CHARACTERIZATION AND EVALUATION OF APPROACHES TO ELICIT
BROADLY REACTIVE NEUTRALIZING ANTIBODIES AGAINST HIV-1
by
ADAM PENN-NICHOLSON
Submitted in partial fulfillment of the requirements
For the degree of Doctor of Philosophy
Dissertation Advisors: Dr. Michael W. Cho and Dr. Michael M. Lederman
Department of Molecular Biology and Microbiology
Program of Molecular Virology
CASE WESTERN RESERVE UNIVERSITY
May, 2008
CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the dissertation of
______Adam Nicholson ______
Candidate for the Ph.D. degree *.
(Signed)____ Eric Arts, Ph.D.______(Chair of the committee)
______Michael W. Cho, PhD ______
______Michael M. Lederman, M.D ______
______Neil S. Greenspan, M.D., Ph.D.______
______Man-Sun Sy, Ph.D.______
(Date) ____Feb 22, 2008______
*We also certify that written approval has been obtained for any proprietary material contained therein.
This work is dedicated in loving memory to my grandfather, Edward Garth Penn
(1923-2004), who instilled within me a deep sense of curiosity and respect for the natural
world, and who taught me above all else to pursue my dreams, never let go, and smile
with each step
and,
to South African President Thabo Mbeki, whose unorthodox policy on AIDS in
the hardest hit nation on the planet during a time of immense crisis, drove me to pursue
my dreams even further
TABLE OF CONTENTS
List of Tables…………………………………………………………………………...…8
List of Figures………………………………………………………………………..……9
Acknowledgements………………………………………………………………………12
Abbreviations……………………………………………………………………….……14
Abstract…………………………………………………………………………………..16
CHAPTER 1:
LITERATURE REVIEW / INTRODUCTION………………………..…….………18
1.A. HIV and AIDS – History, epidemiology, impact and disease……....…19
A. History of discovery……………………………………………..19
B. Impact……………………………………………………………20
C. HIV/AIDS and disease………………………….…….....………20
1.B. Retrovirus Structure and Replication………………….………….……23
1. Nomenclature and phylogeny………………….……..………….23
2. HIV-1 Genome and Virion Structure. ……………………....…...26
3. Transmission and replication dynamics………………………….28
4. Cell Tropism. ……………………………………………………28
5. HIV-1 Lifecycle………………………………………………….30
a. Entry and Integration. ……………………………...……31
b. Production, release and dissemination…………………...31
1.C. Antiretroviral therapy…………………………………………....……..34
1.D. HIV-1 Envelope protein and receptor interactions……………….……38
1 1. HIV-1 envelope structure. ……………………………………….38
2. Mechanisms of receptor binding, conformational change……….39
and the fusion process
1.E. Vaccines and Antibodies……………………………………………….43
1. How vaccines work…………………………………….……...…43
2. B-cell biology. …………………………………………………...46
3. Broadly neutralizing antibodies………………….………………46
a. Overall antibody response………..………………………46
b. Variable regions……….…………………………………50
V3 loop. ……………………………………...……50
V1/V2 Loop………………………………………..51
V4 and V5. ………………………………………...51
c. CD4 binding site (CD4bs) ………………………………52
d. CD4-induced (CD4i) epitopes…………………...………53
e. Carbohydrate-dependent epitopes………………………..54
f. Epitopes on gp41…………………………………………55
g. Fusion Intermediate epitopes…………………….………59
h. Conclusion………………………………….……………61
1.F. Obstacles in eliciting broadly reactive neutralizing antibodies………...63
1. Overview…………………………………………………………63
2. Extensive glycosylation. …………………………...……………63
3. High antigenic variability…………………………..……………64
4. Envelope Tertiary and Quaternary Structures……………………64
2 5. Predicative Experimental Animal Models……………………….66
a. Non-human primate models……………….……………..66
b. Small animal models……………………………………..67
6. Conclusion. ……………………………………………………...68
1.G. Current Strategies to elicit broadly reactive neutralizing antibodies…..69
1. Types of Antigens. ………………………………………………69
2. Variable loop deleted and glycan-deficient envelopes…………..69
3. Fusion Intermediates. ……………………………………………70
4. Mimotopes…….…………………………………………………71
5. Center-of-tree, Consensus and Ancestral envelopes…………….72
6. Polyclonal neutralizing antibody responses………………..…….74
1.H. Summary of thesis work……………………………………………….77
CHAPTER 2:
ASSESSMENT OF ANTIBODY RESPONSES AGAINST GP41 IN HIV-1-
INFECTED PATIENTS USING SOLUBLE GP41 FUSION PROTEINS AND
PEPTIDES DERIVED FROM M GROUP CONSENSUS ENVELOPE
1. Abstract………………………………………………………………………81
2. Introduction………………………………………………………………..…82
3. Methods and Materials
Cloning GST-gp41 fusion constructs…………………………………….85
Protein Expression and Purification……………………………………...85
Immunoprecipitation and Western blot……………………………...…..87
3 ELISA……………………………………………………………………87
Neutralization Assays…………………………………………...……….88
Statistical Analyses………………………………………………………90
4. Results
Design, expression and purification of GST-gp41 fusion proteins………91
Characterization of antigenic properties of GST-gp41 fragments…….....95
Characterization of antibody responses against gp41 in HIV-1-………...97
infected patients.
Antisera from HIV-1-infected patients with stronger reactivity…………99
against gp41 MPER exhibit broader and more potent neutralizing
activity.
Identification of immunogenic linear epitopes within the………..…….106
C-terminal half of gp41 ectodomain
5. Discussion…………………………………………………………………..115
6. Acknowledgements…………………………………………………………121
CHAPTER 3:
DEVELOPMENT OF TRANSGENIC MICE EXPRESSING HIV-1 ENTRY
RECEPTORS ON B CELLS SURFACE FOR USE IN GENERATING
ANTIBODIES AGAINST SHORT-LIVED FUSION INTERMEDIATE
EPITOPES.
1. Abstract……………………………………………………………………..123
2. Introduction…………………………………………………………………124
4 3. Methods and Materials
Cell lines………………………………………………………………..132
Proteins and immunogens………………………………………………132
Plasmids………………………………………………………………...133
Cloning………………………………………………………………….133
Electroporation of Mouse A20 B-cells…………………………………134
Flow Cytometry Analysis of huCD4 and huCCR5 expression on……..134
mouse B-cells
Cell-cell fusion experiments……………………………………………135
Vaccinia Virus………………………………………………………….136
Generating Transgenic Mice……………………………………………137
Identification of Transgenic Mice………………………………………137
Establishment of Homozygous Breeding Lines……...…………………138
Mouse Immunizations……….………………………………………….138
Infection of cells with virus incubated with QS-21…..………………...139
ELISA……………………………………………………………...….. 140
Western Blot……………………………………………………………141
Hybridoma Development…………….…………………………………141
Neutralization Assays………………………..…………………………141
4. Results
Plasmid construction and in vitro expression of human CD4 and……...143
CCR5 on the surface of mouse B cells.
HIV-1 receptors expressed on the surface of mouse B- cells……...... 147
5 support membrane fusion.
Generation of Transgenic Mice…………………………………...……148
Humoral Immune responses against antigens in transgenic mice……...152
Immunization Set A…………………….………………………………155
Immunization Set B……………………….……………………………159
Immunization Set C…………………….………………………………165
Immunization Set D………………….…………………………………173
5. Discussion …………………………………………………………...……..178
6. Acknowledgements………..……………………………………………..…187
CHAPTER 4:
GENERATING HIV-1 ENVELOPES WITH ANTIGENICALLY DIVERSE V3
LOOPS: APPLICATION FOR A POLYVALENT V3 VACCINE.
1. Abstract…………………………………………………………………..…189
2. Introduction…………………………………………..…………………..…190
3. Methods and Materials
Cloning…………………….……………………………………………194
Generating polyvalent V3 loop sequences……………………….…..…195
Protein Expression…………………………………………………...…197
Western blot analysis………………………..………………………….198
Protein purification………………………….………………………….198
4. Results
Generating V3 diversity………………………………………………...200
6 Generating MCON6gp120 and outer domain……………….…...……..205
Representation of V3 loops…………………………………..………....206
Variant analysis…………………………………………………………206
Protein expression and purification…………………………………….213
5. Discussion………………………………………………..…………………215
6. Acknowledgements…………………………………………………………220
CHAPTER 5:
GENERAL DISCUSSION
1. Conclusions and future prospects for HIV-1 vaccine research……….……222
APPENDIX
Section 1: Development of HIV-1 envelope constructs with shortened ………….239
V1/V2 loops that retain fusion competence.
Section 2: gp120 Outer Domain………………………………………...…………249
LIST OF REFERENCES……………………………………………………………..252
7 LIST OF TABLES
Table 1-1: HIV-1 genes, gene products and functions……………..………...…………33
Table 1-2: Prototypic HIV-1 Neutralizing antibodies………………...………………...62
Table 2-1: Neutralizing activity of plasma samples from patient CWRU-4…….….....105
Table 4-1: Poly V3 primer-encoded amino acid sequences..……………………..…207
Table 4-2: Frequency of each amino acid in the pool of proportional and….…...…...211
equivalent polyV3 sequences.
Table 5-1: A list of all ongoing clinical trials of preventative HIV vaccines……..…225
8 LIST OF FIGURES
CHAPTER 1
Figure 1-1. Relationship of HIV-1 virus load and CD4+ T cell counts….………...……22
Figure 1-2: HIV-1 Genetic Diversity and Group M subtype global prevalence………..24
Figure 1-3: Schematic representation of HIV-1 genome and a mature HIV-1…………27
virion illustrating major viral components
Figure 1-4: Schematic representation of HIV-1 replication and target sites…………...36
for antiretroviral drugs.
Figure 1-5: HIV-1 Entry……………………………………………………..………….41
Figure 1-6: How vaccines work……………………………………………………...…45
Figure 1-7: Antibody epitopes of HIV-1 Envelope protein…………………….………48
Figure 1-8: Schematic figures of trimeric HIV-1 envelope spike showing the …….….57
location of conserved epitopes recognized by the few existing BR-NAbs.
Figure 1-9: Ancestral, Consensus and Center-of-Tree HIV-1 sequences………………76
CHAPTER 2
Figure 2-1. Construction and expression of GST-gp41 fusion proteins………………..92
Figure 2-2. Purification and immunoprecipitation analyses of GST-gp41……………..94
Fusion Proteins
Figure 2-3. Characterization of antigenic properties of GST-gp41 fusion proteins……96
Figure 2-4. Antibody responses against gp41 in individual patients………………….100
Figure 2-5. Neutralizing activity of patient plasma samples………………………….103
9 Figure 2-6. Identification of immunogenic linear epitopes targeted by……………….110
patients using overlapping peptide ELISA
Figure 2-7. Identification of immunogenic linear epitopes in gp41 by……….………113
ELISA using M group consensus peptides
CHAPTER 3
Figure 3-1: Presentation of HIV-1 fusion intermediate epitopes to surface-…………..130
bound immunoglobulins on naïve B-cells
Figure 3-2. Transgene constructs express huCD4 and huCCR5 on mouse…………...144
B-cell surfaces
Figure 3-3: Human CD4 and CCR5 expressed on mouse B cells are………………...146
fusion-competent.
Figure 3-4: Identification of transgenic mice and establishment of…………………..149
homozygous breeding lines.
Figure 3-5. FACS analyses of lymphocytes from wild type and transgenic mice…….151
Figure 3-6. Transgenic mice are immunologically competent and………………….... 153
physiologically normal
Figure 3-7: Comparison of antibody responses against HIV-1 Envelope…………….156
Figure 3-8: QS-21 effect of virus infectivity, and silver stain of AT-2 HIV-1BAL.…....160
Figure 3-9: Immunization Set B………………………………………………………162
Figure 3-10: Antibody response against recombinant trimeric envelope from a…...... 166
Consensus Group M envelope sequence
Figure 3-11: B-cell hybridomas……………………………………………………….169
Figure 3-12: Immunized mouse sera reactivity against gp41…………………………172
10 Figure 3-13: Transgenic mice develop diminished humoral response against both...... 174
HIV-1 envelope and control protein lacking the ability to bind human CD4 or CCR5
CHAPTER 4
Figure 4-1: Schematic figure detailing selection of V3 loop amino acid………...….. 201
residues required for coverage of 85% of subtype B isolates
Figure 4-2: Schematic of methodology used to generate polyvalent V3 loop……..…203
DNA sequences.
Figure 4-3: V3 variant schematic, PCR products and gp120 expression………….….209
Figure 4-4: Table and graph showing percentage of polyV3 variants in...... …212
proportional and equivalent groups with amino acid mutations
at each of nine possible residue positions of consensus subtype B V3
sequence targeted in our study
groups showing amino acid mutations at each of nine residue positions
of consensus subtype B V3 sequence targeted in our study.
Figure 4-5: Purification of polyV3 gp120H and OD……………………………….....214
CHAPTER 5
Figure 5-1: HIV-1 envelope evolution showing virus tropism and parallel…………236
changes in sensitivity of those viruses to neutralizing antibody.
APPENDIX
Figure Appendix-1: V1/V2 deletion…………………………………………………245
Figure Appendix-2: V1 and V2 loop deletion mutant fusion competence……….…247
Figure Appendix-3: Structure of gp120 and gp120 outer domain…………………..251
11 ACKNOWLEDGEMENTS
I am deeply indebted to my mentors, Michael W. Cho and Michael M. Lederman for their inspiration, motivation and patience. None of this would have been possible without your continued support and interest in my development as a scientist and a human being. I’d also like to thank my committee members and lab members who have contributed to this work and have played an integral part in my scientific endeavors. I’d like to thank the Department of Molecular Biology and Microbiology, the Center for
AIDS Research and Case Western Reserve University for giving me an opportunity to work in such a world-class institution.
All my gratitude must go to my Mum and Dad, Claire and Edward, who raised me against the odds through some turbulent times, and who never ceased to remind me I was deeply loved. Without the support, freedom and trust you have given me, none of this work would have been possible. I’d also like to thank each of my grandparents who did such a splendid job of raising each of my parents through what I’m sure were even more taxing situations. Your presence is greatly missed. And I’d like to thank my younger brother, Simon, who, in his own right, has raised both my parents and me.
I’d like to thank my many supportive friends in Cleveland, especially the interesting group of folk from the far reaches of the globe that I’ve been fortunate enough to live with over the years, especially on Thursdays. Particular gratitude must be extended to George and Becky Dunn for their immense support and friendship.
Ultimate respect and consideration must be extended to the millions of lab animals who have selflessly given their lives in the name of scientific advancement and
12 mankind’s pursuit of better health. To say we owe our lives, or at very least our improved health, to these animals is an understatement.
Finally, if it were not for Jill King, my life wouldn’t be near as sunny. Thank you, Jill, for your amazing patience, perseverance, friendship and love. Together, our prospects on the horizon ahead look warm and exciting.
13 LIST OF ABBREVIATIONS
Abs Antibodies AIDS Acquired immunodeficiency syndrome AT-2 Aldrithiol-2 B-cell B lymphocytes -gal -galactosidase BR-NAbs Broadly Reactive Neutralizing Antibodies C1-C5 Constant regions 1-5 CA Capsid CD4, 8, 19 Cluster of Differentiation-4, 8, 19 etc. CD4bs CD4 binding site CD4i CD4 induced CDR H3 Third complementarity-determining region of antibody heavy chain CRF Circulating Recombinant Form CCR5 CCR5 C-C Chemokine Receptor-5 CHO Chinese Hamster Ovary Cell CMV Cytomegalovirus CpG ODN Adjuvant of Cytosine-phosphate-Guanine oligodeoxynucleotide repeat sequence CTL Cytotoxic T-lymphocyte CXCR4 CXC (Chemokine) receptor-4 DC-SIGN Dendritic cell-specific intercellular adhesion molecule (ICAM)- grabbing non-integrin DMEM Dulbecco’s modified Eagle’s medium EDTA Ethylenediaminetetra – acetic acid ELISA Enzyme-Linked Immunosorbent Assay Env Envelope glycoprotein Fab Antibody binding fragment FITC Fluorescein isothiocyanate Gag Group-specific antigen GFP Green fluorescent protein GM-CSF Granulocyte macrophage colony- stimulating factor GST Glutathione S-Transferase gp41/120/160/140 Envelope Glycoprotein components HAART Highly Active Antiretroviral Therapy HIV-1 Human immunodeficiency virus type 1 HR1, 2 Heptad Repeat region 1, 2 Hu Human IAVI International AIDS vaccine initiative IN Integrase Ig Immunoglobulin LacZ Lac operon encoding -gal LANL Los Alamos National Laboratory LTR Long Terminal Repeat LTNP Long term non-progressor Luc Luciferase ID50 50% inhibitory dose I.P. Intraperitoneal I.S. Intrasplenic I.V. Intravenous MA Matrix mAb Monoclonal Antibody
14 MHC Major histocompatibility complex MIP-1 Macrophage Inflammatory Protein-1 MOI Multiplicity of infection MPER Membrane-proximal external region Mu Murine MuLv Murine leukemia virus MVA Modified vaccinia Ankara NAb Neutralizing antibodies NC Nucleocapsid Nef Negative factor NIH National Institute of Health NNRTI Non-Nucleoside Reverse Transcription Inhibitor NRTI Nucleoside Reverse Transcription Inhibitor OD gp120 outer domain PAGE Polyacrylamide gel electrophoresis PBL Peripheral blood lymphocytes PCR Polymerase chain reaction PE Phycoerytherin PerCP Peridinin chlorophyl protein PFU Plaque forming units PI Protease Inhibitor p.i. Post-immunization PND Principle neutralizing determinant of V3 PR Protease QS-21 Saponin adjuvant derived from Quillaja saponaria R5 Chemokine receptor CCR5 RANTES Regulated Upon Activation Normally T-cell Expressed and Secreted Rev Regulatory viral protein RLU Relative light units RT Reverse transcriptase S.C. Subcutaneous scFv Single-chain variable fragment antibody SDF-1 Stromal Derived Factor-1 SHIV Simian/human immunodeficiency virus, chimeric virus SIV Simian immunodeficiency virus SU Surface unit T-20 Enfuvirtide, Fuzeon Tat Transactivator T-cell T lymphocytes TM Transmembrane TCLA T-cell line adapted UNAIDS Joint United Nations Program on HIV/AIDS V1-V5 Hypervariable region 1-5 Vif Viral infectivity factor Vpr Viral protein r Vpu Viral protein u VLP Virus-Like Particle VV Vaccinia virus VSV-G Vesicular Stomatitis Virus G Glycoprotein WHO World Health Organization WT Wild type X4 Chemokine receptor CXCR4 X-Gal 5-bromo-4-chloro-3-indolyl-D-galactopyranoside
15
Characterization and evaluation of approaches to elicit Broadly Reactive
Neutralizing Antibodies against HIV-1
Abstract
by
ADAM PENN-NICHOLSON
Despite 25 years of research, an HIV-1 vaccine remains elusive. Eliciting broadly reactive-neutralizing antibodies (BR-NAbs) against HIV-1 envelope glycoprotein is hindered by high sequence variability, epitope masking, decoy epitopes and low immunogenicity. Accurate assessment of the immunogenic properties of envelope is an important goal towards designing antigens capable of eliciting BR-Nabs. This body of work consists of three projects aimed at characterizing humoral immune responses in
HIV-1 infected patients, and evaluation of novel approaches to elicit BR-NAbs. In the first component, five soluble GST-fusion proteins were generated, each encompassing fragments of gp41 ectodomain, an important vaccine target for several BR-NAbs. These proteins allowed characterization of patient antibody response profiles against different regions of gp41, and neutralizing activity was compared among patients with variant responses. We identified several patients who mounted antibodies against epitopes near or overlapping with those targeted by BR-NAbs 2F5 or 4E10, suggesting such antibodies may not be as rare in patients as has previously been thought.
16 Today, vaccines target either conserved structures common across all viruses, or diverse neutralizing domains to generate a broad polyclonal response. During HIV-1 entry, envelope protein undergoes major conformational changes, exposing conserved epitopes. In the second project, we generated two transgenic mouse lines expressing human CD4 with or without CCR5 on the surface of murine B-cells. These mice were used to target humoral responses against conserved fusion-intermediate structures of envelope exposed transiently during receptor binding events.
Lastly, we hypothesize that polyclonal antibodies against multiple epitopes from diverse viral strains will exhibit greater breadth of neutralizing activity against HIV-1 isolates than any single immunogen. In this study, a novel technique was established to generate considerable diversity within the hypervariable V3 sequence, representing the majority of circulating subtype B HIV-1 variants. These polyvalent V3 sequences were represented either proportionally or equivalently to the observed frequency of V3 amino acids in primary isolates. This diversity is extensive yet specific, and can be easily manipulated.
Taken together, the panel of soluble gp41-fusion proteins, transgenic mice and polyvalent V3 antigens will be important tools for better understanding humoral immune responses against envelope and for future vaccine development.
17
CHAPTER 1
LITERATURE REVIEW
AND
INTRODUCTION
18
CHAPTER 1: INTRODUCTION
1.A. HIV and AIDS – History, epidemiology, impact and disease.
1.A.1, History of discovery: The first identification of patients presenting symptoms of what is today known as Acquired Immunodeficiency Syndrome (AIDS) occurred in Los Angeles in 1981. On June 5th, 1981, the Center for Disease Control and
Prevention issued a report in MMWR of several young, sexually active previously healthy homosexual men with by Pneumocystis carinii pneumonia (PCP), and reported case histories suggesting "cellular-immune dysfunction related to a common exposure" and a "disease acquired through sexual contact" (MMWR, 1981). It was not until 1983 that Luc Montagnier’s group at the Pasteur Institute in France discovered the responsible retrovirus (Barre-Sinoussi et al., 1983), and later verified by Robert Gallo and Jay Levy’s groups (Gallo et al., 1984; Levy et al., 1984; Popovic et al., 1984; Sarngadharan et al.,
1984; Schupbach et al., 1984). Initially given three different names (lymphadenopathy- associated virus (LAV), human T lymphotropic virus type III (HTLV-III), AIDS- associated retrovirus (ARV)), the retrovirus was renamed Human Immunodeficiency
Virus (HIV) in 1986 (Coffin et al., 1986). The first antiretroviral drug, azidothymidine
(AZT), became publicly available in 1987, but did little to prevent the course of disease except to delay the onset of AIDS. By 1996, combination therapy with additional drugs that target and inhibit HIV-1 replication at multiple sites was introduced. Highly Active
Anti-Retroviral Therapy (HAART) has done much to delay the onset of AIDS and boost the quality of life of HIV-1 infected patients, but drug-resistant mutant viruses inevitably arise and as such, no cure to AIDS yet exists.
19
1.A.2. Impact: Today, the HIV-1 pandemic has infected about 33 million people
worldwide, and without intervention, 4.1 million new infections can be expected each
year (UNAIDS 2007 AIDS epidemic update). Most of these infections occur in
developing nations where access to antiretroviral drugs is limited. The largest burden is
carried by Sub-Saharan Africa with over 64% of the global HIV-1 infection cases
occurring here, with some nations showing infection prevalence of over 30% of the
population, predominantly in young adults and children. The social and economic impact
is devastating. With poor nations ill-equipped to deal with the effects of the pandemic
and unable to afford expensive antiretroviral drugs without assistance, there is a dire need to prevent infection and develop a vaccine against HIV-1.
1.A.3. HIV/AIDS and disease: HIV-1 induced immunodeficiency is characterized by a depletion of CD4+ T-cells and the development of opportunistic infections that eventually lead to chronic disease and death (Figure 1-1). AIDS is classically defined as a state when cellular immunity declines as a result of CD4+ T-cells dropping below 200 cells/μl of blood, leaving the individual susceptible to opportunistic infections. After HIV-1 infection, the median time of progression to AIDS is nine to ten years, but can vary widely among individuals from a few months to 20 years or more.
Both host factors, such as age or genetic differences among individuals, and viral factors such as the rate of replication and susceptibility to immune and drug control of the individual strain of virus influence the rate and severity of HIV disease expression in different people (Fauci, 1993; Pantaleo, Graziosi, and Fauci, 1993).
Other factors such as co-infection with other microbes and environmental factors may also have important contributions. The first cellular targets of virus replication are
20
memory CD4+ T cells that are mostly found in the mucosa of gut–associated lymphoid
tissue (GALT) (Li et al., 2005b; Mattapallil et al., 2005; Veazey and Lackner, 2005).
Within days of infection, massive numbers of memory T-cells are selectively infected
and killed by direct, virally mediated destruction. The acute phase of infection is
characterized by a rapid increase in viral replication and wide dissemination of virus, and a correlative decline in CD4+T cells in the peripheral blood. This decrease is probably a result of both HIV-1 mediated cell killing and re-trafficking of infected cells to lymphoid tissue, but the GALT remains a major site for infection and killing of newly generated
CD4+ T cells. With immune control, the asymptomatic phase of clinical latency establishes as steady-state level of viremia and a rebound of circulating CD4+ T-cells
amidst chronic high-level virus production. Persistent HIV infection and replication in
mucosal and lymphoid tissues, driven by continual turnover of memory CD4+ T cells in the GALT is likely to be the major source of viral replication, persistence and continual
CD4+ T cell loss in HIV-infected individuals. With disease progression and the onset of
AIDS, CD4+ T- cells decline substantially, viremia increases and opportunistic infections
set in, leading ultimately to death.
21
Figure 1-1. Relationship of HIV-1 virus load and CD4+ T cell counts over time following HIV-1 infection. HIV-1 RNA (copies per ml of plasma) is represented in red, and CD4 T cell count (per ml of venous blood) is shown in blue. After exposure, an acute phase of infection occurs when rapid viral replication results in a dramatic decrease of CD4+ T cells, particularly evident in the mucosal tissue of the GALT. CD8+ T cells help to control infection by killing infected cells, and within 6 weeks, humoral immune responses result in production of antibodies in the infected individual. The immunological control of viremia results in a chronic viral load set point, and CD4+ T cell counts rebound. The asymptomatic phase can last for months to years. Chronic immune activation and CD4+ T- cell turnover eventually lead to a decrease of CD4+ T cells and a rise in viral load which eventually results in AIDS, opportunistic infections and death.
22
1.B. Retrovirus Structure and Replication
1.B.1. Nomenclature and Phylogeny: HIV and Simian Immunodeficiency
Virus (SIV) are species within the genus of lentivirus, of the family Retroviridae, viruses of which are characterized by the requirement to reverse transcribe their single-stranded
RNAs into double stranded DNAs to allow infection, and their long incubation period, persistent infection and ability to infect non-dividing cells. Cells can be actively or latently infected. Two species of HIV infect humans: HIV-1 and HIV-2. HIV-1 is considerably more pathogenic than HIV-2 and escapes immune control easier leading to higher viral loads. Although both HIV-1 and HIV-2 are sexually transmitted, the higher viral load of HIV-1 likely contributes to a greater frequency of transmission and, as such, contributes to the majority of global infections (Reeves and Doms, 2002). HIV-1 is further divided into Group M (main), Group O (outlier) and Group N (non-M –non-O) clades, based on genetic divergence (Figure 1-2). Group M HIV-1 is further divided into multiple subtypes based on 30% and 14% intersubtype genetic divergence in the env and gag regions, respectively (Hu et al., 1996).
