. Bwayo, F. Plummer, M. Clerici, and J. Hinkula. 2000. Mucosal and plasma IgA from HIV-1-exposed uninfected individuals inhibit HIV-1 transcytosis across human epithelial cells. J Immunol 165:5170-5176. 392. Broliden, K., J. Hinkula, C. Devito, P. Kiama, J. Kimani, D. Trabbatoni, J. J. Bwayo, M. Clerici, F. Plummer, and R. Kaul. 2001. Functional HIV-1 specific IgA antibodies in HIV-1 exposed, persistently IgG seronegative female sex workers. Immunol Lett 79:29-36. 393. Horton, R. E., T. B. Ball, C. Wachichi, W. Jaoko, W. J. Rutherford, L. McKinnon, R. Kaul, A. Rebbapragada, J. Kimani, and F. A. Plummer. 2009. Cervical HIV- specific IgA in a population of commercial sex workers correlates with repeated exposure but not resistance to HIV. AIDS Res Hum Retroviruses 25:83-92. 394. Dorrell, L., A. J. Hessell, M. Wang, H. Whittle, S. Sabally, S. Rowland-Jones, D. R. Burton, and P. W. Parren. 2000. Absence of specific mucosal antibody responses in HIV-exposed uninfected sex workers from the Gambia. Aids 14:1117-1122. 395. Van de Perre, P. 2003. Transfer of antibody via mother's milk. Vaccine 21:3374- 3376. 396. Newburg, D. S., R. P. Viscidi, A. Ruff, and R. H. Yolken. 1992. A human milk factor inhibits binding of human immunodeficiency virus to the CD4 receptor. Pediatr Res 31:22-28. 397. Van de Perre, P., A. Simonon, D. G. Hitimana, F. Dabis, P. Msellati, B. Mukamabano, J. B. Butera, C. Van Goethem, E. Karita, and P. Lepage. 1993. Infective and anti-infective properties of breastmilk from HIV-1-infected women. Lancet 341:914-918. 398. Mok, J. 1993. Breast milk and HIV-1 transmission. Lancet 341:930-931. 399. Bongertz, V., C. I. Costa, V. G. Veloso, B. Grinsztejn, E. C. Joao Filho, G. Calvet, J. H. Pilotto, M. L. Guimaraes, and M. G. Morgado. 2001. Vertical HIV-1 transmission: importance of neutralizing antibody titer and specificity. Scand J Immunol 53:302-309. 400. Lynch, J. B., R. Nduati, C. A. Blish, B. A. Richardson, J. M. Mabuka, Z. Jalalian- Lechak, G. John-Stewart, and J. Overbaugh. 2011. The breadth and potency of passively acquired human immunodeficiency virus type 1-specific neutralizing antibodies do not correlate with the risk of infant infection. J Virol 85:5252-5261.

244 401. Walter, J., L. Kuhn, and G. M. Aldrovandi. 2008. Advances in basic science understanding of mother-to-child HIV-1 transmission. Curr Opin HIV AIDS 3:146-150. 402. Becquart, P., H. Hocini, M. Levy, A. Sepou, M. D. Kazatchkine, and L. Belec. 2000. Secretory anti-human immunodeficiency virus (HIV) antibodies in colostrum and breast milk are not a major determinant of the protection of early postnatal transmission of HIV. J Infect Dis 181:532-539. 403. Kittinunvorakoon, C., M. K. Morris, K. Neeyapun, B. Jetsawang, G. C. Buehring, and C. V. Hanson. 2009. Mother to child transmission of HIV-1 in a Thai population: role of virus characteristics and maternal humoral immune response. J Med Virol 81:768-778. 404. Lyimo, M. A., A. L. Howell, E. Balandya, S. K. Eszterhas, and R. I. Connor. 2009. Innate factors in human breast milk inhibit cell-free HIV-1 but not cell- associated HIV-1 infection of CD4+ cells. J Acquir Immune Defic Syndr 51:117- 124. 405. Scarlatti, G., J. Albert, P. Rossi, V. Hodara, P. Biraghi, L. Muggiasca, and E. M. Fenyo. 1993. Mother-to-child transmission of human immunodeficiency virus type 1: correlation with neutralizing antibodies against primary isolates. J Infect Dis 168:207-210. 406. Dickover, R., E. Garratty, K. Yusim, C. Miller, B. Korber, and Y. Bryson. 2006. Role of maternal autologous neutralizing antibody in selective perinatal transmission of human immunodeficiency virus type 1 escape variants. J Virol 80:6525-6533. 407. Gauduin, M. C., P. W. Parren, R. Weir, C. F. Barbas, D. R. Burton, and R. A. Koup. 1997. Passive immunization with a human monoclonal antibody protects hu-PBL-SCID mice against challenge by primary isolates of HIV-1. Nat Med 3:1389-1393. 408. Andrus, L., A. M. Prince, I. Bernal, P. McCormack, D. H. Lee, M. K. Gorny, and S. Zolla-Pazner. 1998. Passive immunization with a human immunodeficiency virus type 1-neutralizing monoclonal antibody in Hu-PBL-SCID mice: isolation of a neutralization escape variant. J Infect Dis 177:889-897. 409. Haigwood, N. L., and L. Stamatatos. 2003. Role of neutralizing antibodies in HIV infection. Aids 17 Suppl 4:S67-71. 410. Baba, T. W., V. Liska, R. Hofmann-Lehmann, J. Vlasak, W. Xu, S. Ayehunie, L. A. Cavacini, M. R. Posner, H. Katinger, G. Stiegler, B. J. Bernacky, T. A. Rizvi, R. Schmidt, L. R. Hill, M. E. Keeling, Y. Lu, J. E. Wright, T. C. Chou, and R. M. Ruprecht. 2000. Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection. Nat Med 6:200-206. 411. Mascola, J. R., G. Stiegler, T. C. VanCott, H. Katinger, C. B. Carpenter, C. E. Hanson, H. Beary, D. Hayes, S. S. Frankel, D. L. Birx, and M. G. Lewis. 2000. Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies. Nat Med 6:207-210. 412. Mascola, J. R., M. G. Lewis, G. Stiegler, D. Harris, T. C. VanCott, D. Hayes, M. K. Louder, C. R. Brown, C. V. Sapan, S. S. Frankel, Y. Lu, M. L. Robb, H. Katinger, and D. L. Birx. 1999. Protection of Macaques against pathogenic

245 simian/human immunodeficiency virus 89.6PD by passive transfer of neutralizing antibodies. J Virol 73:4009-4018. 413. Haigwood, N. L., D. C. Montefiori, W. F. Sutton, J. McClure, A. J. Watson, G. Voss, V. M. Hirsch, B. A. Richardson, N. L. Letvin, S. L. Hu, and P. R. Johnson. 2004. Passive immunotherapy in simian immunodeficiency virus-infected macaques accelerates the development of neutralizing antibodies. J Virol 78:5983-5995. 414. Shibata, R., T. Igarashi, N. Haigwood, A. Buckler-White, R. Ogert, W. Ross, R. Willey, M. W. Cho, and M. A. Martin. 1999. Neutralizing antibody directed against the HIV-1 envelope glycoprotein can completely block HIV-1/SIV chimeric virus infections of macaque monkeys. Nat Med 5:204-210. 415. Veazey, R. S., R. J. Shattock, M. Pope, J. C. Kirijan, J. Jones, Q. Hu, T. Ketas, P. A. Marx, P. J. Klasse, D. R. Burton, and J. P. Moore. 2003. Prevention of virus transmission to macaque monkeys by a vaginally applied monoclonal antibody to HIV-1 gp120. Nat Med 9:343-346. 416. Parren, P. W., P. A. Marx, A. J. Hessell, A. Luckay, J. Harouse, C. Cheng-Mayer, J. P. Moore, and D. R. Burton. 2001. Antibody protects macaques against vaginal challenge with a pathogenic R5 simian/human immunodeficiency virus at serum levels giving complete neutralization in vitro. J Virol 75:8340-8347. 417. Haigwood, N. L., A. Watson, W. F. Sutton, J. McClure, A. Lewis, J. Ranchalis, B. Travis, G. Voss, N. L. Letvin, S. L. Hu, V. M. Hirsch, and P. R. Johnson. 1996. Passive immune globulin therapy in the SIV/macaque model: early intervention can alter disease profile. Immunol Lett 51:107-114. 418. Conley, A. J., J. A. Kessler, 2nd, L. J. Boots, P. M. McKenna, W. A. Schleif, E. A. Emini, G. E. Mark, 3rd, H. Katinger, E. K. Cobb, S. M. Lunceford, S. R. Rouse, and K. K. Murthy. 1996. The consequence of passive administration of an anti-human immunodeficiency virus type 1 neutralizing monoclonal antibody before challenge of chimpanzees with a primary virus isolate. J Virol 70:6751- 6758. 419. Emini, E. A., W. A. Schleif, J. H. Nunberg, A. J. Conley, Y. Eda, S. Tokiyoshi, S. D. Putney, S. Matsushita, K. E. Cobb, C. M. Jett, and et al. 1992. Prevention of HIV-1 infection in chimpanzees by gp120 V3 domain-specific monoclonal antibody. Nature 355:728-730. 420. Mascola, J. R., M. G. Lewis, T. C. VanCott, G. Stiegler, H. Katinger, M. Seaman, K. Beaudry, D. H. Barouch, B. Korioth-Schmitz, G. Krivulka, A. Sambor, B. Welcher, D. C. Douek, D. C. Montefiori, J. W. Shiver, P. Poignard, D. R. Burton, and N. L. Letvin. 2003. Cellular immunity elicited by human immunodeficiency virus type 1/ simian immunodeficiency virus DNA vaccination does not augment the sterile protection afforded by passive infusion of neutralizing antibodies. J Virol 77:10348-10356. 421. Poignard, P., R. Sabbe, G. R. Picchio, M. Wang, R. J. Gulizia, H. Katinger, P. W. Parren, D. E. Mosier, and D. R. Burton. 1999. Neutralizing antibodies have limited effects on the control of established HIV-1 infection in vivo. Immunity 10:431-438. 422. Binley, J. M., B. Clas, A. Gettie, M. Vesanen, D. C. Montefiori, L. Sawyer, J. Booth, M. Lewis, P. A. Marx, S. Bonhoeffer, and J. P. Moore. 2000. Passive

246 infusion of immune serum into simian immunodeficiency virus-infected rhesus macaques undergoing a rapid disease course has minimal effect on plasma viremia. Virology 270:237-249. 423. Stiegler, G., C. Armbruster, B. Vcelar, H. Stoiber, R. Kunert, N. L. Michael, L. L. Jagodzinski, C. Ammann, W. Jager, J. Jacobson, N. Vetter, and H. Katinger. 2002. Antiviral activity of the neutralizing antibodies 2F5 and 2G12 in asymptomatic HIV-1-infected humans: a phase I evaluation. Aids 16:2019-2025. 424. Trkola, A., H. Kuster, P. Rusert, B. Joos, M. Fischer, C. Leemann, A. Manrique, M. Huber, M. Rehr, A. Oxenius, R. Weber, G. Stiegler, B. Vcelar, H. Katinger, L. Aceto, and H. F. Gunthard. 2005. Delay of HIV-1 rebound after cessation of antiretroviral therapy through passive transfer of human neutralizing antibodies. Nat Med 11:615-622. 425. Gilbert, P. B., M. L. Peterson, D. Follmann, M. G. Hudgens, D. P. Francis, M. Gurwith, W. L. Heyward, D. V. Jobes, V. Popovic, S. G. Self, F. Sinangil, D. Burke, and P. W. Berman. 2005. Correlation between immunologic responses to a recombinant glycoprotein 120 vaccine and incidence of HIV-1 infection in a phase 3 HIV-1 preventive vaccine trial. J Infect Dis 191:666-677. 426. Nitayaphan, S., P. Pitisuttithum, C. Karnasuta, C. Eamsila, M. de Souza, P. Morgan, V. Polonis, M. Benenson, T. VanCott, S. Ratto-Kim, J. Kim, D. Thapinta, R. Garner, V. Bussaratid, P. Singharaj, R. el-Habib, S. Gurunathan, W. Heyward, D. Birx, J. McNeil, and A. E. Brown. 2004. Safety and immunogenicity of an HIV subtype B and E prime-boost vaccine combination in HIV-negative Thai adults. J Infect Dis 190:702-706. 427. Karnasuta, C., R. M. Paris, J. H. Cox, S. Nitayaphan, P. Pitisuttithum, P. Thongcharoen, A. E. Brown, S. Gurunathan, J. Tartaglia, W. L. Heyward, J. G. McNeil, D. L. Birx, and M. S. de Souza. 2005. Antibody-dependent cell-mediated cytotoxic responses in participants enrolled in a phase I/II ALVAC- HIV/AIDSVAX B/E prime-boost HIV-1 vaccine trial in Thailand. Vaccine 23:2522-2529. 428. Forthal, D. N., G. Landucci, and E. S. Daar. 2001. Antibody from patients with acute human immunodeficiency virus (HIV) infection inhibits primary strains of HIV type 1 in the presence of natural-killer effector cells. J Virol 75:6953-6961. 429. Chung, A. W., E. Rollman, R. J. Center, S. J. Kent, and I. Stratov. 2009. Rapid degranulation of NK cells following activation by HIV-specific antibodies. J Immunol 182:1202-1210. 430. Sullivan, N., Y. Sun, J. Binley, J. Lee, C. F. Barbas, 3rd, P. W. Parren, D. R. Burton, and J. Sodroski. 1998. Determinants of human immunodeficiency virus type 1 envelope glycoprotein activation by soluble CD4 and monoclonal antibodies. J Virol 72:6332-6338. 431. Huisman, W., B. E. Martina, G. F. Rimmelzwaan, R. A. Gruters, and A. D. Osterhaus. 2009. Vaccine-induced enhancement of viral infections. Vaccine 27:505-512. 432. Massanella, M., I. Puigdomenech, C. Cabrera, M. T. Fernandez-Figueras, A. Aucher, G. Gaibelet, D. Hudrisier, E. Garcia, M. Bofill, B. Clotet, and J. Blanco. 2009. Antigp41 antibodies fail to block early events of virological synapses but inhibit HIV spread between T cells. Aids 23:183-188.