Group M subtypes are classified and named A, B, C, D, F, G, H, J and K with circulating recombinant forms named CRF01 through CRF04. These clades and subtypes have some degree of geographical association, with HIV-1 Group M subtype B being the most widely distributed across the globe, and subtype C being the most predominant form globally, accounting for 47.2% of all infections (East and Southern Africa, India). The geographic distribution of circulating HIV-1 subtypes is of particular importance in the development of regional vaccine candidates and HIV diagnostics.
23
A.
B.
Figure 1-2: HIV-1 Genetic Diversity and Group M subtype global prevalence.
24
Figure 1-2: HIV-1 Genetic Diversity and Group M subtype global prevalence.
(A) Primate lentivirus phylogenetic tree illustrating relationships between HIV-1 (M, N and O groups), HIV-2 and SIV and between subtypes of HIV-1 group M. The pol gene
sequence was used to illustrate genetic relationships as it is relatively conserved. The
small arrows indicate where the sequence would branch in an env gene reconstruction.
Figure adapted from (Kuiken et al., 1999). (B) Global HIV-1 Group M Diversity. The
frequency of each HIV-1 subtype and recombinant form was estimated in each country
based on published findings. Countries are color-coded based on the dominant HIV-1
group main (M) subtype. The countries coloured grey have a low level of HIV-1
prevalence or were not represented in the scientific literature related to HIV-1 subtype
prevalence. The pie charts depict the proportion of each subtype or recombinant form in
each geographical region. The size of the pies is proportional to the number of HIV-1
infected individuals in that particular region. (Figure adapted from (Arien, Vanham, and
Arts, 2007)).
25
1.B.2. HIV-1 Genome and Virion Structure: The HIV-1 genome consists of two identical, single stranded, positive-sense RNAs of approximately 9.3 kb which encode for the viruses nine genes in three overlapping reading frames (Figure 1-3). These nine genes lie between two Long Terminal Repeat (3’ LTR and 5’ LTR) promoter sequences, partly regulated by the HIV-1 Tat protein. Two genes (gag and env) are responsible for production of structural proteins, one (pol) for enzymatic proteins and the six remaining regulatory and accessory genes (tat, rev, vif, vpr, vpu and nef) encode proteins involved in viral infection and replication (Table 1-1).
The two RNA copies are bound to nucleocapsid proteins (p7, NC) and, with enzymes reverse transcriptase (p66/p51, RT), protease (p15, PR), ribonuclease (RNAse H) and intergrase (p31, IN), are enclosed within the conically shaped core capsid structure (p24,
CA). A matrix (p17, MA) surrounds the capsid, and allows membrane anchoring with the outer phospholipid bilayer membrane, or envelope, that is derived from the human host cell as virus particles bud from the cell. Embedded in the viral membrane are envelope proteins or spikes, consisting of trimers of gp120/gp41 heterodimer glycoproteins (gp120 surface protein and gp41 transmembrane protein). Each virion has approximately 10-15 envelope spikes that are distributed in what appears to be clusters across the viral membrane (Zanetti et al., 2006; Zhu et al., 2003). The entire virion is approximately 110nm in size, spherical but somewhat pleomorphic.
26
Figure 1-3: Schematic representation of HIV-1 genome and a mature HIV-1 virion
illustrating major viral components. The HIV-1 genome encodes nine genes in three
overlapping reading frames. The double stranded viral RNA is associated with
nucleocapsid and encased by the viral capsid core along with the enzymes reverse
transcriptase and integrase. Matrix protein lies beneath the viral envelope derived from
the host cellular lipid membrane. This membrane is studded with trimeric envelope protein, or spikes, consisting of transmembrane glycoprotein gp41 and surface envelope glycoprotein gp120.
27
1.B.3. Transmission and replication dynamics: HIV-1 is spread from one individual to another through contact with contaminated blood, semen, vaginal fluid or breast milk. Unprotected sexual intercourse accounts for the predominant mode of transmission(Walker et al., 2003), although vertical transmission from an infected mother to her child, either through childbirth or breastfeeding, is the major cause of infection in children worldwide. Although screening of blood supplies for virus has eliminated transmission through blood transfusion, the sharing of needles by injection drug users plays a significant role in transmission in several developed nations (UNAIDS 2006
Report on the global AIDS epidemic).
The primary cellular targets of HIV-1 include CD4+ T-cells and macrophages.
Although HIV-1 is primarily transmitted through vaginal and rectal mucosa during sexual intercourse, the first major cellular targets for HIV-1 infection are memory T-cells bearing CD4 and CCR5 found in high numbers in the mucosa of the gut-associated lymphoid tissue (GALT). Infection of GALT cells leads to rapid depletion of CD4 T- cells and is a major site of viral replication(Guadalupe et al., 2003; Li et al., 2005b;
Mattapallil et al., 2005; Mehandru et al., 2007; Veazey and Lackner, 2005). This decrease in the number of CD4 T-cells is achieved through direct viral killing of the infected cell, increased cellular apoptosis and through killing of infected cells by HIV-1 specific CD8+ cytotoxic T lymphocytes. Continuous viral replication and the resultant chronic immune stimulation eventually lead to an exhaustion of CD4 T-cell turnover.
Loss of CD4 T-cells leads towards immune dysfunction, the onset of AIDS related diseases and ultimately results in death.
1.B.4. Cell Tropism: In 1984, the retrovirus linked as the etiological agent of AIDS was discovered to utilize T4 (or CD4) as the major receptor for entry of human T-
28
lymphocytes (Dalgleish et al., 1984; Klatzmann et al., 1984). Yet, expression of human
CD4 on the cell surface was not sufficient to permit HIV entry into mouse cells or murine-human hybrid cells (Jasin, Page, and Littman, 1991; Tersmette et al., 1989;
Weiner et al., 1991), suggesting interplay with a possible secondary factor involved in entry events. In addition, while all HIV-1 strains replicated well in primary T-cell cultures, not all viruses had the ability to replicate in both T-cell lines and macrophage cultures, demonstrating some cellular specificity, possibly defined by an entry coreceptor.
Viruses with the ability to grow in T-cell lines were observed to form giant multinucleated cells, called syncitia. Thus, viruses were classified upon their ability to grow in T-cell lines (T-tropic) or in macrophages (M-tropic), or in both (dual-tropic).
It was only when soluble factors secreted from CD8 T-cells (Brinchmann,
Gaudernack, and Vartdal, 1990) showed inhibition of virus replication that some clues arose to the identity of the receptor. In 1995, these suppressive factors were identified as the chemokines RANTES, MIP-1 and MIP-1 (Arenzana-Seisdedos et al., 1996;
Cocchi et al., 1995), and SDF-1 (Bleul et al., 1996). Shortly after this finding was reported, chemokine receptors CCR5 (the major receptor for ligands RANTES, MIP-1 and MIP-1) and CXCR4 (receptor for ligand SDF-1) were identified as the key coreceptors for HIV-1 entry (Alkhatib et al., 1996; Choe et al., 1996; Deng et al., 1996;
Dragic et al., 2000; Feng et al., 1996). In rare circumstances, other chemokine receptors such as CCR2b, CCR3, CCR7, CCR8, STRL33/BONZO, gpr15/BOB may facilitate HIV entry (Unutmaz, KewalRamani, and Littman, 1998), but CXCR4 and CCR5 are considered the critical coreceptors for viral entry in human infection.
For reasons poorly understood, transmission of T-tropic virus using CXCR4 for cell entry (X4-tropic) is particularly poor. Almost all new infections are caused by HIV using
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CCR5 as the coreceptor (R5 tropic virus). During the course of infection, about 50% of patients’ virus population switch from using CCR5 to those using CXCR4 as coreceptor for infection (Koot et al., 1993; Schuitemaker et al., 1992). This tropism switch is associated with a more rapid CD4 T-cell loss and the progression towards AIDS (Connor et al., 1997; Schuitemaker et al., 1992; Tersmette et al., 1988), although it is unclear whether the tropism switch is the cause or consequence of immune deterioration (Koot et al., 1993). Several viruses can use both CCR5 and CXCR4 for cell entry, and are termed dual-tropic, or R5X4 viruses.
The astounding discovery of a group of individuals considered at high risk for infection who remained persistently seronegative, led to identification of a 32 base pair deletion in the CCR5 gene (CCR532) that resulted in the chemokine receptor not being expressed on the cell surface(Liu et al., 1996). Individuals who are homozygous for this mutation are not infected by HIV using CCR5 as a receptor but can, on rare occasions, become infected by X4-tropic virus. CCR532 homozygous individuals lacking expression of CCR5 appear healthy and show no obvious unfavorable phenotype, except for a recently observed increased susceptibility to serious infection by West Nile
Virus(Glass et al., 2006). Nevertheless, given that almost all new infections are caused by R5 tropic virus, in conjunction with the fact that CCR5 appears to be largely dispensable, CCR5 has become a major target for compounds that act as HIV-1 entry inhibitors.
1.B.5. HIV-1 Lifecycle: This section acts as a general overview of HIV-1 lifecycle and replication events. Sections relating to viral entry and interactions between envelope protein and cellular receptors are discussed in far greater detail in Chapter 1.D.2.
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1.B.5.a. Entry and Integration: Entry of the virus is mediated by binding of the envelope glycoprotein to the primary receptor CD4 (Dalgleish et al., 1984; Klatzmann et al., 1984) and subsequent binding to chemokine receptor CXCR4 or CCR5, found on the surface of both CD4+ T-cells, and macrophages and monocytes (Alkhatib et al., 1996;
Dragic et al., 2000; Feng et al., 1996). Both CCR5 and CXCR4 are members of the G protein-coupled receptor superfamily. Following penetration of the cellular membrane by gp41, viral and cellular membranes fuse, allowing the virus capsid core to enter into the cytoplasm of the target cell. The capsid uncoats and releases the + sense viral RNA and reverse transcriptase copies it into a complementary – sense DNA, which is further copied into + sense DNA by the DNA-dependent DNA polymerase activity of RT. The double-stranded DNA intermediate migrates across the cytoplasm, crosses the nuclear membrane in a process known as nuclear translocation and is randomly integrated into the host genome by the enzyme intergrase, to yield a stable proviral genome.
1.B.5.b. Production, release and dissemination: At this stage, the provirus may lie dormant and establish a latent infection, which may escape immune detection, or may be activated and establish productive infection characterized by extensive virus replication. Host transcription factors activate transcription from the LTR promoter, leading to production of short transcripts which are blocked from further elongation until
HIV-1 encoded transactivator protein Tat, removes this block. Tat binds to a secondary structure of LTR RNA known as the trans-activation response element (TAR), and recruits the cellular transcription elongation factor b (pTEFb) complex including cyclin
T1 and CDK9. pTEFb phosphorylates RNA pol II, allowing the production of full-length viral RNA transcripts, which are spliced and exported (Karn, 1999). The Rev protein plays an important role in export of full-length RNA out of the nucleus by binding to a
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secondary RNA structure of transcripts known as the Rev response element (RRE). From these spliced and unspliced transcripts, HIV-1 accessory proteins (Vif, Vpu, Nef, Vpr), regulatory proteins (Tat, Rev), structural proteins (Gag, Env), and enzymatic proteins
(Pol) are generated (Table 1-1). Some transcripts serve as new viral genomes, bind to gag and are packaged into the virion as it is assembled at the host inner-membrane surface. Viral envelope glycoproteins are generated in the endoplasmic reticulum, transported to the Golgi complex where gp160 is cleaved by the endoproteases, furin, into surface unit gp120 and transmembrane glycoprotein gp41 (Moulard and Decroly,
2000). Envelope proteins are transported to the plasma membrane where they occur on the membrane surface as trimers, forming spikes. Gag and gag-pol associate at the membrane where gag multimerizes, incorporates viral RNA and buds from the plasma membrane. As the immature virus particle buds, it envelops its spherical core within the host plasma membrane containing the viral envelope spikes. Alternatively, cells bearing envelope proteins may fuse to other receptor-bearing T-cells to form syncitia. Following virion budding, HIV-1 protease, a gene product of pol, cleaves the polyproteins into individual, functional HIV-1 proteins and enzymes to yield a mature HIV-1 particle with a conical core that can disseminate and infect subsequent cells or be transmitted to a new host. The remaining infected host cell either undergoes lysis due to extensive viral replication, is targeted for destruction by CD8+ cytolytic T-cells, or in the case of latently infected memory cells, serve as a latent viral reservoir that escapes immune defenses and anti-retroviral drugs.
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Name Size Function
Structural genes
Gag p55 Matrix (MA) p17 membrane anchoring; gp41 cytoplasmic tail interaction, nuclear transport of core Capsid (CA) p24 core capsid Nucleocapsid (NC) p7 binds RNA p6 binds Vpr Pol Protease (PR) p11 Gag/Pol polyprotein cleavage and viral maturation Reverse Transcriptase p66 and (RT), p51 transcribes RNA to DNA RNase H p15 RNase H activity Intergrase p31 DNA provirus integration
Env Env (gp160) gp120 surface glycoprotein gp120 binds CD4 and coreceptor gp41 transmembrane glycoprotein gp41 anchors envelope; forms fusion pore
Regulatory genes
Tat Tat p16/p14 viral transcriptional activator Rev Rev p19 RNA transport, stability and utilization factor
Accessory genes
Vif Vif p23 promotes virion maturation and infectivity Vpu Vpu p16 Promotes virion budding; degrades CD4 Nef Nef p25-p27 CD4 and MHC class I downregulation Vpr Vpr p10-15 Nuclear localization of preintegration complex; inhibits cell division and arrests cells at G2/M phase
Table 1-1: HIV-1 genes, gene products and functions. Adapted from (Leitner et al.,
2007)
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1.C. Antiretroviral therapy
There is no cure and no vaccine for HIV-1. However, with the advent of highly
active antiretroviral therapy (HAART) in 1996, life expectancy of HIV-1 infected
individuals increased dramatically and fundamental changes in the management of HIV
infection occurred. HAART acts to delay the onset of disease and death by using a
“cocktail therapy“ approach incorporating combinations of different antiretroviral drugs, thereby lowering viral load to undetectable limits and allowing an increase in CD4+ T- cell numbers. Although virus is undetectable, patients are not cleared of infection and virus may remain dormant in latently infected cells. Combination therapy or HAART was developed partly in an effort to overcome the rapid development of drug-resistant mutants seen in previous single-drug therapy approaches. The strength of HAART in preventing development of drug resistant viruses is that a combination of antiretroviral drugs act against several different viral targets concurrently.
Typically, two nucleoside analog reverse transcriptase inhibitors (NRTI) are used with one protease inhibitor (PI) or a non-nucleoside reverse transcriptase inhibitor
(NNRTI) (Figure 1-4). With greatly reduced viral replication, the development of mutants that can escape activities of all drugs simultaneously is much slower than the development of mutants against a single drug treatment. Nucleoside analog reverse transcriptase inhibitors act by competing with natural deoxynucleotides for incorporation into the newly formed viral DNA strand during HIV reverse transcription of RNA.
Incorporated NRTI block chain elongation by preventing natural nucleotides from forming 5’-3’ phosphodiester bonds needed to extend the DNA chain, which is detrimental to the viral replication cycle (Furman et al., 1986; Mitsuya et al., 1985).
Non-nucleotide reverse transcriptase inhibitors act non-competitively by binding directly
34
to the reverse transcriptase molecule and preventing movement of domains required for
DNA synthesis. Other drugs, known as protease inhibitors, are incorporated into the
budding immature virion at the plasma membrane and act by inhibiting maturation of virus. Protease inhibitors prevent viral protease enzymes from cleaving Gag-Pol polyprotein, thereby reducing infectivity of virions by preventing maturation of viruses with ordered matrix, capsid and nucleocapsid structural proteins.
The most recent class of antiretroviral drugs includes entry inhibitors. The only such drugs approved for use by the United States Food and Drug Administration is the fusion inhibitor T-20, or enfuvirtide (Kilby et al., 2002), and the very recently approved
CCR5 antagonist Maraviroc (Dorr et al., 2005; Fatkenheuer et al., 2005). T-20 acts by
binding to the HR1 region of gp41 and blocking formation of the 6 helix-bundle involved
in promoting membrane fusion events (Kilby et al., 1998). Maraviroc binds to a
hydrophobic pocket of CCR5 and prevents binding by HIV-1 gp120. Other entry
inhibitors in the pipeline include attachment inhibitors that block binding of envelope to
CD4, and antibodies against CD4 and CCR5. Currently, there is considerable
investigation into the use of entry inhibitors as microbicidal agents that prevent
acquisition of HIV-1 infection during sexual intercourse.
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Figure 1-4: Schematic representation of HIV-1 replication and target sites for antiretroviral drugs
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Figure 1-4: Schematic representation of HIV-1 replication and target sites for
antiretroviral drugs. Entry of HIV-1 into the human CD4+ T-cell or macrophage begins with binding of virus envelope glycoprotein to CD4 and chemokine coreceptor,
CCR5 or CXCR4, promoting fusion of virus and cell membranes. The virus core is
released into the cytoplasm and capsid disassembles, releasing the viral RNA genome.
RNA is reverse transcribed into DNA by the viral enzyme Reverse Transcriptase (RT),
and the double-stranded DNA is transported into the nucleus and integrated into a
random location of the host genome. Here, the stable proviral DNA may lie dormant, or
may be transcribed by cellular machinery. RNA is spliced and exported out of the
nucleus, where some full-length transcripts are maintained to become new viral genomes
and others are translated into virion proteins and targeted to the plasma membrane by
cellular proteins. Gag and Gag-Pol monomers multimerize at the inner membrane,
promoting budding and release of new virions. These immature virions are enveloped in
the host cell plasma membrane studded with viral envelope glycoproteins gp120 and gp41. The viral enzyme protease cleaves the Gag-Pol polyproteins, producing mature virions with matrix and a capsid core, which are fully infectious and disseminate throughout the body. Antiretroviral drugs in use primarily target several sites during the
HIV-1 lifecycle, numbers 1-4. These include (1) entry inhibitors, (2) reverse transcriptase inhibitors (both nucleoside, and non-nucleoside RT inhibitors), (3) integrase inhibitors, and (4) protease inhibitors.
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1.D. HIV-1 Envelope protein and receptor interactions
1.D.1. HIV-1 envelope structure: The HIV-1 env gene yields a highly glycosylated envelope glycoprotein with a molecular mass of about 160kD, known as gp160, which, when presented as a trimer, functions to mediate viral entry. At the Golgi complex, this protein is cleaved by host furin proteases into two subunit proteins that remain non-covalently associated. This Envelope complex, consisting of surface unit glycoprotein gp120 and transmembrane glycoprotein gp41 is transported to the cell surface and embedded into the plasma membrane, where it is incorporated into budding virions. Envelope mediates virus entry in a three-step process, namely 1) binding to primary receptor molecule CD4, 2), binding to co-receptor molecules CCR5 or CXCR4, and 3) formation of a membrane pore and fusion of viral and cell membranes. Gp120 is primarily involved in attachment and binding to the host cell, and gp41 mediates fusion events, but entry itself is only possible when the gp120-gp41 envelope complex exists in a trimeric form known as an envelope spike. This quaternary structure is crucial for envelope function. Gp120 consists of five conserved or constant regions (C1-C5) and five hypervariable regions (V1-V5) which form domains that tend to cover large portions of the conserved regions of envelope involved in receptor binding. These V1-V4 loops result from disulphide bonds that form between highly conserved cysteine residues that lie adjacent to the variable regions. Both gp120 and gp41 contain multiple N-linked glycosylation sites within their conserved regions, and almost 50% of gp120’s molecular mass can be accounted for by glycans. Gp41 mediates fusion through two important structural domains: the fusion domain, which consist of a 15 amino acid stretch of hydrophobic residues at the amino terminus, and two complementary heptad repeat
38
regions (HR1 and HR2) which, upon self-annealing, bring the viral and cellular
membranes close together and induce fusion.
1.D.2. Mechanisms of receptor binding, conformational change and the
fusion process: HIV-1 entry is dependent on binding to two receptor molecules, each of
which induces considerable conformational reconfiguration in the protein structure.
Initial binding of gp120 to the N-terminus Domain 1 (D1) of CD4 occurs at a recessed
CD4 binding pocket of the gp120, partially overlapped by the large V1/V2 loop (Jameson
et al., 1988; Kwong et al., 1998; McDougal et al., 1986; Sattentau and Moore, 1991;
Zhou et al., 2007). This induces a conformational change, involving shifting of the
V1/V2 and V3 hypervariable loops and formation of the bridging sheet, that results in the
exposure of the highly conserved coreceptor binding site (Bieniasz et al., 1997; Rizzuto
et al., 1998; Trkola et al., 1996a). Amino acids in the tip of the V3 loop are believe to interact with coreceptor molecules, either CCR5 (on the surface of monocytes and macrophages) or CXCR4 (on the surface of T-cells) (Chesebro et al., 1992; De Jong et al., 1992; Fouchier et al., 1992; Shioda, Levy, and Cheng-Mayer, 1992). The utilization
of either or both of these coreceptors determines the cellular tropism of the HIV-1 isolate,
as has been discussed in Chapter 1.B.4.
Binding to coreceptor induces subsequent conformational changes that allow the
gp120 molecules of the trimer to swing outwards and expose a previously buried N-
terminal region of gp41, known as the fusion peptide (Bosch et al., 1989; Gallaher, 1987)
(Figure 1-5). Due to the hydrophobic nature of this fusion domain, it is embedded into
the plasma membrane of the target cell, destabilizing it. Subsequently, the loosely
structured HR2 domains coil into the grooves of the trimeric HR1 domains in a process
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known as HR2 zipping, forming a stable six-helix bundle. This acts to bring the viral and cellular membranes in close proximity to each other (Chan et al., 1997; Weissenhorn et al., 1997b). This promotes destabilization of the membranes and the formation of a fusion pore which allows host and viral membranes to mix, thereby permitting entry of the HIV-1 capsid into the cell cytoplasm (Gallaher et al., 1989; Melikyan et al., 2000;
Myszka et al., 2000; Weissenhorn et al., 1997b).
While the presence of CD4 and chemokine receptors are required for virus entry, attachment of virus to the cell surface can be facilitated by other molecules, including cellular proteins incorporated into the virus membrane that interact with their physiologic ligands on the cell surfaces (Arthur et al., 1992). Additionally, other cell types including dendritic cells bind HIV gp120 with high affinity through the C-type lectin DC-SIGN
(dendritic cell-specific ICAM3 grabbing nonintegrin) (Curtis, Scharnowske, and Watson,
1992). It has been proposed that dendritic cells may capture virions via DC-SIGN without necessarily being infected, and transport and present infectious virus to a permissive target cell in a process known as infection in trans (Feinberg et al., 2001;
Geijtenbeek et al., 2000; Speck et al., 1999; Turville et al., 2002). This is of particular importance given that DC-SIGN positive cells are abundant in both human and rhesus macaque rectal and vaginal mucosa and all HIV-1, HIV-2 and SIV strains studied bind
DC-SIGN (Geijtenbeek et al., 2000; Jameson et al., 2002).
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Figure 1-5: HIV-1 Entry. Figure HIV-1 1-5:
41
Figure 1-5: HIV-1 Entry. Each HIV-1 Envelope protein is composed of surface glycoprotein gp120 and transmembrane glycoprotein gp41, which form functional units as trimers (three gp120 and three gp41 molecules), called spikes. Initial binding of gp120 to CD4 receptor induces a conformational change in gp120 (Panel 1) that exposes the chemokine coreceptor binding site. This allows CCR5 or CXCR4 to bind gp120 (Panel
2), inducing further conformational changes that expose the trimeric gp41 (Panel 3). The hydrophobic N-terminus of gp41, known as the fusion peptide, embeds itself into the host cell membrane and the helical second heptad repeat (HR2) region of the prehelix bundle coils into the opposite and complementary grooves of the first heptad repeat (HR1), forming what is known as a 6-helix bundle (Panel 4). These short-lived structures through each conformational change are known as fusion intermediates. Formation of the 6-helix bundle brings the viral and cellular membranes within close proximity of each other and promotes the formation of a fusion pore (Panel 5). This pore promotes mixing of membrane lipids, and the pore grows large enough for the virus core to pass through, and into the cytoplasm (Panel 6).
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1.E. Vaccines and Antibodies
1.E.1. How vaccines work: Vaccines work by inducing an immune response via the stimulation of stimulating lymphocytes. After initial encounter with antigen, immune cells known as memory cells retain the ability to recognize and quickly respond to antigen thereby preventing infection by pathogens bearing those antigenic markers and controlling disease (Figure 1-6). The two main arms of the immune response are controlled by either B-cell or T-cells, which initiate a humoral or cellular immune response, respectively. B-cells produce antibodies that act to block initial infection of microorganisms by neutralization, opsonization or complement activation, whereas CD8
T-cells recognize and kill infected host cells after initial infection has generally already been established, thereby helping to limit spread and transmission of infection and control disease. CD8 T cells are also known as cytotoxic T lymphocytes (CTL).
However, it is widely agreed that neutralizing antibodies are the principal component required for sterilizing immunity, and have shown an important role in protection against infection and in controlling virus replication post-infection (Baba et al., 2000; Cho et al.,
2001; Emini et al., 1992; Mascola et al., 2000; Shibata et al., 1999; Trkola et al., 2005).
Both naïve CD8 T-cells and B-cells are dependent on growth and differentiation factors provided by CD4 helper T-cells for activation and maturation, the very cells targeted by HIV-1. As such, it is clear why HIV-1 induced CD4 T- cell depletion eventually leads to immunodeficiency.
Due to extensive gene rearrangement, naïve B-cells and T-cells show a broad repertoire of receptors capable of recognizing and binding epitopes. Each cell has a receptor with paratopes recognizing specific epitopes, each of which is essentially
43
randomly generated. Upon binding of these receptors to a specific epitope, such as one
that might be included in a vaccine, cells present that antigen to CD4 T-cells, which
stimulate B-cells or CD8 T-cells to proliferate and differentiate in a process of clonal expansion. This increases the number of cells that recognize that specific epitope and initiates an immune response.
Host CD8+ CTL responses are generated during the early stages of protection, prior to the development of neutralizing antibodies, which generally appear within 4-8 weeks. However, in the case of HIV-1 infection, these early antibodies may be of little clinical relevance (Richman et al., 2003; Wei et al., 2003) as substantial time is needed for antibody maturation. Maturation allows for development of high-affinity antibody
with optimized binding to antigen and generally results in the development of
neutralizing antibodies. Eventually, during infection of a single host, HIV-1 may evolve
to escape both antibody and CTL recognition.
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Figure 1-6: How vaccines work. Response of immune cells to a vaccine (blue shaded area) and then to a challenge infection (orange shaded area) is shown. The primary response to the vaccine requires several weeks, whereas the anamnestic response to the challenge occurs within days. Upon clearance of vaccine antigens or challenge microbes, immune response declines but immunological memory remains for years.
45
1.E.2. B-cell biology: Activation of naïve B-cells is initiated by binding of
antigen to specific membrane-bound immunoglobulins IgM or IgD found on the naïve B-
cell surface. This complex is internalized by endocytosis and is proteolytically cleaved
into peptides via the MHC class II processing pathway. Epitopes are presented to helper
T-cells via MHC II, which in turn facilitates clonal expansion of B-cells on induction of
cytokine secretion by helper T-cells. Isotype switching occurs whereby B-cells
expressing IgM or IgD on their surfaces may secrete other antibody isotypes (IgG, IgA or
IgE), undergo affinity maturation and may become memory cells. However, for the
development of neutralizing antibodies, it is crucial that epitope recognition occur via
IgM or IgD on the surface of B-cells. In the sections ahead, I shall discuss several
mechanisms HIV-1 has developed to prevent exposure of conserved epitopes to naïve B-
cells, limiting the ability to develop broadly neutralizing antibodies.
1.E.3. Broadly neutralizing antibodies
1.E.3.a. Overall antibody response: Multiple epitopes on HIV-1 gp160
protein elicit antibodies, but most of these antibodies are either non-neutralizing or strain
specific. The Los Alamos National Laboratory, as of December 14th, 2007, has 1402 polyclonal and monoclonal antibodies on record. Of these, 467 have defined epitopes on gp160, representing approximately 217 distinct epitopes on gp160 under 21 amino acids
(see Figure 1-7). There are also considerable polyvalent antibody responses, elicited either during natural infection or through immunization strategies. The variable regions are some of the most immunodominant epitopes on the envelope complex, with a particularly strong immune response targeted against the V3 loop. Additional antibodies
46
against an immunodominant region of gp41 that lies between the two heptad repeat
domains, known as cluster I epitopes, appear to be strongly elicited.
Despite over two decades of active research, only a handful of potent and broadly
reactive neutralizing antibodies (BR-Nabs) have been identified (see Table 1-2 on page
62). Such Nabs are rare even in virus-infected patients (Braibant et al., 2006; Burton et al., 2004b; Richman et al., 2003; Wei et al., 2003); nevertheless, their existence suggests that it is possible to induce such antibodies. Monoclonal antibodies (mAb) b12, 2G12 and 447-52D bind to gp120 (Burton et al., 2004b; Gorny et al., 1992; Roben et al., 1994;
Trkola et al., 1996b). 2F5, 4E10, and recently identified m48 recognize adjacent but distinct epitopes on the membrane-proximal external region (MPER) of gp41 (Muster et al., 1993; Stiegler et al., 2001; Zhang et al., 2006; Zwick et al., 2001b), a highly
conserved domain required for HIV-1 fusion with the cell membrane (Salzwedel, West, and Hunter, 1999; Suarez et al., 2000) (Figure 1-8). These antibodies, and possible BR-
NAb targets, will be discussed below.
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Figure 1-7: Antibody epitopes of HIV-1 Envelope protein. protein. Envelope HIV-1 of epitopes Antibody Figure 1-7:
48
Figure 1-7: Antibody epitopes of HIV-1 Envelope protein. Determined from both monoclonal and polyclonal antibodies in the Los Alamos National Laboratory HIV-
1 Immunology database, April 11th, 2007. The location of well characterized linear binding sites of 21 amino acids or less. This map is meant to provide the relative location of epitopes on gp160. The dotted line shows an outline of epitopes listed, which roughly represents the number of antibodies. These antibodies are derived from various sources, including HIV-1-infected patients and envelope-immunized animals. SP = Signal peptide; V1-V5 = variable regions 1 through 5; FP = fusion peptide; TM = gp41 transmembrane domain. Amino acids positions based on HIV-1 HXB-2 are listed below.
49
1.E.3.b. Variable regions
V3 loop: The primary immune response is aimed at epitopes within the V3 loop.
Many anti-V3 antibodies are potently neutralizing against primary isolates, but remain
limited in their breadth of neutralizing activity. Due to this potency, V3 is classically
referred to as the principal neutralizing determinant (PND) and much attention was
turned towards this region in early vaccine research (Javaherian et al., 1989; LaRosa et al., 1990; Putney et al., 1986). However, since virus rapidly evolves to escape NAbs, it has not been strongly considered as a vaccine candidate. It was also shown that the development of potent anti-V3 antibodies required prolonged antigenic stimulation to allow for maturation of antibodies with greater breadth, as seen by their slowness to develop in HIV-1 infected patients (Pilgrim et al., 1997). But with the discovery of mAb
447-52D (Gorny et al., 1992) attention has yet again turned towards eliciting antibodies that target V3. 447-52D is a V3 specific neutralizing antibody that interacts with 14 residues at the crown of V3 (its core GPxR epitope is highly conserved among clade B viruses (Gorny et al., 1992; Sharon et al., 2003; Stanfield et al., 2004), and shows considerable potency and breadth of activity (Subtypes A, B, F (potent); C, D (weakly), E
(none) (Gorny et al., 1992)). Other anti-V3 mAbs do not recognize the same epitope as
447-52D, yet are also cross-neutralizing against several primary isolates (Gorny et al.,
2004; Gorny et al., 2002). This reflects the view that although there is considerable V3 sequence variation, there is some structural conservation and as such, at least one conserved epitope. Furthermore, V3 involvement in binding to chemokine receptors
illustrates functional constraints imposed on V3 structure. Interestingly, the V3 crown
has been seen to mimic the structure of residues involved in binding of the natural ligands
of CCR5 (MIP-1, MIP-1, RANTES) and CXCR4 (SDF-1). The V3 loop is generally
50
constrained to approximately 33-35 amino acids in length, with very few deletions or insertions, but frequent substitutions, further illustrating structural significance (Hartley et al., 2005). This is in contrast to the V1/V2 loop, which varies widely in both sequence and size, ranging from 65-85 amino acids in length.
V1/V2 Loop: Although there is considerable diversity in both sequence and size of the V1/V2 loop, the stem of V1/V2 appears relatively well conserved. This region is involved in the formation of the chemokine binding site and interaction with CD4
(Kwong et al., 1998; Rizzuto et al., 1998), suggesting a possible target for the induction
of NAbs (Srivastava, Ulmer, and Barnett, 2005). However, antibodies against this region
have not yet been identified. Interestingly, all of the anti-V1 mAbs in the Los Alamos
HIV-1 Immunology database (each of which target the V1 tip and possess potent but
strain specific-neutralizing activity) are derived from humanized transgenic mice
possessing human IgG genes that had been immunized with gp120, and not from
infected human patients. The lack of detected V1-specific antibodies in HIV-1-infected
patients is likely related to the extreme diversity of this region and an inability to
adequately select anti-V1 antibodies from infected patients with hetrologous gp120.
Regardless, the anti-V1 mAb obtained from humanized rodents display only strain-
specific neutralizing activity. Anti-V2 NAbs are generally weak and remain limited in
breadth of activity, yet there are some neutralizing antibodies that target a semi-
conserved region of V2 (Honnen et al., 2007). Given the variation of both size and
sequence, the V2 loop is not typically considered a promising vaccine target.
V4 and V5. The V4 and V5 regions are genetically variable in sequence and
structure and tolerate large deletions and insertions while still maintaining functionality.
Removal or shortening of V4 and V5 aims to expose conserved epitopes that exist below
51
but are masked by the variable loops. Removal of three N-linked glycosylation residues
on HIV-1 V4 and V5 resulted in a mutant virus that was used as a vaccine antigen. The
antibodies raised in response neutralized autologous virus with greater potency than did
sera from animals immunized with wild type virus. However, no increase in breadth of
neutralization was observed (Bolmstedt et al., 1996). Few antibodies against V4 have
been identified that are neutralizing. Interestingly, an unrelated peptide inserted into the
V4 loop, when targeted by an antibody specific for that peptide, neutralized the mutant
virus (Ren, Sodroski, and Yang, 2005). Although the V4 loop plays no function in entry,
it appears that the specific target need not contribute functionally to the process of virus
entry in order to contribute to neutralization of the virus as whole. Such antibodies may be involved in targeting virus or virus-infected cells for lysis or phagocytosis via opsonization, induction of the complement cascade and/or antibody-dependent cell- mediated cytotoxicity.
1.E.3.c. CD4 binding site (CD4bs) CD4 acts as the primary receptor involved in attachment of HIV-1 to the cell surface. Consequently, antibodies that interfere with this attachment would be expected to neutralize virus (Srivastava, Ulmer, and Barnett, 2005).
Many CD4-binding site (CD4bs) Abs have been developed that inhibit binding of soluble
CD4 (sCD4) to gp120 and whole virus, and effectively neutralize T-cell line-adapted
(TCLA) viruses, yet primary isolates, surprisingly, are not well neutralized (D'Souza et al., 1997; McDougal et al., 1986; Sullivan et al., 1995). One exception to this is mAb
IgG1b12 (Barbas et al., 1992; Roben et al., 1994)), which has potent and considerable breadth of neutralizing activity against primary isolates. Monoclonal antibody b12 potently neutralized 50% of 90 isolates from multiple clades tested (Binley et al., 2004;
52
Burton et al., 2004b; Trkola et al., 1995). The b12 binding-site is not linear but
recognizes a highly conserved, discontinuous epitope consisting of multiple residues of
C2, C3 and C4, and may also be somewhat dependent on regions within the V2 domain
(Pinter et al., 2004; Zwick et al., 2003). The difficulty in eliciting antibodies that
neutralize primary isolates hints at the complexity of the CD4 binding site as a target.
1.E.3.d. CD4-induced (CD4i) epitopes: Upon binding of gp120 to CD4, extensive conformational change is undergone that either creates or exposes additional epitopes known as CD4-induced, or CD4i. Several antibodies in HIV-1 infected individuals better react with gp120 in this CD4-complexed form, and are thought to recognize regions near or involved in binding to the chemokine receptors (Moulard et al., 2002; Thali et al.,
1993; Xiang et al., 2002). These epitopes are part of the gp120 bridging sheet, a beta- sheet consisting of four anti-parallel beta-strands contributed by the CD4 region and the
V1/V2 stem, overlapping, or in close proximity to, the coreceptor binding site (Kwong et al., 1998; Xiang et al., 2002). Interestingly, CD4i mAbs fail to neutralize primary virus as whole IgG molecules, but do have considerable breadth and potency when presented in smaller forms, either as antigen-binding fragments (Fabs) or single-chain variable fragments (scFv) (Darbha et al., 2004; Gershoni et al., 1993; Moulard et al., 2002; Thali
et al., 1993; Xiang et al., 2002). This is likely a result of steric hindrance by which the
space between the CD4i epitope of gp120 and the cell membrane is too small to
accommodate the large size of an entire IgG (Dey, Del Castillo, and Berger, 2003;
Labrijn et al., 2003; Moulard et al., 2002; Sullivan et al., 1998). Antibodies most likely were generated against soluble, monomeric gp120 that was released during shedding in a
manner similar to most antibodies against envelope protein (Parren et al., 1997).
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Nevertheless, the remarkable breadth of neutralization of such antibody fragments, in particular Fab X5(Moulard et al., 2002) and 17b(Thali et al., 1993), against both R5, X4 and dual-tropic R5X4 primary isolates, suggests that the CD4i epitopes are highly conserved and sensitive targets for inhibition.
1.E.3.e. Carbohydrate-dependent epitopes: Approximately 50% of the mass of gp120 is from carbohydrate moieties (Leonard et al., 1990), and these glycans are believed to play key roles in maintaining gp120 in a functional conformation that supports CD4 binding, as unglycosylated gp120 does not interact with CD4 (Haigwood et al., 1990). As such, several glycans are structurally conserved. Furthermore, carbohydrate moieties on viral proteins essentially camouflage non-self proteins from immune detection, establishing what is known as “glycan shield” which is poorly immunogenic. Nevertheless, but quite surprisingly, there is a single human mAb that specifically recognizes conserved and densely clustered oligomannose sugars on the exposed surface of gp120 that is both potent and broadly neutralizing against both TCLA
HIV-1 and primary isolates from multiple clades (Binley et al., 2004; Burton et al.,
2004b; Scanlan et al., 2002; Trkola et al., 1996b). Although MAb 2G12 does not bind to either CD4 or coreceptor binding sites, it does interfere with the gp120-CCR5 interaction
(Sanders et al., 2002; Trkola et al., 1996a), possibly through an indirect, steric effect.
2G12 has an unusual and unique structure whereby the arms of the IgG swap variable heavy domains and the two Fabs assemble into an interlocked domain-swapped dimer, forming an extended binding site (Calarese et al., 2003). Antibodies with similar domain-swapped conformations as seen in 2G12 are incredibly rare in patient sera, and
54
coupled with the unusual epitope and low immunogenicity of oligomannose residues,
2G12-like antibodies are unlikely to be elicited in a vaccine strategy.
1.E.3.f. Epitopes on gp41: Compared to gp120, gp41 has fewer glycosylation sites and is less variable in both sequence and structure. Several antibodies against gp41 have been identified that show considerable breadth of neutralization, including 2F5,
4E10, Fab Z13 (and Z13’s binding optimized form, Z13e1), and recently identified m48
(Nelson et al., 2007; Stiegler et al., 2001; Zhang et al., 2006; Zwick et al., 2001b). Of great interest is that all of these antibodies target adjacent but distinct epitopes of a highly conserved region of gp41 know as the membrane proximal external region (MPER), a 30 amino acid domain close to the host membrane that forms the “foot” of the viral spike. It is believed that these mAbs neutralize virus by interfering with late membrane fusion events (de Rosny et al., 2004; Salzwedel, West, and Hunter, 1999; Srivastava, Ulmer, and
Barnett, 2005; Suarez et al., 2000). 2F5 has been well characterized and can potently neutralize 67% of 90 isolates tested from diverse subtypes (Binley et al., 2004; Burton et al., 2004b; Trkola et al., 1995). 4E10 binds to an epitope only four residues downstream of the core 2F5 epitope, and neutralized 100% of 90 isolates tested (Binley et al., 2004;
Burton et al., 2004b; Parker et al., 2001; Zwick et al., 2001b). Although 4E10 exhibits exceptional breadth of neutralization, far more than any other HIV-1 mAb yet discovered, it is less potent than most other antibodies discussed thus far. Interestingly, both 2F5 and
4E10 have long CDR H3 domains that make contact with residues downstream of their core epitopes, and the H3 domains may be important in the neutralizing activity of these monoclonal antibodies. Fab Z13, and its affinity-enhanced full-size variant IgG Z13e1, binds to a distinct epitope between and overlapping both the 2F5 and 4E10 core epitopes,
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it extremely broad in neutralizing activity, yet is an order of magnitude less potent than
mAb 4E10 (Binley et al., 2004; Nelson et al., 2007; Zwick et al., 2001b). Monoclonal
antibody m48 neutralized a panel of primary isolates from multiple clades with greater
potency than either 4E10 or Fab Z13, and binds to a MPER epitope that likely overlaps,
but is distinct from those recognized by either 2F5, 4E10 or Fab Z13 (Zhang et al., 2006).
The great breadth of neutralizing activity of antibodies that target the gp41 MPER
presents this region very favorably as a target for vaccine studies. A greater
understanding and characterization of immune responses against MPER is needed. The
MPER is exposed on both intact virions and the membrane surface of infected cells, yet
epitopes for the above mAbs remain poorly antigenic, as illustrated by the rare
occurrence of such antibodies in HIV-1 infected individuals (Alam et al., 2007b; Coeffier et al., 2000; Zwick et al., 2001b). Furthermore, although 2F5 and 4E10 binding epitopes are available on both functional trimers of envelope and during the fusion process, they may be better exposed upon virus envelope binding to receptors (Burton et al., 2004b;
Reeves et al., 2005). Other epitopes surrounding the mAb epitopes are highly
immunogenic, especially a 19 amino acid region adjacent to the MPER known as cluster
II, that partially overlaps the 2F5 core epitope. Cluster I, a 25 amino acid region that lies
between the two heptad repeat regions, is considered immunodominant, yet no antibodies
against either cluster I or cluster II, appear neutralizing, except for one named clone 3
(CL3). CL3 binds to epitope GCSGKLICTT at the C-terminal end of cluster I and can
neutralize 3 clade B TCLA isolates and 3 primary isolates from Group O (Cotropia et al.,
1996; Ferrantelli et al., 2004a; Viveros et al., 2000). This antibody, to date, has been
poorly characterized.
56
A.
B.
Z13
Figure 1-8: Schematic figures of trimeric HIV-1 envelope spike showing the location of conserved epitopes recognized by the few identified BR-NAbs.
57
Figure 1-8: Schematic figures of trimeric HIV-1 envelope spike showing the location of conserved epitopes recognized by the few identified broadly reactive neutralizing antibodies (BR-NAbs). (A) Arrangement of the HIV-1gp120 glycoproteins in a trimeric complex. The perspective is from the target-cell membrane.
Blue arrows indicate the CD4-binding pockets, and the conserved chemokine-receptor- binding regions are in red. Areas shaded in light green indicate the more variable, glycosylated surfaces of the gp120 cores and areas shaded in red indicate the more conserved coreceptor binding sites formed after binding to CD4. The approximate locations of the V1/V2 and V3 loops, and V4 and V5 regions are indicated. Sites for complex-carbohydrates are shown in navy blue. Figure adapted from (Wyatt et al.,
1998). (B) Three monomers of gp120 (shown in blue) and gp41 (green) assemble into a functional, trimeric form. Epitopes on the membrane-proximal external region (MPER) of gp41 recognized by BR-NAbs 2F5, 4E10 and Z13 are shown in yellow, orange and red. These epitopes may only be exposed subsequent to conformational changes induced in envelope following receptor engagement. Similarly, CD4 induced (CD4i) epitopes, shown in red, are only presented after CD4 binding which induces changes in envelope conformation including shifting of the V1/V2 and V3 hypervariable loops, formation of the bridging sheet and exposure of the highly conserved coreceptor binding site.
Monoclonal antibody 447-52D binds to the tip of the V3 loop (purple) and neutralizes several viruses from subtypes A, B, and F potently, and subtypes C and D weakly. The epitope recognize by potent and broadly neutralizing antibody IgG1 b12 (shown in yellow) covers the CD4 binding site. Glycan residues recognized by 2G12 are shown in white. (Figure adapted from (Burton et al., 2004b)).
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1.E.3.g. Fusion Intermediate epitopes: The importance of presenting functional envelope to B-cells in a native setting has long been identified as a favorable approach to inducing cross-reactive NAbs against primary isolates compared to non-functional, monomeric recombinant gp120. In 1999, LaCasse et al. (LaCasse et al., 1999) conducted a study designed to expose native conformation dependent epitopes of envelope as it underwent the fusion process. These fusion intermediates were captured by formaldehyde fixation of cell-to-cell fusions, and these whole-cell vaccines elicited antibodies in mice transgenic for CD4 and CCR5 that were capable of neutralizing 23 of 24 primary HIV-1 isolates of different subtypes. This activity, however, was later reported to be due to a non-specific cytotoxicity of an unknown nature (Nunberg, 2002), but may result from antibodies elicited against human cellular proteins during immunization of mice with human whole cells. Since then, other such approaches to target fusion intermediate epitopes have been pursued, using Env-CD4 (Celada et al., 1990; Devico et al., 1996;
Fouts et al., 2002; Fouts et al., 2000; Gershoni et al., 1993; He, D'Agostino, and Pinter,
2003; Kang et al., 1994) and Env-CD4-CCR5 complexes (Xiao et al., 2003; Zipeto et al.,
2006). Although better quality and more potent neutralizing activity has been observed, these antibodies are not broad in activity.
Other epitopes that may be better exposed following receptor and coreceptor binding are those bound by BR-NAbs 2F5 and 4E10 (Burton et al., 2004b; Reeves et al.,
2005). As of yet, elicitation of such antibodies in a vaccine has not been achieved. This may be due to the difficulty in targeting these conformation dependent epitopes using linear peptides with undefined structures, spatial constraints and the short-lived nature of fusion intermediate conformations. As has been seen, proteins or peptides that can bind a known antibody do not necessarily elicit that antibody. As such, trimeric, native,
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membrane-bound envelope spikes that are functional may be a better antigen than short
peptides or monomeric recombinant proteins (Beddows et al., 2006; Binley et al., 2000a;
Dey et al., 2007).
The prehairpin intermediate, exposed subsequent to CCR5 binding in a step prior to
6-helix bundle formation (Luftig et al., 2006; Miller et al., 2005), has recently been
identified as a fusion intermediate target for neutralizing antibody IgG1 D5. MAb D5
binds to a hydrophobic pocket on the HR1 groove, in a similar fashion to the fusion-
inhibitor peptide T-20, or enfuvirtide, and possesses significant breadth and moderate
potency of neutralizing activity. The fact that both scFv and whole IgG1 D5 antibodies
possess similar potency of neutralization suggests that the prehairpin fusion intermediate
structure is an ideal target for neutralizing antibodies and that, surprisingly, full-size
immunoglobulins do have access to this short-lived epitope during fusion events.
This conserved region of HR1 has long been known as a target for entry-inhibition,
and interestingly, viruses generate escape mutants during therapy with enfuvirtide within
14 days in vivo. The resistant viruses show mutations that map to the HR1 domain (Wei
et al., 2002). These mutants show reduced replicative fitness, likely due to reduced
kinetics of 6-helix bundle formation (Lu et al., 2004; Reeves et al., 2005). Furthermore, these mutant viruses are more sensitive to neutralizing antibodies against gp41, likely as a result of prolonged exposure of these fusion intermediate epitopes (Reeves et al., 2005).
Of interest is the relative lack of neutralizing antibodies against the 6-helix bundle, yet this region is particularly immunogenic, as seen in HIV-1 infected patient sera
(Opalka et al., 2004). Likely, antibodies against 6-helix are elicited against antigenic, non-functional gp41 membrane stumps on the surface of infected cells post fusion, or on virions following shedding of gp120 (Moore et al., 2006). Due the short-lived nature of
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fusion intermediates, neutralizing epitopes that prevent 6-helix bundle formation, such as
that targeted by D5, are unlikely to be efficiently presented to naïve B-cells for long
enough for IgM molecules to bind, limiting elicitation of neutralizing antibodies of high
enough titer to be effective in vivo.
There may exist several as-yet-undefined fusion intermediate epitopes that could
act as potential targets for neutralizing antibodies, but exposure of these epitopes is likely
rare due to the transient nature of the fusion event and as a result of sterically limited
accessibility of membrane-bound immunoglobulins on naïve B-cell surfaces to these
epitopes.
1.E.3.h. Conclusion: Current monoclonal BR-NAbs have several limitations,
including reactivity with poorly immunogenic and relatively inaccessible epitopes, low
potency and shallow breadth of cross-reactivity. There is an important need to identify new epitopes or new approaches to elicit a protective immune response.
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Target Target Site Antibody Reference Potency/breadth gp120 CD4BS IgGb12 (Burton et al., 1991) Strong/broad F105 (Posner et al., 1991) Weak/narrow Glycan 2G12 (Buchacher et al., 1994), Moderate/broad (Trkola et al., 1996b) V3 447-52D (Gorny et al., 1992) Strong/narrow CD4i 17b (Thali et al., 1993) Weak/narrow 48D (Thali et al., 1993) Weak/narrow X5 (Moulard et al., 2002) Weak/narrow gp41 MPER 2F5 (Muster et al., 1993) Moderate/broad 4E10 (Zwick et al., 2001b) Weak/broad Z13, Z13e1 (Zwick et al., Weak/narrow 2001b),(Nelson et al., 2007) m48 (Zhang et al., 2006) Weak/narrow HR1 D5 (Miller et al., 2005) Weak/narrow
Table 1-2. Prototypic HIV-1 Neutralizing antibodies. Adapted from (Phogat, Wyatt, and Karlsson Hedestam, 2007)
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1.F. Obstacles in eliciting broadly reactive neutralizing antibodies
1.F.1. Overview: The difficulty in eliciting BR-NAbs to HIV-1 in both natural
infection and through vaccination stem from multiple mechanisms HIV-1 uses to evade
host immune responses. These include a high degree of antigenic variety, masking of
conserved epitopes, low immunogenicity of some domains of envelope and preferential
exposure of non-neutralizable domains, and employing complex tertiary and quaternary
structures to mediate fusion events.
Much of envelope protein appears poorly antigenic. In early antigenicity
experiments, recombinant gp120 was unable to induce high-level antibody response in
human clinical studies (Belshe et al., 1993; Keefer et al., 1994). More recent phase III
HIV vaccine trials report suboptimal antibody responses against envelope even though
volunteers received seven immunizations of recombinant gp120 (Gilbert et al., 2005a;
Gilbert et al., 2005b). Although this phenomenon is poorly understood, Moore and
Burton(Moore and Burton, 2004) propose heavy glycosylation and complex
oligomerization contribute to the poor immunogenicity.
1.F.2. Extensive glycosylation: Roughly 50% of the molecular weight of gp120
can be accounted for by glycans(Leonard et al., 1990). These carbohydrate moieties are
poorly immunogenic and, while covering most of the exposed surface of the trimeric
envelope structure, help HIV-1 evade immune detection by establishing an “immuno-
silent face” of the envelope (Kwong et al., 2000). Beyond lowering overall
immunoreactivity of envelope, these complex carbohydrates and terminal mannose sugars act as a “glycan shield” that masks or protects critical neutralizing epitopes that lie
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below. Indeed, heavy glycosylation is suggested to protect the CD4 binding site, the V3
loop, the co-receptor binding site and part of gp41 from immune detection.
1.F.3. High antigenic variability: Regions of gp120 that are not heavily
glycosylated exhibit further protective masking of conserved elements through
hypervariable regions. In particular, variable loops V1/2 and V3 overlap crucial domains
involved in the CD4 and coreceptor binding sites (Poignard et al., 2001; Wyatt et al.,
1998). These regions are particularly immunogenic, eliciting a broad range of antibodies.
Many antibodies against the V3 loop are potently neutralizing, hence this region of gp120
is known as the principal neutralizing determinant (PND), part of what has been described as gp120’s “neutralizing face.” However, these NAbs remain highly strain- specific as a result of extensive sequence and structural variability. Viral escape mutants rapidly evolve, requiring the immune system to adapt to control infection, leading to chronic immune stimulation, which is a key factor in the progression towards AIDS. Yet,
even with this observed variability in sequence, regions of both V1/V2 stem and V3
crown remain conserved in structure, being required for co-receptor binding. Elicitation
of BR-NAbs to these conserved structures is rare and requires prolonged antigenic
stimulation to allow antibody maturation.
1.F.4. Envelope Tertiary and Quaternary Structures: The conserved CD4
binding site of gp120 lies in a deep pocket or groove, partially overlapped by both
carbohydrate moieties and the V1/V2 loop (Kwong et al., 1998; Wyatt et al., 1998). This
recessed pocket makes elicitation of CD4bs NAbs extremely difficult. Furthermore,
conserved coreceptor binding sites form and are exposed to antibodies only upon CD4
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binding-induced conformational changes. Additional conserved epitopes are exposed
upon coreceptor binding-induced structural changes, yet these fusion intermediate
structures are transient epitopes for antibodies, and recognition is further complicated by
steric limitations. Fusion intermediate epitopes may be presented on both gp120 and
gp41 structures and some well-known neutralizable sites, such as those on the MPER,
may show enhanced exposure during the fusion process. Yet recognition of epitopes such as CD4i epitopes or those of the pre-fusogenic gp41 are unlikely to ever be presented to naïve B cells. Quaternary structure of envelope severely restricts antibody accessibility to conserved fusion domains. In addition, binding to receptors disrupts the non-covalent association of gp120 to gp41, releasing monomeric gp120. These monomers present epitopes that are normally occluded within the trimeric envelope structure. Although conserved, these epitopes generate antibodies to what is referred to as the “non-neutralizing face” of gp120’s inner domain (Wyatt and Sodroski, 1998).