247 433. Martin, N., S. Welsch, C. Jolly, J. A. Briggs, D. Vaux, and Q. J. Sattentau. 2010. Virological synapse-mediated spread of human immunodeficiency virus type 1 between T cells is sensitive to entry inhibition. J Virol 84:3516-3527. 434. Fouts, T. R., J. M. Binley, A. Trkola, J. E. Robinson, and J. P. Moore. 1997. Neutralization of the human immunodeficiency virus type 1 primary isolate JR- FL by human monoclonal antibodies correlates with antibody binding to the oligomeric form of the envelope glycoprotein complex. J Virol 71:2779-2785. 435. Parren, P. W., I. Mondor, D. Naniche, H. J. Ditzel, P. J. Klasse, D. R. Burton, and Q. J. Sattentau. 1998. Neutralization of human immunodeficiency virus type 1 by antibody to gp120 is determined primarily by occupancy of sites on the virion irrespective of epitope specificity. J Virol 72:3512-3519. 436. Moore, J. P., and J. Sodroski. 1996. Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein. J Virol 70:1863-1872. 437. Ruprecht, C. R., A. Krarup, L. Reynell, A. M. Mann, O. F. Brandenberg, L. Berlinger, I. A. Abela, R. R. Regoes, H. F. Gunthard, P. Rusert, and A. Trkola. 2011. MPER-specific antibodies induce gp120 shedding and irreversibly neutralize HIV-1. J Exp Med 208:439-454. 438. Scanlan, C. N., R. Pantophlet, M. R. Wormald, E. O. Saphire, D. Calarese, R. Stanfield, I. A. Wilson, H. Katinger, R. A. Dwek, D. R. Burton, and P. M. Rudd. 2003. The carbohydrate epitope of the neutralizing anti-HIV-1 antibody 2G12. Adv Exp Med Biol 535:205-218. 439. Thali, M., J. P. Moore, C. Furman, M. Charles, D. D. Ho, J. Robinson, and J. Sodroski. 1993. Characterization of conserved human immunodeficiency virus type 1 gp120 neutralization epitopes exposed upon gp120-CD4 binding. J Virol 67:3978-3988. 440. Ofek, G., M. Tang, A. Sambor, H. Katinger, J. R. Mascola, R. Wyatt, and P. D. Kwong. 2004. Structure and mechanistic analysis of the anti-human immunodeficiency virus type 1 antibody 2F5 in complex with its gp41 epitope. J Virol 78:10724-10737. 441. Buchacher, A., R. Predl, K. Strutzenberger, W. Steinfellner, A. Trkola, M. Purtscher, G. Gruber, C. Tauer, F. Steindl, A. Jungbauer, and et al. 1994. Generation of human monoclonal antibodies against HIV-1 proteins; electrofusion and Epstein-Barr virus transformation for peripheral blood lymphocyte immortalization. AIDS Res Hum Retroviruses 10:359-369. 442. Muster, T., Steindl, F, Buchacher, A, Purtscher, M., Trkola, A., Maiwald, G., Himmler, G., Ruker, F., Jungbauer, A., Katinger, H. 1992. A conserved neutralizing epitope on gp41 of HIV-1. In Advances in AIDS vaccine Development Groups, Chantilly, Va. 44. 443. Zwick, M. B., A. F. Labrijn, M. Wang, C. Spenlehauer, E. O. Saphire, J. M. Binley, J. P. Moore, G. Stiegler, H. Katinger, D. R. Burton, and P. W. Parren. 2001. Broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41. J Virol 75:10892-10905. 444. Walker, L. M., S. K. Phogat, P. Y. Chan-Hui, D. Wagner, P. Phung, J. L. Goss, T. Wrin, M. D. Simek, S. Fling, J. L. Mitcham, J. K. Lehrman, F. H. Priddy, O. A.

248 Olsen, S. M. Frey, P. W. Hammond, S. Kaminsky, T. Zamb, M. Moyle, W. C. Koff, P. Poignard, and D. R. Burton. 2009. Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science 326:285-289. 445. Barbas, C. F., 3rd, E. Bjorling, F. Chiodi, N. Dunlop, D. Cababa, T. M. Jones, S. L. Zebedee, M. A. Persson, P. L. Nara, E. Norrby, and et al. 1992. Recombinant human Fab fragments neutralize human type 1 immunodeficiency virus in vitro. Proc Natl Acad Sci U S A 89:9339-9343. 446. Burton, D. R., C. F. Barbas, 3rd, M. A. Persson, S. Koenig, R. M. Chanock, and R. A. Lerner. 1991. A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals. Proc Natl Acad Sci U S A 88:10134-10137. 447. Barbas, C. F., 3rd, T. A. Collet, W. Amberg, P. Roben, J. M. Binley, D. Hoekstra, D. Cababa, T. M. Jones, R. A. Williamson, G. R. Pilkington, and et al. 1993. Molecular profile of an antibody response to HIV-1 as probed by combinatorial libraries. J Mol Biol 230:812-823. 448. Binley, J. M., T. Wrin, B. Korber, M. B. Zwick, M. Wang, C. Chappey, G. Stiegler, R. Kunert, S. Zolla-Pazner, H. Katinger, C. J. Petropoulos, and D. R. Burton. 2004. Comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type 1 monoclonal antibodies. J Virol 78:13232-13252. 449. Zwick, M. B., M. Wang, P. Poignard, G. Stiegler, H. Katinger, D. R. Burton, and P. W. Parren. 2001. Neutralization synergy of human immunodeficiency virus type 1 primary isolates by cocktails of broadly neutralizing antibodies. J Virol 75:12198-12208. 450. Xu, W., B. A. Smith-Franklin, P. L. Li, C. Wood, J. He, Q. Du, G. J. Bhat, C. Kankasa, H. Katinger, L. A. Cavacini, M. R. Posner, D. R. Burton, T. C. Chou, and R. M. Ruprecht. 2001. Potent neutralization of primary human immunodeficiency virus clade C isolates with a synergistic combination of human monoclonal antibodies raised against clade B. J Hum Virol 4:55-61. 451. Verrier, F., A. Nadas, M. K. Gorny, and S. Zolla-Pazner. 2001. Additive effects characterize the interaction of antibodies involved in neutralization of the primary dualtropic human immunodeficiency virus type 1 isolate 89.6. J Virol 75:9177- 9186. 452. Roben, P., J. P. Moore, M. Thali, J. Sodroski, C. F. Barbas, 3rd, and D. R. Burton. 1994. Recognition properties of a panel of human recombinant Fab fragments to the CD4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1. J Virol 68:4821-4828. 453. Parren, P. W., H. J. Ditzel, R. J. Gulizia, J. M. Binley, C. F. Barbas, 3rd, D. R. Burton, and D. E. Mosier. 1995. Protection against HIV-1 infection in hu-PBL- SCID mice by passive immunization with a neutralizing human monoclonal antibody against the gp120 CD4-binding site. Aids 9:F1-6. 454. D'Souza, M. P., D. Livnat, J. A. Bradac, and S. H. Bridges. 1997. Evaluation of monoclonal antibodies to human immunodeficiency virus type 1 primary isolates by neutralization assays: performance criteria for selecting candidate antibodies

249 for clinical trials. AIDS Clinical Trials Group Antibody Selection Working Group. J Infect Dis 175:1056-1062. 455. Kessler, J. A., 2nd, P. M. McKenna, E. A. Emini, C. P. Chan, M. D. Patel, S. K. Gupta, G. E. Mark, 3rd, C. F. Barbas, 3rd, D. R. Burton, and A. J. Conley. 1997. Recombinant human monoclonal antibody IgG1b12 neutralizes diverse human immunodeficiency virus type 1 primary isolates. AIDS Res Hum Retroviruses 13:575-582. 456. Li, A., T. W. Baba, J. Sodroski, S. Zolla-Pazner, M. K. Gorny, J. Robinson, M. R. Posner, H. Katinger, C. F. Barbas, 3rd, D. R. Burton, T. C. Chou, and R. M. Ruprecht. 1997. Synergistic neutralization of a chimeric SIV/HIV type 1 virus with combinations of human anti-HIV type 1 envelope monoclonal antibodies or hyperimmune globulins. AIDS Res Hum Retroviruses 13:647-656. 457. Trkola, A., T. Ketas, V. N. Kewalramani, F. Endorf, J. M. Binley, H. Katinger, J. Robinson, D. R. Littman, and J. P. Moore. 1998. Neutralization sensitivity of human immunodeficiency virus type 1 primary isolates to antibodies and CD4- based reagents is independent of coreceptor usage. J Virol 72:1876-1885. 458. Ugolini, S., I. Mondor, P. W. Parren, D. R. Burton, S. A. Tilley, P. J. Klasse, and Q. J. Sattentau. 1997. Inhibition of virus attachment to CD4+ target cells is a major mechanism of T cell line-adapted HIV-1 neutralization. J Exp Med 186:1287-1298. 459. Xiang, S. H., P. D. Kwong, R. Gupta, C. D. Rizzuto, D. J. Casper, R. Wyatt, L. Wang, W. A. Hendrickson, M. L. Doyle, and J. Sodroski. 2002. Mutagenic stabilization and/or disruption of a CD4-bound state reveals distinct conformations of the human immunodeficiency virus type 1 gp120 envelope glycoprotein. J Virol 76:9888-9899. 460. Trkola, A., A. B. Pomales, H. Yuan, B. Korber, P. J. Maddon, G. P. Allaway, H. Katinger, C. F. Barbas, 3rd, D. R. Burton, D. D. Ho, and et al. 1995. Cross-clade neutralization of primary isolates of human immunodeficiency virus type 1 by human monoclonal antibodies and tetrameric CD4-IgG. J Virol 69:6609-6617. 461. Ruprecht, R. M., F. Ferrantelli, M. Kitabwalla, W. Xu, and H. M. McClure. 2003. Antibody protection: passive immunization of neonates against oral AIDS virus challenge. Vaccine 21:3370-3373. 462. Mantis, N. J., J. Palaia, A. J. Hessell, S. Mehta, Z. Zhu, B. Corthesy, M. R. Neutra, D. R. Burton, and E. N. Janoff. 2007. Inhibition of HIV-1 infectivity and epithelial cell transfer by human monoclonal IgG and IgA antibodies carrying the b12 V region. J Immunol 179:3144-3152. 463. van Montfort, T., A. A. Nabatov, T. B. Geijtenbeek, G. Pollakis, and W. A. Paxton. 2007. Efficient capture of antibody neutralized HIV-1 by cells expressing DC-SIGN and transfer to CD4+ T lymphocytes. J Immunol 178:3177-3185. 464. Mo, H., L. Stamatatos, J. E. Ip, C. F. Barbas, P. W. Parren, D. R. Burton, J. P. Moore, and D. D. Ho. 1997. Human immunodeficiency virus type 1 mutants that escape neutralization by human monoclonal antibody IgG1b12. off. J Virol 71:6869-6874. 465. Thali, M., C. Furman, D. D. Ho, J. Robinson, S. Tilley, A. Pinter, and J. Sodroski. 1992. Discontinuous, conserved neutralization epitopes overlapping the CD4-

250 binding region of human immunodeficiency virus type 1 gp120 envelope glycoprotein. J Virol 66:5635-5641. 466. Pantophlet, R., E. Ollmann Saphire, P. Poignard, P. W. Parren, I. A. Wilson, and D. R. Burton. 2003. Fine mapping of the interaction of neutralizing and nonneutralizing monoclonal antibodies with the CD4 binding site of human immunodeficiency virus type 1 gp120. J Virol 77:642-658. 467. Saphire, E. O., P. W. Parren, C. F. Barbas, 3rd, D. R. Burton, and I. A. Wilson. 2001. Crystallization and preliminary structure determination of an intact human immunoglobulin, b12: an antibody that broadly neutralizes primary isolates of HIV-1. Acta Crystallogr D Biol Crystallogr 57:168-171. 468. Saphire, E. O., P. W. Parren, R. Pantophlet, M. B. Zwick, G. M. Morris, P. M. Rudd, R. A. Dwek, R. L. Stanfield, D. R. Burton, and I. A. Wilson. 2001. Crystal structure of a neutralizing human IGG against HIV-1: a template for vaccine design. Science 293:1155-1159. 469. Herrera, C., C. Spenlehauer, M. S. Fung, D. R. Burton, S. Beddows, and J. P. Moore. 2003. Nonneutralizing antibodies to the CD4-binding site on the gp120 subunit of human immunodeficiency virus type 1 do not interfere with the activity of a neutralizing antibody against the same site. J Virol 77:1084-1091. 470. Zhou, T., I. Georgiev, X. Wu, Z. Y. Yang, K. Dai, A. Finzi, Y. Do Kwon, J. Scheid, W. Shi, L. Xu, Y. Yang, J. Zhu, M. C. Nussenzweig, J. Sodroski, L. Shapiro, G. J. Nabel, J. R. Mascola, and P. D. Kwong. 2010. Structural Basis for Broad and Potent Neutralization of HIV-1 by Antibody VRC01. Science. 471. Wyatt, R., J. Moore, M. Accola, E. Desjardin, J. Robinson, and J. Sodroski. 1995. Involvement of the V1/V2 variable loop structure in the exposure of human immunodeficiency virus type 1 gp120 epitopes induced by receptor binding. J Virol 69:5723-5733. 472. Stamatatos, L., and C. Cheng-Mayer. 1995. Structural modulations of the envelope gp120 glycoprotein of human immunodeficiency virus type 1 upon oligomerization and differential V3 loop epitope exposure of isolates displaying distinct tropism upon virion-soluble receptor binding. J Virol 69:6191-6198. 473. Sattentau, Q. J., J. P. Moore, F. Vignaux, F. Traincard, and P. Poignard. 1993. Conformational changes induced in the envelope glycoproteins of the human and simian immunodeficiency viruses by soluble receptor binding. J Virol 67:7383- 7393. 474. Moore, J. P., Q. J. Sattentau, R. Wyatt, and J. Sodroski. 1994. Probing the structure of the human immunodeficiency virus surface glycoprotein gp120 with a panel of monoclonal antibodies. J Virol 68:469-484. 475. Mbah, H. A., S. Burda, M. K. Gorny, C. Williams, K. Revesz, S. Zolla-Pazner, and P. N. Nyambi. 2001. Effect of soluble CD4 on exposure of epitopes on primary, intact, native human immunodeficiency virus type 1 virions of different genetic clades. J Virol 75:7785-7788. 476. Richardson, N. E., N. R. Brown, R. E. Hussey, A. Vaid, T. J. Matthews, D. P. Bolognesi, and E. L. Reinherz. 1988. Binding site for human immunodeficiency virus coat protein gp120 is located in the NH2-terminal region of T4 (CD4) and requires the intact variable-region-like domain. Proc Natl Acad Sci U S A 85:6102-6106.

251 477. Jameson, B. A., P. E. Rao, L. I. Kong, B. H. Hahn, G. M. Shaw, L. E. Hood, and S. B. Kent. 1988. Location and chemical synthesis of a binding site for HIV-1 on the CD4 protein. Science 240:1335-1339. 478. Pantophlet, R., I. A. Wilson, and D. R. Burton. 2003. Hyperglycosylated mutants of human immunodeficiency virus (HIV) type 1 monomeric gp120 as novel antigens for HIV vaccine design. J Virol 77:5889-5901. 479. Pantophlet, R., and D. R. Burton. 2003. Immunofocusing: antigen engineering to promote the induction of HIV-neutralizing antibodies. Trends Mol Med 9:468- 473. 480. Beaumont, T., E. Quakkelaar, A. van Nuenen, R. Pantophlet, and H. Schuitemaker. 2004. Increased sensitivity to CD4 binding site-directed neutralization following in vitro propagation on primary lymphocytes of a neutralization-resistant human immunodeficiency virus IIIB strain isolated from an accidentally infected laboratory worker. J Virol 78:5651-5657. 481. Pantophlet, R., I. A. Wilson, and D. R. Burton. 2004. Improved design of an antigen with enhanced specificity for the broadly HIV-neutralizing antibody b12. Protein Eng Des Sel 17:749-758. 482. Pinter, A., W. J. Honnen, Y. He, M. K. Gorny, S. Zolla-Pazner, and S. C. Kayman. 2004. The V1/V2 domain of gp120 is a global regulator of the sensitivity of primary human immunodeficiency virus type 1 isolates to neutralization by antibodies commonly induced upon infection. J Virol 78:5205- 5215. 483. Zwick, M. B., R. Kelleher, R. Jensen, A. F. Labrijn, M. Wang, G. V. Quinnan, Jr., P. W. Parren, and D. R. Burton. 2003. A novel human antibody against human immunodeficiency virus type 1 gp120 is V1, V2, and V3 loop dependent and helps delimit the epitope of the broadly neutralizing antibody immunoglobulin G1 b12. J Virol 77:6965-6978. 484. Zwick, M. B., P. W. Parren, E. O. Saphire, S. Church, M. Wang, J. K. Scott, P. E. Dawson, I. A. Wilson, and D. R. Burton. 2003. Molecular features of the broadly neutralizing immunoglobulin G1 b12 required for recognition of human immunodeficiency virus type 1 gp120. J Virol 77:5863-5876. 485. Ashish, A. K. Solanki, C. D. Boone, and J. K. Krueger. 2010. Global structure of HIV-1 neutralizing antibody IgG1 b12 is asymmetric. Biochem Biophys Res Commun 391:947-951. 486. Pietzsch, J., J. F. Scheid, H. Mouquet, F. Klein, M. S. Seaman, M. Jankovic, D. Corti, A. Lanzavecchia, and M. C. Nussenzweig. 2010. Human anti-HIV- neutralizing antibodies frequently target a conserved epitope essential for viral fitness. J Exp Med 207:1995-2002. 487. Corti, D., J. P. Langedijk, A. Hinz, M. S. Seaman, F. Vanzetta, B. M. Fernandez- Rodriguez, C. Silacci, D. Pinna, D. Jarrossay, S. Balla-Jhagjhoorsingh, B. Willems, M. J. Zekveld, H. Dreja, E. O'Sullivan, C. Pade, C. Orkin, S. A. Jeffs, D. C. Montefiori, D. Davis, W. Weissenhorn, A. McKnight, J. L. Heeney, F. Sallusto, Q. J. Sattentau, R. A. Weiss, and A. Lanzavecchia. 2010. Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-infected individuals. PLoS One 5:e8805.