Although immunogenic when presented as gp120 monomers, antibodies elicited against the gp120 inner domain are non-neutralizing due to restricted accessibility of these antibodies to this region on the native functional trimer. Monomeric gp120 is also suggested to posses a highly flexible structure in its free state, showing several different conformations, which presents the immune system with a considerable number of epitopes irrelevant to virus neutralization (Grundner et al., 2005). This immune diversion tactic generates many antibodies to a particularly immunogenic inner surface that never bind to the functional envelope trimer (Wyatt and Sodroski, 1998). A similar such immune diversion tactic is seen in gp41, whereby several epitopes between the heptad repeat domains, known as cluster I, and not shown to exhibit any neutralizing activity, are particularly immunodominant over other gp41 epitopes. This lowers antigenicity of gp41
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MPER epitopes targeted by BR-NAbs 2F5 and 4E10. Furthermore, HIV-1 may use a
tactic of autoantigen mimicry whereby epitopes mimic human self-structures, such as
cardiolipin (Alam et al., 2007a; Haynes et al., 2005; Sanchez-Martinez et al., 2006), which induced immune tolerance. However, this aspect of cardiolipin cross-reactivity to
2F5 and 4E10 is considered somewhat controversial (Ferrantelli et al., 2004a; Joos et al.,
2006; Scherer et al., 2007; Trkola et al., 2005).
1.F.5. Predictive Experimental Animal Models: A significant obstacle has arisen in developing an appropriate animal model in which to test vaccine constructs.
1.F.5.a Non-human primate models: The only animals susceptible to HIV infection are chimpanzees, Pan troglodytes, and pigtail macaques, Macaca nemestrina,
yet neither species develops AIDS-like symptoms. African monkeys are the natural hosts
to SIV, but do not develop clinical disease following infection. In contrast, rhesus
macaques from Asia are extensively used in research as they are highly susceptible to
SIV infection and progress towards AIDS, closely mimicking human disease (Levy,
1996). Significant advances were made when SIV/HIV hybrid viruses were created
(SHIV), incorporating HIV envelope in the context of an SIV background. After serial
passage of these viruses, highly pathogenic SHIV variants capable of replication in
rhesus macaques emerged (Li et al., 1992; Reimann et al., 1996). To date, most SHIVs
used in protection studies use SHIV-89.6P with an X4 tropic HIV-1 envelope, and
questions have arisen as to whether R5 tropic viruses might be more appropriate given
the comparative susceptibility of X4 tropic virus to neutralization (Feinberg and Moore,
2002). However, a recently developed R5 tropic SHIV, derived from passage of X4
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tropic SHIVSF162 in macaques and known as SHIVSF162P3, has found application in
pathogenesis and challenge studies in macaques (Harouse et al., 2001).
Problems arise as, in order to achieve 100% infection of non-human primates, doses
of challenge virus generally used are vastly higher that would typically occur in natural infection, by as much as 1000 fold. However, evidence has shown that repeated low dose
mucosal challenge of monkeys with SIV has shown similar viral and immunological
kinetics as high dose challenges and may have important applications in future
preventative vaccine research (McDermott et al., 2004; Subbarao et al., 2007).
1.F.5.b. Small animal models: Non-human primate models suffer a
significant drawback in that availability is restricted, they are costly and difficult to house
and handle, and primates are not susceptible to transgenic manipulation. In contrast, small animal models overcome many of these hurdles, and such models have been developed, but only with limited success. Transgenic rats expressing human CD4 and
CCR5 or CXCR4 were effectively infected with HIV-1 and released infectious virions, but levels of viremia were particularly low (Goffinet et al., 2007; Keppler et al., 2002).
Likewise, transgenic rabbits are susceptible to infection but show inefficient virus spread
(Dunn et al., 1995). Mouse cells exhibit multiple blocks to infection by HIV at the level of receptor binding, genome replication and release. Even when some of these blocks are overcome, a significant assembly block still remains, the precise nature of which has not been elucidated. This significantly reduces viremia in some murine cells. Xenograft models such as severely compromised immunodeficiency (SCID) mice reconstituted with human peripheral blood lymphocytes (hu-PBL-SCID) (Mosier et al., 1991) or human thymus and liver tissues (SCID-hu(Thy/Liv)) (McCune et al., 1988; Namikawa et al.,
67
1988) mice have been extensively used, and allow for rapid virus replication and high
viremia and T-cell depletion. Yet inoculation of SCID-hu mice with HIV elicit only
weak, if any, immune responses, resulting in uncontrolled virus replication in the absence
of cellular or humoral responses. This is likely due to failure of lymphocytes to mature
adequately in xenograft rodent models. However, development of the Trimera human
IgG rodent model does allow for effective virus replication at high titers for 3 months
(Reisner and Dagan, 1998). In this model, the normal hematopoietic system of each
mouse is eradicated by total body irradiation, and the immune system is reconstituted by
transplantation of murine SCID bone marrow and intraperitoneal injection of human
PBLs. These mice have been used in evaluating vaccines against HIV, Influenza and
HBV. Yet the cost and difficulty in generating these mice and their limited lifespan
decreases their appeal overall to vaccine research.
1.F.6 Conclusion: Taken together, HIV-1 employs multiple innovative
techniques to protect neutralizing epitopes from antibodies that include immune
camouflage, diversion and masking. Additionally conserved, hidden epitopes that are
only exposed temporally during the fusion process are unlikely to be presented to
membrane bound immunoglobulins on the surface of naïve B-cells due to steric
limitations. Several methods to overcome these obstacles to eliciting BR-NAbs are
discussed below.
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1.G. Current Strategies to elicit broadly reactive neutralizing antibodies:
1.G.1. Types of Antigens: Experimental HIV-1 vaccines can typically be
categorized into eight different forms of antigen: (1) Subunit or recombinant protein
vaccines; (2) Whole-killed or inactivated virus vaccines; (3) Live-attenuated vaccines;
(4) Virus-Like Particles; (5) Pseudovirions; (6) Recombinant viral vectors; (7) Peptide
vaccines; (8) DNA vaccines.
These antigens are frequently utilized in combinations to generate additive or
synergistic immune responses, either administered simultaneously or successively. Viral
vector prime followed by protein boost have shown enhanced antibody responses
(Clements-Mann et al., 1998; Cooney et al., 1993; Giavedoni et al., 1993; Graham et al.,
1993; Hu et al., 1991), as have DNA vaccine prime and viral vector boost (Letvin et al.,
1997; Richmond et al., 1998; Wang et al., 2005; Wang et al., 2006).
Several different approaches to generate an effective HIV-1 vaccine have been
investigated, but none yet have succeeded in eliciting potent BR-Nabs.
1.G.2. Variable loop deleted and glycan-deficient envelopes: An intensively
investigated line of research hypothesizes that deletion of either variable loops (Barnett et
al., 2001; Gzyl et al., 2004; Kim et al., 2003; Lu et al., 1998; Xu et al., 2006) or glycan
residues (Bolmstedt et al., 1996; Quinones-Kochs, Buonocore, and Rose, 2002; Reitter,
Means, and Desrosiers, 1998) will expose underlying conserved epitopes and promote
elicitation of BR-NAbs. Although limited success has been observed, the general conclusion is that this approach is unable to elicit BR-NAbs against primary isolates.
69
Interestingly, variable loop- and glycan-deficient envelope-bearing viruses exhibit
increased sensitivity to neutralizing antibodies and HIV-1 patient plasma.
Little to no benefit on cross-reactive Nab titer or breadth was seen from complete deletion of variable loops (Gzyl et al., 2004). Interestingly, partial deletions of variable loops appeared to redirect immune responses towards more conserved epitopes of the gp120 outer domain (Gzyl et al., 2004; Kang et al., 2005; Kim et al., 2003). A similar approach has been employed in hyper-glycosylation of envelope to redirect immune response against known neutralizing domains (Garrity et al., 1997; Muller, 2004; Nara and Garrity, 1998; Pantophlet, Wilson, and Burton, 2003). How this hyperglycosylation might affect overall structure is still unknown. In one study, a vast reduction of binding by non-neutralizing or weakly neutralizing antibodies was observed using envelopes showing 6-7 extra N-linked glycosylation sites, while maintaining the ability to bind BR-
NAbs b12 and 2G12 (Pantophlet, Wilson, and Burton, 2003; Pantophlet, Wilson, and
Burton, 2004).
1.G.3. Fusion Intermediates: Other approaches attempt to expose conserved epitopes that are normally protected from immune detection by complex tertiary and quaternary structures. The inner domain of gp120, although immunogenic, elicits antibodies that cannot access these epitopes on native, functional trimeric envelope
(Wyatt et al., 1998). Studies to capture envelope in distinct conformations as it undergoes the fusion process, know as fusion intermediates, have attempted to generate antibodies against transitional state conformations of native, trimeric envelope.
Envelopes mimicking fusion intermediates include CD4-independent envelopes
(Hoffman et al., 1999; Zhang et al., 2007), CD4-gp120 complexes (Celada et al., 1990;
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Devico et al., 1996; Fouts et al., 2002; Fouts et al., 2000; Gershoni et al., 1993; He,
D'Agostino, and Pinter, 2003; Kang et al., 1994; Varadarajan et al., 2005), fusion
intermediates (Chakrabarti et al., 2002; LaCasse et al., 1999; Nunberg, 2002; Xiao et al.,
2003; Zipeto et al., 2006), and envelopes that halt fusion (Abrahamyan et al., 2003;
Sanders et al., 2002).
Although some promising results have been presented, there is considerable
concern that recombinant proteins fail to maintain a native structure that will be effective against primary isolate virus. Also, when membrane bound gp160 is used as antigen, surface–expressed IgM on naïve B-cells has limited accessibility to the viral-cell membrane where fusion occurs, limiting epitope detection and B-cell maturation.
Additionally, concerns of cytotoxicity and antibodies against human cellular proteins
have been reported in several studies (Celada et al., 1990; Devico et al., 1996; Fouts et
al., 2002; LaCasse et al., 1999; Nunberg, 2002).
To this end, quaternary virus structures that might better represent native trimeric
envelope structures have been used, including aldrithiol-2 (AT-2) inactivated whole virions (Lifson et al., 2004), VLPs or pseudoviruses (Devitt et al., 2007; Doan et al.,
2005; Hammonds et al., 2005) and trimeric, soluble gp140 (Bower et al., 2006; Earl et al.,
2001; Kim et al., 2005; Srivastava et al., 2002).
1.G.4. Mimotopes: Mimotopes are paratope-binding macromolecules that mimic the structural configuration of a specific desired epitope which, when incorporated into a vaccine, act as antigens that may elicit antibodies similar to those that recognize the desired epitope. Peptide mimotopes have been identified from random linear or structurally constrained peptides on the surface of bacteriophage by direct interaction
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with antibodies of the required epitope specificity. Successful application of the phage display strategy has helped to identify peptide mimotopes for BR-NAbs 2G12
(Agadjanyan et al., 1997; Krauss et al., 2007; Monzavi-Karbassi et al., 2003; Pashov et al., 2005a; Pashov et al., 2005b; Pashov et al., 2006) and IgG1b12 (Dorgham et al., 2005;
Saphire et al., 2007; Zwick et al., 2001a), gp41 (Enshell-Seijffers et al., 2001;
McGaughey et al., 2003; Palacios-Rodriguez et al., 2007) and CCR5 binding inhibitor agents (Konigs et al., 2007). Yet, immunizations using these mimotopes fail to elicit the desired BR-Nabs. Nevertheless, mimotopes are an attractive immunogen in vaccine development. In one study, Chen et al.(Chen et al., 2001) showed that although rhesus macaques immunized with a pool of HIV-1 specific mimotopes from both gp120 and gp41 did become infected upon challenge with virus, the monkeys experienced lower peak levels of viremia than naïve or mock immunized controls.
1.G.5. Center-of-tree, Consensus and Ancestral envelopes: With more than
30% sequence diversity seen within HIV-1 envelope protein among different viral subtypes, the prospect of a centralized envelope sequence instilled some hope in finding a single sequence that minimizes the genetic distance between vaccine strains and contemporary strains. By using evolutionary models, sequences are artificially reconstructed to form a consensus, ancestral or center-of-tree sequence (Gao et al., 2004;
Gaschen et al., 2002; Kesturu et al., 2006; Nickle et al., 2003; Rolland et al., 2007). The consensus (CON) sequence corresponds to the most frequent amino acid or nucleotide at each site within a gene sequence alignment. An ancestral (ANC) sequence represents the most common ancestor to all viruses (Figure 1-9). Some concern arises in that the consensus sequence may not represent an actual virus isolate, unlike the ancestral
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sequence that is derived from an actual virus sequence that existed in the past. Thereby,
the ancestral virus will have a conformation with native folding and function, although
consensus Envelope from Group M is functional and binds to both CD4 and CCR5. The
third approach, know as center-of-tree (COT), identifies a central position on the HIV-1
phylogenic tree where the average evolutionary distance to each tip on the phylogenic
tree is minimized, while still residing on the evolutionary path.
To date, six centralized envelope immunogens have been developed and tested as
vaccines. Two envelopes are derived from the entire M group (MCon6, ConS) and four
from subtype specific consensus (ConB, ConC) and ancestral (An1-EnvB, AncC) genes.
(Doria-Rose et al., 2005; Gao et al., 2005; Kothe et al., 2007; Kothe et al., 2006; Liao et al., 2006; Weaver et al., 2006). Most of these envelopes elicited only low titer, if any,
neutralizing antibody responses against HIV-1 after vaccination, although increased
immunogenicity was seen when compared to wild type envelope. Only the group M
ConS Env elicited a significantly high titer of neutralizing antibodies with breadth across
3 different group M clades(Liao et al., 2006). However, this envelope was delivered as a
secreted trimeric recombinant gp140, lacking the gp41 transmembrane domain, the
gp120/gp41 cleavage site, and the fusion peptide, and containing shortened consensus
variable loops. The improved immunogenicity and neutralizing activity observed using
ConSgp140 may be due, in part, to these gene modifications, although the considerable
breadth observed is likely a result of the consensus sequence of the envelope.
Given that the V3 loop has been labeled as the Principal Neutralizing Determinant
(PND), consensus sequences for V3 have also been generated (Sirivichayakul et al.,
2004; Tian, Lan, and Chen, 2002) and tested as peptides in vaccination studies (Cruz et
al., 2004; Haynes et al., 2006). Haynes et al.(Haynes et al., 2006) showed that a single
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peptide that clustered around the subtype B V3 consensus sequence elicited antibodies upon immunization that were capable of neutralizing 31% of subtype B isolates with high potency. The author suggests that this may be the maximum attainable percentage of viral isolates neutralized by anti-V3 peptide antibodies based on consensus sequence.
1.G.6. Polyclonal neutralizing antibody responses: To develop a vaccine, considerable challenge lies in overcoming viral genetic diversity. One such mechanism involves artificially generating a consensus, ancestral or center-of-tree envelope sequences. Another approach suggested by several research groups involves the design
and development of multivalent vaccines, or vaccines that are based on inclusion of
multiple epitopes of different strains, thus broadening the specificities of the antibodies
produced (see (Hurwitz et al., 2005; Hurwitz et al., 2008; McBurney and Ross, 2007;
Slobod et al., 2005) for reviews). The hypothesis is that a greater number of strain-
specific neutralizing epitopes from multiple diverse virus isolates incorporated into a
vaccine cocktail will elicit a polyclonal antibody response with greater breadth of
neutralization than any antigen from a single isolate might do. Support for the use of
multi-strain based vaccines stems from established effective vaccines against
antigenically diverse organisms such as poliovirus, influenza virus and Streptococcus
pneumoniae, and human papilloma virus (reviewed in (Hurwitz et al., 2005)).
Multivalent cocktail vaccines for HIV-1 vaccine may consist of multiple epitopes
stemming from different virus components (such as gag, pol, env, nef) (Brave et al.,
2007; Fischer et al., 2007; Shinoda et al., 2004), or a single gene product from multiple
different viral isolates or clades (Cho et al., 2001; Ljungberg et al., 2002; Lockey et al.,
2000; Pal et al., 2005; Rollman et al., 2004; Wang et al., 2006; Zolla-Pazner et al., 1998),
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with some studies focused specifically on multiple V3 epitopes (Cruz et al., 2004;
Haynes et al., 1995; Hewer and Meyer, 2003; Hewer and Meyer, 2005; Montero et al.,
1997; Schilling et al., 2006). Indeed, animals immunized with gp120 or V3 peptides from multiple HIV-1 isolates developed antibodies with greater breadth of neutralizing activity than animals immunized with a single envelope protein. (Cho et al., 2001;
Haynes et al., 1995; Montero et al., 1997; Pal et al., 2005; Rollman et al., 2004; Shinoda et al., 2004; Wang et al., 2006). However, there may be a trade-off between breadth of protection and potency of specific antibody response. Some researchers may choose to narrow the scope of their polyvalent vaccines by including epitopes from viruses limited to certain geographic regions, thereby allowing for development of region specific vaccines. While protection against all circulating strains may not be adequately achieved, protection against the most prevalent virus strains would be highly desirable. This form of limited coverage may not be good enough, but for now, it may be our only choice.
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A B
Figure 1-9: Ancestral, Consensus and Center-of-Tree HIV-1 sequences. (A) Adapted from (Nickle et al., 2003). Four possible phylogenetic shapes and the resulting reconstructed sequences. The Ancestor (Anc) and the Center Of the Tree (COT) can only fall on an evolutionary path (i.e., on a branch of the phylogeny), whereas the Consensus
(Con) may not. (B) Western blot analysis of envelope proteins from multiple virus subtypes using sera raised against different subtypes. Figure taken from (Gao et al.,
2004). Equal amounts of envelope from each virus strain is loaded on 10% SDS polyacrylamide gels, and tested for recognition by sera from individual HIV-1 infected patients, each infected by a specific virus subtype. Subtypes are indicated by single letters after Env protein and serum designations. Consensus envelope from group M
(CON6) is recognized by sera raised against different virus subtypes.
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1.H. Summary of thesis work
From this literature review, it is apparent that the mechanisms HIV-1 employs to
avoid immune responses and clearance are intricate and manifold. Despite over 20 years of innovate research, an effective vaccine that elicits broadly reactive neutralizing antibodies is nowhere in sight. The classic approaches to developing vaccines have failed, even after large scale Phase III clinical testing and billions of research dollars spent. Nevertheless, the discovery of BR-NAbs in HIV-1 patient sera, although rare,
suggests that the development of an effective vaccine that may elicit such antibodies is
indeed feasible. To accomplish these goals, it may require establishing a fresh
perspective towards the problem by breaking away from unsuccessful molds, and
undertaking exploration of typically unconventional, yet innovative approaches. Given
the devastating global impacts of the HIV-1 epidemic and the inability of researcher to develop an effective vaccine, large rewards may require large risks.
To this end, we conducted three research projects with different approaches but each with the same primary objective: to better understand how to elicit broad and potently neutralizing antibodies against HIV-1 envelope.
In Chapter 2, we aimed to better assess the immunogenic properties of gp41, with particular attention to the membrane proximal external region (MPER) which contains epitopes recognized by BR-NAbs 2F5, 4E10 and Z13. Although this region is considered a favorable region to include in any vaccine, no groups have succeeded in eliciting antibodies with neutralizing capacity similar to mAbs 2F5 or 4E10. A more thorough understanding of the immunogenicity of this region and human antibody responses against gp41 would greatly help in the design of effective antigens. We chose to
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characterize HIV-1 patient immune responses against five soluble Glutathione S-
Transferase (GST)-gp41 fusion proteins, each including the MPER. We discovered
tremendous diversity among patient sera with regards to antibody recognition of gp41,
with several patient sera including antibodies with similar binding properties to mAbs
2F5 ad 4E10. Furthermore, patients with strong antibody recognition against gp41
MPER exhibit broader and more potent neutralizing activity against HIV-1.
The second project hypothesizes that HIV-1 envelope fusion intermediate epitopes
may elicit BR-NAbs against conserved epitopes if we can promote a favorable interaction of naïve B-cell membrane-bound immunoglobulin with envelope as it undergoes receptor
binding and viral entry events. In a natural setting, where infection occurs on the surface
of CD4+ T cells or macrophages, membrane-bound immunoglobulins on B cells are
unlikely to encounter these short-lived fusion intermediate structures due to steric
hindrance. To this end, we generated two transgenic mouse lines that express huCD4
with or without CCR5 on the surface of B cells. Experiments described in Chapter 3
detail characterization of these mice and measure humoral immune responses against
several HIV-1 envelope antigens that bind to HIV-1 receptors, and against non-CD4-
binding control protein. Although we did not isolate any BR-NAbs, these mice may yet
prove useful as a tool to generate antibodies to short-lived envelope fusion intermediates.
In Chapter 4 we establish a technique that successfully generates considerable
sequence diversity within the V3 region to represent 85% of subtype B primary isolate
V3 regions through multiple antigens. On account of the V3 region being highly
immunogenic, well exposed, structurally semi-conserved and capable of eliciting potent
neutralizing antibodies, some of which (e.g. mAb 447-52D) can neutralize primary
isolates from different subtypes, V3 is an attractive vaccine candidate. However, even
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though the V3 region is known as the principal neutralizing determinant, extensive V3
sequence diversity poses a considerable challenge to developing an antibody response
with the ability to neutralize multiple virus isolates. We hypothesize that polyclonal
antibodies against a polyvalent V3 vaccine including multiple epitopes from diverse viral strains will exhibit greater breadth of neutralizing activity against HIV-1 isolates than any single immunogen, and may prevent the rapid evolution of escape mutants. Our technique could be easily utilized to generate a large pool of diverse V3 loops for use in a polyvalent vaccine approach, characterizing immune responses or investigation of functional role of V3 in binding, entry and antibody resistance.
The generation of better HIV-1 antigens, a thorough understanding of immune responses against HIV-1 envelope proteins during natural infection and exploration of novel approaches towards elicitation of antibodies may help to delineate requirements of
protection and promote future AIDS vaccine research.
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CHAPTER 2
Assessment of antibody responses against gp41 in HIV-1-
infected patients using soluble gp41 fusion proteins and
peptides derived from M group consensus envelope
Adam Penn-Nicholson1,3, Dong P. Han1, Soon J. Kim1, Hanna Park1, Rais Ansari1,
David C. Montefiori,4 and Michael W. Cho*1,2,3
Departments of 1Medicine, 2Biochemistry, 3Molecular Biology and Microbiology
Case Western Reserve University School of Medicine, Cleveland, OH 44106
Department of Surgery4, Duke University, Durham, NC
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Abstract
Accurate assessment of immunogenic properties of HIV-1 (human
immunodeficiency virus type 1) envelope glycoprotein is an important goal towards
designing antigens that can elicit broadly reactive neutralizing antibodies (BR-Nabs)
against the virus. Transmembrane glycoprotein gp41 is targeted by a number of BR-
Nabs, including 2F5 and 4E10. Accordingly, gp41 is an attractive target antigen for
vaccine development. In spite of this, its immunogenic properties are not fully
understood, due in part to difficulties in generating large fragments of the protein in soluble forms. In this study, we generated five soluble glutathione S-transferase (GST)-
fusion proteins that encompass C-terminal 30, 64, 100, 142, or 172 (full-length) amino
acids of gp41 ectodomain from an M group consensus envelope sequence (MCON6).
These proteins, in conjunction with overlapping peptides, were used to evaluate antibody
responses in HIV-1-infected patients. We found (i) tremendous variation in antibody
response profiles against different regions of gp41 among individual patients, (ii) patients
with stronger antibody responses against the membrane-proximal external region
(MPER) exhibit broader and more potent neutralizing activity than those with weaker
anti-MPER response, and (iii) patients who mount antibodies against epitopes that are
near, or overlap with, those targeted by 2F5 or 4E10 may not be as rare as has previously
been thought. Taken together, the panel of soluble gp41 fusion proteins we generated
will be important tools for better understanding humoral immune responses against gp41
and for future vaccine development.
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Introduction
During human immunodeficiency virus type 1 (HIV-1) infection, many antibodies
are elicited against viral envelope glycoprotein. The vast majority of these antibodies are
non-neutralizing, and those that neutralize are mostly isolate-specific (for reviews, see
(Burton, Stanfield, and Wilson, 2005; Poignard et al., 2001)). Results from both active
and passive immunization studies indicate that pre-existing neutralizing antibodies
(Nabs) can confer protection against HIV-1 infection(Baba et al., 2000; Cho et al., 2001;
Emini et al., 1992; Mascola et al., 2000; Shibata et al., 1999; Trkola et al., 2005).
However, the precise role of humoral immunity in controlling natural HIV-1 infection is not yet clear. Better understanding of antibody responses against HIV-1 envelope glycoproteins in virus-infected patients may facilitate development of a protective
vaccine against the virus.
Antisera that exhibit broadly neutralizing activity against diverse HIV-1 isolates
have been observed in some long-term non-progressors (LTNP) (Braibant et al., 2006;
Cecilia et al., 1999; Pilgrim et al., 1997). However, they are rare; despite over two
decades of AIDS research, only a handful of broadly reactive Nabs (BR-Nabs) have been
identified, including monoclonal antibodies (mAbs) b12, 2G12, 447-52D, 2F5, 4E10 and
m48 (Gorny et al., 1992; Muster et al., 1993; Roben et al., 1994; Stiegler et al., 2001;
Trkola et al., 1996b; Zhang et al., 2006; Zwick et al., 2001b). While the first three
antibodies are gp120-specific, the latter three target gp41. Antibodies that target gp41 are
of great interest from a vaccine development standpoint because they are more cross-
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reactive against viruses from different clades than those directed against gp120 (Binley et
al., 2004; Opalka et al., 2004; Yuste et al., 2006; Zwick et al., 2001b).
Monoclonal antibodies 2F5 and 4E10 target adjacent, but distinct, linear epitopes
in a highly conserved region of gp41 near the viral membrane known as the membrane-
proximal external region (MPER) (Muster et al., 1993; Stiegler et al., 2001; Zhang et al.,
2006; Zwick et al., 2001b), a determinant that plays a critical role in HIV-1 fusion with the cell membrane (Salzwedel, West, and Hunter, 1999; Suarez et al., 2000). Epitope mapping studies of 2F5 with synthetic peptides (Barbato et al., 2003; Biron et al., 2002;
Joyce et al., 2002), phage displayed peptide libraries (Menendez et al., 2004; Muster et al., 1993; Zwick et al., 2001b), and protease protection assays (Parker et al., 2001) have identified ELDKWA as the core antibody binding site. 4E10 binds primarily to the hexapeptide NWFNIT, which lies just four amino acids downstream of the 2F5 epitope
(Brunel et al., 2006; Cardoso et al., 2005; Stiegler et al., 2001; Zwick et al., 2001b). The epitope for m48 has not yet been precisely defined although it is thought to be distinct from those recognized by 2F5 or 4E10, and highly conformational, requiring a proper disulfide bond formation (Zhang et al., 2006).
During the past two decades, much of HIV-1 vaccine development efforts have focused on gp120. Consequently, much less is known about the immunological properties of gp41. Efforts to evaluate immunogenicity of gp41 have been hampered by the fact that the protein, either as a whole or in part, is difficult to express in soluble forms in the absence of gp120 (Gairin et al., 1991; Luo et al., 2006; Qiao et al., 2005;
Scholz et al., 2005; Weissenhorn et al., 1997a). Moreover, the large number of highly immunogenic epitopes on gp120 renders gp160 or gp140 unsuitable for assessing
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immunogenic properties of gp41, particularly against the MPER (Pantophlet and Burton,
2003; Wei et al., 2003). In addition, assessment of antibody responses against gp41 in
virus-infected patients has been done mostly in the context of short, synthetic peptides
(Calarota et al., 1996; Gnann, Nelson, and Oldstone, 1987; Goudsmit, Meloen, and
Brasseur, 1990; Horal et al., 1991; Schrier et al., 1988). While peptides are suitable for
identifying linear epitopes, antibody responses to non-contiguous, conformational
epitopes cannot be assessed.
To date, most efforts to express soluble forms of gp41 have been limited to small
fragments of the protein, including the heptad repeat (HR) regions (Root, Kay, and Kim,
2001), the immunodominant loop between HR1 and HR2 known as cluster I (Gnann,
Nelson, and Oldstone, 1987), a region between HR2 and the 2F5 epitope known as
cluster II (Binley et al., 1996; Goudsmit, Meloen, and Brasseur, 1990; Xu et al., 1991),
and the MPER (Luo et al., 2006; Opalka et al., 2004). Although Scholz et al.(Scholz et
al., 2005) have reported generating a larger 146 amino acid gp41 fragment (residues 536-
681) fused to E. coli chaperone SlyD and showed its immunoreactivity with HIV-1 patient sera, no other immunoprobing was performed using mAbs to confirm antigenic integrity of the protein.
In this study, we systematically generated five glutathione S-transferase (GST)- fusion proteins, which consist of C-terminal 30, 64, 100, 142, or 172 (full-length) amino acid residues of the gp41 ectodomain from an M group consensus envelope sequence
(MCON6). They were expressed in E. coli, solubilized and purified. These proteins, in conjunction with consensus overlapping peptides, were used to evaluate antibody responses against gp41 in HIV-1-infected patients.
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Materials and Methods
Cloning GST-gp41 fusion constructs.
To generate plasmid constructs expressing five GST-gp41 fusion proteins, regions encoding C-terminal 30, 64, 100, 142 or 172 amino acids of M group consensus gp41 ectodomain were PCR-amplified from pcDNA-MCON6gp160 (kindly provided by Dr.
Beatrice Hahn, University of Alabama; (Gao et al., 2005)). PCR reactions were carried
out using a common reverse primer 5’-GAATTCTTAATGGTGATGATGGTGATGC-
TTGATGTACCA-CAGCCAGTTGG-3’ for all five constructs, and individual forward
primers, 5’-CGCGGATCCGAGAAGAACGAGCAGGAGC-3’ (for -30); 5’-
CGCGGATCCGACGAGATCTGGGACAACATGACC-3’ (for -64); 5’-CGC-
GGATCCGAGCGCTACCTGAAGGACC-AGC-3’ (for -100); 5’-CGCGGATCCC-
GCCAGCTGCTGTCCGGCATC G-3’ (for -142); 5’-CGCGGATCCGCCGTGGGC-
ATCGGCGCC-3’ (for -172). Underlined and double underlined sequences indicate restriction enzyme sites BamHI and EcoRI, respectively. Amplified DNA fragments were digested with BamHI and EcoRI and ligated into corresponding sites on vector
pGEX-2T (GE Healthcare Life Sciences). GST-gp41-64 contains inadvertent,
inconsequential I649V mutation (numbering based on MCON6).
Protein Expression and Purification.
E.coli BL21(DE3) was transformed with recombinant plasmid or pGEX-2T and
cultured overnight at 37°C in superbroth containing ampicillin (50 g/ml). Cells were diluted 1:100 in fresh superbroth and cultured to 0.6 OD600 at 37°C, at which time fusion
protein expression was induced with 1mM IPTG (isopropyl-beta-D-
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thiogalactopyranoside) for 2 h. Cells were harvested, washed and lysed in PBS
(phosphate-buffered saline) by sonication. The cell lysate was subjected to centrifugation
at 10,000 rpm (HB4 rotor) for 20 min in a Sorvall Superspeed centrifuge. Pellets
containing inclusion bodies were washed with PBS and solubilized in PBS containing 8
M urea. Insoluble debris was removed by centrifugation and soluble proteins were bound
to Ni-NTA resin (QIAGEN) on a column. GST-gp41-30, -64 and -100 were renatured
through serial incubations with 10 bed volumes of PBS containing a decreasing step
gradient of urea at 8 M, 6 M, 4 M, 3 M, 2 M, 1 M and 0 M. Renaturation of GST-gp41-
142 and -172 fusion proteins required much slower, continuous gradient of reduction in
urea concentration, particularly below 4 M. The process typically took place over the
period of 2-3 days. The column was washed with PBS containing 20 mM imidazole and
proteins were eluted in PBS containing 200 mM of imidazole. The eluted proteins were
dialyzed against PBS, concentrations were determined by Bradford assay, and purity was assessed by SDS-PAGE followed by silver staining.
Non-fusion GST was expressed similarly. To purify the protein, cells were lysed in PBS by sonication and the lysate was subjected to centrifugation at 5,000 rpm (HB4 rotor) for 30 min. GST was purified according to the manufacturers protocol (Novagen).
Briefly, GST•Bind™ Resin was added to the supernatant and incubated at 4oC for 60 min. GST-bound resin was pelleted by centrifugation, washed twice with PBS, loaded onto a column, and washed again with GST Binding/Wash buffer (4.3 mM Na2HPO4,
1.47 mM KH2PO4, 0.137 M NaCl, 2.7 mM KCl, pH 7.3). GST was eluted in a buffer (50
mM Tris-HCl, pH 8.0) containing 10 mM reduced glutathione. The eluted proteins were
dialyzed against PBS, and concentrations were determined by a Bradford assay.
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Immunoprecipitation and Western blot.
HIV-1 gp41 mAbs 2F5 (Buchacher et al., 1994; Purtscher et al., 1996; Purtscher et al., 1994) and 4E10 (Stiegler et al., 2001) were obtained from Dr. Hermann Katinger through the NIH AIDS Research and Reference Reagent Program (NIH ARRRP).
Immunoprecipitation (IP) was done by incubating GST or GST-gp41 fusion proteins with either 2F5 or 4E10 and protein A agarose in IP buffer (10 mM Tris, pH 7.5, 200 mM
NaCl, 1 mM EDTA, 0.5% Triton X-100). The mixture was agitated overnight at 4°C.
Subsequently, antigen-antibody complex bound to protein A agarose was washed three times with IP buffer. Proteins were denatured and eluted from the resin by heating in boiling water for 3 min in 2X SDS-PAGE sample buffer. Immunoprecipitated or purified proteins were subjected to SDS-PAGE (10% acrylamide), followed by Western blot analyses using rabbit anti-GST IgG (Molecular Probes) and goat anti-rabbit IgG conjugated horseradish peroxidase (Pierce). Protein bands were visualized with
SuperSignal chemiluminescent substrates (Pierce) according to the manufacturer’s protocol.
ELISA.
Plasma samples from 44 HIV-1-infected patients were obtained through the Case
Western Reserve University Center for AIDS Research Clinical Core. Samples were heat inactivated at 56°C for 30 min. prior to use. 6-helix and 5-helix bundle proteins (Root,
Kay, and Kim, 2001) were kindly provided by Dr. Michael Root of Thomas Jefferson
University. The following reagents were obtained through the NIH ARRRP: HIV-Ig from NABI and NHLBL; HIV-1 M group consensus envelope overlapping peptides (Cat
# 9487), HIV-1 IIIB N36 and C34 Peptides ((Gallo et al., 2004); Cat # 9822 and 9824,
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respectively) from DAIDS; mAb 98-6 (Gorny et al., 1989; Robinson et al., 1991; Tyler et
al., 1990; Xu et al., 1991) from Dr. Susan Zolla-Pazner. SARS-CoV S protein peptide
(PFYSNVTGFHTINHTF; Cat #9605) was obtained through the NIH Biodefense and
Emerging Infections Research Resources Repository.
Purified GST-gp41 fusion proteins or overlapping peptides (0.5 or 20 pmoles per
well, respectively) were coated onto 96-well Nunc-Immuno Plates (Nunc; Cat # 439454)
using antigen coating buffer (150 mM Na2CO3, 350 mM NaHCO3, 30 mM NaN3, pH 9.6)
at 4°C overnight. N36, C34, 5-Helix and 6-Helix antigens were coated at 33 ng/well.
Wells were blocked with PBS (pH 7.5) containing 2.5% skim milk and 25% FBS at 37°C for 1 h, then washed four times with 0.1% Tween 20 in PBS. NP-40 was added to patient plasma samples (0.1% final) before dilution in blocking buffer. Antibodies and plasma samples were diluted as indicated, added to wells and incubated for 2 h at 37°C in 200 l blocking buffer. Wells were washed 4 times, and secondary antibody goat anti-human
IgG conjugated to horseradish peroxidase (Pierce; Cat # 31410) was incubated at 1:3000 dilution at 37°C for 1hr. Wells were washed 4 times, and developed by adding 100 l
TMB HRP-substrate (Bio-Rad) for 5-10 min. Reactions were stopped with 50 l of 2 N
H2SO4. Plates were read on a microplate reader (Versamax by Molecular Devices) at
450nm. Experiments were done in duplicates.
Neutralization Assays.
Single round infection assays in TZM-bl cells (Derdeyn et al., 2000; Platt et al.,
1998; Wei et al., 2002) using pseudoviruses were performed as we and others have
previously described (Derdeyn et al., 2000; Kim et al., 2001; Wei et al., 2002; Wei et al.,
2003). Assays were done in two laboratories with slightly different protocols. In Cho
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lab, pseudoviruses were generated using pNL4-3.Luc.R-E- (Connor et al., 1995; He et al.,
1995), pCMV-Tat (Jeang et al., 1987), and pLTR-gp140 constructs encoding gp140 of
HIV-1 isolates BAL, 89.6, DH-12, and AD8 (Kim et al., 2001) or pHCMV-G encoding
VSV-G protein (Burns et al., 1993). Briefly, 3x107 293T cells at 85% confluency in
T225 flask were co-transfected by calcium phosphate method with 150 g pNL4-3Luc.R-
E-, 150 g pLTR-gp140 or pHCMV-G constructs, and 25 g pCMV-Tat. Transfected cells were incubated for 16 h before replacing medium. Cells were cultured for two more days, at which time culture medium was collected and clarified by centrifugation. Cell- free virus stocks were aliquoted and stored at -80°C. Pseudoviruses were titered in TZM- bl cells by -galactosidase staining as previously described (Kim et al., 2001; Wei et al.,
2002). All cells were cultured at 37°C, 5% CO2, in DMEM supplemented with 10%
FBS, penicillin/streptomycin, and glutamine.
Heat-inactivated plasma samples or mAbs diluted in serum-free DMEM at indicated concentrations were mixed with about 100-150 infectious units of pseudoviruses. The mixture was incubated at 37°C for 1hr, and then added to TZM-bl target cells in 96-well plates in 50 l. After 1 h adsorption, the virus inoculum was removed and 200 l of fresh medium was added. Two days-post infection, cells were lysed and virus infectivity was determined using -Glo luminescence assay as per manufacturer’s protocol (Promega). Relative luminescence units (RLU) were measured using a luminometer (Bio-Rad). Assays were performed in duplicates or in quadruplicates. Uninfected TZM-bl cells were used to determine background luminescence and mean background was subtracted from all readings. Virus infectivity was determined as a percentage of no-serum controls (i.e. virus only).
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In Montefiori lab, assays were also done in TZM-bl cells as described (Li et al.,
2005a; Montefiori, 2004). Briefly, 200 TCID50 of virus was incubated with serial 3-fold
dilutions of serum sample in triplicate in a total volume of 150 μl for 1 hr at 37oC in 96-
well flat-bottom culture plates. Freshly trypsinized cells (10,000 cells in 100 μl of
growth medium containing 75 μg/ml DEAE dextran) were added to each well. One set
of control wells received cells + virus (virus control) and another set received cells only
(background control). After a 48-hour incubation, 100 μl of cells was transferred to 96- well black solid plates (Costar) for measurements of luminescence using the Britelite
Luminescence Reporter Gene Assay System (PerkinElmer Life Sciences). Assay stocks of Env-pseudotyped viruses were prepared by transfection in 293T cells and were titrated in TZM-bl cells as described (Li et al., 2005a).
Statistical Analyses.
For the purpose of comparing high and low GST-gp41-30-binding patient groups for the breadth of neutralizing activity, summary % infectivity for individual patients was calculated as the average % infectivity observed for each individual across the four viruses. These statistically independent summary values of % infectivity were then used to compare groups using both the two group t-test and the non-parametric
Wilcoxon Rank Sum Test. Observed % neutralization of all four viruses was significantly higher (p<0.01) in the high GST-gp41-30 antibody binding group for both tests.
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Results
Design, expression and purification of GST-gp41 fusion proteins.
To assess antibody responses against conformational, non-contiguous epitopes on
gp41 in HIV-1-infected patients, we constructed five gp41 fragments fused to gluthione-
S-transferase (GST). GST was chosen as a fusion partner to facilitate protein refolding
process. Proteins are truncated N-terminally, each about 30-40 amino acids apart,
containing the C-terminal 30, 64, 100, 142, or the entire 172 a.a. of the protein (Fig. 2-
1A). Therefore, each is designed to encompass the MPER containing the epitopes
recognized by mAbs 2F5 and 4E10. The truncation sites were chosen to be either just
up- or down-stream of HR domains. In addition, sites were chosen so that the N-terminal
ends of the gp41 fragments would be charged amino acids (i.e. RQ for -142, ER for -100,
DE for -64, and EK for -30). Proteins were tagged with six-histidine residues at the C-
terminus to facilitate protein renaturation and purification. In order to enhance protein
recognition by antisera from patients infected with a wide range of primary isolates from
different clades, we chose to generate our gp41 fragments from an envelope from the M
group consensus sequence (MCON6).
All five fusion proteins were expressed efficiently in E. coli BL21(DE3) upon
induction with IPTG (Fig. 2-1B and 2-1C). As expected, all five proteins were insoluble.
Anticipating difficulty in solubilizing two larger gp41 fragments, our initial efforts focused on GST-gp41-30, -64 and -100. Bacterial pellets were sonicated, and inclusion bodies were isolated and denatured in 8 M urea. Solubilized proteins were bound to Ni-
NTA resin. Subsequently, proteins were renatured gradually by sequential incubation
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Figure 2-1. Construction and expression of GST-gp41 fusion proteins. (A) A schematic diagram of gp41 and the five GST-gp41 fusion proteins generated. Key features of gp41 are indicated. Linear B cell epitopes identified to date are shown above gp41, and 2F5 and 4E10 binding sites are indicated. FP = Fusion Peptide; HR = Heptad
Repeat; ID = Immunodominant region; TM = Transmembrane domain. (B and C) SDS-
PAGE analyses of GST-gp41 fusion protein expression in uninduced (U) and induced (I)
E.coli cells. Arrows indicate GST-gp41 fusion proteins. Gels were stained with
Coomassie Blue. (D) Sequence of MCON6 gp41 ectodomain with 6x-His tag. The starting residues of each construct are indicated by inverted triangle.
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with decreasing concentrations of urea (8 M, 6 M, 4 M, 3 M, 2 M and 1 M) before a final
wash with PBS and elution from the resin using imidazole. Eluted proteins were finally
dialyzed in PBS. This single-step purification/renaturation procedure yields about 37, 40, and 20 mg per liter for GST-gp41-30, -64, and -100, respectively, with >85-90% purity
(Fig. 2-2A). The identities of the purified proteins were confirmed by Western
immunoblot using anti-GST antibody (Fig. 2-2B). A minor band of unknown identity
(~23 kD) was co-purified with GST-gp41-64 (Fig. 2-2A). The contaminant is likely a
cleavage product of GST since it is immunoreactive to anti-GST-antibody (Fig. 2-2B).
To verify that our fusion proteins are antigenically correct, they were subjected to
immunoprecipitation analyses using BR-Nabs 2F5 and 4E10, followed by Western
immunoblot with anti-GST antibody. As shown in Figures 2-2C and 2-2D, all three
proteins were recognized by 2F5 and 4E10, respectively. Recognition of GST-gp41-30
by 4E10, however, appeared somewhat weaker than that seen against GST-gp41-64 and -
100. No binding was observed for GST protein, demonstrating specific recognition of
gp41 MPER by 2F5 and 4E10.
Having successfully generated soluble GST-gp41-30, -64, and -100, we pursued
generating soluble GST-gp41-142 and -172. Solubilization of the two larger proteins was
more difficult. Initially, we followed the same protocol used to solubilize the smaller
proteins. However, this resulted in precipitation of the proteins even at 6 M urea. We
hypothesized that a slower transition from 8 M to 6 M should provide more time needed
for the protein to refold into the conformation that would render the protein soluble.
Using a continuous, shallow gradient (see Materials and Methods section for details), we
were able to maintain a significant portion of the protein soluble (Fig. 2-2E).
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Figure 2-2. Purification and immunoprecipitation analyses of GST-gp41 Fusion
Proteins. GST and three gp41 variants (-30, -64 and -100) were initially expressed and purified. Proteins were analyzed by silver stain (A), Western blot with anti-GST antibody (B), and immunoprecipitation with 2F5 (C) or 4E10 (D) followed by Western blot with anti-GST antibody. (E) Coomassie Blue-stained SDS-PAGE analyses of purified GST-gp41-142 and -172.
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Typical final yields for GST-gp41-142 and -172 were about 5.4 and 4.5 mg/liter,
sufficient for our studies. In addition to the difficulty in solubilizing these proteins, we
observed that these proteins would precipitate upon repeated freezing-and-thawing. To
prevent protein precipitation, working stocks of the proteins were kept at near 0 °C (ice
bath) in a cold room.
Characterization of antigenic properties of GST-gp41 fragments.
ELISA was performed to evaluate the antigenic properties of purified gp41 fusion
proteins more quantitatively. Proteins were probed with BR-Nabs 2F5 and 4E10,
polyclonal HIV-Ig (from pooled HIV-1 patient sera), and mAb 98-6, which recognizes
the coiled-coil structure of the HR1 and HR2 regions (5-helix or 6-helix bundle) (Gorny
et al., 1989; Taniguchi et al., 2000). Wells were coated with equimolar amounts of the
proteins to assess relative antigenicity.
2F5 was similarly reactive against all five gp41 protein fragments, indicating that
the epitope recognized by the antibody is conformationally similar and equally exposed among all five proteins (Fig. 2-3A). In contrast, some variations in recognition of the proteins by 4E10 were observed despite the fact that all proteins contain the same epitope recognized by the antibody (Fig. 2-3B). Compared to GST-gp41-64, -142, and -172, which were equally reactive, GST-gp41-30 and -100 were about 20- and 5-fold less reactive, respectively. This was somewhat surprising considering that the 4E10 binding site is only 4 amino acids downstream of 2F5 epitope. In addition, 4E10 binding strength was significantly weaker against the five proteins compared with 2F5, requiring approximately 5 to 100 fold higher concentrations (Fig. 2-3B). This was not unexpected
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Figure 2-3. Characterization of antigenic properties of GST-gp41 fusion proteins.
Purified GST and GST-gp41 fusion proteins were analyzed by ELISA using mAbs 2F5
(A), 4E10 (B), 98-6 (C) and polyclonal HIV-Ig (D and E). Equimolar amounts (0.5 pmoles) of GST or GST-gp41 fusion proteins were coated in each well. To detect three smaller fusion proteins by HIV-Ig, higher concentrations of the antibody were used with extended enzymatic reaction time in panel (E).
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since 4E10 exhibits relatively weak, albeit broad, neutralizing activity (Binley et al.,
2004).
98-6 reacted strongly against GST-gp41-142 and -172, indicating that these proteins exist in post-hairpin formation configuration (Fig. 2-3C). As predicted, the three smaller proteins were not detected by 98-6 since they do not contain both heptad repeat regions required to form the coiled-coil structure. Similar to 98-6, HIV-Ig reacted most strongly against GST-gp41-172 and -142 (Fig. 2-3D). Although GST-gp41-100, -64 and
-30 fusion proteins were significantly less reactive with HIV-Ig compared to -172 and -
142, antibodies against them could be detected using higher concentrations of the antibody and longer enzymatic reaction time (Fig. 2-3E). Among the three smaller proteins, HIV-Ig reacted most strongly against GST-gp41-100, which was expected since it contains the immunodominant domain of gp41 (Fig 2-1A). GST-gp41-64 was also reactive, albeit less than GST-gp41-100. In contrast, GST-gp41-30 reacted very weakly, supporting the notion that the MPER is poorly immunogenic in the context of natural
HIV-1 infection.
Characterization of antibody responses against gp41 in HIV-1-infected patients.
Evaluating antibody responses against HIV-1 envelope glycoproteins using HIV-
Ig provides only an overall picture because antibodies are prepared from a large number of virus-infected patient sera. We hypothesized that we might see some differences amongst individual patients considering significant polymorphism in the host immune system and/or variation in viral genome. The results from studies described above demonstrated that the soluble GST-fusion proteins we generated are antigenically intact
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and suitable for assessing immune responses against gp41 in HIV-1-infected patients.
Since all of the epitopes targeted by BR-Nabs identified to date map within the C- terminal 100 amino acids, we focused our efforts on characterizing antibody responses against GST-gp41-30, -64, and -100. Archived plasma samples from 44 HIV-1-infected patients were evaluated by ELISA using equimolar amounts of the three GST fusion proteins. Purified GST protein was used as a negative control. Results are shown in
Figure 2-4. Patient samples were arranged in descending order of magnitude of antibody reactivity against GST-gp41-30 (numbered CWRU-1 though -44). Of all forty-four patients, only two (CWRU-19 and -41) were on anti-retroviral therapy at the time when the plasma samples were taken.
Overall, the strongest antibody responses were detected using GST-gp41-100, followed by -64 and -30, consistent with what we observed using HIV-Ig (Fig. 2-3E); the mean A450 values were 1.8, 1.3 and 0.4 (Fig. 2-4A), and the median values were 1.9, 1.0 and 0.3 (Fig. 2-4B), respectively. Interestingly, we observed tremendous variation amongst individual patients in antibody reactivity against our three gp41 fragments, both in terms of the magnitude and binding pattern, as we hypothesized. Some of the patients exhibited very low antibody titers against all three gp41 fragments (e.g. CWRU-28, -34, -
40 and -42), whereas others mounted strong antibody responses against all of them (e.g.
CWRU-1, -3 and -5). While some patients showed good binding against only the GST- gp41-100 (e.g. CWRU-22, -25, -32, -36, -39, -43), other patients exhibited good reactivity against both GST-gp41-100 and -64, but not -30 (e.g. CWRU-29, -33, -35, -
38). In general, antibody responses against GST-gp41-64 were most variable with standard deviation of 0.98, compared to 0.35 and 0.55 of GST-gp41-30 and -100,
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respectively (Fig. 2-4B). Interestingly, markedly greater antibody responses were
observed against GST-gp41-64 than against -100 in seven patients (CWRU-1, -3, -5, -8, -
10, -13 and -17), despite the fact that the latter protein is larger. One possible explanation
is that immunogenic epitopes recognized on GST-gp41-64 in these particular patients are
buried in the context of GST-gp41-100.
Perhaps the most intriguing aspect of this study was that there were many patients
who mounted strong antibody responses against gp41 MPER, three of them in particular,
as demonstrated by antibody reactivity to GST-gp41-30. Retrospective analyses of the
patients showed that many patients who might be considered as “slow progressors”
(infected longer than 5 years, CD4 T-cell count >500, and never been on anti-retroviral
therapy; indicated by black bars on top of Fig. 2-4A) generally exhibited higher antibody
reactivity against GST-gp41-30. Previous studies have shown that BR-Nabs are stronger
and more frequent in LTNPs than other infected patients (Cecilia et al., 1999; Pilgrim et al., 1997). This result raised a possibility that some of these patients could have BR-Nabs targeting the MPER, some of which could exhibit 2F5- or 4E10-like properties.
Antisera from HIV-1-infected patients with stronger reactivity against gp41 MPER exhibit broader and more potent neutralizing activity.
To test our hypothesis that patients who mount stronger antibody responses
against gp41-MPER might have broader neutralizing activity, we compared neutralizing
activity of antisera from six patients with the highest reactivity against GST-gp41-30
(CWRU-1 to -6) and six other patients with lower reactivity (CWRU-19, -29, -36, -38, -
41 and -43) indicated by dots and asterisks in Fig. 2-4A, respectively. The latter samples
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Figure 2-4. Antibody responses against gp41 in individual patients.
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Figure 2-4. Antibody responses against gp41 in individual patients. (A) ELISA was
performed with plasma samples from HIV-1-infected patients at a dilution factor of
1:300. Plasma from an uninfected individual (HIV-ve) and no plasma were used as
controls. Plasma samples are arranged in descending order of reactivity to GST-gp41-30.
Six samples exhibiting the highest reactivity (•) and six samples with lower reactivity (*), which are further evaluated in Figs. 5 and 6, are indicated. Patients who have been infected longer than 5 years with CD4 counts >500 and never been on anti-retroviral therapy are indicated by black bars on the top. For clarity, average A450 value of GST only is shown as a line indicated as GST. Arrowheads indicate average A450 values against each fusion protein for all patients. Equimolar amounts (0.5 pmoles) of GST or
GST-gp41 fusion proteins were coated in each well. (B) Dot plot analyses of ELISA data showing distribution of antibody reactivity against each fusion protein. Median values are shown.
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were chosen semi-randomly to make sure they had good antibody reactivity against GST-
gp41-100. Neutralization assays were performed against four primary R5 or R5/X4 HIV-
1 isolates (BAL, AD8, DH12 and 89.6) using a single-round pseudovirus infection assay.
Viruses pseudotyped with vesicular stomatitis virus G protein (VSV-G) were used as a
negative control.
Although there was no absolute correlation between antibody reactivity against
GST-gp41-30 and breadth or potency of neutralizing activity on an individual patient
basis, overall, the plasma from patients with higher reactivity did exhibit broader and
more potent activity (Fig. 2-5A; p<0.01). This was particularly true for patients CWRU-
3, -4 and -6. It is also noteworthy that plasma samples from these patients efficiently
neutralized HIV-1AD8, which is one of the most difficult viruses to elicit neutralizing antibodies against (Bower et al., 2006; Cho et al., 2001; Kim et al., 2005; Kim et al.,
2003); none of the six samples with lower GST-gp41-30 reactivity exhibited strong
neutralizing activity against this virus. In contrast, plasma samples from all twelve
patients neutralized HIV-189.6 quite effectively, suggesting that this virus might be particularly sensitive to neutralization despite being a primary isolate. Titration analyses of plasma samples from six patients with strong reactivity against GST-gp41-30 confirmed potent neutralizing activity of those from patients CWRU-3, -4 and -6 (Fig. 2-
5B). The analyses showed that patient CWRU-5 also exhibited quite potent neutralizing activity against all viruses, albeit somewhat less effectively against HIV-1AD8. None of the plasma samples neutralized viruses pseudotyped with VSV-G (data not shown), indicating specific, antibody-mediated neutralization of HIV-1.
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Fig 2-5. Neutralizing activity of patient plasma samples. plasma patient of activity Neutralizing Fig 2-5.
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Fig 2-5. Neutralizing activity of patient plasma samples. HIV-1 Env pseudotyped viruses (BAL, AD8, DH12 and 89.6) were used to assess neutralizing activity in a TZM- bl cell-based assay. Data is shown as a percentage of virus infectivity in the absence of plasma. (A) Twelve patients identified as having either high or low reactivity to GST- gp41-30 were analyzed at a single plasma dilution factor of 1:90. MAb b12 (12.5 g/ml) and uninfected patient (HIV-ve) were used as controls. None of the plasma samples neutralized VSV-G pseudotyped virus (data not shown). (B) Titration analyses of neutralizing activity of plasma samples from six patients who exhibited strong reactivity to GST-gp41-30.