252 488. Wu, X., Z. Y. Yang, Y. Li, C. M. Hogerkorp, W. R. Schief, M. S. Seaman, T. Zhou, S. D. Schmidt, L. Wu, L. Xu, N. S. Longo, K. McKee, S. O'Dell, M. K. Louder, D. L. Wycuff, Y. Feng, M. Nason, N. Doria-Rose, M. Connors, P. D. Kwong, M. Roederer, R. T. Wyatt, G. J. Nabel, and J. R. Mascola. 2010. Rational Design of Envelope Identifies Broadly Neutralizing Human Monoclonal Antibodies to HIV-1. Science. 489. Wu, X., T. Zhou, S. O'Dell, R. T. Wyatt, P. D. Kwong, and J. R. Mascola. 2009. Mechanism of human immunodeficiency virus type 1 resistance to monoclonal antibody B12 that effectively targets the site of CD4 attachment. J Virol 83:10892-10907. 490. Leaman, D. P., H. Kinkead, and M. B. Zwick. 2010. In-solution virus capture assay helps deconstruct heterogeneous antibody recognition of human immunodeficiency virus type 1. J Virol 84:3382-3395. 491. Parren, P. W., P. Fisicaro, A. F. Labrijn, J. M. Binley, W. P. Yang, H. J. Ditzel, C. F. Barbas, 3rd, and D. R. Burton. 1996. In vitro antigen challenge of human antibody libraries for vaccine evaluation: the human immunodeficiency virus type 1 envelope. J Virol 70:9046-9050. 492. Poignard, P., M. Moulard, E. Golez, V. Vivona, M. Franti, S. Venturini, M. Wang, P. W. Parren, and D. R. Burton. 2003. Heterogeneity of envelope molecules expressed on primary human immunodeficiency virus type 1 particles as probed by the binding of neutralizing and nonneutralizing antibodies. J Virol 77:353-365. 493. Li, Y., S. A. Migueles, B. Welcher, K. Svehla, A. Phogat, M. K. Louder, X. Wu, G. M. Shaw, M. Connors, R. T. Wyatt, and J. R. Mascola. 2007. Broad HIV-1 neutralization mediated by CD4-binding site antibodies. Nat Med 13:1032-1034. 494. Sanders, R. W., M. Venturi, L. Schiffner, R. Kalyanaraman, H. Katinger, K. O. Lloyd, P. D. Kwong, and J. P. Moore. 2002. The mannose-dependent epitope for neutralizing antibody 2G12 on human immunodeficiency virus type 1 glycoprotein gp120. J Virol 76:7293-7305. 495. Gram, G. J., A. Hemming, A. Bolmstedt, B. Jansson, S. Olofsson, L. Akerblom, J. O. Nielsen, and J. E. Hansen. 1994. Identification of an N-linked glycan in the V1-loop of HIV-1 gp120 influencing neutralization by anti-V3 antibodies and soluble CD4. Arch Virol 139:253-261. 496. Trkola, A., M. Purtscher, T. Muster, C. Ballaun, A. Buchacher, N. Sullivan, K. Srinivasan, J. Sodroski, J. P. Moore, and H. Katinger. 1996. Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1. J Virol 70:1100-1108. 497. Armbruster, C., G. M. Stiegler, B. A. Vcelar, W. Jager, N. L. Michael, N. Vetter, and H. W. Katinger. 2002. A phase I trial with two human monoclonal antibodies (hMAb 2F5, 2G12) against HIV-1. Aids 16:227-233. 498. Astronomo, R. D., H. K. Lee, C. N. Scanlan, R. Pantophlet, C. Y. Huang, I. A. Wilson, O. Blixt, R. A. Dwek, C. H. Wong, and D. R. Burton. 2008. A glycoconjugate antigen based on the recognition motif of a broadly neutralizing human immunodeficiency virus antibody, 2G12, is immunogenic but elicits antibodies unable to bind to the self glycans of gp120. J Virol 82:6359-6368.

253 499. Luallen, R. J., J. Lin, H. Fu, K. K. Cai, C. Agrawal, I. Mboudjeka, F. H. Lee, D. Montefiori, D. F. Smith, R. W. Doms, and Y. Geng. 2008. An engineered Saccharomyces cerevisiae strain binds the broadly neutralizing human immunodeficiency virus type 1 antibody 2G12 and elicits mannose-specific gp120-binding antibodies. J Virol 82:6447-6457. 500. Doores, K. J., Z. Fulton, M. Huber, I. A. Wilson, and D. R. Burton. 2010. Antibody 2G12 Recognizes Di-Mannose Equivalently in Domain- and Non- Domain-Exchanged Forms, but only binds the HIV-1 Glycan Shield if Domain- Exchanged. J Virol. 501. Calarese, D. A., C. N. Scanlan, M. B. Zwick, S. Deechongkit, Y. Mimura, R. Kunert, P. Zhu, M. R. Wormald, R. L. Stanfield, K. H. Roux, J. W. Kelly, P. M. Rudd, R. A. Dwek, H. Katinger, D. R. Burton, and I. A. Wilson. 2003. Antibody domain exchange is an immunological solution to carbohydrate cluster recognition. Science 300:2065-2071. 502. Muster, T., F. Steindl, M. Purtscher, A. Trkola, A. Klima, G. Himmler, F. Ruker, and H. Katinger. 1993. A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1. J Virol 67:6642-6647. 503. Zwick, M. B., R. Jensen, S. Church, M. Wang, G. Stiegler, R. Kunert, H. Katinger, and D. R. Burton. 2005. Anti-human immunodeficiency virus type 1 (HIV-1) antibodies 2F5 and 4E10 require surprisingly few crucial residues in the membrane-proximal external region of glycoprotein gp41 to neutralize HIV-1. J Virol 79:1252-1261. 504. Alam, S. M., M. Morelli, S. M. Dennison, H. X. Liao, R. Zhang, S. M. Xia, S. Rits-Volloch, L. Sun, S. C. Harrison, B. F. Haynes, and B. Chen. 2009. Role of HIV membrane in neutralization by two broadly neutralizing antibodies. Proc Natl Acad Sci U S A 106:20234-20239. 505. Sanchez-Martinez, S., M. Lorizate, H. Katinger, R. Kunert, and J. L. Nieva. 2006. Membrane association and epitope recognition by HIV-1 neutralizing anti-gp41 2F5 and 4E10 antibodies. AIDS Res Hum Retroviruses 22:998-1006. 506. Matyas, G. R., L. Wieczorek, Z. Beck, C. Ochsenbauer-Jambor, J. C. Kappes, N. L. Michael, V. R. Polonis, and C. R. Alving. 2009. Neutralizing antibodies induced by liposomal HIV-1 glycoprotein 41 peptide simultaneously bind to both the 2F5 or 4E10 epitope and lipid epitopes. Aids 23:2069-2077. 507. Xu, H., L. Song, M. Kim, M. A. Holmes, Z. Kraft, G. Sellhorn, E. L. Reinherz, L. Stamatatos, and R. K. Strong. 2009. Interactions between lipids and human anti- HIV antibody 4E10 can be reduced without ablating neutralizing activity. J Virol 84:1076-1088. 508. Ofek, G., K. McKee, Y. Yang, Z. Y. Yang, J. Skinner, F. J. Guenaga, R. Wyatt, M. B. Zwick, G. J. Nabel, J. R. Mascola, and P. D. Kwong. 2009. Relationship between antibody 2F5 neutralization of HIV-1 and hydrophobicity of its heavy chain third complementarity-determining region. J Virol 84:2955-2962. 509. Dennison, S. M., S. M. Stewart, K. C. Stempel, H. X. Liao, B. F. Haynes, and S. M. Alam. 2009. Stable Docking of Neutralizing Hiv-1 Gp41 Membrane Proximal External Region Monoclonal Antibodies 2f5 and 4e10 Is Dependent on the Membrane Immersion Depth of Their Epitope Regions. J Virol.

254 510. Parker, C. E., L. J. Deterding, C. Hager-Braun, J. M. Binley, N. Schulke, H. Katinger, J. P. Moore, and K. B. Tomer. 2001. Fine definition of the epitope on the gp41 glycoprotein of human immunodeficiency virus type 1 for the neutralizing monoclonal antibody 2F5. J Virol 75:10906-10911. 511. Cardoso, R. M., F. M. Brunel, S. Ferguson, M. Zwick, D. R. Burton, P. E. Dawson, and I. A. Wilson. 2007. Structural basis of enhanced binding of extended and helically constrained peptide epitopes of the broadly neutralizing HIV-1 antibody 4E10. J Mol Biol 365:1533-1544. 512. Nelson, J. D., F. M. Brunel, R. Jensen, E. T. Crooks, R. M. Cardoso, M. Wang, A. Hessell, I. A. Wilson, J. M. Binley, P. E. Dawson, D. R. Burton, and M. B. Zwick. 2007. An affinity-enhanced neutralizing antibody against the membrane-proximal external region of human immunodeficiency virus type 1 gp41 recognizes an epitope between those of 2F5 and 4E10. J Virol 81:4033-4043. 513. Crooks, E. T., P. L. Moore, D. Richman, J. Robinson, J. A. Crooks, M. Franti, N. Schulke, and J. M. Binley. 2005. Characterizing anti-HIV monoclonal antibodies and immune sera by defining the mechanism of neutralization. Hum Antibodies 14:101-113. 514. Gray, E. S., N. Taylor, D. Wycuff, P. L. Moore, G. D. Tomaras, C. K. Wibmer, A. Puren, A. DeCamp, P. B. Gilbert, B. Wood, D. C. Montefiori, J. M. Binley, G. M. Shaw, B. F. Haynes, J. R. Mascola, and L. Morris. 2009. Antibody specificities associated with neutralization breadth in plasma from human immunodeficiency virus type 1 subtype C-infected blood donors. J Virol 83:8925-8937. 515. Gray, E. S., M. C. Madiga, P. L. Moore, K. Mlisana, S. S. Abdool Karim, J. M. Binley, G. M. Shaw, J. R. Mascola, and L. Morris. 2009. Broad neutralization of human immunodeficiency virus type 1 mediated by plasma antibodies against the gp41 membrane proximal external region. J Virol 83:11265-11274. 516. Haynes, B. F., J. Fleming, E. W. St Clair, H. Katinger, G. Stiegler, R. Kunert, J. Robinson, R. M. Scearce, K. Plonk, H. F. Staats, T. L. Ortel, H. X. Liao, and S. M. Alam. 2005. Cardiolipin polyspecific autoreactivity in two broadly neutralizing HIV-1 antibodies. Science 308:1906-1908. 517. Scherer, E. M., M. B. Zwick, L. Teyton, and D. R. Burton. 2007. Difficulties in eliciting broadly neutralizing anti-HIV antibodies are not explained by cardiolipin autoreactivity. Aids 21:2131-2139. 518. Nabel, G. J. 2005. Immunology. Close to the edge: neutralizing the HIV-1 envelope. Science 308:1878-1879. 519. Xiang, S. H., L. Wang, M. Abreu, C. C. Huang, P. D. Kwong, E. Rosenberg, J. E. Robinson, and J. Sodroski. 2003. Epitope mapping and characterization of a novel CD4-induced human monoclonal antibody capable of neutralizing primary HIV-1 strains. Virology 315:124-134. 520. Labrijn, A. F., P. Poignard, A. Raja, M. B. Zwick, K. Delgado, M. Franti, J. Binley, V. Vivona, C. Grundner, C. C. Huang, M. Venturi, C. J. Petropoulos, T. Wrin, D. S. Dimitrov, J. Robinson, P. D. Kwong, R. T. Wyatt, J. Sodroski, and D. R. Burton. 2003. Access of antibody molecules to the conserved coreceptor binding site on glycoprotein gp120 is sterically restricted on primary human immunodeficiency virus type 1. J Virol 77:10557-10565.

255 521. Gustchina, E., J. M. Louis, C. Frisch, F. Ylera, A. Lechner, C. A. Bewley, and G. M. Clore. 2009. Affinity maturation by targeted diversification of the CDR-H2 loop of a monoclonal Fab derived from a synthetic naive human antibody library and directed against the internal trimeric coiled-coil of gp41 yields a set of Fabs with improved HIV-1 neutralization potency and breadth. Virology 393:112-119. 522. Salzwedel, K., E. D. Smith, B. Dey, and E. A. Berger. 2000. Sequential CD4- coreceptor interactions in human immunodeficiency virus type 1 Env function: soluble CD4 activates Env for coreceptor-dependent fusion and reveals blocking activities of antibodies against cryptic conserved epitopes on gp120. J Virol 74:326-333. 523. Decker, J. M., F. Bibollet-Ruche, X. Wei, S. Wang, D. N. Levy, W. Wang, E. Delaporte, M. Peeters, C. A. Derdeyn, S. Allen, E. Hunter, M. S. Saag, J. A. Hoxie, B. H. Hahn, P. D. Kwong, J. E. Robinson, and G. M. Shaw. 2005. Antigenic conservation and immunogenicity of the HIV coreceptor binding site. J Exp Med 201:1407-1419. 524. Gorny, M. K., L. Stamatatos, B. Volsky, K. Revesz, C. Williams, X. H. Wang, S. Cohen, R. Staudinger, and S. Zolla-Pazner. 2005. Identification of a new quaternary neutralizing epitope on human immunodeficiency virus type 1 virus particles. J Virol 79:5232-5237. 525. Pejchal, R., L. M. Walker, R. L. Stanfield, S. K. Phogat, W. C. Koff, P. Poignard, D. R. Burton, and I. A. Wilson. 2010. Structure and function of broadly reactive antibody PG16 reveal an H3 subdomain that mediates potent neutralization of HIV-1. Proc Natl Acad Sci U S A 107:11483-11488. 526. Moore, P. L., E. S. Gray, D. Sheward, M. Madiga, N. Ranchobe, Z. Lai, W. J. Honnen, M. Nonyane, N. Tumba, T. Hermanus, S. Sibeko, K. Mlisana, S. S. Abdool Karim, C. Williamson, A. Pinter, and L. Morris. 2011. Potent and broad neutralization of HIV-1 subtype C viruses by plasma antibodies targeting a quaternary epitope including residues in the V2 loop. J Virol. 527. Fung, M. S., C. R. Sun, W. L. Gordon, R. S. Liou, T. W. Chang, W. N. Sun, E. S. Daar, and D. D. Ho. 1992. Identification and characterization of a neutralization site within the second variable region of human immunodeficiency virus type 1 gp120. J Virol 66:848-856. 528. Rusche, J. R., K. Javaherian, C. McDanal, J. Petro, D. L. Lynn, R. Grimaila, A. Langlois, R. C. Gallo, L. O. Arthur, P. J. Fischinger, and et al. 1988. Antibodies that inhibit fusion of human immunodeficiency virus-infected cells bind a 24- amino acid sequence of the viral envelope, gp120. Proc Natl Acad Sci U S A 85:3198-3202. 529. Gorny, M. K., A. J. Conley, S. Karwowska, A. Buchbinder, J. Y. Xu, E. A. Emini, S. Koenig, and S. Zolla-Pazner. 1992. Neutralization of diverse human immunodeficiency virus type 1 variants by an anti-V3 human monoclonal antibody. J Virol 66:7538-7542. 530. Gunthard, H. F., P. L. Gowland, J. Schupbach, M. S. Fung, J. Boni, R. S. Liou, N. T. Chang, P. Grob, P. Graepel, D. G. Braun, and et al. 1994. A phase I/IIA clinical study with a chimeric mouse-human monoclonal antibody to the V3 loop of human immunodeficiency virus type 1 gp120. J Infect Dis 170:1384-1393.