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Based on neutralization assays against four HIV-1 isolates, CWRU-4 exhibited
the broadest and the most potent neutralizing activity. To determine further the extent of
its breadth, neutralization assays were performed against 24 virus isolates, including
sixteen from clade B, four from clade C, and four from clade A. As shown in Table 1,
plasma samples from patient CWRU-4 were able to neutralize all of the viruses tested,
albeit at different potency. Some isolates such as MN and SF162.LS are historically
easily neutralizable “Tier 1” viruses (Mascola et al., 2005a), whereas other viruses from a
Standard Reference Panel of Subtype B HIV-1 Env Clones (NIH ARRRP Cat no. 11227)
and Subtype A and C designed for Tier 2 and 3 studies are considered neither unusually
sensitive nor resistant to neutralization. Based on our past experience (D. Montefiori),
very few patient samples have this level of breadth and potency of neutralizing activity.
At present, the epitope(s) targeted by these neutralizing antibodies are not known.
Table 2-1. Neutralizing activity of plasma samples from patient CWRU-4
Virus Clade ID50 Virus Clade ID50 Virus Clade ID50 MN B 7,103 TRO.11 B 828 Du156.12 C 511 Bal.26 B 1,093 AC10.0.29 B 912 Du172.17 C 969 SF162.LS B 43,740 RHPA4259.7 B 1,229 ZM197M.PB7 C 176 SS1196.1 B 807 THRO4156.18 B 311 CAP210.2.00.E8 C 175 6535.3 B 1,091 REJO4541.67 B 1,517 Q842.d12 A 139 QH0692.42 B 129 TRJO4551.58 B 262 Q168.a2 A 216 SC422661.8 B 144 WITO4160.33 B 60 Q461.e2 A 37 PVO.4 B 457 CAAN5342.A2 B 215 Q769.d22 A 181
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Identification of immunogenic linear epitopes within the C-terminal half of gp41
ectodomain.
Despite the lack of absolute correlation between antibody reactivity against GST-
gp41-30 and breadth or potency of neutralizing activity on an individual patient basis, the
fact that four of six patients (CWRU-3, -4, -5 and -6) with the highest reactivity against
the protein exhibited strong neutralizing activity suggested that something might be
unique about these patients. To further characterize antibody responses in these patients, immunogenic linear epitopes were examined by ELISA using 30 overlapping peptides
(15-mers, 11 a.a. overlap) spanning amino acid residues 572 through 702 (numbering based on MCON6; 569-699 on HxB2).
Out of 30 peptides, three were recognized strongly by all six patients (Fig. 2-
6A). Two peptides (596-610: LGIWGCSGKLICTTT, and 600-614:
GCSGKLICTTTVPWN) were within the immunodominant domain of cluster I
(designated a.a. 579-604 on HxB2: RVLAVERYLKDQQLLGIWGCSGKLIC; (Binley et al., 1996)). Since both peptides exhibited very similar antibody reactivity profile, the
core epitope likely consist of residues GCSGKLICTTT, which contains both cysteine
residues previously reported to be critical for recognition (Gnann, Nelson, and Oldstone,
1987).
Four peptides upstream and one peptide downstream of the two immunodominant
peptides in cluster I were also reactive, but only for certain patient plasma: 580-594
(CWRU-3), 584-598 and 588-602 (CWRU-4, -5 and -6), 592-606 (CWRU-3 and -5), and
604-618 (CWRU-3, -4 and -5). It is interesting to note that these peptides are recognized
only by patient plasma that exhibit broader, more potent neutralizing activity. That is,
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patients CWRU-1 and -2 did not mount detectable antibodies against epitopes within these peptides. It has been shown that antibodies that target the immunodominant epitopes do not neutralize HIV-1. It remains to be determined whether any of the antibodies that bind to epitopes adjacent to these immunodominant epitopes have neutralizing activity or not.
The third highly immunoreactive peptide (656-670: QEKNEQELLALDKWA) was found in a region that overlaps cluster II (designated a.a. 644-663 on HxB2:
RLIEESQNQQEKNEQELLAL; (Binley et al., 1996)). This peptide contains residues
“ALDKWA”, which is the core epitope for mAb 2F5 (ELDKWA) (Muster et al., 1993) and not considered to be a part of cluster II (Binley et al., 1996; Goudsmit, Meloen, and
Brasseur, 1990). The fact that a peptide just upstream of it (652-666:
SQNQQEKNEQELLAL) is recognized by none of the patient plasma indicates critical participation of and/or contribution from residues “DKWA” in forming the correct conformation of epitope(s) within the peptide. This is interesting for two reasons.
Firstly, most patients (i.e. all except CWRU-2) also recognized a peptide just downstream of it (660-674: EQELLALDKWASLWN). Secondly, direct ELISA analyses with 2F5 showed that the antibody binds to both peptides 656-670 and 660-674 (Fig. 2-6C).
Therefore, we believe some of these patients may have mounted antibodies similar to 2F5 in epitope recognition, if not neutralizing activity, during natural infection.
One of the most striking findings of this study was that antibodies from patient
CWRU-6 bound strongly to peptides 672-686 (LWNWFDITNWLWYIK) and 680-694
(NWLWYIKIFIMIVGG) (Fig. 2-6A), the same two peptides recognized by mAb 4E10
(Fig. 2-6C). Although 4E10 binds primarily to residues “NWFDIT”, the antibody is
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known to also interact with sequences downstream of it (e.g. L682 and W683; L679 and
W680 for HxB2; (Brunel et al., 2006)). This strongly suggests, although does not prove, that CWRU-6 may have mounted 4E10-like antibodies. Moreover, antibodies from patient CWRU-6 bound strongly to peptide 664-678 (LALDKWASLWNWFDI), which contains epitopes recognized by both 2F5 and 4E10 in almost complete entirety. In this regard, the antibody that recognizes this peptide might have Z13-like properties, a Fab fragment that has been shown to bind to an epitope overlapping residues of both 2F5 and
4E10 epitopes (Nelson et al., 2007; Zwick et al., 2001b).
Another interesting observation is that antibodies from patient CWRU-4, which exhibited the most potent and broad neutralizing activity, strongly reacted with peptide
624-638 (EIWDNMTWMEWEREI). This peptide encompasses a motif highly homologous to caveolin-1-binding domain (WNNMTWMEW). Caveolin-1 is a scaffolding protein that organizes and concentrates specific ligands within the caveolae membranes. The caveolin-1 binding site has recently been shown to play an important role during the formation of a fusion pore or endocytosis of HIV-1 (Huang et al., 2007).
What draws our interest is the finding that rabbits immunized with a peptide encompassing the caveolin-1 binding domain (SLEQIWNNMTWMQWDK) mounted quite broad neutralizing activity against multiple primary HIV-1 isolates from different clades (Hovanessian et al., 2004). Therefore, one possible source of broadly neutralizing activity for patient CWRU-4 is antibodies directed against the caveolin-1 binding domain. As shown in Fig. 2-4, antibody reactivity against GST-gp41-64 was significantly greater than that against GST-gp41-30 for most patients. This was the case even for the six patients with the highest reactivity against GST-gp41-30, except for CWRU-2 and -6,
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who showed similar reactivity. However, ELISA analyses using 15 a.a. peptides revealed almost no antibody reactivity against those in C-terminal 64 residues upstream of the
MPER (i.e. a.a. 623-656; Fig. 2-6A). Antibody reactivity was not observed even when much longer 34 a.a. peptide was used (C34), which encompasses the entire HR2 domain
(Fig. 2-6B, second panel). These results indicate that antibodies that specifically recognize GST-gp41-64 most likely bind to highly conformational and/or non-contiguous epitopes, which are formed only in the presence of the C-terminal 30 residues (i.e.
MPER). This interpretation is consistent with a previous study, which reported that antibodies directed against cluster III (a.a. 617-646; 614-643 for HxB2) are highly conformational (Binley et al., 1996). Although none of the 12 patients (6 high- and 6 low-reactivity against GST-gp41-30) we analyzed mounted detectable antibody responses against eitherN36 or C34 peptides corresponding to HR1 and HR2 regions, respectively
(Fig. 2-6B, first and second panel), all of them showed equally strong reactivity against a
5-helix bundle protein complex (Fig. 2-6B, third panel), a monomeric polypeptide consisting of three HR1 and two HR2 segments connected by linker sequences designed to mimic a trimeric, HR1-HR2 coiled-coil structure (Root, Kay, and Kim, 2001). This strong response is likely a result of antibodies against non-functional, trimeric gp41 stumps on the surface of virions exposed after gp120 shedding (Moore et al., 2006). As expected, mAb 98-6 recognized 5-helix bundle, but not N36 or C34 peptides (Fig. 2-6B, fourth panel).
ELISA results (Fig. 2-6A) revealed that plasma samples from some of the patients exhibited peptide reactivity profiles that resembled those of 2F5 or 4E10 antibodies (i.e. recognition of peptides 656-670 and 660-674 by 2F5, and peptides 672-686 and 680-694
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Figure 2-6. Identification of immunogenic linear epitopes targeted by patients using overlapping peptide ELISA
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Figure 2-6. Identification of immunogenic linear epitopes targeted by
patients using overlapping peptide ELISA. (A) Plasma samples (1:100 dilution) from six patients that exhibited the strongest reactivity against GST-gp41-30 were further
analyzed by peptide ELISA (20 pmoles/well). A schematic diagram of a section of gp41
is shown on the top and the key regions are indicated, including the binding sites of
caveolin-1, 2F5 and 4E10, and three immunogenic clusters as defined by Binley et al.
(Binley et al., 1996). Aligned amino acid sequences of peptides that are immunoreactive
are shown. Peptides are numbered based on MCON6 envelope. Those recognized by
mAbs 2F5 and 4E10 are indicated in red and blue, respectively (see panel C). (B) Plasma
samples were further evaluated with larger peptides encompassing HR1 (N36:
SGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL) and HR2 (C34:
WMEWDREINN-YTSLIHSLIEESQNQQEKNEQELL), and 5-helix bundle. The plot
shows average reactivity of six patients in either high or low GST-gp41-30-reactive
groups. MAb 98-6 was used as a positive control. (C) Immunoreactivity of peptides to
2F5 (1 g/ml) and 4E10 (20 g/ml).
111
by 4E10). Considering that some of these patients might indeed have 2F5- or 4E10-like
antibodies, we were curious as to the prevalence of patient antisera that exhibit similar
profiles. We conducted ELISA analyses of all 44 patient samples simultaneously (except
for CWRU-5 due to insufficient amount) using eight peptides that span the entire MPER.
Two peptides from the cluster I immunodominant domain (596-610 and 600-614) were
also evaluated, and an unrelated peptide (SARS-CoV spike glycoprotein a.a. 61-76) was
used as a negative control.
Although the two immunodominant peptides were highly reactive for most
individuals, about one-fourth of the patients mounted surprisingly weak antibody
responses against the region (A450 values less than 0.5; Fig. 2-7). In general, antibody
reactivity against peptide 656-670 correlated with that against GST-gp41-30. However,
there were several notable exceptions for which antibody reactivity was very weak (e.g.
patients CWRU-8, -16, -18, -19 and -20). Plasma samples from eight patients (indicated by triangles) reacted substantially above background levels for both peptides 656-760 and
660-674, suggesting these patients might have mounted 2F5-like antibodies. All but one of these patients had fairly strong antibody reactivity against GST-gp41-30. Plasma from several patients reacted substantially to peptide 664-678 (e.g. patients CWRU-3, -6, -8, -
9, -17 and -26), suggesting that they might have Z13-like antibodies (Nelson et al., 2007;
Zwick et al., 2001b). Finally, plasma from four patients (CWRU-3, -6, -11 and -12) reacted against peptides 672-686 and 680-694, which are recognized by 4E10. Very weak or near background levels of antibody reactivity were observed for peptides 668-
682, 676-690, and 684-698. Together, these results suggest that several patients develop antibodies against epitopes that are near, or overlap with, those targeted by 2F5 or 4E10.
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Fig 2-7. Identification of immunogenic linear epitopes in gp41 by ELISA using M group consensus peptides.
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Fig 2--7. Identification of immunogenic linear epitopes in gp41 by ELISA using M group consensus peptides. Immunoreactivity of plasma samples from 43 patients were analyzed against seven peptides from cluster I region (a.a. 580-618), eight peptides from the MPER (a.a. 656-698), and one peptide encompassing the caveolin-1-binding site (a.a.
624-638). A negative control peptide derived from SARS-CoV S protein showed antibody reactivity profile similar to peptide 684-698 (data not shown). CWRU-5 was not included due to insufficient amount of the sample. Peptides recognized by mAbs 2F5 and 4E10 are indicated. Patient samples that reacted significantly against both peptides recognized by 2F5 or 4E10 (arbitrarily defined as A450 value greater than twice the sum of average and standard deviation of SARS peptide background reactivity) are indicated by triangles or dots, respectively. It should be noted that Y-axis for seven peptides from the MPER are in different scale.
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Discussion
In view of the limited number of BR-Nabs that have been identified to date, better
characterization of antibody responses in HIV-1-infected patients and identification of
new and potent BR-Nabs is critical in designing vaccine immunogens. As a first step in our efforts toward achieving these goals, we have systematically generated and purified five soluble fusion proteins containing the C-terminal 30, 64, 100, 142 or 172 amino acids of gp41 ectodomain from an M group consensus sequence. These recombinant proteins appear to have intact antigenic structures based on immunoprobing analyses with
BR-Nabs 2F5 and 4E10, 98-6, and antisera from HIV-1-infected patients.
Characterization of antibody responses against gp41 had been largely limited to short peptides because the protein has been difficult to produce in soluble forms. Therefore, the set of gp41 fragments we have generated will be invaluable reagents for characterizing antibody responses against HIV-1 gp41.
Expression of eukaryotic or viral proteins in bacteria typically results in misfolding and aggregation of proteins, which accumulate in inclusion bodies. Our gp41 protein fragments were no exception despite the fact that they were fused to GST.
However, we were successful in denaturing the proteins, refolding, and renaturing them into soluble forms in the absence of any detergent, although we experienced greater difficulties in solubilizing the two larger fragments and obtained substantially lower yields. Although we did not attempt to express gp41 protein fragments without GST, we are quite certain that GST played a critical role in allowing the proteins to renature into soluble forms. This conclusion is based in part from our observation that attempts to
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remove GST by cleaving between the two fusion partners with thrombin resulted in immediate precipitation of gp41 (data not shown). We speculate that GST could be functioning in a way analogous to gp120 by masking some of the hydrophobic patches on gp41 away from aqueous solvent to prevent non-specific aggregation.
Since GST-gp41 fusion proteins are antigenically intact, shown by their ability to bind not only BR-Nabs 4E10 and 2F5, but also conformational antibodies that bind to cluster III, they could be potential candidates for vaccine development. However, these proteins are not ideal as antigens for a vaccine as the large GST portion is likely to be immunodominant over smaller gp41 fragments. On the other hand, these proteins are highly suitable for structural studies, especially GST-gp41-64 since it is well recognized by both 2F5 and 4E10 and we can easily produce up to 40 mg of the protein from one liter of bacterial culture. To date, the only solved crystal structures of gp41 are HR1/HR2 coiled-coil core and peptide epitopes that bind to 2F5 and 4E10 (Cardoso et al., 2005;
Chan et al., 1997; Ofek et al., 2004; Tan et al., 1997; Weissenhorn et al., 1997b).
Determining the structure of the entire MPER with the HR2 domain would facilitate better understanding of gp41 function and designing antigens that can elicit antibodies such as 2F5 or 4E10.
All five GST-gp41 fusion proteins were equally well recognized by 2F5, indicating that the epitope is conformationally identical and similarly exposed amongst all proteins (Fig. 2-3A). Although all five proteins were also recognized by 4E10, GST- gp41-100 and -30 were about 5- and 20-fold less reactive, respectively, when compared to the other three proteins (Fig. 2-3B). This result was somewhat unexpected taking into account that the 4E10 epitope is considered to be linear and is only four amino acids
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downstream of the 2F5 epitope. One likely explanation is that although the epitope is
linear, it is highly conformational, requiring other regions of gp41 for proper formation of
the antigenic epitope structure. In this regard, it has previously been reported that the
4E10 epitope assumes a helical conformation (Cardoso et al., 2005) and modifications
that enhance helical properties increase antibody-binding affinity (Brunel et al., 2006;
Cardoso et al., 2007). Based on these findings, our current hypotheses are (1) sequences
within GST-gp41-64 upstream of the MPER (i.e. between C-terminal residues 64 and 30)
provide constraints on the helical conformation of 4E10 epitope, thereby enhancing
antibody binding compared to GST-gp41-30; (2) additional sequences between HR1 and
HR2 within GST-gp41-100 are either disrupting or masking the epitope partially to
reduce 4E10 binding; and (3) this inhibition is reversed when the HR2 domain forms
coiled-coil structure with HR1, as demonstrated by reactivity to mAb 98-6 (Fig. 2-3C), in
GST-gp41-142 and -172. More detailed biochemical and structural analyses are needed
to test these hypotheses.
We observed tremendous variation in antibody responses to gp41 amongst
different patients, not only with respect to the magnitude, but also the pattern of antibody reactivity against different gp41 fragments and peptides. One parameter that could affect the magnitude of antibody responses is plasma viral load. Greater antigenic stimulation might be expected to induce stronger antibody responses. In this regard, the duration of infection since seroconversion and possible treatment of patients with anti-retroviral therapy could potentially influence antibody levels (Binley et al., 2000b; Lafeuillade et
al., 1997; Morris et al., 2001). An important contribution may also stem from the overall
responsiveness of the patients’ immune system to antigenic stimulation; higher viral load
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could also mean greater deterioration of helper T cell immunity, and therefore weaker B cell response. In the present study, the patients were chosen randomly, without considerations to these parameters, because we were primarily interested in studying differential epitope targeting in individual patients and identifying those who mounted strong antibody responses against potential neutralization epitopes (viz. MPER).
The variation in antibody responses against different gp41 regions or peptides in different patients could be attributable to differences in patients’ immune system (e.g. immunoglobulin gene repertoire) and/or HIV-1 isolates the patients are infected with
(e.g. envelope protein sequence). We saw that many patients who mounted strong antibody responses against the MPER (i.e. reactivity against GST-gp41-30) exhibited broader, more potent neutralizing activity compared to those who did not. It should be emphasized, however, that we presently do not know the epitope(s) targeted by Nabs.
They could be directed against the MPER, other regions of gp41, or epitopes within gp120. Furthermore, the breadth of neutralizing activity could be polyclonal with multiple Nabs targeting different epitopes. In any event, further characterization of B cell repertoire, virus isolates from these patients, and mAbs generated from the patients could provide clues as to how BR-Nabs could be elicited.
Considering that plasma samples from four of six patients that showed strong antibody reactivity against GST-gp41-30 had potent neutralizing activity, this fusion protein could be used as a tool for rapidly screening patient sera to identify those who might have BR-Nabs. In this regard, we were somewhat disappointed with the fact that patients CWRU-1 and -2, whose antisera reacted most strongly against the protein, did not exhibit potent neutralizing activity as we had hoped. One possible explanation is that
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there might be epitopes in the MPER that elicit non-neutralizing antibodies. It is
conceivable that these antibodies could prevent binding of Nabs that target epitopes in close proximity due to steric hindrance (e.g. 2F5 or 4E10). In this case, observed neutralizing activity would be determined by relative antibody titers and affinity between neutralizing and non-neutralizing antibodies that compete for the adjacent epitopes. If this hypothesis is true, then it would have strong implication in terms of vaccine design; not only does the antigen have to have correct neutralization epitopes, the antigen should not have competing non-neutralizing epitopes nearby.
Attempts to elicit antibodies with similar properties to 2F5 and 4E10 in animals immunized with antigens containing the gp41 MPER have not been successful. It has been proposed that this difficulty is due to autoantigen mimicry by HIV-1 based on observations that 2F5 and 4E10 cross-react with phospholipid cardiolipin (Alam et al.,
2007a; Haynes et al., 2005; Sanchez-Martinez et al., 2006). This view that 2F5 and 4E10 have properties of autoantibodies is quite controversial since they have been used in passive immunization studies without any complication (Ferrantelli et al., 2004b; Joos et al., 2006; Trkola et al., 2005). In addition, a more recently published study reports that
2F5 fails to exhibit any cardiolipin reactivity under their set of experimental conditions
(Scherer et al., 2007). Also, while 4E10 does have general affinity to lipids, this reactivity resembles that of anti-phospholipid antibodies elicited during many infections rather than that of autoimmune antiphospholipid syndrome. Thus, the inability to elicit antibodies with similar properties to 2F5 and 4E10 might not be attributable to immune tolerance mechanisms. In this study, we have identified many patients who mounted antibodies against the same peptides recognized by 2F5 and, to a lesser extent, by 4E10
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(Fig. 2-7). Although we have yet to determine the precise epitopes and specificities of
these antibodies, the results of our study suggest that patients who mount antibodies
against epitopes that are near, or overlap with, those targeted by 2F5 or 4E10 may not be
as rare as has previously been thought. In agreement with our findings, Gray et al. (Gray et al., 2007) have recently reported up to one-third of HIV-1-infected patients mount
Nabs against the MPER. Additional studies with a larger panel of patient samples and detailed biochemical analyses of purified antibodies that target the MPER could provide more definitive answers. In this regard, the fusion proteins we generated could be ideal reagents for rapid assessment of antibody responses against gp41 and for affinity purification of MPER-directed antibodies.
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Acknowledgments
We are grateful to Dr. Beatrice Hahn and Dr. Michael Root for providing invaluable reagents. We also thank Dr. Michael Lederman, Dr. Benigno Rodriguez, and
Dr. Barb Gripshover for their assistance in obtaining patient plasma samples, and Ms.
Wilma Mackay for statistical analyses. Dr. Dong P. Han contributed to construct design, expression and immunoprecipitation experiments. Rais Ansari assisted in GST-gp41 fusion protein purification steps. Hanna Park assisted in expression of GST-gp41-142 and -172. We would like to acknowledge Dr. Syed Mohsin Waheed for his assistance in cloning GST-gp41 constructs. Soon J. Kim was responsible for peptide ELISA of
CWRU-5. Adam Penn-Nicholson performed all patient sera and mAb ELISA analyses against fusion proteins and overlapping peptides. Antibody neutralization of virus was measured by Adam Penn-Nicholson and David Montefiori. The following reagents were obtained through the NIH AIDS Research and Reference Reagent Program, Division of
AIDS, NIAID, NIH: pNL4-3.Luc.R-E- from Dr. Nathaniel Landau; TZM-bl from Dr.
John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc.; mAbs 2F5 and 4E10 from Dr.
Hermann Katinger; mAb 98-6 from Dr. Susan Zolla-Pazner; HIV-Ig from NABI and
NHLBL; and HIV-1 M group consensus envelope overlapping peptides, and HIV-1 IIIB
N36 and C34 Peptides from DAIDS. SARS-CoV S protein peptide was obtained through the NIH Biodefense and Emerging Infections Research Resources Repository. This work was supported by the NIH grants AI-55340, AI-60503, American Foundation for AIDS
Research, and Developmental Grants from CWRU Center for AIDS Research (AI-36219) to M.W.C, and by AI30034 to D.C.M.
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CHAPTER 3
Development of transgenic mice expressing HIV-1 entry
receptors on B cell surfaces for use in generating antibodies
against short-lived fusion intermediate epitopes.
Adam Penn-Nicholson1,2, Michael W. Cho1,2 and Michael M. Lederman1,3
Departments of 1Medicine, 2Biochemistry, 3Molecular Biology and Microbiology
Case Western Reserve University School of Medicine, Cleveland, OH 44106
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Abstract
HIV-1 entry is mediated by sequential binding of gp120 to CD4 and chemokine
receptors. Each binding event of HIV envelope to CD4 and chemokine receptors is
thought to induce conformational changes in envelope, which result in exposure of conserved regions of the protein to immune detection. In a natural setting, where infection occurs on the surface of CD4+ T cells or macrophages, membrane-bound immunoglobulins on B-cells are unlikely to encounter these short-lived fusion intermediate structures. We hypothesize that better antibody responses could be mounted against fusion intermediates if fusion events occur on the B cell surface where envelope proteins are in close proximity to surface immunoglobulins. To this end, we generated two transgenic mouse lines that express hCD4 or huCD4 and huCCR5 on the surface of
B-cells. These molecules are recognized by conformation sensitive antibodies, and bind to both huCD4 and, subsequently, huCCR5. Transgenic mouse huCCR5 also binds the natural ligand derivative PSC-RANTES. B-cells expressing transgenes are fusion competent and permit HIV-1 entry. Transgenic mice are physiologically normal, generate antibody upon inoculation and can clear vaccinia virus infection. Overall, transgenic mice appear to produce lower titers of antibody against both HIV-1 envelope and control protein compared to wild type mice, but antibody titers increase given
additional boosts. Although we did not identify any broadly neutralizing antibodies
against HIV-1, selection of alternative fusion competent antigens may assist future
studies in achieving this goal. With new antigens and better adjuvants that do not disrupt
tertiary and quaternary protein structure, these mice may yet prove useful as a tool to generate broadly neutralizing antibodies to short-lived envelope fusion intermediates.
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Introduction
Despite over 20 years of research on Human Immunodeficiency Virus type 1 (HIV-
1), only a handful of broadly reactive neutralizing antibodies (BR-Nabs) against the virus have been identified. Yet eliciting such antibodies are considered a crucial component of any protective vaccine that hopes to provide sterilizing immunity (Baba et al., 2000;
Mascola et al., 2000; Shibata et al., 1999; Trkola et al., 2005). The vast majority of antibodies elicited against HIV-1 envelope protein (gp120/gp41) are either non- neutralizing or are highly strain-specific (for reviews see (Burton, Stanfield, and Wilson,
2005; Poignard et al., 2001)). The limited capacity of these antibodies is achieved through a multitude of unusual strategies HIV-1 employs to evade immune detection, including 1) high sequence diversity and the presence of large hypervariable immunogenic loops covering much of envelope, 2) extensive glycosylation to generate what has been termed an “immunosilent face” of gp120, and 3) complex tertiary and quaternary structures which act to limit exposure, both spatially and temporally, of conserved epitopes to immune detection.
Nevertheless, although rare, several BR-NAbs have been discovered in HIV-1 infected patients, including monoclonal antibodies b12, 2G12, 447-52D, 2F5, 4E10 and m48 (Burton et al., 1991; Gorny et al., 1992; Muster et al., 1993; Roben et al., 1994;
Stiegler et al., 2001; Trkola et al., 1996b; Zhang et al., 2006; Zwick et al., 2001b), giving hope that a vaccine to elicit such antibodies could indeed be feasible. The first three antibodies listed target epitopes on gp120, the last three target epitopes on the membrane- proximal external region (MPER) of gp41. However, no such antibodies have yet been elicited through immunization. The discovery of any additional BR-NAbs and
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characterization of their epitopes would be an important development in vaccine research.
HIV-1 infects host cells through a membrane fusion event mediated by binding of
the envelope glycoproteins gp120/gp41 to the human CD4 receptor followed by binding to chemokine coreceptor molecules CCR5 or CXCR4. Each binding event induces extensive conformational changes, creating new, transient structural conformations that promote events leading towards membrane fusion and viral entry (Hart et al., 1991;
Jones, Korte, and Blumenthal, 1998; Matthews et al., 1994; Sattentau et al., 1993). Upon
Env binding to CD4, the essential co-receptor binding site, which remains largely occluded in native envelope, is exposed as the V2 loop shifts to expose and stabilize the bridging sheet, allowing discontinuous conformational structures to bind to CCR5 or
CXCR4 (Kwong et al., 1998; Wyatt et al., 1995). Following coreceptor binding and the subsequent conformational rearrangements, a dramatic disassociation of gp120 from its non-covalently bonded gp41 occurs, in a process known as gp120 shedding (Moore et al.,
1990). This event releases steric constraints of gp41 and membrane fusion events are triggered. The hydrophobic fusion peptide (FP) of gp41 embeds itself in the host cell membrane and two helical heptad repeat regions (HR1 and HR2) of gp41, that make up the prehairpin intermediate structure, form a stable six-helix bundle (a trimer of HR1 and antiparallel HR2) by each HR2 coiling into the opposite and complementary groves of the opposite HR1 (Kliger et al., 2000; Melikyan et al., 2000; Tan et al., 1997;
Weissenhorn et al., 1997b). This brings the viral and cellular membranes close together, promotes membrane fusion through mixing of the lipid bilayers and formation of a fusion pore and allows entry of virus nucleocapsid and RNA into the host cell cytoplasm.
Each binding event and subsequent conformational change creates new fusion- intermediates that could possibly act as targets for neutralizing antibodies (Dimitrov et
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al., 2001; Jones, Korte, and Blumenthal, 1998). Although epitopes for well known BR-
NAbs 2F5 and 4E10 are exposed on functional timers of envelope, they may be better
exposed following binding of gp120 to receptors (Burton et al., 2004b; Reeves et al.,
2005). Furthermore, studies have reported the formation of new short-lived fusion
intermediate structures on the gp41 surface of trimeric envelope bound to CD4 and CCR5
(Finnegan et al., 2001; Finnegan et al., 2002).
A class of antibodies targeting conserved fusion intermediate epitopes exposed after
CD4 binding, called CD4-induced (CD4i) epitopes, have been identified (e.g. 17b, 21c,
23e, 48d, 49e, X5) (Moulard et al., 2002; Thali et al., 1993; Xiang et al., 2002). These
antibodies engage epitopes overlapping, or in close proximity to, the coreceptor binding
site (Kwong et al., 1998). Although initially promising, these antibodies do not show
neutralizing activity against primary HIV-1 isolates, likely as a result of spatial
limitations and steric hindrance. When Fab or single chain variable fragments (scFv) of
17b, 48d, and X5 were generated, the smaller antibody fragments showed potent and
broad neutralizing activity against primary HIV-1 isolates, but failed to show this activity
as full-size IgG1 molecules (Labrijn et al., 2003). Since these anti-CD4i antibodies were
discovered and isolated from HIV-1 infected patients, yet still have major steric problems in binding their epitopes, the likelihood that they were generated against the native, trimeric envelope complex bound to CD4 at the interface of viral and cellular membranes is low. Instead, like most antibodies against envelope, they were likely generated against soluble, monomeric gp120 released during shedding (Parren et al., 1997). This is supported by the finding that mAbs against CD4i epitopes bind monomeric gp120 (Thali et al., 1993) or envelope on infected cells (Wyatt et al., 1998) after incubation with soluble CD4, yet fail to bind in cell-cell or virus-cell fusion systems (Finnegan et al.,
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2001; Gershoni et al., 1993).
The recent discovery of antibody D5 draws attention to another fusion intermediate
epitope of gp41 known as the prehairpin intermediate, exposed subsequent to CCR5
binding in a step prior to 6-helix bundle formation (Luftig et al., 2006; Miller et al.,
2005). D5 binds to a hydrophobic pocket on the HR1 groove, in a similar fashion to the
fusion-inhibitor peptide T20 (enfuvirtide) and possess significant breadth and potency of
neutralizing activity. The fact that both scFv and whole IgG1 D5 antibodies possess
similar potency of neutralization suggests that the prehairpin fusion intermediate structure
is an ideal target for neutralizing antibodies and that full-size immunoglobulins do have
access to this short-lived epitope during fusion events. Further support for the concept of
gp41 fusion intermediate epitope accessibility to antibodies is provided by Chow et al.
(Chow et al., 2002) who inhibited fusion with gp140-gp41 complexes connected by long, flexible linkers.
Strategies to generate antibodies against fusion intermediate epitopes employing antigens deigned to expose conserved domains through deletion of variable loops
(Barnett et al., 2001; Kim et al., 2003; Lu et al., 1998) or mutation of glycan residues
(Bolmstedt et al., 1996; Quinones-Kochs, Buonocore, and Rose, 2002) have yielded mixed results, but suggest these modified envelopes do not necessarily induce a greater breadth of neutralizing activity. Efforts to mimic a conformational state of envelope as it undergoes fusion include using CD4-independent HIV-1 envelopes (where the coreceptor binding site is already exposed) (Hoffman et al., 1999; Zhang et al., 2007), CD4-envelope complexes (Celada et al., 1990; Devico et al., 1996; Fouts et al., 2002; Fouts et al., 2000;
Gershoni et al., 1993; He, D'Agostino, and Pinter, 2003; Kang et al., 1994) fusion
intermediates (Chakrabarti et al., 2002; LaCasse et al., 1999; Nunberg, 2002; Xiao et al.,
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2003; Zipeto et al., 2006) and envelopes that halt during fusion (Abrahamyan et al., 2003;
Sanders et al., 2002). However, in several studies where broad antibody neutralization was observed in animals immunized with envelope fusion mimics, neutralizing activity was not directed against envelope protein, but specifically against human CD4 receptor
protein (Celada et al., 1990; Devico et al., 1996; Fouts et al., 2002).
While generating BR-NAbs is a critical objective of HIV prophylactic vaccine
strategies, no strategy tested so far has made a significant breakthrough. There are
several difficulties in targeting fusion-intermediate epitopes, the least of which involves
the transient nature of these epitopes. Furthermore, these short-lived fusion intermediate
epitopes are rarely, if ever, presented to membrane bound immunoglobulins on the
surface of naïve B cells when natural viral infection (or cell-cell syncitium) takes place on either the T-cell or monocyte/macrophage plasma membrane. In addition, the complex tertiary and quaternary structure of native trimeric envelope limits exposure of conserved epitopes until receptor engagement. These fusion intermediate epitopes are particularly rare in relation to epitopes on native envelope, gp120 monomers and debris
(Parren et al., 1997).
To circumvent these hurdles, we developed two transgenic mouse lines expressing either human CD4 (huCD4) or huCD4 and human CCR5 (huCCR5) on the surface of mouse B-cells. We hypothesized that by inducing HIV-1 receptor engagement and fusion events in close proximity to membrane bound IgM and IgD on naïve B-cell surfaces, we would increase the likelihood of maturation and clonal expansion of B-cells that secrete antibodies against fusion intermediate epitopes. By generating mice with huCD4 alone on the B cell surface, the fusion process would halt, allowing the membrane to realign itself and prolonging exposure of intermediate structures, whereas if both
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huCD4 and huCCR5 are present, the fusion process will proceed to expose additional
fusion intermediates that could be targeted. Furthermore, transgenic mice would not
develop antibodies against self-proteins huCD4 or huCCR5. In this study, we show mouse B-cells expressing functional huCD4 and huCCR5 receptors are fusion competent with HIV-1 envelope protein, and mice are immunologically competent and without any other apparently deleterious phenotype. Transgenic mice generate antibodies against
HIV-1 envelope protein and SARS-Spike (S) transmembrane deleted (TM) protein, although at lower titers than are generated by wild type mice. Although we failed to detect any broadly reactive neutralizing antibodies from either mouse sera or B-cell hybridomas prepared from splenocytes, these mice may still be valuable tools for generating fusion-intermediate epitopes in mice immunized with slow fusion-competent, membrane bound trimeric envelope antigens.
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A.
B.
Figure 3-1: Presentation of HIV-1 fusion intermediate epitopes to surface bound immunoglobulin on naïve B-cells.
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Figure 3-1: Presentation of HIV-1 fusion intermediate epitopes to surface bound immunoglobulin on naïve B-cells. (A) Binding and fusion events initiate structural changes that expose transient epitopes of fusion intermediates. Due to the rarity of these epitopes, their temporal nature and physical constraints, they are highly unlikely to be presented to naïve B-cell surface immunoglobulins. (B) By developing transgenic mice in which B-cells are directly targeted by HIV-1, we hope to increase the likelihood of presenting conserved epitopes of fusion intermediates to naïve B-cell surface immunoglobulins. Only one gp120 molecule is shown for simplicity.
Immunization of transgenic mice expressing human CD4 with/out CCR5 coreceptor on naïve B cell surface with fusion competent HIV-1 Env immunogens may lead to development of broadly neutralizing antibodies.
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Materials and Methods
Cell Lines
The A20 murine B-cell line (Cat no. TIB-208); Sp2/0 myeloma cells were kindly
provided by Dr. Man-Sun Sy of Case Western Reserve University; 293 T-cells and the
4VA1 CD8 T-cell hybridoma line was kindly provided by Dr. Clifford Harding of Case
Western Reserve University; BSC-1 African Green monkey kidney cells and human
epithelial HeLa cells were obtained from Dr. Michael Cho; NIH 3T3 cells were obtained
from Dr. Koh Fujinaga of Case Western Reserve University.
Proteins and Immunogens
Aldrithiol-2 (AT-2) inactivated HIV-1 strains ADA and Bal were kindly provided
as a gift from Dr. Jeff Lifson of the AIDS Vaccine Program, SAIC/Frederick, Inc.,
National Cancer Institute, Frederick, Maryland, USA. HIV-1Bal gp120 was obtained
from NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID,
NIH: HIV-1DH12 gp120 was generated in house, as described in Cho et al. (Cho et al.,
2001). Purified protein MCON6gp140CF was kindly provided as a gift from Dr. Barton
Haynes (Duke Human Vaccine Institute, Duke University School of Medicine, Durham
NC, USA). The following reagent was obtained through the NIH Biodefense and
Emerging Infectious resources Repository, NIADS, NIH: SARS-CoV Spike (S) (TM),
Recombinant from baculovirus, NR-722. Production, expression and characterization of
each GST-gp41-30, -64 and -100 fusion protein was described in detail in the methods
and materials section of Chapter 2 “Assessment of antibody responses against gp41 in
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HIV-1-infected patients using soluble gp41 fusion proteins and peptides derived from M
group consensus envelope.”
Plasmids
Plasmid VLPE2.13 was kindly provided by James Hagman of the National
Jewish Medical and Research Center, University of Colorado, Denver, CO (Wagner et al., 1996); huCD4 plasmid pT4 was kindly provided by Dr. Robert Doms (University of
Pennsylvania) and huCCR5 plasmid pcCCR5 was obtained from John Moore (Weiss
Cornell Medical College, NY, NY). Plasmid pcDNA-MCON6gp160 was kindly
provided by Dr. Beatrice Hahn of the University of Alabama at Birmingham. Plasmid
pTM-GFP, expressing Green Fluorescent Protein (GFP) under control of the T7-
polymerase (T7pol) promoter, was produced in house (unpublished).
Cloning
Plasmid VLPE2.13, containing a B-cell specific promoter/enhancer element
(normally involved in the expression of mouse immunoglobulin variable lambda (V) light chain domain) was digested with PmeI to generate blunt ends. pT4 was digested with BssSI and XbaI, 5’ overhangs were filled with Large Klenow fragment and insert fragments were ligated into PmeI blunted vector VLPE2.13 with T4 DNA ligase. DH5
E. coli cells were transformed and DNA samples were screened for correct size and insert
orientation by digestion with Nhe I and Kpn I, and Eco RI and Bam HI. Protein
expression in mouse A20 B-cells was detected by flow cytometry. Plasmid pcCCR5 was
digested with Hind III and Xho I, 5’ overhangs were filled with Large Klenow Fragment
and purified insert fragments were ligated into blunted PmeI digested vector VLPE2.13.
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Miniprep samples were analyzed for correct orientation and size by digestion with KpnI and NotI and protein expression was tested in mouse A20 B-cells by flow cytometry.
Electroporation of Mouse A20 B-cells:
Approximately 10-15x106 mouse A20 B-cells (ATCC no. TIB-208) per transfection were prepared. Healthy cells were washed in phosphate buffered saline
(PBS) three times by centrifugation for 5 min at 1000 rpm (Sorvall TH3000), and resuspended in 450ul 1X PBS containing 1mM each CaCl2 and MgCl2. Cells were incubated for 5min at room temperature with 20 μg of each expression plasmid (either
VLPE2.13-huCD4 or both VLPE2.13-huCD4 and VLPE2.13-huCCR5) and with 3 μg selection plasmid pcDNA3.1. Control experiments involved co-transfection of
VLPE2.13 with pcDNA3.1, or expression plasmids without the inclusion of selection plasmid pcDNA3.1. Four hundred microlitres of the transfection mix was transferred to a
0.4cm electroporation curvette and pulsed once at 280V, 960ufd for 15-17msec. Cells were gently mixed by flicking the curvette and left to stand at room temperature for
10min before resuspension and transfer to a flask containing 25ml of complete RPMI
(10% FBS, Penicillin/Streptomycin). After 24hrs incubation at 37°C, 5% CO2, 400
μg/ml G418 was added to the culture flask. Cells were selected in complete RPMI with
400 μg/ml G418 for 2 weeks, and maintained in complete RPMI containing 200 μg/ml for a further 6 weeks.
Flow Cytometry Analysis of huCD4 and huCCR5 expression on mouse B-cells
Surface molecule expression was monitored by staining cells with the following monoclonal antibodies: anti-CD4-fluorescein isothiocyanate, anti-CCR5 –phycoerythrin,
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anti-CD19-peridinin-chlorophyll-protein (PerCP, and the appropriate isotype control monoclonal antibodies (BD PharMingen). Cells were stained and then washed in wash buffer (phosphate-buffered saline with 1% bovine serum albumin and 0.1% sodium azide), fixed in 1% formaldehyde, and analyzed using a dual-laser flow cytometer
(FACSCaliber; Becton Dickinson, San Jose, CA) and CellQuest software (BD
Bioscience, San Diego, CA). A20 mouse B cells expressing huCCR5 were incubated with 125nM PSC-RANTES (kindly provided as a gift from Dr. Oliver Hartley,
University of Geneva, Switzerland) overnight, prior to FACS analysis.
Cell-cell fusion experiments
Approximately 3x10e6 HeLa cells at 80% confluency were transfected by a standard calcium phosphate precipitation technique, with 30 μg pTM-GFP. Briefly, 0.5 ml of 0.25 M CaCl2 solution containing 30 μg of plasmids was slowly mixed with 2x
HEPES buffered saline (HBS) (50 mM HEPES, 1.5 mM Na2HPO4, 280 mM NaCl, pH
7.1) and the mixture was added to cells. After an overnight incubation, the transfection medium was removed and cells were washed three times with serum free DMEM. Cells were infected with recombinant vaccinia virus vvDHEnv expressing HIV-1DH12 Env (Cho et al., 2001; Cho et al., 1998) at an moi of 5 in 2ml medium at 37°C for 1hr.
Concurrently, 3 x10e6 A20 mouse B cell cultures identified as expressing huCD4 and hCCR5 on their cell surface were washed twice with serum free RPMI and infected with
T7pol expressing Vaccinia Virus vTF7-3 (Fuerst et al., 1986) at a multiplicity of infection of 5 at 37°C in 2ml of serum free medium, with rocking every 15min. After
1hr, both the HeLa and the A20 infected cells were gently washed, and medium was replaced with 4.5ml complete DMEM (10% FBS, Penicillin/Streptomycin) and incubated
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for a further 5hr at 37°C. Cells were gently washed in serum free medium three times, infected A20 cells were resuspended in complete DMEM containing 80 μg/ml Cytosine
Arabinoside (to inhibit vaccinia virus replication) and added to pTM-GFP transfected
HeLa cells that had been infected with vvDHEnv, or uninfected controls. Upon fusion of
HIV-1 envelope expressing cells with mouse B-cells expressing huCD4 and huCCR5 transgenes, T7 polymerase from the vTF7-3 infected mouse B-cell binds to the T7pol promoter of pTM-GFP in the HeLa cell, and induces expression of GFP. The mixed cells were incubated at 37°C for 12hr before fusion was measured as readout of GFP detection by fluorescent microscopy.
Vaccinia Virus
Vaccinia virus vTF7-3 was provided as a gift (Fuerst et al., 1986); Recombinant vaccinia viruses that drive expression of HIV-1 envelopes from different strains
(vvDHEnv, vvADEnv and vTM-Balgp160) were described previously (Cho et al., 2001;
Cho et al., 1998). vvMCON6gp140CF was provided as a gift from Barton Haynes (Duke
Human Vaccine Institute, Duke University School of Medicine, Durham NC, USA).
Vaccinia virus was amplified in HeLa cells and purified by sucrose gradient centrifugation. To generate crude virus stock, infected HeLa cells were scraped from the plate, and cells and cellular debris were collected by centrifugation (Heraeus/Sorvall swinging bucket rotor 75006445, 2,500 rpm for 5 min) and released by freeze-thawing 3-
4 times in sterile H2O by and incubation with trypsin. After centrifugation, Vaccinia virus contained in the crude stock supernatant was purified by centrifugation (Sorvall rotor TH-641) on a 36% sucrose cushion at 26,500 rpm for 80 min at 4°C. The pellet was resuspended in PBS and centrifuged at 18,750 g for 45 min on a step gradient of
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sucrose with PBS (40%, 35%, 30%, 20%, 10%, 0%). A band containing vaccinia virus
was clearly visible and collected, and was pelleted by centrifugation at 25, 000 g for 60
min at 4°C before resuspension in PBS. Vaccinia virus was titered by infection of BSC-1
cells at limiting dilution of stock, and counting plaques by staining cells with crystal
violet. Virus was stored at -80°C.
Generating Transgenic Mice
Plasmids VLPE2.13-hCCR5 and VLPE2.13-huCD4 were linearized by
digestion with Sal I, and the Ampicillin resistance gene was removed. The smaller DNA
fragment containing the V2 promoter and either huCD4 or huCCR5 gene were gel
extracted and purified. Either VLPE2.13-hCD4 DNA or both VLPE2.13-hCD4 and
VLPE2.13-hCCR5 DNA was microinjected into the male pronuclei of fertilized mouse
eggs, with assistance from the Case Transgenic and Targeting Facility. Mouse strain
C57BL6/SJL was used for generating transgenic mice.
Identification of Transgenic Mice
Tail cuts of mice were subjected to PCR gene analysis for incorporation of
huCD4 or huCCR5 genes. Primer sets used for identification of VLPE2.13-huCD4 were
sense 5’- CTAGAGAATTCGGGAAAGACGC-3’, and antisense 5’-
GACGCCCCCCAGCACAATC-AGG-3’. Primer sets used for identification of
VLPE2.13-hCCR5 were sense 5’-CCCA-GGTTTAGCTTAACAAGATGG-3’, and
antisense 5’-CCGTTTTCGAGTCCGTGTC-3’. A20 murine B-cell DNA and whole
genomic wild type mouse tail cut DNA were used as negative controls for PCR detection.
As positive controls for detection, A20 mouse B-cell DNA spiked with VLPE2.13-hCD4
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or VLPE2.13-hCCR5 DNA was used. Gene positive mice were confirmed for B-cell surface protein expression by flow cytometry.
Establishment of Homozygous Breeding Lines
Transgenic mice identified both as gene positive and protein expression positive for huCD4 or huCD4 and huCCR5 were mated with wild type mates. The F1 gene positive pups were backcrossed to founder parents to establish single breeding lines.
Subsequent male and female gene positive pups were identified, isolated and independently bred with wild type mates to enable delineation of homozygous and heterozygous status. All resultant pups were screened by PCR for genomic incorporation of huCD4 or huCCR5, and the transgenic parent yielding 100% of pups born gene positive was deemed homozygous. This experiment was repeated with another wild type mate to confirm homozygosity, and protein expression was tested by Flow Cytometry.
Homozygous mating pairs were used to establish permanent inbred breeding lines.
Mouse Immunizations
Several different immunization strategies were undertaken in this project and are explained in detail in the results section. In summary, all recombinant vaccinia virus prime-boost immunizations used 1x107 pfu purified virus, delivered
intraperitoneally in 200 μl total volume with PBS. Recombinant protein was mixed with
adjuvant (either QS-21 or CpG) in PBS for a final volume of 200 μl for intra-peritoneal immunizations or 100 μl for subcutaneous and intrasplenic immunizations. CpG ODN
1826 Class B for use in mice was purchased from Coley Pharmaceuticals (Wellesley,
MA). QS-21 was kindly provided as a gift from Antigenics, Inc (New York, NY). For
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intrasplenic immunizations, mice were anaesthetized by intraperitoneal injection with 0.2 ml rodent anesthesia cocktail (15 mg ketamine, 3mg xylazine, 0.5mg acepromaxine in 1.7
5 ml saline), and a small incision in the skin on the left flank of the mice was performed after fur was shaved and skin was swabbed with 70% ethanol. The peritoneal membrane was freed and through the incision, by means of forceps, the spleen was pulled out gently from the peritoneal cavity and onto the peritoneal membrane. While the spleen was held by forceps, 50 μl of antigen was injected through the capsule inside the spleen. As the spleen swelled, the suspension was seen to diffuse both under the capsule and deeper.
The spleen was gently slipped back inside the peritoneal cavity, the peritoneal membrane closed with glue (Histoacryl, Braun, Melsungen, Germany) and the skin sutured. The mice were monitored under special care for 6 hr to ensure recovery and the wound healed within 48 h. Mice were bled 10-14 days after each immunization, and blood was collected and allowed to coagulate at 4°C before centrifugation and serum was collected.
Serum was heat inactivated at 56°C for 30 min before use.
Infection of cells with virus incubated with QS-21
An infectious molecular clone HIV-1AD8 was incubated with various concentrations of QS-21 (25 μg/ml, 12.5 μg/ml, 6.25 μg/ml, 3.125 μg/ml, 0 μg/ml.) in serum free DMEM for 1h at 37ºC. Virus-QS-21 mix was added to PBS and mixed well before infection of TZM-bl cells at 85% confluency in a 24 well plate. At this point, the final concentration of QS-21 with cells is 8 fold diluted with PBS (i.e. 3.125 μg/ml; 1.56
μg/ml; 0.7825 μg/ml; 0.390 μg/ml; 0 μg/ml). Infection was allowed to proceed for 4 hr at
37ºC, medium was removed and 2ml of 10% FBS containing DMEM was added to the cells. Cells were incubated at 37ºC for 44 h before X-Gal staining.
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ELISA
Mouse immunoglobulin isotype profiles were established using BD Pharmingen
ELISA kit catalog no. 550487 according to the manufacturers protocol. Recombinant
proteins and GST-gp41 fusion proteins were coated onto Nunc immunosorbent 96 well
plates (cat # 439454) using ELISA binding buffer (150 mM Na2CO3, 350 mM NaHCO3,
30 mM NaN3, pH 9.6) at in 50 μl final volume per well, at 4°C overnight. 20 ng/well of
HIV-1DH12 gp120 generated from HeLa cells in-house (Cho et al., 2001), purified
recombinant HIV-1BaL gp120, or MCON6gp140CF protein were used for detection of
anti-envelope antibodies by ELISA. An equivalent number of moles (0.5
picomoles/well) of GST-gp41-30, -64 or -100 (described in detail in Chapter 2) were
used for detection of anti-gp41 antibodies by ELISA. Wells were blocked with 2.5%
skim milk, 25% FBS, 1x PBS, pH7.5 at 37°C for 1hr, then washed four times with 0.1%
Tween 20 in PBS. Mouse serum was heat inactivated at 56°C for 30 min before being
added to wells in 3 fold serial dilutions in blocking buffer, and incubated for 2 hr at 37°C in 200 μl final volume. Wells were washed 4 times, and secondary antibody goat anti- mouse IgG horseradish peroxidase (HRP) (Cat no. 31430; Pierce, Rockford, Ill.) was added at 1:3000 dilution and incubated at 37°C for 1hr. Wells were washed 4 times, and developed by adding 100 μl Bio-Rad TMB peroxidase EIA substrate (cat no 172-1067,
Bio-Rad, Hercules, CA) for 5-10 min. Reactions were stopped with 50 μl of 2N H2SO4.
Plates were read on a microplate reader (Versamax by Molecular Devices Sunnyvale,
CA) at 450 nm. Results are representative of samples in duplicate, assayed a minimum
of three times.
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Western Blot
Following 2 days of infection, cells were lysed with a hypotonic cell lysis buffer
(10 mM Tris, pH 8.0, 10 mM NaCl, 1.5 mM MgCl2, 1% NP-40). Insoluble cell debris
and nuclei were removed by a brief centrifugation in a microfuge. Cell lysates were subjected to SDS-PAGE and Western blot. Envelope proteins were detected with rabbit
anti-gp160 polyclonal antibody (Willey et al., 1991) at 1:1000-2000 dilution, followed by
goat anti-rabbit IgG conjugated horseradish peroxidase (Pierce). Protein bands were visualized with SuperSignal chemiluminescent substrates (Pierce) according to the manufacturer’s protocol.
Hybridoma Development
Four days post-immunization, splenocytes from immunized mice whose sera showed anti-gp120 antibodies as determined by ELISA, were fused with 8-
Azaguanine (20 μg/ml) pre-selected Sp2/0 myeloma cells at a ratio of
approximately 3 splenocytes to 1 myeloma cell. Cells fused with 50%
PolyEtyhlene Glycol (PEG) are plated into ten 96 well plates per spleen, precoated
with a feeder cell layer from a nude mouse. After hypoxzanthine-aminopterin-
thymidine (HAT) selective culture, the hybridoma culture supernatants were
analyzed for antibody with ELISA and neutralization assays. Cells from positive
wells were subcloned in limiting dilutions to generate mAbs.
Neutralization Assays
Single round infection assays in TZM-bl cells (Derdeyn et al., 2000; Platt et al.,
1998; Wei et al., 2002) using pseudoviruses were performed as we and others have
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previously described (Derdeyn et al., 2000; Kim et al., 2001; Wei et al., 2002; Wei et al.,
2003). Assays were done in two laboratories with slightly different protocols. In Cho lab, pseudoviruses were generated using pNL4-3.Luc.R-E- (Connor et al., 1995; He et al.,
1995), pCMV-Tat (Jeang et al., 1987), and pLTR-gp140 constructs encoding gp140 of
HIV-1 isolates BAL, 89.6, DH-12, and AD8 (Kim et al., 2001) or pHCMV-G encoding
VSV-G protein (Burns et al., 1993). Briefly, 3x107 293T cells at 85% confluency in
T225 flask were co-transfected by calcium phosphate method with 150 g pNL4-3Luc.R-
E-, 150 g pLTR-gp140 or pHCMV-G constructs, and 25 g pCMV-Tat. Transfected cells were incubated for 16 h before replacing medium and cultured for two more days before medium was collected and clarified by centrifugation and stored at -80°C.