256 531. Kwong, P. D. 2004. The 447-52D antibody: hitting HIV-1 where its armor is thickest. Structure 12:173-174. 532. Burton, D. R., R. C. Desrosiers, R. W. Doms, W. C. Koff, P. D. Kwong, J. P. Moore, G. J. Nabel, J. Sodroski, I. A. Wilson, and R. T. Wyatt. 2004. HIV vaccine design and the neutralizing antibody problem. Nat Immunol 5:233-236. 533. Korber, B., B. Gaschen, K. Yusim, R. Thakallapally, C. Kesmir, and V. Detours. 2001. Evolutionary and immunological implications of contemporary HIV-1 variation. Br Med Bull 58:19-42. 534. Canducci, F., M. Sampaolo, M. C. Marinozzi, E. Boeri, V. Spagnuolo, A. Galli, A. Castagna, A. Lazzarin, M. Clementi, and N. Gianotti. 2009. Dynamic patterns of human immunodeficiency virus type 1 integrase gene evolution in patients failing raltegravir-based salvage therapies. Aids 23:455-460. 535. Li, H., C. F. Xu, S. Blais, Q. Wan, H. T. Zhang, S. J. Landry, and C. E. Hioe. 2009. Proximal glycans outside of the epitopes regulate the presentation of HIV-1 envelope gp120 helper epitopes. J Immunol 182:6369-6378. 536. Zwick, M. B., and D. R. Burton. 2007. HIV-1 neutralization: mechanisms and relevance to vaccine design. Curr HIV Res 5:608-624. 537. Yuan, W., J. Bazick, and J. Sodroski. 2006. Characterization of the multiple conformational States of free monomeric and trimeric human immunodeficiency virus envelope glycoproteins after fixation by cross-linker. J Virol 80:6725-6737. 538. Moore, J. P., Y. Cao, L. Qing, Q. J. Sattentau, J. Pyati, R. Koduri, J. Robinson, C. F. Barbas, 3rd, D. R. Burton, and D. D. Ho. 1995. Primary isolates of human immunodeficiency virus type 1 are relatively resistant to neutralization by monoclonal antibodies to gp120, and their neutralization is not predicted by studies with monomeric gp120. J Virol 69:101-109. 539. Dalgleish, A. G., J. B. Marriott, B. Souberbielle, C. Montesano, J. Sheikh, D. Sidebottom, M. Westby, and B. Austen. 1999. The role of HIV gp120 in the disruption of the immune system. Immunol Lett 66:81-87. 540. Plotkin, S. A. 2008. Vaccines: correlates of vaccine-induced immunity. Clin Infect Dis 47:401-409. 541. Lambert, P. H., M. Liu, and C. A. Siegrist. 2005. Can successful vaccines teach us how to induce efficient protective immune responses? Nat Med 11:S54-62. 542. Edghill-Smith, Y., H. Golding, J. Manischewitz, L. R. King, D. Scott, M. Bray, A. Nalca, J. W. Hooper, C. A. Whitehouse, J. E. Schmitz, K. A. Reimann, and G. Franchini. 2005. Smallpox vaccine-induced antibodies are necessary and sufficient for protection against monkeypox virus. Nat Med 11:740-747. 543. Roukens, A. H., and L. G. Visser. 2008. Yellow fever vaccine: past, present and future. Expert Opin Biol Ther 8:1787-1795. 544. Co, M. D., E. D. Kilpatrick, and A. L. Rothman. 2009. Dynamics of the CD8 T- cell response following yellow fever virus 17D immunization. Immunology 128:e718-727. 545. Tajiri, K., T. Ozawa, A. Jin, Y. Tokimitsu, M. Minemura, H. Kishi, T. Sugiyama, and A. Muraguchi. 2010. Analysis of the epitope and neutralizing capacity of human monoclonal antibodies induced by hepatitis B vaccine. Antiviral Res 87:40-49.

257 546. Kemp, T. J., A. Hildesheim, M. Safaeian, J. G. Dauner, Y. Pan, C. Porras, J. T. Schiller, D. R. Lowy, R. Herrero, and L. A. Pinto. 2011. HPV16/18 L1 VLP vaccine induces cross-neutralizing antibodies that may mediate cross-protection. Vaccine. 547. Lee, S. A., R. Orque, P. A. Escarpe, M. L. Peterson, J. W. Good, E. M. Zaharias, P. W. Berman, H. W. Sheppard, and R. Shibata. 2001. Vaccine-induced antibodies to the native, oligomeric envelope glycoproteins of primary HIV-1 isolates. Vaccine 20:563-576. 548. Flynn, N. M., D. N. Forthal, C. D. Harro, F. N. Judson, K. H. Mayer, and M. F. Para. 2005. Placebo-controlled phase 3 trial of a recombinant glycoprotein 120 vaccine to prevent HIV-1 infection. J Infect Dis 191:654-665. 549. Pitisuttithum, P., P. Gilbert, M. Gurwith, W. Heyward, M. Martin, F. van Griensven, D. Hu, J. W. Tappero, and K. Choopanya. 2006. Randomized, double- blind, placebo-controlled efficacy trial of a bivalent recombinant glycoprotein 120 HIV-1 vaccine among injection drug users in Bangkok, Thailand. J Infect Dis 194:1661-1671. 550. Graham, B. S., and J. R. Mascola. 2005. Lessons from failure--preparing for future HIV-1 vaccine efficacy trials. J Infect Dis 191:647-649. 551. Silbermann, B., M. Tod, C. Desaint, G. Pialoux, K. Petitprez, L. Slama, H. Poncelet, C. Moreau, V. Mazarin, F. Heshmati, D. Salmon-Ceron, J. G. Guillet, and O. Launay. 2008. Long-Term Persistence of Vaccine-Induced HIV Seropositivity among Healthy Volunteers. AIDS Res Hum Retroviruses. 552. Vaine, M., S. Wang, Q. Liu, J. Arthos, D. Montefiori, P. Goepfert, M. J. McElrath, and S. Lu. 2010. Profiles of human serum antibody responses elicited by three leading HIV vaccines focusing on the induction of Env-specific antibodies. PLoS One 5:e13916. 553. Wang, S., J. S. Kennedy, K. West, D. C. Montefiori, S. Coley, J. Lawrence, S. Shen, S. Green, A. L. Rothman, F. A. Ennis, J. Arthos, R. Pal, P. Markham, and S. Lu. 2008. Cross-subtype antibody and cellular immune responses induced by a polyvalent DNA prime-protein boost HIV-1 vaccine in healthy human volunteers. Vaccine 26:1098-1110. 554. Hanke, T. 2008. STEP trial and HIV-1 vaccines inducing T-cell responses. Expert Rev Vaccines 7:303-309. 555. Gaucher, D., R. Therrien, N. Kettaf, B. R. Angermann, G. Boucher, A. Filali- Mouhim, J. M. Moser, R. S. Mehta, D. R. Drake, 3rd, E. Castro, R. Akondy, A. Rinfret, B. Yassine-Diab, E. A. Said, Y. Chouikh, M. J. Cameron, R. Clum, D. Kelvin, R. Somogyi, L. D. Greller, R. S. Balderas, P. Wilkinson, G. Pantaleo, J. Tartaglia, E. K. Haddad, and R. P. Sekaly. 2008. Yellow fever vaccine induces integrated multilineage and polyfunctional immune responses. J Exp Med 205:3119-3131. 556. Querec, T. D., R. S. Akondy, E. K. Lee, W. Cao, H. I. Nakaya, D. Teuwen, A. Pirani, K. Gernert, J. Deng, B. Marzolf, K. Kennedy, H. Wu, S. Bennouna, H. Oluoch, J. Miller, R. Z. Vencio, M. Mulligan, A. Aderem, R. Ahmed, and B. Pulendran. 2009. Systems biology approach predicts immunogenicity of the yellow fever vaccine in humans. Nat Immunol 10:116-125.

258 557. Miller, J. D., R. G. van der Most, R. S. Akondy, J. T. Glidewell, S. Albott, D. Masopust, K. Murali-Krishna, P. L. Mahar, S. Edupuganti, S. Lalor, S. Germon, C. Del Rio, M. J. Mulligan, S. I. Staprans, J. D. Altman, M. B. Feinberg, and R. Ahmed. 2008. Human effector and memory CD8+ T cell responses to smallpox and yellow fever vaccines. Immunity 28:710-722. 558. Buchbinder, S. P., D. V. Mehrotra, A. Duerr, D. W. Fitzgerald, R. Mogg, D. Li, P. B. Gilbert, J. R. Lama, M. Marmor, C. Del Rio, M. J. McElrath, D. R. Casimiro, K. M. Gottesdiener, J. A. Chodakewitz, L. Corey, and M. N. Robertson. 2008. Efficacy assessment of a cell-mediated immunity HIV-1 vaccine (the Step Study): a double-blind, randomised, placebo-controlled, test-of-concept trial. Lancet 372:1881-1893. 559. Cranage, M. P., C. A. Fraser, Z. Stevens, J. Huting, M. Chang, S. A. Jeffs, M. S. Seaman, A. Cope, T. Cole, and R. J. Shattock. 2010. Repeated vaginal administration of trimeric HIV-1 clade C gp140 induces serum and mucosal antibody responses. Mucosal Immunol 3:57-68. 560. Ching, L., and L. Stamatatos. 2010. Alterations in the immunogenic properties of soluble trimeric human immunodeficiency virus type 1 envelope proteins induced by deletion or heterologous substitutions of the V1 loop. J Virol 84:9932-9946. 561. Wu, L., T. Zhou, Z. Y. Yang, K. Svehla, S. O'Dell, M. K. Louder, L. Xu, J. R. Mascola, D. R. Burton, J. A. Hoxie, R. W. Doms, P. D. Kwong, and G. J. Nabel. 2009. Enhanced exposure of the CD4-binding site to neutralizing antibodies by structural design of a membrane-anchored human immunodeficiency virus type 1 gp120 domain. J Virol 83:5077-5086. 562. Selvarajah, S., B. Puffer, R. Pantophlet, M. Law, R. W. Doms, and D. R. Burton. 2005. Comparing antigenicity and immunogenicity of engineered gp120. J Virol 79:12148-12163. 563. Emileh, A., and C. F. Abrams. 2011. A mechanism by which binding of the broadly neutralizing antibody b12 unfolds the inner domain alpha1 helix in an engineered HIV-1 gp120. Proteins 79:537-546. 564. Bowley, D. R., A. F. Labrijn, M. B. Zwick, and D. R. Burton. 2007. Antigen selection from an HIV-1 immune antibody library displayed on yeast yields many novel antibodies compared to selection from the same library displayed on phage. Protein Eng Des Sel 20:81-90. 565. Berry, J. D., J. Rutherford, G. J. Silverman, R. Kaul, M. Elia, S. Gobuty, R. Fuller, F. A. Plummer, and C. F. Barbas, 3rd. 2003. Development of functional human monoclonal single-chain variable fragment antibody against HIV-1 from human cervical B cells. Hybrid Hybridomics 22:97-108. 566. Zhang, M. Y., Y. Shu, S. Phogat, X. Xiao, F. Cham, P. Bouma, A. Choudhary, Y. R. Feng, I. Sanz, S. Rybak, C. C. Broder, G. V. Quinnan, T. Evans, and D. S. Dimitrov. 2003. Broadly cross-reactive HIV neutralizing human monoclonal antibody Fab selected by sequential antigen panning of a phage display library. J Immunol Methods 283:17-25. 567. Goodglick, L., N. Zevit, M. S. Neshat, and J. Braun. 1995. Mapping the Ig superantigen-binding site of HIV-1 gp120. J Immunol 155:5151-5159. 568. Raska, C. S., C. E. Parker, S. W. Sunnarborg, R. M. Pope, D. C. Lee, G. L. Glish, and C. H. Borchers. 2003. Rapid and sensitive identification of epitope-containing

259 peptides by direct matrix-assisted laser desorption/ionization tandem mass spectrometry of peptides affinity-bound to antibody beads. J Am Soc Mass Spectrom 14:1076-1085. 569. Papac, D. I., J. Hoyes, and K. B. Tomer. 1994. Epitope mapping of the gastrin- releasing peptide/anti-bombesin monoclonal antibody complex by proteolysis followed by matrix-assisted laser desorption ionization mass spectrometry. Protein Sci 3:1485-1492. 570. Parker, C. E., D. I. Papac, S. K. Trojak, and K. B. Tomer. 1996. Epitope mapping by mass spectrometry: determination of an epitope on HIV-1 IIIB p26 recognized by a monoclonal antibody. J Immunol 157:198-206. 571. Jeyarajah, S., C. E. Parker, M. T. Summer, and K. B. Tomer. 1998. Matrix- assisted laser desorption ionization/mass spectrometry mapping of human immunodeficiency virus-gp120 epitopes recognized by a limited polyclonal antibody. J Am Soc Mass Spectrom 9:157-165. 572. Hochleitner, E. O., M. K. Gorny, S. Zolla-Pazner, and K. B. Tomer. 2000. Mass spectrometric characterization of a discontinuous epitope of the HIV envelope protein HIV-gp120 recognized by the human monoclonal antibody 1331A. J Immunol 164:4156-4161. 573. Parker, C. E., and K. B. Tomer. 2000. Epitope mapping by a combination of epitope excision and MALDI-MS. Methods Mol Biol 146:185-201. 574. Burton, D. R., J. Pyati, R. Koduri, S. J. Sharp, G. B. Thornton, P. W. Parren, L. S. Sawyer, R. M. Hendry, N. Dunlop, P. L. Nara, and et al. 1994. Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody. Science 266:1024-1027. 575. Parren, P. W., T. W. Geisbert, T. Maruyama, P. B. Jahrling, and D. R. Burton. 2002. Pre- and postexposure prophylaxis of Ebola virus infection in an animal model by passive transfer of a neutralizing human antibody. J Virol 76:6408- 6412. 576. Maruyama, T., L. L. Rodriguez, P. B. Jahrling, A. Sanchez, A. S. Khan, S. T. Nichol, C. J. Peters, P. W. Parren, and D. R. Burton. 1999. Ebola virus can be effectively neutralized by antibody produced in natural human infection. J Virol 73:6024-6030. 577. Posner, M. R., T. Hideshima, T. Cannon, M. Mukherjee, K. H. Mayer, and R. A. Byrn. 1991. An IgG human monoclonal antibody that reacts with HIV-1/GP120, inhibits virus binding to cells, and neutralizes infection. J Immunol 146:4325- 4332. 578. Prieto, I., S. Hervas-Stubbs, M. Garcia-Granero, C. Berasain, J. I. Riezu-Boj, J. J. Lasarte, P. Sarobe, J. Prieto, and F. Borras-Cuesta. 1995. Simple strategy to induce antibodies of distinct specificity: application to the mapping of gp120 and inhibition of HIV-1 infectivity. Eur J Immunol 25:877-883. 579. Charbit, A., P. Martineau, J. Ronco, C. Leclerc, R. Lo-Man, V. Michel, D. O'Callaghan, and M. Hofnung. 1993. Expression and immunogenicity of the V3 loop from the envelope of human immunodeficiency virus type 1 in an attenuated aroA strain of Salmonella typhimurium upon genetic coupling to two Escherichia coli carrier proteins. Vaccine 11:1221-1228.

260 580. Little, S. F., B. E. Ivins, P. F. Fellows, and A. M. Friedlander. 1997. Passive protection by polyclonal antibodies against Bacillus anthracis infection in guinea pigs. Infect Immun 65:5171-5175. 581. Graziano, F., C. Haley, L. Gundersen, and P. W. Askenase. 1981. IgE antibody production in guinea pigs treated with cyclophosphamide. J Immunol 127:1067- 1070. 582. Mach, V., K. Ohno, H. Kokubo, and Y. Suzuki. 1996. The Drosophila fork head factor directly controls larval salivary gland-specific expression of the glue protein gene Sgs3. Nucleic Acids Res 24:2387-2394. 583. Casal, J. I., J. P. Langeveld, E. Cortes, W. W. Schaaper, E. van Dijk, C. Vela, S. Kamstrup, and R. H. Meloen. 1995. Peptide vaccine against canine parvovirus: identification of two neutralization subsites in the N terminus of VP2 and optimization of the amino acid sequence. J Virol 69:7274-7277. 584. Wei, X., J. M. Decker, H. Liu, Z. Zhang, R. B. Arani, J. M. Kilby, M. S. Saag, X. Wu, G. M. Shaw, and J. C. Kappes. 2002. Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy. Antimicrob Agents Chemother 46:1896-1905. 585. Platt, E. J., K. Wehrly, S. E. Kuhmann, B. Chesebro, and D. Kabat. 1998. Effects of CCR5 and CD4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1. J Virol 72:2855-2864. 586. Li, M., F. Gao, J. R. Mascola, L. Stamatatos, V. R. Polonis, M. Koutsoukos, G. Voss, P. Goepfert, P. Gilbert, K. M. Greene, M. Bilska, D. L. Kothe, J. F. Salazar- Gonzalez, X. Wei, J. M. Decker, B. H. Hahn, and D. C. Montefiori. 2005. Human immunodeficiency virus type 1 env clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies. J Virol 79:10108-10125. 587. Seaman, M. S., H. Janes, N. Hawkins, L. E. Grandpre, C. Devoy, A. Giri, R. T. Coffey, L. Harris, B. Wood, M. G. Daniels, T. Bhattacharya, A. Lapedes, V. R. Polonis, F. E. McCutchan, P. B. Gilbert, S. G. Self, B. T. Korber, D. C. Montefiori, and J. R. Mascola. 2010. Tiered categorization of a diverse panel of HIV-1 Env pseudoviruses for assessment of neutralizing antibodies. J Virol 84:1439-1452. 588. Mascola, J. R., P. D'Souza, P. Gilbert, B. H. Hahn, N. L. Haigwood, L. Morris, C. J. Petropoulos, V. R. Polonis, M. Sarzotti, and D. C. Montefiori. 2005. Recommendations for the design and use of standard virus panels to assess neutralizing antibody responses elicited by candidate human immunodeficiency virus type 1 vaccines. J Virol 79:10103-10107. 589. Loboda, A. V., A. N. Krutchinsky, M. Bromirski, W. Ens, and K. G. Standing. 2000. A tandem quadrupole/time-of-flight mass spectrometer with a matrix- assisted laser desorption/ionization source: design and performance. Rapid Commun Mass Spectrom 14:1047-1057. 590. Ayed, A., A. N. Krutchinsky, W. Ens, K. G. Standing, and H. W. Duckworth. 1998. Quantitative evaluation of protein-protein and ligand-protein equilibria of a large allosteric enzyme by electrospray ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 12:339-344.

261 591. Crooks, E. T., P. L. Moore, M. Franti, C. S. Cayanan, P. Zhu, P. Jiang, R. P. de Vries, C. Wiley, I. Zharkikh, N. Schulke, K. H. Roux, D. C. Montefiori, D. R. Burton, and J. M. Binley. 2007. A comparative immunogenicity study of HIV-1 virus-like particles bearing various forms of envelope proteins, particles bearing no envelope and soluble monomeric gp120. Virology 366:245-262. 592. Fu, Y., Q. Yang, R. Sun, D. Li, R. Zeng, C. X. Ling, and W. Gao. 2004. Exploiting the kernel trick to correlate fragment ions for peptide identification via tandem mass spectrometry. Bioinformatics 20:1948-1954. 593. Kyte, J., and R. F. Doolittle. 1982. A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105-132. 594. Grantham, R. 1974. Amino acid difference formula to help explain protein evolution. Science 185:862-864. 595. Ditzel, H. J., K. Itoh, and D. R. Burton. 1996. Determinants of polyreactivity in a large panel of recombinant human antibodies from HIV-1 infection. J Immunol 157:739-749. 596. Wikman, M., E. Rowcliffe, M. Friedman, P. Henning, L. Lindholm, S. Olofsson, and S. Stahl. 2006. Selection and characterization of an HIV-1 gp120-binding affibody ligand. Biotechnol Appl Biochem 45:93-105. 597. Chen, Y. H., and M. P. Dierich. 1996. Identification of a second site in HIV-1 gp41 mediating binding to cells. Immunol Lett 52:153-156. 598. Moore, J. P., Y. Cao, A. J. Conley, R. Wyatt, J. Robinson, M. K. Gorny, S. Zolla- Pazner, D. D. Ho, and R. A. Koup. 1994. Studies with monoclonal antibodies to the V3 region of HIV-1 gp120 reveal limitations to the utility of solid-phase peptide binding assays. J Acquir Immune Defic Syndr 7:332-339. 599. Korber, B. T., E. E. Allen, A. D. Farmer, and G. L. Myers. 1995. Heterogeneity of HIV-1 and HIV-2. Aids 9 Suppl A:S5-18. 600. Haynes, B. F., M. A. Moody, L. Verkoczy, G. Kelsoe, and S. M. Alam. 2005. Antibody polyspecificity and neutralization of HIV-1: a hypothesis. Hum Antibodies 14:59-67. 601. Argov, S., A. Schattner, R. Burstein, Z. T. Handzel, Y. Shoenfeld, and Z. Bentwich. 1991. Autoantibodies in male homosexuals and HIV infection. Immunol Lett 30:31-35. 602. Lucchese, G., A. Stufano, B. Trost, A. Kusalik, and D. Kanduc. 2007. Peptidology: short amino acid modules in cell biology and immunology. Amino Acids 33:703-707. 603. Kanduc, D., R. Serpico, A. Lucchese, and Y. Shoenfeld. 2008. Correlating low- similarity peptide sequences and HIV B-cell epitopes. Autoimmun Rev 7:291-296. 604. Zimmerman, J. M., N. Eliezer, and R. Simha. 1968. The characterization of amino acid sequences in proteins by statistical methods. J Theor Biol 21:170-201. 605. Diamandis, E. P., and T. K. Christopoulos. 1991. The biotin-(strept)avidin system: principles and applications in biotechnology. Clin Chem 37:625-636. 606. Vermeire, K., K. Van Laethem, W. Janssens, T. W. Bell, and D. Schols. 2009. Human immunodeficiency virus type 1 escape from cyclotriazadisulfonamide- induced CD4-targeted entry inhibition is associated with increased neutralizing antibody susceptibility. J Virol 83:9577-9583.

262 607. Rusert, P., A. Mann, M. Huber, V. von Wyl, H. F. Gunthard, and A. Trkola. 2009. Divergent effects of cell environment on HIV entry inhibitor activity. Aids 23:1319-1327. 608. Binley, J. M., R. Wyatt, E. Desjardins, P. D. Kwong, W. Hendrickson, J. P. Moore, and J. Sodroski. 1998. Analysis of the interaction of antibodies with a conserved enzymatically deglycosylated core of the HIV type 1 envelope glycoprotein 120. AIDS Res Hum Retroviruses 14:191-198. 609. Kang, S. M., F. S. Quan, C. Huang, L. Guo, L. Ye, C. Yang, and R. W. Compans. 2005. Modified HIV envelope proteins with enhanced binding to neutralizing monoclonal antibodies. Virology 331:20-32. 610. Dorgham, K., I. Dogan, N. Bitton, C. Parizot, V. Cardona, P. Debre, O. Hartley, and G. Gorochov. 2005. Immunogenicity of HIV type 1 gp120 CD4 binding site phage mimotopes. AIDS Res Hum Retroviruses 21:82-92. 611. Zwick, M. B., L. L. Bonnycastle, A. Menendez, M. B. Irving, C. F. Barbas, 3rd, P. W. Parren, D. R. Burton, and J. K. Scott. 2001. Identification and characterization of a peptide that specifically binds the human, broadly neutralizing anti-human immunodeficiency virus type 1 antibody b12. J Virol 75:6692-6699. 612. Burton, D. R. 1995. Phage display. Immunotechnology 1:87-94. 613. Sundberg, E. J., and R. A. Mariuzza. 2002. Molecular recognition in antibody- antigen complexes. Adv Protein Chem 61:119-160. 614. Dunfee, R. L., E. R. Thomas, and D. Gabuzda. 2009. Enhanced macrophage tropism of HIV in brain and lymphoid tissues is associated with sensitivity to the broadly neutralizing CD4 binding site antibody b12. Retrovirology 6:69. 615. Douagi, I., M. N. Forsell, C. Sundling, S. O'Dell, Y. Feng, P. Dosenovic, Y. Li, R. Seder, K. Lore, J. R. Mascola, R. T. Wyatt, and G. B. Karlsson Hedestam. 2010. Influence of novel CD4 binding-defective HIV-1 envelope glycoprotein immunogens on neutralizing antibody and T-cell responses in nonhuman primates. J Virol 84:1683-1695. 616. Moore, P. L., E. T. Crooks, L. Porter, P. Zhu, C. S. Cayanan, H. Grise, P. Corcoran, M. B. Zwick, M. Franti, L. Morris, K. H. Roux, D. R. Burton, and J. M. Binley. 2006. Nature of nonfunctional envelope proteins on the surface of human immunodeficiency virus type 1. J Virol 80:2515-2528. 617. Mouquet, H., J. F. Scheid, M. J. Zoller, M. Krogsgaard, R. G. Ott, S. Shukair, M. N. Artyomov, J. Pietzsch, M. Connors, F. Pereyra, B. D. Walker, D. D. Ho, P. C. Wilson, M. S. Seaman, H. N. Eisen, A. K. Chakraborty, T. J. Hope, J. V. Ravetch, H. Wardemann, and M. C. Nussenzweig. 2010. Polyreactivity increases the apparent affinity of anti-HIV antibodies by heteroligation. Nature 467:591-595. 618. Cardoso, R. M., M. B. Zwick, R. L. Stanfield, R. Kunert, J. M. Binley, H. Katinger, D. R. Burton, and I. A. Wilson. 2005. Broadly neutralizing anti-HIV antibody 4E10 recognizes a helical conformation of a highly conserved fusion- associated motif in gp41. Immunity 22:163-173. 619. Quakkelaar, E. D., F. P. van Alphen, B. D. Boeser-Nunnink, A. C. van Nuenen, R. Pantophlet, and H. Schuitemaker. 2007. Susceptibility of recently transmitted subtype B human immunodeficiency virus type 1 variants to broadly neutralizing antibodies. J Virol 81:8533-8542.

263 620. Stiegler, G., R. Kunert, M. Purtscher, S. Wolbank, R. Voglauer, F. Steindl, and H. Katinger. 2001. A potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp41 of human immunodeficiency virus type 1. AIDS Res Hum Retroviruses 17:1757-1765. 621. Seddon, A. M., P. Curnow, and P. J. Booth. 2004. Membrane proteins, lipids and detergents: not just a soap opera. Biochim Biophys Acta 1666:105-117. 622. Saito, S., and T. Tsuchiya. 1984. Characteristics of n-octyl beta-D- thioglucopyranoside, a new non-ionic detergent useful for membrane biochemistry. Biochem J 222:829-832. 623. Barbas, C. F., 3rd, D. Hu, N. Dunlop, L. Sawyer, D. Cababa, R. M. Hendry, P. L. Nara, and D. R. Burton. 1994. In vitro evolution of a neutralizing human antibody to human immunodeficiency virus type 1 to enhance affinity and broaden strain cross-reactivity. Proc Natl Acad Sci U S A 91:3809-3813. 624. Peters, P. J., M. J. Duenas-Decamp, W. M. Sullivan, R. Brown, C. Ankghuambom, K. Luzuriaga, J. Robinson, D. R. Burton, J. Bell, P. Simmonds, J. Ball, and P. R. Clapham. 2008. Variation in HIV-1 R5 macrophage-tropism correlates with sensitivity to reagents that block envelope: CD4 interactions but not with sensitivity to other entry inhibitors. Retrovirology 5:5. 625. Chang, S. Y., B. H. Bowman, J. B. Weiss, R. E. Garcia, and T. J. White. 1993. The origin of HIV-1 isolate HTLV-IIIB. Nature 363:466-469. 626. Willey, R. L., R. A. Rutledge, S. Dias, T. Folks, T. Theodore, C. E. Buckler, and M. A. Martin. 1986. Identification of conserved and divergent domains within the envelope gene of the acquired immunodeficiency syndrome retrovirus. Proc Natl Acad Sci U S A 83:5038-5042. 627. Obaro, S. K. 2002. The new pneumococcal vaccine. Clin Microbiol Infect 8:623- 633. 628. Group, T. F. I. S. 2007. Quadrivalent vaccine against human papillomavirus to prevent high-grade cervical lesions. N Engl J Med 356:1915-1927. 629. Wu, T. T., and G. Johnson. 2004. HIV vaccine candidates. Drugs Today (Barc) 40:949-955. 630. Wang, J., J. Sen, L. Rong, and M. Caffrey. 2008. Role of the HIV gp120 conserved domain 1 in processing and viral entry. J Biol Chem 283:32644-32649. 631. Binley, J. M., R. W. Sanders, B. Clas, N. Schuelke, A. Master, Y. Guo, F. Kajumo, D. J. Anselma, P. J. Maddon, W. C. Olson, and J. P. Moore. 2000. A recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure. J Virol 74:627-643. 632. McKeating, J. A., M. Thali, C. Furman, S. Karwowska, M. K. Gorny, J. Cordell, S. Zolla-Pazner, J. Sodroski, and R. A. Weiss. 1992. Amino acid residues of the human immunodeficiency virus type I gp120 critical for the binding of rat and human neutralizing antibodies that block the gp120-sCD4 interaction. Virology 190:134-142. 633. el Ahmar, W., P. Poumbourios, D. A. McPhee, and B. E. Kemp. 1991. N-terminal residues 105-117 of HIV-1 gp120 are not involved in CD4 binding. AIDS Res Hum Retroviruses 7:855-858.

264 634. Moore, J. P., R. L. Willey, G. K. Lewis, J. Robinson, and J. Sodroski. 1994. Immunological evidence for interactions between the first, second, and fifth conserved domains of the gp120 surface glycoprotein of human immunodeficiency virus type 1. J Virol 68:6836-6847. 635. Kropelin, M., C. Susal, V. Daniel, and G. Opelz. 1998. Inhibition of HIV-1 rgp120 binding to CD4+ T cells by monoclonal antibodies directed against the gp120 C1 or C4 region. Immunol Lett 63:19-25. 636. Nakamura, G. R., R. Byrn, K. Rosenthal, J. P. Porter, M. R. Hobbs, L. Riddle, D. J. Eastman, D. Dowbenko, T. Gregory, B. M. Fendly, and et al. 1992. Monoclonal antibodies to the extracellular domain of HIV-1IIIB gp160 that neutralize infectivity, block binding to CD4, and react with diverse isolates. AIDS Res Hum Retroviruses 8:1875-1885. 637. Reeves, J. P., C. Y. Lo, D. M. Klinman, and S. L. Epstein. 1995. Mouse monoclonal antibodies to human immunodeficiency virus glycoprotein 120 generated by repeated immunization with glycoprotein 120 from a single isolate, or by sequential immunization with glycoprotein 120 from three isolates. Hybridoma 14:235-242. 638. Vossenkamper, A., and J. Spencer. 2011. Transitional B Cells: How Well Are the Checkpoints for Specificity Understood? Arch Immunol Ther Exp (Warsz). 639. Ito, M., M. Baba, A. Sato, R. Pauwels, E. De Clercq, and S. Shigeta. 1987. Inhibitory effect of dextran sulfate and heparin on the replication of human immunodeficiency virus (HIV) in vitro. Antiviral Res 7:361-367. 640. Watkins, D. I., D. R. Burton, E. G. Kallas, J. P. Moore, and W. C. Koff. 2008. Nonhuman primate models and the failure of the Merck HIV-1 vaccine in humans. Nat Med 14:617-621. 641. Patterson, L. J., N. Malkevitch, D. Venzon, J. Pinczewski, V. R. Gomez-Roman, L. Wang, V. S. Kalyanaraman, P. D. Markham, F. A. Robey, and M. Robert- Guroff. 2004. Protection against mucosal simian immunodeficiency virus SIV(mac251) challenge by using replicating adenovirus-SIV multigene vaccine priming and subunit boosting. J Virol 78:2212-2221. 642. Sallusto, F., A. Lanzavecchia, K. Araki, and R. Ahmed. 2010. From vaccines to memory and back. Immunity 33:451-463. 643. Hart, T. K., R. Kirsh, H. Ellens, R. W. Sweet, D. M. Lambert, S. R. Petteway, Jr., J. Leary, and P. J. Bugelski. 1991. Binding of soluble CD4 proteins to human immunodeficiency virus type 1 and infected cells induces release of envelope glycoprotein gp120. Proc Natl Acad Sci U S A 88:2189-2193. 644. di Marzo Veronese, F., R. Rahman, R. Pal, C. Boyer, J. Romano, V. S. Kalyanaraman, B. C. Nair, R. C. Gallo, and M. G. Sarngadharan. 1992. Delineation of immunoreactive, conserved regions in the external glycoprotein of the human immunodeficiency virus type 1. AIDS Res Hum Retroviruses 8:1125- 1132. 645. Mascola, J. R., S. W. Snyder, O. S. Weislow, S. M. Belay, R. B. Belshe, D. H. Schwartz, M. L. Clements, R. Dolin, B. S. Graham, G. J. Gorse, M. C. Keefer, M. J. McElrath, M. C. Walker, K. F. Wagner, J. G. McNeil, F. E. McCutchan, and D. S. Burke. 1996. Immunization with envelope subunit vaccine products elicits neutralizing antibodies against laboratory-adapted but not primary isolates of

265 human immunodeficiency virus type 1. The National Institute of Allergy and Infectious Diseases AIDS Vaccine Evaluation Group. J Infect Dis 173:340-348. 646. Francis, D. P., W. L. Heyward, V. Popovic, P. Orozco-Cronin, K. Orelind, C. Gee, A. Hirsch, T. Ippolito, A. Luck, M. Longhi, V. Gulati, N. Winslow, M. Gurwith, F. Sinangil, and P. W. Berman. 2003. Candidate HIV/AIDS vaccines: lessons learned from the World's first phase III efficacy trials. Aids 17:147-156. 647. Hessell, A. J., E. G. Rakasz, D. M. Tehrani, M. Huber, K. L. Weisgrau, G. Landucci, D. N. Forthal, W. C. Koff, P. Poignard, D. I. Watkins, and D. R. Burton. 2010. Broadly neutralizing monoclonal antibodies 2F5 and 4E10 directed against the human immunodeficiency virus type 1 gp41 membrane-proximal external region protect against mucosal challenge by simian-human immunodeficiency virus SHIVBa-L. J Virol 84:1302-1313. 648. Hessell, A. J., E. G. Rakasz, P. Poignard, L. Hangartner, G. Landucci, D. N. Forthal, W. C. Koff, D. I. Watkins, and D. R. Burton. 2009. Broadly neutralizing human anti-HIV antibody 2G12 is effective in protection against mucosal SHIV challenge even at low serum neutralizing titers. PLoS Pathog 5:e1000433. 649. Center, R. J., A. K. Wheatley, S. M. Campbell, A. J. Gaeguta, V. Peut, S. Alcantara, C. Siebentritt, S. J. Kent, and D. F. Purcell. 2009. Induction of HIV-1 subtype B and AE-specific neutralizing antibodies in mice and macaques with DNA prime and recombinant gp140 protein boost regimens. Vaccine 27:6605- 6612. 650. Derby, N. R., Z. Kraft, E. Kan, E. T. Crooks, S. W. Barnett, I. K. Srivastava, J. M. Binley, and L. Stamatatos. 2006. Antibody responses elicited in macaques immunized with human immunodeficiency virus type 1 (HIV-1) SF162-derived gp140 envelope immunogens: comparison with those elicited during homologous simian/human immunodeficiency virus SHIVSF162P4 and heterologous HIV-1 infection. J Virol 80:8745-8762. 651. Srivastava, I. K., L. Stamatatos, H. Legg, E. Kan, A. Fong, S. R. Coates, L. Leung, M. Wininger, J. J. Donnelly, J. B. Ulmer, and S. W. Barnett. 2002. Purification and characterization of oligomeric envelope glycoprotein from a primary R5 subtype B human immunodeficiency virus. J Virol 76:2835-2847. 652. Li, Y., K. Svehla, N. L. Mathy, G. Voss, J. R. Mascola, and R. Wyatt. 2006. Characterization of antibody responses elicited by human immunodeficiency virus type 1 primary isolate trimeric and monomeric envelope glycoproteins in selected adjuvants. J Virol 80:1414-1426. 653. Garrity, R. R., G. Rimmelzwaan, A. Minassian, W. P. Tsai, G. Lin, J. J. de Jong, J. Goudsmit, and P. L. Nara. 1997. Refocusing neutralizing antibody response by targeted dampening of an immunodominant epitope. J Immunol 159:279-289. 654. Morner, A., I. Douagi, M. N. Forsell, C. Sundling, P. Dosenovic, S. O'Dell, B. Dey, P. D. Kwong, G. Voss, R. Thorstensson, J. R. Mascola, R. T. Wyatt, and G. B. Karlsson Hedestam. 2009. Human immunodeficiency virus type 1 env trimer immunization of macaques and impact of priming with viral vector or stabilized core protein. J Virol 83:540-551. 655. Moore, J. P., J. A. McKeating, Y. X. Huang, A. Ashkenazi, and D. D. Ho. 1992. Virions of primary human immunodeficiency virus type 1 isolates resistant to

266 soluble CD4 (sCD4) neutralization differ in sCD4 binding and glycoprotein gp120 retention from sCD4-sensitive isolates. J Virol 66:235-243. 656. Moore, J. P., and D. D. Ho. 1995. HIV-1 neutralization: the consequences of viral adaptation to growth on transformed T cells. Aids 9 Suppl A:S117-136. 657. Vaine, M., M. Duenas-Decamp, P. Peters, Q. Liu, J. Arthos, S. Wang, P. Clapham, and S. Lu. 2011. Two Closely Related Env Antigens from the Same Patient Elicited Different Spectra of Neutralizing Antibodies against Heterologous HIV-1 Isolates. J Virol 85:4927-4936. 658. McKinnon, L. R., T. B. Ball, J. Kimani, C. Wachihi, L. Matu, M. Luo, J. Embree, K. R. Fowke, and F. A. Plummer. 2005. Cross-clade CD8(+) T-cell responses with a preference for the predominant circulating clade. J Acquir Immune Defic Syndr 40:245-249. 659. Yin, J., A. Dai, J. Lecureux, T. Arango, M. A. Kutzler, J. Yan, M. G. Lewis, A. Khan, N. Y. Sardesai, D. Montefiore, R. Ruprecht, D. B. Weiner, and J. D. Boyer. 2010. High antibody and cellular responses induced to HIV-1 clade C envelope following DNA vaccines delivered by electroporation. Vaccine. 660. Brave, A., D. Hallengard, M. Malm, V. Blazevic, E. Rollman, I. Stanescu, and K. Krohn. 2009. Combining DNA technologies and different modes of immunization for induction of humoral and cellular anti-HIV-1 immune responses. Vaccine 27:184-186. 661. Selvarajah, S., B. A. Puffer, F. H. Lee, P. Zhu, Y. Li, R. Wyatt, K. H. Roux, R. W. Doms, and D. R. Burton. 2008. Focused dampening of antibody response to the immunodominant variable loops by engineered soluble gp140. AIDS Res Hum Retroviruses 24:301-314. 662. Phogat, S. K., S. M. Kaminsky, and W. C. Koff. 2007. HIV-1 rational vaccine design: molecular details of b12-gp120 complex structure. Expert Rev Vaccines 6:319-321. 663. Irving, M. B., O. Pan, and J. K. Scott. 2001. Random-peptide libraries and antigen-fragment libraries for epitope mapping and the development of vaccines and diagnostics. Curr Opin Chem Biol 5:314-324. 664. Saphire, E. O., M. Montero, A. Menendez, N. E. van Houten, M. B. Irving, R. Pantophlet, M. B. Zwick, P. W. Parren, D. R. Burton, J. K. Scott, and I. A. Wilson. 2007. Structure of a high-affinity "mimotope" peptide bound to HIV-1- neutralizing antibody b12 explains its inability to elicit gp120 cross-reactive antibodies. J Mol Biol 369:696-709. 665. Cao, J., L. Bergeron, E. Helseth, M. Thali, H. Repke, and J. Sodroski. 1993. Effects of amino acid changes in the extracellular domain of the human immunodeficiency virus type 1 gp41 envelope glycoprotein. J Virol 67:2747- 2755. 666. Vincent, N., C. Genin, and E. Malvoisin. 2002. Identification of a conserved domain of the HIV-1 transmembrane protein gp41 which interacts with cholesteryl groups. Biochim Biophys Acta 1567:157-164. 667. Li, H., and V. Papadopoulos. 1998. Peripheral-type benzodiazepine receptor function in cholesterol transport. Identification of a putative cholesterol recognition/interaction amino acid sequence and consensus pattern. Endocrinology 139:4991-4997.

267 668. Aloia, R. C., H. Tian, and F. C. Jensen. 1993. Lipid composition and fluidity of the human immunodeficiency virus envelope and host cell plasma membranes. Proc Natl Acad Sci U S A 90:5181-5185. 669. Peisajovich, S. G., and Y. Shai. 2003. Viral fusion proteins: multiple regions contribute to membrane fusion. Biochim Biophys Acta 1614:122-129. 670. Eckert, D. M., and P. S. Kim. 2001. Mechanisms of viral membrane fusion and its inhibition. Annu Rev Biochem 70:777-810. 671. Bouma, P., M. Leavitt, P. F. Zhang, I. A. Sidorov, D. S. Dimitrov, and G. V. Quinnan, Jr. 2003. Multiple interactions across the surface of the gp120 core structure determine the global neutralization resistance phenotype of human immunodeficiency virus type 1. J Virol 77:8061-8071. 672. Zhang, M. Y., X. Xiao, I. A. Sidorov, V. Choudhry, F. Cham, P. F. Zhang, P. Bouma, M. Zwick, A. Choudhary, D. C. Montefiori, C. C. Broder, D. R. Burton, G. V. Quinnan, Jr., and D. S. Dimitrov. 2004. Identification and characterization of a new cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody. J Virol 78:9233-9242. 673. Binley, J. M., C. S. Cayanan, C. Wiley, N. Schulke, W. C. Olson, and D. R. Burton. 2003. Redox-triggered infection by disulfide-shackled human immunodeficiency virus type 1 pseudovirions. J Virol 77:5678-5684. 674. Abrahamyan, L. G., R. M. Markosyan, J. P. Moore, F. S. Cohen, and G. B. Melikyan. 2003. Human immunodeficiency virus type 1 Env with an intersubunit disulfide bond engages coreceptors but requires bond reduction after engagement to induce fusion. J Virol 77:5829-5836. 675. Law, M., R. M. Cardoso, I. A. Wilson, and D. R. Burton. 2007. Antigenic and immunogenic study of membrane-proximal external region-grafted gp120 antigens by a DNA prime-protein boost immunization strategy. J Virol 81:4272- 4285. 676. Kelker, H. C., D. Schlesinger, and F. T. Valentine. 1994. Immunogenic and antigenic properties of an HIV-1 gp120-derived multiple chain peptide. J Immunol 152:4139-4148.

268 Section 8.0 Appendices

Appendix A List of Abbreviations aa Amino acids Ab Antibody ABTS 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) AC After coupling AD After dialysis Ad5hr Adenovirus type 5 host range mutant ADCC Antibody-dependent cellular cytotoxicity ADCVI Antibody-dependent cell-mediated viral inhibition AIDS Acquired Immunodeficiency Syndrome APC Antigen presenting cell APOBEC apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like ARRRP NIH AIDS Research and Reference Reagent Program ART Antiretroviral AZT Azidothymidine BCA Bicinchoninic acid BCR B cell receptor BD Before dialysis BNAbs Broadly neutralizing monoclonal antibodies BOG n-Octyl-beta-D-glucopyranoside BSA Bovine serum albumin C region Constant region of gp120 C terminus Carboxy terminus C3 Complement protein 3 CAVD The Collaboration for AIDS Vaccine Discovery CCAC Canadian Council on Animal Care CCR or CXCR Chemokine receptor CD Cluster of differentiation CD40L CD40 ligand CD4BS CD4 binding site CD4i CD4-induced epitope overlapping the co-receptor binding site CDC US Centre for Disease Control and Prevention CDR Complementarity determining region CLR C-type lectin receptors CRF Circulating recombinant form CSR Class switch recombination CTL Cytotoxic T lymphocytes D Domain of CD4

269 D# Donor # Da Daltons DC Dendritic cells DC-SIGN dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin ddH20 double distilled water DDM n-dodecyl-D-maltoside DEA Diethanolamine DEAE Diethylaminoethyl DHB Dihydroxy benzoate DNA Deoxyribonucleic acid EC Elite controller EDTA Ethylenediaminetetraacetic acid ELISA Enzyme-linked immunosorbant assay EMLA Eutectic mixture of Local Anesthetic Env Envelope ER Endoplasmic reticulum fab Fragment antigen-binding domain (antibody) Fc Fragment Crystallizable FcR Fragment Crystallizable Receptor FCS Fetal calf serum FDC Follicular dendritic cell Fv Antibody variable domain ga Gauge Gag Group-specific antigen gene GM Complete growth media gp Glycoprotein Gp Guinea pig H Heavy chain (antibody) HBV Hepatitis B Virus HCl Hydrochloric acid HESN HIV-exposed seronegative HIV Human Immunodeficiency Virus HIV+ HIV-1 positive, or HIV-1 infected HIV-1 or HIV-2 HIV type 1 or HIV type 2 HLA Human leukocyte antigen HPV Human Papilloma Virus HRP Horseraddish peroxidase HSV-2 Herpes Simplex Virus type 2 IC Immune comples IC Inhibitory concentration

270 ID50 Infectious dose 50% IFN-g Interferon-g Ig Immunoglobulin IL Interleukin IRF Interferon regulatory factor L Light chain (antibody) LTNP Long-term non-progressor LTR Long terminal repeat m/z Mass-to-charge ratio mAb monoclonal antibody mac Macaque MALDI Matrix-assisted laser desorption/ionization MHC Major histocompatability complex MIP macrophage inflammatory protein MOI Multiplicity of infection MPER, MPR membrane-proximal region MS Mass spectrometry MS/MS Tandem mass spectrometry sequencing Mu Murine, or mouse N terminus Amino terminus NCBI National Center for Biotechnology Information Nef Negative factor NK Natural killer cell NHP Non-human primate NLR NOD-like receptor NMR Nuclear magnetic resonance NNRTI Non-nucleoside reverse transcriptase inhibitor NOD Nucleotide-binding oligomerization domain NRTI Nucleoside reverse transcriptase inhibitor Nt Nterm epitope peptide/immunogen Nterm gp120 amino terminal epitope identified by mass spectrometry OD Optical density p value Probability value pAb polyclonal antibody PAGE Polyacrylamide gel electrophoresis PAMPS Pattern-associated molecular patterns PBMC peripheral blood mononuclear cell PBS Phosphate buffered saline PCR Polymerase chain reaction PHA Phytohaemagglutinin pNPP Alkaline phosphatase yellow

271 Pol HIV polymerase protein PRR Pattern recognition receptor QqTOF Manitoba/SCIEX prototype hybrid quadrupole-time of flight mass spectrometer R Rabbit RANTES Regulated upon Activation, Normal T-cell Expressed, and Secreted Rev Regulator of virion RIG Retinoic acid-inducible gene RLR RIG-1-like receptor RLU Relative light unit RNA Ribonucleic acid RPMI Roswell Park Memorial Institute media RT Reverse transcriptase SAMT S-acyl-2-mercaptobenzamide thioester sCD4 Soluble CD4 scFv Single chain antibody SCID Severe compromised immunodeficiency Scr Scrambled peptide/immunogen SDS Sodium dodecyl sulphate SHIV Simian/Human Immunodeficiency Virus SIV Simian Immunodeficiency Virus STI Sexually transmitted infection Tat Transactivator of transcription TBS Tris-buffered saline TCID50 Tissue culture infectious dose 50% TCLA T cell line-adapted TCR T cell receptor TGF-b Transforming growth factor-b Th T helper cell TLR Toll-like receptor TNF Tumor necrosis factor Treg Regulatory T cell TRIM5a tripartite interaction motif 5 a UNAIDS United Nations on HIV/AIDS V region Variable region of gp120 VC Viremic controller Vif Virion infectivity factor Vpr Viral protein R Vpu Viral protein U WHO World Health Organization ZDV 3’-azido-3’-deoxythymidine

272

Appendix B Amino acids

G Gly Glycine A Ala Alanine C Cys Cysteine I Ile Isoleucine L Leu Leucine V Val Valine P Pro Proline M Met Methionine F Phe Phenylalanine W Trp Tryptophan Y Tyr Tyrosine S Ser Serine T Thr Threonine D Asp Aspartic acid R Arg Arginine K Lys Lysine E Glu Glutamic acid H His Histidine N Asp Asparagine Q Gln Glutamine

273 Appendix C Nterm amino acid sequences for neutralization-tested viruses

Strain Sequence at Nterm1 IIIB TEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTE SF162 VEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTE 6535.3 TEKLWVTVYYGVPVWKEATTTLFCASEAKAYDTE QH0692 AENLWVTVYYGVPVWKEATTTLFCASDAKAYETE SC422661.9 TEKLWVTVYYGVPVWKEATTTLFCASDAKAYETE PVO.4 EEKLWVTVYYGVPVWKEATTTLFCASDAKAYNTE RHPA4259.7 ADQLWVTVYYGVPVWKEANTTLFCASDAKAYDTE THRO4156.18 TDKLWVTVYYGVPVWKEAVTTLFCASDAKAYDTE SVA-MLV -murine retroviral envelope- Bal TEKLWVTVYYGVPVWKEATTTLFCASDAKAYDTE 1. Red bolded amino acids are those that vary from the consensus Nterm sequence; blue bolded indicates the presence of a putative N-linked glycosylation site; bold indicates the immunogen sequence.

274 Appendix D Animal serum and IgG neutralization data

Table D1. PBMC neutralizations with HIV IIIB and D074. HIV-1 IIIB was pre- incubated with sample and a MOI of 0.1 was tested for growth on D074 PBMC. Neutralization-positive results are bolded. Rabbit Sample Sample IC dilution-1 or IgG Maximum % 50 Neutralizationd sample type day (D) concentration* (p24)a inhibition*,b (p24)c PBS D3 <100 (<100) 0 (0) n (n) pre- Serum D7 <100 (6400) 31.0 (51.4) n (y) bleed D12 <100 (nd) 7.5 (nd)e n (nd) D3 <100 (<100) 22.9 (0) n (n) Serum D7 <100 (<100) 20.6 (43.1) n (n) RPBS D12 <100 (nd) 15.8 (nd) n (nd) #1 D3 10 (>10) 63.4 (0) y (n) IgG D7 >10 (>10) 0 (0) n (n) D12 >10 (nd) 20.9 (nd) n (nd) D3 <100 (<100) 0 (7.9) n (n) Serum D7 <100 (<100) 0 (0) n (n) RPBS D12 <100 (nd) 22.7 (nd) n (nd) #2 D3 >10 (bdl)f 24.8 (bdl) n (n/a) IgG D7 >10 (>10) 0 (0) n (n) D12 >10 (nd) 9.8 (nd) n (nd)

Rabbit Sample Sample IC dilution-1 or IgG Maximum % 50 Neutralization sample type day (D) concentration (p24) inhibition (p24) Gp120 D3 <100 (bdl) 9.0 (bdl) n (n) pre- Serum D7 <100 (<100) 16.1 (43.4) n (n) bleed D12 <100 (nd) 44.3 (nd) n (nd) D3 <100 (bdl) 10.5 (bdl) n (n) Serum D7 <100 (400) 40.0 (59.1) n (y) R120 D12 <100 (nd) 31.2 (nd) n (nd) #3 D3 >10 (bdl) 39.2 (bdl) n (n) IgG D7 >10 (0.15625) 42.4 (58.5) n (y) D12 >10 (nd) 40.9 (nd) n (nd) D3 6 400 (bdl) 83.4 (bdl) y (n/a) Serum D7 1 600 (1 600) 91.9 (90.8) y (y) R120 D12 400 (nd) 66.24 (nd) y (nd) #4 D3 >10 (bdl) 0 (bdl) n (n) IgG D7 2.5 (2.5) 54.8 (bdl-19.5)g y (n) D12 10 (nd) 88.6 (nd) y (nd)

275 Table D1 (continued). Rabbit Sample Sample IC dilution-1 or IgG Maximum % 50 Neutralization sample type day (D) concentration (p24) inhibition (p24) Nterm D3 25 600 (bdl) 54.2 (bdl) y (n) pre- Serum D7 <100 (<100) 28.1 (40.4) n (n) bleed D12 <100 (nd) 44.1 (nd) n (nd) D3 1 600 (bdl) 44.1 (bdl) n (n) Serum D7 6 400 (<100) 60.5 (0) y (n) D12 <100 (nd) 33.8 (nd) n (nd) RNt #5 D3 >10 (bdl) 0 (bdl) n (n) IgG D7 >10 (>10) 0 (0) n (n) D12 >10 (nd) 25.1 (nd) n (nd) D3 <100 (bdl) 0 (bdl) n (n) Serum D7 <100 (<100) 0 (0) n (n) D12 <100 (nd) 5.9 (nd) n (nd) RNt #6 D3 0.0098 (bdl) 72.0 (bdl) y (n) IgG D7 >10 (>10) 0 (10.3) n (n) D12 >10 (nd) 19.5 (nd) n (nd)

IC dilution-1 or Rabbit Sample Sample 50 Maximum % IgG concentration Neutralization sample type day (D) inhibition (p24) (p24) D3 <100 (bdl) 0 (bdl) n (n) Scrambled Serum D7 <100 (<100) 26.2 (42.1) n (n) pre-bleed D12 <100 (nd) 0 (nd) n (nd) D3 <100 (<100) 0 (bdl) n (n) Serum D7 <100 (bdl) 5.1 (0) n (n) D12 <100 (nd) 14.2 (nd) n (nd) RScr #7 D3 >10 (bdl) 0 (bdl) n (n) IgG D7 >10 (>10) 0 (0) n (n) D12 >10 (nd) 0 (nd) n (nd) D3 <100 (bdl) 0 (bdl) n (n) Serum D7 <100 (<100) 0 (0) n (n) D12 <100 (nd) 0 (nd) n (nd) RScr #8 D3 0.039 (bdl) 62.9 (bdl) y (n) IgG D7 >10 (>10) 0 (0) n (n) D12 >10 (nd) 0 (nd) n (nd) * Are based on OD405 values where <100=no detectable inhibition below highest dilution (1/100) a. IgG neutralizing concentration based on concentration of detectable p24, derived from standard curve where >10=no detectable neutralization below highest concentration (10 µg/ml) b. at last neutralizing titre or highest % neutralization ever reached across tested dilutions c. % inhibition by p24 concentration d. 50% neutralization y=yes n=no e. nd = not done f. bdl = below detection levels g. down to 0.25ug/ml R120 2 IgG inhibited p24 growth to below the level of detection and the first value above the level of detection indicated a 19% inhibition (at 0.625ug/ml IgG)

276 The dataset for the infection of D074 PBMC with IIIB HIV is shown in Table D1. The different immunization groups are separated. The 3rd column shows the 50% inhibition concentration of each sample. For the serum samples, the 50% inhibition number indicates the reciprocal dilution, while for the IgG, the 50% inhibition number indicates the IgG concentration to neutralize by 50% in µg/ml. Any values that represent 50% neutralization minimum are bolded. Although the infection protocol was optimized to a

7-day incubation period, HIV p24 supernatants were collected on three days (days 3, 7, and 12) to ensure that neutralization of viral growth is captured. The 4th column on the table shows the inhibition attained by the lowest neutralizing titre, and, if non- neutralizing at 50%, the highest achieved % of neutralization ever. The first block of

Table D1 shows the neutralization profile for the PBS-immunized serum and purified

IgG. In only one sample at one time point tested positive for neutralization – RPBS 1 on day three at the lowest dilution (0.1). This likely represents a background blip and not actual specific neutralization of HIV by specific antibodies. The second block from

Table D1 shows the neutralization profile of the gp120-immunized serum and IgG. Here there are some more substantial neutralizations seen in R120 #3 serum on day three (at

2.5x10-3 dilution) accompanied by R120 #3 IgG neutralization on the same day (at 0.16

µg/ml IgG). R120 #4 serum showed neutralization titres on all three harvest days (day three at 1.6x10-4 dilution, day seven at 6.3x10-4 dilution, day twelve at 2.5x10-3). The

R120 #4 serum neutralization reached over 90%. Purified IgG from R120 #4 showed some neutralization of IIIB at both days 7 (at 2.5 µg/ml) and 12 (at 10 µg/ml), but no IgG dilution was able to neutralize >90%. This reflects the pseudovirus assays whereby sera samples neutralize better than purified IgG, and whereby R120 #4 neutralized IIIB virus

277 better than R120 #3. Block three of Table D1 shows the Nterm peptide-immunized sera neutralization profile. All samples, pre-bleed, RNt #5, and RNt #6, show one positive neutralization result over the study (pre-bleed serum day three a 3.9x10-5, RNt #5 serum day 7 at 1.6x10-4, and RNt #6 IgG day 3 at 9.8x10-3 µg/ml). The inconsistent neutralizations between the two tested animals, the serum and IgG tests, the three days of sampling, the dose-independence of the neutralizations (Figure 33), and the lack of responses that exceed the pre-bleed neutralization levels suggest these positive results are not specific. The fourth block of Table D1 shows the results of Scrambled peptide- immunized samples. The only result positive for neutralization, RScr #8 day three IgG at

0.039 µg/ml, suggests that this result, too is an aberrant non-specific result. Together, these data match the pseudovirus study; gp120 immunization elicits autologous neutralizing antibody responses against IIIB gp120.

278 Table D2. PBMC neutralizations with HIV Bal and D074. HIV-1 Bal was pre-incubated with sample and a MOI of 0.1 was tested for growth on D074 PBMC. Neutralization- positive results are bolded. IC dilution-1 or Rabbit Sample Sample 50 % inhibitionb IgG concentration Neutralization sample type day (D) (p24)c (p24)a PBS D3 <100 (bdld) 24.5 (bdl) n (n/a) pre- Serum D7 <100 (<100) 36.6 (44.4) n (n) bleed D12 <100 (6400) 7.7 (56.9) n (y) D3 <100 (bdl) 35.4 (bdl) n (n/a) Serum D7 <100 (6400) 49.1 (53.2) n (y) RPBS D12 <100 (6400) 36.4 (70.3) n (y) #1 D3 bdl (bdl) bdl (bdl) n/a (n/a) IgG D7 >10 (0.625) 46.7 (52.1) n (y) D12 >10 (0.15625) 27.0 (67.1) n (y) D3 bdl (bdl) bdl (bdl) n/a (n/a) Serum D7 <100 (<100) 27.2 (35.3) n (n) RPBS D12 <100 (1600) 19.7 (59.5) n (y) #2 D3 >10 (bdl) 0 (bdl) n (n/a) IgG D7 >10 (0.15625**) 28.6 (52.2) n (y) D12 >10 (0.039**) 21.5 (52.0) n (y)

IC dilution-1 or Rabbit Sample Sample 50 IgG concentration % inhibition (p24) Neutralization sample type day (D) (p24) Gp120 D3 100 (bdl) 58.3 (bdl) y (n/a) pre- Serum D7 <100 (<100) 23.5 (38.0) n (n) bleed D12 <100 (400) 12.6 (54.5) n (y) D3 <100 (bdl) 38.3 (bdl) n (n/a) Serum D7 <100 (<100) 25.9 (48.3) n (n) R120 D12 <100 (25600) 14.0 (52.7) n (y) #3 D3 >10 (bdl) 0 (bdl) n (n/a) IgG D7 >10 (2.5) 33.4 (54.1) n (y) D12 <100 (0.0098) 0 (56.7) n (y) D3 400 (bdl) 69.84 (bdl) y (n/a) Serum D7 100 (100) 53.2 (53.8) y (y) R120 D12 <100 (25600) 0 (50.6) n (y) #4 D3 bdl (bdl) bdl (bdl) n/a (n/a) IgG D7 0.0098 (0.0098) 57.0 (57.4) y (y) D12 >10 (>10) 13.3 (45.6) n (n)

279 Table D2. continued IC dilution-1 or Rabbit Sample Sample 50 IgG concentration % inhibition (p24) Neutralization sample type day (D) (p24) Nterm D3 400 (bdl) 53.2 (bdl) y (n/a) pre- Serum D7 <100 (6400) 42.5 (60.4) n (y) bleed D12 <100 (1600) 8.0 (58.4) n (y) D3 bdl (bdl) bdl (bdl) n/a (n/a) Serum D7 102400 (102400) 60.9 (60.9) y (y) D12 <100 (6400) 19.4 (55.9) n (y) RNt #5 D3 bdl (bdl) bdl (bdl) n/a (n/a) IgG D7 0.0098 (0.0098) 73.7 (67.4) y (y) D12 >10 (>10) 22.4 (47.6) n (n) D3 bdl (bdl) bdl (bdl) n/a (n/a) Serum D7 <100 (<100) 20.9 (45.0) n (n) D12 <100 (400) 0 (54.8) n (y) RNt #6 D3 2.5 (bdl) 65.7 (bdl) y (n/a) IgG D7 >10 (>10) 0 (0) n (n) D12 >10 (0.625) 5.9 (58.4) n (y)

IC dilution-1 or Rabbit Sample Sample 50 % inhibition IgG concentration Neutralization sample type day (D) (p24) (p24) D3 bdl (bdl) bdl (bdl) n/a (n/a) Scrambled Serum D7 <100 (<100) 0 (0) n (n) pre-bleed D12 <100 (<100) 9.0 (0) n (n) D3 <100 (dbl) 39.0 (bdl) n (n/a) Serum D7 <100 (<100) 0 (0) n (n) D12 <100 (102400**) 13.3 (60.1) n (y) RScr #7 D3 bdl (bdl) bdl (bdl) n/a (n/a) IgG D7 >10 (>10) 0 (0) n (n) D12 >10 (>10) 34.8 (48.0) n (n) D3 bdl (bdl) bdl (bdl) n/a (n/a) Serum D7 <100 (<100) 0 (0) n (n) D12 <100 (<100) 34.0 (46.9) n (n) RScr #8 D3 bdl (bdl) bdl (bdl) n/a (n/a) IgG D7 >10 (>10) 0 (0) n (n) D12 >10 (>10) 27.6 (40.6) n (n) * are based on OD405 values, ** did not dilute out a. based on concentration of p24, derived from standard curve, in µg/ml IgG b. at lowest neutralizing titre or highest % neutralization ever reached across tested dilutions c. % inhibition by p24 concentration d. bdl = below detection levels

280 In the same donor, 074, the rabbit samples were tested for neutralization of Bal HIV. The first block of Table D2 shows the neutralizations by the PBS-immunized samples. There appears to be higher levels of non-specific neutralization by these samples. The pre- bleed day twelve, RPBS 1 serum and IgG days seven and twelve and RPBS #2 serum day

12 and IgG days 7 and twelve are all positive for >50% neutralization, although the is only reflected in the calculated p24 concentrations and not the OD values. Since these animals were not introduced to HIV antigens, this neutralization panel indicates that HIV

Bal neutralizations have a high background, with spontaneous neutralizations from 52-

70% reduction in growth, when represented as concentration of p24 detected. The second block of Table D2 shows the neutralization of HIV Bal in D074 by gp120- immunized samples. Again, there is some background neutralization of virus by the pre- bleed serum samples at days three and twelve (at dilutions of 0.1 and 2.5x10-3, respectively). R120 #3 serum shows neutralization responses at day 12 at a dilution of

3.9x10-5. The R120 #3 IgG neutralization on day twelve reflects this with >50% neutralization seen at 0.0098 µg/ml of antibody. There was also some IgG day seven neutralization at 2.5 µg/m. While these neutralizations must be taken with a grain of salt considering the background neutralization level, it is noteworthy that the serum neutralization is seen at a dilution 2 logs lower than the background neutralization level.

The R120 #4 serum samples show neutralization at all three harvest days (day three at

2.5x10-3, day seven at 0.1, and day twelve at 3.9x10-5). The R120 #4 IgG was positive only at day seven, also following the trend that the serum neutralizations are more potent than the IgG neutralizations. Block three of Table D2 show the neutralization profile of

Nterm peptide-immunized samples. As with the PBS neutralization profile, there is some

281 seemingly some random neutralization events across all samples. All three harvest days of pre-bleed samples exerted some non-specific neutralization (day three at 2.5x10-3 dilution, day seven at 1.6x10-4 dilution, and day twelve at 6.3x10-4 dilution). Day seven of RNt #5 serum neutralized at 9.8x10-6. This is very strong neutralization and is reflected in the IgG day seven sample neutralization at 9.8x10-3 µg/ml. This result, neutralization on day seven by RNt #5 serum and IgG, was also seen in the D074 IIIB infections RNt #5 serum sample. Together, these data do not confirm that the Nterm epitope elicited neutralizing antibodies, but does suggest that there may be some level of protection afforded. This is why the rabbit samples need to be tested for neutralization with the same viruses, in another donor. Some inhibition by RNt #6 serum (day twelve at

2.5x10-3 dilution) and IgG (days three at 2.5 µg/ml and twelve at 0.6 µg/ml) but none of these neutralizations are very potent and only the day three IgG neutralization was reflected in the IIIB D074 test. Block four of Table D2 shows the neutralization profile of the Scrambled peptide-immunized samples. In this panel, there is one measurable instance of non-specific neutralization on day twelve by RSc #7 (at 9.8x10-6). This second study of the neutralization of Bal virus in donor 074 PBMC had more background inhibition, but nonetheless re-enforced that gp120 was able to induce neutralizing antibodies in rabbits. Also, these data hint that there may be some neutralization by RNt

#5 in for both IIIB and Bal.

282 Table D3. PBMC neutralizations with HIV IIIB and D039. HIV-1 IIIB was pre- incubated with sample and a MOI of 0.1 was tested for growth on D039 PBMC. Neutralization-positive results are bolded. IC dilution-1 or Rabbit Sample Sample 50 % inhibitionb IgG concentration Neutralization sample type day (D) (p24)c (p24)a D3 >10 (>10) 35.6 (17.8) n (n) RPBS IgG D7 >10 (adld) 0 (adl) n (n/a) #1 D12 >10 (adl) 0 (adl) n (n/a) D3 <100 (25600) 38.9 (54.7) n (y) Serum D7 <100 (<100) 2.1 (0) n (n) RPBS D12 <100 (<100) 0 (0) n (n) #2 D3 >10 (>10) 0 (36.0) n (n) IgG D7 >10 (>10) 5.6 (44.65) n (n) D12 >10 (>10) 0 (0) n (n)

IC dilution-1 or Rabbit Sample Sample 50 % inhibition IgG concentration Neutralization sample type day (D) (p24) (p24) Gp120 D3 <100 (<100) 29.8 (39.1) n (n) pre- Serum D7 <100 (adl) 18.8 (adl) n (n/a) bleed D12 1600 (1600) 68.2 (88.2) y (y) D3 102400 (102400) 59.7 (64.0) y (y) Serum D7 <100 (adl) 4.0 (adl) n (n/a) D12 6400 (6400) 89.5 (83.5) y (y) R120 #3 D3 0.0098 (0.0098) 91.6 (53.8) y (y) IgG D7 >10 (adl) 0 (adl) n (n/a) D12 0.15625 (0.0391) 61.6 (80.8) y (y) D3 102400 (102400) 71.8 (74.7) y (y) Serum D7 <100 (adl) 42.0 (adl) n (n/a) D12 100 (100) 69.4 (60.7) y (y) R120 #4 D3 0.15625 (0.039) 62.6 (64.4) y (y) IgG D7 >10 (adl) 38.6 (adl) n (n/a) D12 >10 (>10) 14.4 (25.1) n (n)

283 Table D3. continued IC dilution-1 or Rabbit Sample Sample 50 % inhibition IgG concentration Neutralization sample type day (D) (p24) (p24) D3 <100 (<100) 37.9 (45.8) n (n) Nterm Serum D7 <100 (adl) 21.3 (adl) n (n/a) pre-bleed D12 <100 (adl) 0 (adl) n (n/a) D3 <100 (<100) 26.2 (37.7) n (n) Serum D7 <100 (adl) 29.5 (adl) n (n/a) D12 <100 (adl) 0 (adl) n (n/a) RNt #5 D3 >10 (>10) 0 (0) n (n) IgG D7 >10 (adl) 26.0 (adl) n (n/a) D12 >10 (adl) 0 (adl) n (n/a) D3 102400 (<100) 52.6 (31.6) y (n) Serum D7 <100 (adl) 0 (adl) n (n/a) D12 <100 (adl) 0 (adl) n (n/a) RNt #6 D3 0.0098 (>10) 61.1 (44.4) y (n) IgG D7 >10 (adl) 0 (adl) n (n/a) D12 >10 (adl) 0 (adl) n (n/a)

IC dilution-1 or Rabbit Sample Sample 50 % inhibition IgG concentration Neutralization sample type day (D) (p24) (p24) D3 25600 (25600) 69.8 (56.4) y (y) Scrambled Serum D7 <100 (adl) 0 (adl) n (n/a) pre-bleed D12 <100 (adl) 0 (adl) n (n/a) D3 6400 (<100) 51.1 (24.0) y (n) Serum D7 <100 (adl) 0 (adl) n (n/a) D12 <100 (adl 0 (adl) n (n/a) RScr #7 D3 0.625 (>10) 61.1 (42.7) y (n) IgG D7 >10 (adl) 0 (adl) n (n/a) D12 >10 (adl) 0 (adl) n (n/a) D3 25600 (<100) 55.8 (36.4) y (n) Serum D7 <100 (adl) 0 (adl) n (n/a) D12 < (adl) 0 (adl) n (n/a) RScr #8 D3 0.0098 (>10) 62.6 (44.9) y (n) IgG D7 >10 (adl) 12.7 (adl) n (n/a) D12 >10 (adl) 0 (adl) n (n/a) * are based on OD405 values a. are based on concentration of p24, derived from standard curve, in µg/ml IgG b. at lowest neutralizing titre or highest % neutralization ever reached across tested dilutions c. % inhibition by p24 concentration d. adl = above detection limit

284 The IIIB and Bal viruses were then tested for neutralization in different donor PBMC,

D039. Table D3 shows the results from IIIB infections. The first block of Table D3 shows the neutralization profile of PBS-immunized sera and IgG. There was only one measurable instance of neutralization >50% in any sample, RPBS #1 on day three (at

3.9x10-6 dilution), which my be a non-specific background blip. The second block of

Table D3 shows the neutralization profile of gp120-immunized samples. There is some appreciable background neutralization by the pre-bleed serum, but only at day twelve (at

3.9x10-6 dilution). Both sera and IgG samples from R120 #3 showed neutralization titres at multiple days (serum – day three at 9.8x10-6 and day twelve at 1.6x10-4 dilutions; IgG - day three at 9.8x10-3 µg/ml and day twelve at between 0.04 and 0.16µg/ml). Both sera and IgG from R120 #4 were also IIIB-neutralizing (serum – day three at 9.8x10-6 and day twelve at 0.01 dilutions; IgG – day three at between 0.16 and 0.039 µg/ml). Each neutralization by the gp120-immunized sera are seen at multiple harvests, are positive by both OD and concentration p24, and are also seen in the IIIB D074 and are therefore representative of true gp120-specific neutralization. Block three of Table D3 shows the neutralizations by Nterm peptide-immunized samples. No other pre-bleed samples showed neutralization of IIIB. Unlike the D074 experiments, RNt #5 serum and IgG were unable to neutralize HIV, suggesting that the Nterm may not elicit neutralizing responses.

There were small amounts of inhibition in serum and IgG (30% and 26% reduction in growth, respectively), but these levels did not reach higher than the small amounts of inhibition by Nterm the pre-bleed serum. Block four of Table D3 shows the Bal neutralizations by Scrambled peptide-immunized samples. In this assay there was a consistent level of neutralization by all samples (pre-bleed serum, RScr #7 serum and

285 IgG, and RScr #8 serum and IgG, between 51-70% inhibition), indicating that this neutralization may be an artifact of the day 3 collection point and not represent true neutralization. The donor 039 IIIB neutralization studies support the findings that gp120- immunized rabbits express autologous neutralizing antibody but do not confirm a neutralizing role for Nterm-specific antibodies.

286 Table D4. PBMC neutralizations with HIV Bal and D039. HIV-1 Bal was pre-incubated with sample and a MOI of 0.1 was tested for growth on D039 PBMC. Neutralization- positive results are bolded. IC dilution-1 or Rabbit Sample Sample 50 % inhibitionb IgG concentration Neutralization sample type day (D) (p24)c (p24)a D3 Bdld (bdl) Bdl (bdl) n/a (n/a) RPBS IgG D7 >10 (>10) 0 (0) n (n/a) #1 D12 >10 (adle) 9.2 (adl) n (n/a) D3 <100 (<100) 0 (0) n (n) Serum D7 <100 (<100) 0 (0) n (n) RPBS D12 <100 (<100) 0 (0) n (n) #2 D3 >10 (>10) 0 (0) n (n) IgG D7 >10 (10) 20.6 (61.0) n (y) D12 >10 (>10) 34.0 (20.3) n (n)

IC dilution-1 or Rabbit Sample Sample 50 % inhibition IgG concentration Neutralization sample type day (D) (p24) (p24) Gp120 D3 Bdl (<100) Bdl (5.3) n/a (n) pre- Serum D7 <100 (<100) 0 (0) n (n) bleed D12 <100 (<100) 0 (0) n (n) D3 Bdl (400) Bdl (53.3) n/a (y) Serum D7 <100 (<100) 0 (0) n (n) D12 <100 (<100) 0 (0) n (n) R120 #3 D3 Bdl (>10) Bdl (49.3) n/a (n) IgG D7 >10 (>10) 0 (0) n (n) D12 >10 (>10) 0 (0) n (n) D3 Bdl (100) Bdl (76.0) n/a (y) Serum D7 <100 (<100) 26.7 (27.6) n (n) D12 <100 (<100) 0 (31.4) n (n) R120 #4 D3 Bdl (bdl) Bdl (bdl) n/a (n/a) IgG D7 >10 (>10) 0 (39.3) n (n) D12 >10 (>10) 12.2 (0) n (n)

287 Table D4. continued IC dilution-1 or Rabbit Sample Sample 50 % inhibition IgG concentration Neutralization sample type day (D) (p24) (p24) D3 Bdl (bdl) Bdl (bdl) n/a (n/a) Nterm pre- Serum D7 <100 (<100) 19.4 (45.8) n (n) bleed D12 <100 (<100) 0 (0) n (n) D3 Bdl (bdl) Bdl (bdl) n/a (n/a) Serum D7 <100 (<100) 13.3 (33.2) n (n) D12 <100 (<100) 0 (0) n (n) RNt #5 D3 Bdl (bdl) Bdl (bdl) n/a (n/a) IgG D7 >10 (>10) 22.4 (36.7) n (n) D12 >10 (>10) 0 (0) n (n) D3 <100 (bdl) 0 (bdl) n (n/a) Serum D7 400 (400) 76.6 (52.3) y (y) D12 <100 (<100) 0 (9.2) n (n) RNt #6 D3 >10 (bdl) 0 (bdl) n (n/a) IgG D7 2.5 (2.5) 86.5 (64.4) y (y) D12 >10 (>10) 8.9 (28.0) n (n)

IC dilution-1 or Rabbit Sample Sample 50 % inhibition IgG concentration Neutralization sample type day (D) (p24) (p24) D3 Bdl (>10) Bdl (0) n/a (n) Scrambled Serum D7 <100 (<100) 0 (0) n (n) pre-bleed D12 <100 (adl) 0 (adl) n (n/a) D3 Bdl (bdl) Bdl (bdl) n/a (n/a) Serum D7 100 (100) 71.5 (56.8) y (y) D12 <100 (100) 35.9 (50.9) n (y) RScr #7 D3 Bdl (bdl) Bdl (bdl) n/a (n/a) IgG D7 0.0098 (0.0098) 91.3 (70.3) y (y) D12 >10 (>10) 20.5 (15.6) n (n) D3 Bdl (bdl) Bdl (bdl) n/a (n/a) Serum D7 <100 (<100) 0 (0) n (n) D12 <100 (adl) 0 (adl) n (n/a) RScr #8 D3 Bdl (>10) Bdl (0) n/a (n) IgG D7 >10 (>10) 22.1 (0) n (n) D12 >10 (adl) 0 (adl) n (n/a) * are based on OD405 values a. are based on concentration of p24, derived from standard curve, in µg/ml IgG b. at lowest neutralizing titre or highest % neutralization ever reached across tested dilutions c. % inhibition by p24 concentration d. bdl = below detection levels e. adl = above detection limit

288 Bal virus was then tested for neutralization by rabbit sera and IgG in donor 039 PBMC

(Table D2). In this experiment, a sampling error limited the amount of samples that could be tested for neutralization and so the RPBS pre-bleeds and RPBS #1 sera were not tested. Therefore, block one of Table D4 shows the neutralization panel of RPBS #1 IgG and RPBS #2 serum and IgG. Only one background level neutralization >50% was seen on day seven by RPBS #2 IgG at the highest dilution, 10 µg/ml. Block two of Table D4 shows the neutralization panel of gp120-immunized samples. This is the first instance where there is almost no appreciable neutralizations. Both R120 #3 and R120 34 day three sera showed some neutralization at higher dilutions (2.5x10-4 and 0.1 dilutions, respectively). This was not reflected in the IgG compartment, but was also not seen in the pre-bleed sera samples, and therefore may represent true moderate neutralizations.

Thus, some heterologous neutralization of Bal by IIIB gp120-immunized rabbit sera was detected. The third block of Table D4 shows the neutralization profile for Bal of Nterm peptide-immunized samples. In this experiment, only RNt #6 showed some neutralization of Bal on day seven. This was reflected in both the serum (at 0.1 dilution) and IgG (at 9.8x10-3 µg/ml), but the inability of these samples to neutralization HIV consistently in the other assays suggest that this may not indicate that Nterm elicited a specific neutralizing response in this animal. Block four of Table D4 shows the neutralization profile of the Scrambled peptide-immunized samples. Very little inhibition of viral growth was seen in this grouping, but there was some detectable background neutralization >50% by RScr #7 on day seven in both the serum (at 0.1 dilution) and IgG

(at 9.8x10-3 µg/ml). These neutralizations were not replicated in RScr #8 and were not seen in the other assay, and therefore do not represent specific neutralization of HIV.

289