Pseudoviruses were titered in TZM-bl cells by -galactosidase staining as previously described (Kim et al., 2001; Wei et al., 2002). All cells were cultured at 37°C, 5% CO2, in DMEM supplemented with 10% FBS, penicillin/streptomycin, and glutamine.
Heat-inactivated plasma samples or mAbs diluted in serum-free DMEM at indicated concentrations were mixed with about 100-150 infectious units of pseudoviruses. The mixture was incubated at 37°C for 1hr, and then added to TZM-bl target cells in 96-well plates in 50 l. After 1 h adsorption, the virus inoculum was removed and 200 l of fresh medium was added. Two days-post infection, cells were lysed and virus infectivity was determined using -Glo luminescence assay as per manufacturer’s protocol (Promega).
Relative luminescence units (RLU) were measured using a luminometer (Bio-Rad).
Assays were performed in duplicates or in quadruplicates. Uninfected TZM-bl cells were used to determine background luminescence and mean background was subtracted from all readings. Virus infectivity was determined as a percentage of no-serum controls (i.e. virus only).
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Results
Fusion intermediate structures of HIV-1 envelope protein are notoriously short-
lived structures. Furthermore, fusion intermediate epitopes are inaccessible to antibodies
due to steric restriction, and through the remote proximity of the fusion reaction to
immunoglobulins on naïve B cell surfaces. We hypothesized that by placing the human
cellular receptors involved in HIV-1 binding and fusion on the surface of mouse B-cells,
we could effectively increase the likelihood of exposure of these fusion intermediates to membrane bound immunoglobulins on the surface of naïve B-cells. Antigen binding would result in clonal expansion, isotype switching and the production of high affinity antibodies. By circumventing B-cell proximity constraints that exist in nature, we hope to elicit antibodies against a full spectrum of fusion intermediate structures that may exist during viral and cellular membrane fusion events. We chose the murine system as an initial model primarily because transgenic founder lines are relatively easy to establish.
Mice are also immunologically and genetically well characterized, both cheap and easy to handle and house in local animal facilities, and require less antigen for immunization than larger animals such as rabbits or guinea pigs.
Plasmid construction and in vitro expression of human CD4 and CCR5 on the surface
of mouse B cells
Human CD4 (huCD4) and huCCR5, the receptors involved in HIV-1 fusion, were
each cloned into plasmid pVLPE2.13 under control of a B-cell specific
promoter/enhancer element (Hagman et al., 1990), normally involved in the expression of
mouse immunoglobulin variable lambda (V) light chain domain.
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A.
B.
Fig. 3-2. Transgene constructs express huCD4 and huCCR5 on mouse B-cell surfaces.
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Fig. 3-2. Transgene constructs express huCD4 and huCCR5 on mouse B-cell surfaces. (A) Plasmids encoding human CD4 or CCR5 under a B cell-specific promoter/enhancer element plasmid (VLPE2.13) were constructed, and tested for expression in A20 mouse B-cells (B) and 4AV1 T-cells (data not shown). A20 B-cells were transfected by electroporation and enriched by selection with antibiotics. Cells were stained with FITC-conjugated anti-CD4 antibody (SK3 clone) or PE-conjugated anti-CCR5 antibody (2D7 clone) and analyzed by flow cytometry. A20 cells expressing
CCR5 were treated with 125 nM PSC-RANTES 16 hours prior to analysis.
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A B
Figure 3-3: Human CD4 and CCR5 expressed on mouse B cells are fusion- competent. HeLa cells expressing HIV-1DH12 gp160 and transfected with pTM-GFP were mixed with either parental (A) or CD4/CCR5-expressing (B) mouse A20 B cells, in a cell-to-cell fusion assay. Cells were electroporated with 30 μg of DNA and infected with recombinant vaccinia virus (either vTF7-3 or vvDHEnv) at moi of 3. Fusion and mixing of cell cytoplasms results in GFP expression as T7-polymerase in B-cells binds to
T7-polymerase promoter of plasmid pTM-GFP in envelope expressing HeLa cells.
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The resultant plasmids, pVLPE2.1-huCD4 and pVLPE2.13-huCCR5 (Fig. 3-2A), were
successful in encoding for surface expression of the HIV-1 cellular receptors when
electroporated into mouse A20 B cells. Figure 3-2B illustrates expression and detection
of huCD4 alone and both huCD4 and huCCR5 together on the surface of mouse A20 B-
cells, as determined by flow-cytometry using conformation specific monoclonal
antibodies SK3 and 2D7. Neither huCD4 or huCCR5 expression was detected on a mouse
T-cell line (4VA1) when electroporated with the same plasmid constructs (data not
shown), illustrating promoter specificity to B-cells. Importantly, downregulation of
surface CCR5 molecules was observed when A20 cells expressing huCCR5 were treated
with 125 nM PSC-RANTES (Pastore et al., 2003), an amino-terminus modified
derivative of RANTES, demonstrating functionality of huCCR5 to interact with its
human ligand on mouse cells.
HIV-1 receptors expressed on the surface of mouse B cells support membrane fusion
It is essential that our constructs be fusion competent when bound by HIV-1 envelope
if they are to undergo conformational changes that expose fusion intermediate epitopes.
To verify this we performed a cell-to-cell fusion assay with our huCD4CCR5 expressing
A20 B cells and HIV-1DH12 gp160 expressing HeLa cells. Mouse cells were infected with
T7pol expressing recombinant vaccinia virus vTF7-3, and envelope expressing HeLa cells were transfected with plasmid pTM-GPF, with Green Fluorescent Protein (GFP) under control of the T7pol promoter. Upon fusion and cytoplasmic mixing, the GFP gene is activated and cell-cell fusion can be determined by fluorescent microscopy (Fig. 3-3B),
whereas control A20 B-cells lacking CD4 and CCR5 expression did not express GFP
(Fig. 3-3A). As shown, CD4 and CCR5 constructs we generated expressed proteins that
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support membrane fusion initiated by HIV-1 envelope binding to CD4 and CCR5.
Generation of Transgenic Mice
We hypothesize that the likelihood of fusion intermediate envelope structures binding to membrane-bound antibody would be greatly enhanced if only CD4 were present, halting the fusion process and prolonging exposure of fusion intermediate conformations. In this regard, we chose to generate two transgenic mouse lines expressing human proteins involved in HIV entry on B-cell surfaces: One line expressing only the primary HIV-1 receptor, huCD4, and a second line expressing both huCD4 and coreceptor molecule huCCR5. Plasmid DNA was linearized and microinjected into the male pronuclei of fertilized mouse eggs. Sixty and sixty-three pups were born for the huCD4 and huCD4/CCR5 experiments, respectively, with a 23% founder rate as established by PCR analysis of tail cuts for transgenes (Fig. 3-4A). Although we identified several transgene positive founders, not all founders expressed huCD4 or huCCR5 proteins. Through
FACS analysis of circulating CD19+ B-cells from blood, we were able to confirm expression of huCD4 and both huCD4 and huCCR5 on mouse B-cell surfaces in approximately 50% of the transgene positive founders with conformation-sensitive, species-specific flourochrome-labelled monoclonal antibodies. Using a single founder for each transgenic line as an example, we saw 94.5% of B-cells express huCD4 alone and
78% express both huCD4 and huCCR5 (Fig 3-5). Inspection of the dot plots of gated mouse B cells reveals a single cell population; thus we expect that expression of these transgenes in circulating B-lymphocytes is actually near universal. Furthermore, CD19
negative non-B cells did not express huCD4 or huCCR5 on their cell surface (data not
shown), further illustrating the cell specificity of our promoter sequence.
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A.
B.
Figure 3-4: Identification of transgenic mice and establishment of homozygous breeding lines.
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Figure 3-4: Identification of transgenic mice and establishment of homozygous breeding lines. (A) Transgenic mice were developed, and mice were identified as gene positive by PCR analysis. Tail cuts were digested and DNA isolated.
Using specific DNA primers overlapping portions of the VLPE2.13 plasmid backbone and either huCD4 or huCCR5 genes, transgenic mice are differentiated from wild type mice. Pups developed from eggs microinjected with huCD4 and huCCR5 are analyzed, alongside A20 mouse B-cells, A20 cells spiked with expression plasmids, or plasmids alone. (B) Transgenic mice identified as gene-positive (through PCR) and protein- positive (through FACS analysis) were bred into homozygous lines. Homozygosity was identified by screening for 100% gene positive pups after mating a wild type mouse with a transgenic mouse. Squares and circles represent male and female mice, and solid colors signify transgene positive mice.
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A B Gate on CD19+ B cells
C D
Figure. 3-5. FACS analyses of lymphocytes from wild type and transgenic mice. PBMC from wild type (A and C), huCD4 (B) or huCD4/CCR5-transgenic mice
(D) were probed with conformation specific monoclonal antibodies against CD4 (SK3 clone), CCR5 (2D7) and B-cell antigen CD19 (ID3). Panels C and D are gated to CD19+
B cells.
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Transgenic mice were physiologically normal (e.g. general health, body weight gain,
physical appearance, reproductive capacity and lifespan) compared with wild type mice.
Importantly, PBMC from both wild type and transgenic mice contained an equivalent
number of CD19+ B lymphocytes. With this knowledge, we chose to mate
transgene/protein positive F1 pups of founders, identify homozygous breeding pairs, and
establish permanent inbred breeding lines (Fig 3-4B).
Humoral Immune responses against antigens in transgenic mice
Since our goal is to elicit HIV-1 neutralizing antibodies in transgenic mice, it is critical
that the expression of human CD4 and CCR5 does not significantly impair mice from
mounting effective antibody responses against antigen. To assess immune profiles of
transgenic mice, antibody isotype and subclass profiles of unimmunized wild type and
transgenic animals (n=3) were compared via ELISA (Fig. 3-6A). Although the levels of
IgA and Ig chain were somewhat lower in transgenic than in wild type animals, no major variations were observed. IgD and IgE levels were not evaluated because of their
low abundance in serum. A preliminary immunization was carried out to determine the
relative ability of mice to mount immune responses and high antibody titers to antigen
(Figure 3-6B). Mice were immunized three times at four-week intervals with 1x107 pfu
recombinant Vaccinia Virus (VV) driving expression of HIV-1DH12 gp160, and bled two
weeks post immunization. Although we did not detect high-level anti-gp160 antibodies in
mouse sera after 3 immunizations, we did detect equal or better immune response against
VV proteins in transgenic mice compared to wild type mice. More importantly,
transgenic mice were able to clear VV infection similarly to wild type mice. These data
suggest that our transgenic mice likely have functional and responsive immune systems.
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A.
B.
Figure 3-6. Transgenic mice are immunologically competent and physiologically normal.
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Figure 3-6. Transgenic mice are immunologically competent and physiologically normal. (A) Mouse immunoglobulin isotyping ELISA Kit from BD
Pharmingen was used according to manufacturer’s protocol. Plasma from three unimmunized animals was diluted 1:1000, and mean values and standard deviations across all mice in each group is shown. (B) Sera from mice immunized three times at four week intervals with 1x107pfu recombinant vaccinia virus encoding expression of
HIV-1DH12 gp160 (vvDHEnv) is used at 1:1000 dilution in a western blot to detect antibody response against vaccinia virus proteins (vTF7-3) and human cellular proteins
(HeLa cell lysate). Both wild type mice and transgenic mice effectively cleared vaccinia virus infection. Transgenic mice also appeared similar in size, weight, lifespan, breeding and behavior compared to wild type mice.
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Next, we undertook a series of different immunization protocols to elicit neutralizing
antibodies to HIV-1 envelope proteins described below as Immunization Set A to Set D.
Immunization Set A
An initial set of immunizations was undertaken to determine the ability of transgenic
mice to generate antibodies against HIV-1 envelope protein. Four different immunization
strategies were conducted, each tested in wild type, huCD4 and huCD4CCR5 mice. Two
different adjuvants (CpG ODN Class B and QS-21) were tested for their ability to elicit
high titer antibodies against Aldrithiol inactivated (AT-2) HIV-1ADA, either with or
without a prime immunization with vvADEnv (a recombinant Vaccinia virus that can
drive expression of HIV-1 gp160 in infected mammalian cells). AT-2 inactivated HIV-1
particles were chosen because prior analysis has shown that the envelope proteins retain
full structural integrity and remain fusion competent, yet due to zinc finger inactivation of
the nucleocapsid, remain non-infectious, making them safer to use during immunizations
(Arthur et al., 1998; Lifson et al., 2004; Rossio et al., 1998). The exact details of each
experiment are shown in Figure 3-7A. A similar strategy has successfully been employed
in macaques using rVV prime, AT-2 HIV-1 boost and QS-21 as an adjuvant (Willey et
al., 2003). Ten micrograms of p24 capsid content was administered intraperitoneally per immunization, which is the equivalent of 1x1011 particles, approximately 2x1012
gp120/gp41 complexes (approximately 7-8 trimers per low Env HIV-1 virion) or a total
of 0.4 μg of gp120 per innoculation. For each immunization, this equated to
administration of 132 μg of total protein. Antibody responses against envelope were
evaluated two weeks post immunization by ELISA against hetrologous HIV-1DH12 gp120,
in order to better identify antibodies against conserved regions of envelope (Fig. 3-7B).
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Figure 3-7: Comparison of antibody responses against HIV-1 Envelope.
(A) Wild type, CD4, and CD4CCR5 transgenic mice were immunized intraperitoneally
three times with 10 μg p24 capsid content AT-2 inactivated HIV-1ADA particles (Groups
A and B), or primed once with 1x107pfu recombinant vaccinia virus expressing gp160 of
HIV-1ADA (vvADenv) and immunized twice with AT-2 HIV-1ADA (Groups C and D).
Either 5 μg QS-21 from Antigenics (Groups B and D) or 10 μg CpG ODN 1826 Class B
from Coley Pharmaceuticals (Groups A and C) was used as adjuvant upon inactivated
HIV-1 particle administration. A fourth immunization of Group D with vvADEnv took
place at week 12. Number of mice used is indicated (n= 2 or 3). (B) ELISA was
performed using purified gp120 of heterologous HIV-1DH12 produced in HeLa cells (Cho
et al., 2001). Serum samples were collected on wks 0 (pre-bleed), 6, 10 and 15. Heat-
inactivated pooled sera were used at 1:300 dilution. (C) Neutralization assay of Group D
transgenic mouse sera against full-length, infectious HIV-1AD8 and HIV-1DH12. Sera from two mice in Group D showing the highest gp120 ELISA binding profile were pooled, diluted 10 fold and tested in a neutralization assay in TZM-bl target cells bearing CD4 and CCR5 receptors. Infected cells are stained for -galactosidase activity. (D) Western blot analyses of antibodies against human cellular proteins, vaccinia virus (VV) proteins and HIV-1 envelope protein in sera from wild type and transgenic mice in Group D after the fourth immunization (week 15). Wells were loaded with cell lysate from HeLa cells transfected with pcDNA3.1, or transfected with empty vector pTM-1 or envelope expression plasmid pTM-MCON6gp160 and infected with vaccinia virus vTF7-3 to induce protein expression. Antisera were diluted to 1:500. Polyclonal rabbit anti-gp160 antiserum at 1:1000 dilution was used as a control for detection.
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Very low antibody levels were observed in all three groups of animals when CpG was
used as adjuvant, irrespective of prime-boost strategy. In comparison, higher titers of
anti-HIV-1DH12 gp120 antibodies were detected in mice using 5 μg QS-21 as adjuvant,
albeit still relatively low. We chose to give the mice initially primed with vvADEnv a
subsequent immunization with vvADEnv at week 12, and observed a substantial
enhancement of antibody response. Antisera from mice given this additional boost were
tested for neutralizing activity against virus from vaccine strain HIV-1AD8 and
hetrologous virus HIV-1DH12 (Fig. 3-7C). Heat inactivated serum at a dilution factor of
10 neutralized neither HIV-1AD8 nor HIV-1DH12, whereas control mouse serum raised against HIV-1DH12 gp120 (Kim YB 2003, Virology) potently neutralized HIV-1DH12 but
not HIV-1AD8. To detect immune responsiveness, we ran a western blot to compare wild type and huCD4CCR5 mouse antibodies against HeLa cell lysate, wild type Vaccinia virus vTF7-3, HIV-1 Group M Consensus Envelope (MCON6) gp160, and purified recombinant HIV-1DH12 gp120 (Fig. 3-7D). All mice exhibited a particularly strong humoral immune response against human cellular proteins and, suggesting that the AT-2
HIV-1ADA contains considerable human cellular protein contaminants. There was a
significant response against vaccinia virus proteins. Of particular interest, both wild type
and huCD4CCR5 transgenic mice elicited antibodies that detect MCON6gp160, but detection of purified Bal gp120 is far weaker. Control rabbit anti-gp160 polyclonal
serum had far stronger recognition of DH12 gp120 than MCON6 gp160, further
illustrating the immense difficulty of eliciting antibodies with activity against a broad
range of HIV-1 isolates. Overall, general immune responsiveness appears greater in the
huCD4CCR5 transgenic mice compared to wild type mice, although MCON6 gp160 is
better recognized by wild type mouse serum. However, although weak, detection of
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DH12 gp120 in ELISA was slightly stronger in transgenic compared to wild type mice.
Nevertheless, we chose not to further explore antibodies or develop mAbs from this
group of animals as, with low titer anti-Env Abs and no neutralizing activity of serum
against autologous virus strain HIV-1AD8, we were unlikely to isolate any BR-NAbs.
Immunization Set B
From Immunization Set A, we determined that QS-21 was an effective vaccine
adjuvant and can be useful in eliciting high titer antibodies against vaccine antigens.
Indeed, QS-21 has been used in several HIV-vaccine studies in rodents, rabbits and
humans (Cho et al., 2001; Cristillo et al., 2006; Evans et al., 2001; Fouts et al., 2002;
Goepfert et al., 2007; Kim et al., 2003). QS-21 is a triterpene glucoside belonging to a
family of saponins derived from the Soap bark tree (Quillaja saponaria). Saponins,
including QS-21, act as mild detergents, as reflected by the Latin root of the word, sapon-
, meaning soap. We questioned whether the surfactant properties of QS-21 might affect
virion structural integrity by disrupting the viral plasma membrane and trimeric envelope
structure. Infectious HIV-1AD8 was mixed with various concentrations of QS-21 and
virus infectivity was determined (Figure 3-8A). The results showed that virus infectivity
was significantly reduced at concentrations of as little as 3 μg/ml, well below the
concentration QS-21 is used as adjuvant in our Set A immunizations (50 μg/ml).
Therefore, QS-21, as well as other commonly used adjuvants based on oil or lipid (e.g.
Freund’s, MF59, and Ribi) may not be suitable for virus particle-based envelope
antigens. Unfortunately, very few studies have been performed on the effects of
adjuvants on tertiary or quaternary structures of antigens. We hypothesized that CpG
ODNs and co-infection with recombinant vaccinia virus could act to stimulate overall
humoral immune responsiveness without compromising virus structural integrity.
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A. B.
Figure 3-8: (A) QS-21 effect of virus infectivity. B) SDS-PAGE Silver Stain of AT-2 inactivated HIV-1BAL.
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Figure 3-8: (A) QS-21 effect of virus infectivity: Molecular clone HIV-1AD8 was
incubated with various concentrations of adjuvant QS-21 in DMEM for 1 h at 37ºC
before infection of TZM-bl target cells. Infection was allowed to proceed for 4 hr at
37ºC, media was removed and replaced with DMEM + 10% FBS. Cells were incubated
at 37ºC for 44 h before X-Gal staining. B) SDS-PAGE Silver Stain of AT-2
inactivated HIV-1BAL. Prominent bands of structural proteins p24 (capsid), p17 (matrix) and p6/p7 (nucleocapsid) are easily identifiable. There is little gp120 or gp160 associated with virus particles, estimated to be around 7 spikes per virion. Concentration of total protein in sample is 15 fold greater than concentration of capsid and 330 fold greater than that of envelope.
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Figure 3-9: Immunization Set B - Comparison of antibody responses against
HIV-1 Envelope using alternate adjuvants and routes of delivery. (A) Four
huCD4CCR5 transgenic mice in each group were primed with recombinant vaccinia
virus vvBAL-Env, and boosted five weeks later with 40 μg capsid content AT-2 HIV-
1BAL. Group A received the boost sub-cutaneously with 5 μg CpG ODN 1826 Class B as
adjuvant, while Group B received the boost intraperitoneally with concurrent
administration of a second vvBAL-Env boost. After a further four weeks, one mouse
from each group (mouse A-1 and mouse B-1) received 20 μg capsid content AT-2 HIV-
1BAL delivered by intrasplenic immunization. (B) ELISA of immunized mouse sera
(1:300 dilution) against purified HIV-1BAL gp120. (C) Virus neutralization assay of
mouse sera at 10 fold dilution against HIV-1 pseudotyped virus with BAL, DH12 or
control VSV-G envelope. Monoclonal antibody b12 (12.5 μg/ml) was used as control for
neutralizing activity. Infectivity was determined by measuring luciferase activity of
pseudotyped virus infection in TZM-bl target cells.
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In this regard, we chose to immunize two groups of huCD4CCR5 mice (n=4) (Fig. 3-9)
with an initial recombinant VV prime (vvBal-Env) followed by boosting with AT-2 HIV-
1 (40 μg capsid content, four-fold greater than Immunization Set A) from well-
characterized strain Bal. One group of transgenic mice received 5 μg Class B CpG ODN
as adjuvant, administered subcutaneously with antigen, and the second group was
boosted concurrently with vvBal-Env intraperitoneally in an attempt to stimulate humoral
response and produce an “adjuvant-like” effect. Finally, one mouse from each of the two
groups was given a final boost with AT-2 HIV-1Bal intrasplenically, in an attempt to
provide antigen directly to B-cells in the spleen - a technique that has shown promising
results in several studies (Cayeux et al., 2001; Moonsom, Khunkeawla, and Kasinrerk,
2001; Ni et al., 2004; Velikovsky et al., 2000; Yoshida et al., 2003; Yu et al., 2005).
These mice were sacrificed four days later and used to develop hybridomas.
Unfortunately, antibody responses from both groups to purified Bal gp120 (as measured
by ELISA) was particularly poor, and did not increase significantly four days post
intrasplenic immunization (Fig. 3-9B). No significant neutralizing activity was observed
in mouse sera (Fig. 3-9C) and after initial hybridoma supernatant screening yielded no
neutralization, the immunization strategy was deemed flawed. Silver staining of AT-2
HIV-1Bal (Fig. 3-8B) results in multiple bands, leading us to believe there are substantial cellular contaminants. In fact, total protein concentration is 15 fold higher than that of capsid concentration. Prominent bands represent HIV-1 p24, p17 and p6, but these whole virions incorporate little envelope, as evident by only weak bands at 120kD and 160kD.
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Immunization Set C
Given the failure of AT-2 HIV-1 to elicit a strong humoral response against trimeric
HIV-1 envelope, we chose to use recombinant protein MCON6gp140CF as antigen,
kindly provided by Dr Barton Haynes of Duke University (Gao et al., 2005).
MCON6gp140CF is a recombinant, trimeric envelope complex from artificially
generated HIV-1 Group M Consensus envelope sequence. It is soluble and secreted from
cells into supernatant as the construct lacks the gp41 transmembrane tail that normally
anchors it to the plasma membrane. Additionally, both the gp120-gp41 cleavage site and
the gp41 fusion peptide have been deleted from the construct. Although this antigen does
not undergo fusion, it is still relevant to our study as it binds both CD4 and CCR5 and
undergoes considerable conformational change upon each binding event. Additionally,
by using HIV-1 Group M consensus envelope, we are more likely to generate antibodies
against conserved epitopes, some of which have shown greater breadth and potency when
compared to wild type antigens (Kothe et al., 2007; Liao et al., 2006).
Four mice from each group (wild type, huCD4 and huCD4CCR5) were primed with
recombinant vvMCON6gp140CF and boosted twice with 5 μg purified MCON6gp140CF
protein with 5 μg QS-21 as adjuvant, at four-week intervals (Fig. 3-10A). One mouse in
the huCD4 group, labeled CD4-d), died after the 3rd immunization at week 8, before
blood could be drawn for serum analysis.
Immunization after the first protein boost resulted in production of antibodies against
MCON6gp140CF (determined by ELISA) in both transgenic mouse groups and the wild type control group (Fig. 3-10B) compared to week 0 pre-immunization sera. This response was stimulated with a second protein boost in all four wild type mice, in two of three huCD4 mice and in only one of four huCD4CCR5 mice (CD4CCR5-d)).
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Immunization Set C.
Immunize: Week 0 ( IP) Week 4 (IP) Week 8 ( IP) Week 12(IP) A.
Bleed: Week 0 Week 6 Week 10 Week 12.5 Mouse groups: n=4 1) Wild type 2) CD4 3) CD4CCR5 Adjuvant Antigens Route
none- Prime: 1x107 pfu vvMCon6gp140CF i.p. 5ug QS-21- Boost: 5ug MCon6gp140CF i.p. 5ug QS-21- Boost: 5ug MCon6gp140CF i.p. 5ug QS-21- Extra Boost: 5ug MCon6gp140CF i.p. (transgenic mice only)
B.
C.
Figure 3-10:
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Figure 3-10: Antibody response against recombinant trimeric envelope from a
Consensus Group M envelope sequence. (A) Four wild type, CD4 and CD4CCR5
mice were each primed with recombinant vaccinia virus vvMCON6gp140CF, and
boosted at weeks four and eight with 5 μg MCON6gp140CF recombinant protein, intra-
peritoneally, using 5 μg QS-21 as adjuvant. One mouse in the CD4 group, labeled
CD4d), died after the second boost at week 8, before blood could be drawn. At week 12,
one CD4 and one CD4CCR5 transgenic mouse was administered a final booster
inoculation of 5 μg recombinant MCON6gp140CF. Serum was collected from blood through tail cuts at weeks 6 and 10, and 4 days after the final boost for transgenic mice.
At this time, transgenic mice that had been boosted three times were sacrificed and
spleens were harvested for use in generating B-cell hybridomas. (B) ELISA of
immunized mouse sera (1:900 dilution) against 20 μg/well purified HIV-1
MCON6gp140CF. (C) Virus neutralization assay of pooled mouse sera at 10 fold
dilution against HIV-1 BAL, DH12 or control VSV-G envelope pseudotyped virus.
Monoclonal antibody b12 (12.5 μg/ml) was used as control for neutralizing activity.
Infectivity was determined by measuring luciferase activity of pseudotyped virus
infection of TZM-bl cells.
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However, mouse CD4CCR5-d) had no apparent antibody response after the first protein
boost, leading us to believe that the antigen mixture may have leaked during I.P.
immunization of that animal. A striking observation from these results is that wild type
mice generated a higher titer antibody response than of that seen in the dual-positive huCD4CCR5 mice and some of the huCD4 mice. This could not be accounted for by
human error alone, and results are in disagreement with our previous observations in
immunocompetence studies. Nevertheless, we were pleased to find our transgenic mice
developed antibodies against our antigen, and were interested to determine if these
antibodies were of better quality in terms of both breadth and potency of HIV-1
neutralization. Serum from mice in each group were pooled and measured for
neutralizing activity against HIV-1 envelope pseudotyped virions (Fig. 3-10C). Serum
from all three groups showed significant neutralization of Bal at 1:10 dilution, and poor
to no neutralization of DH12. Wild type mouse sera neutralized Bal more potently than
huCD4 and huCD4CCR5 sera, but this is likely due differential titers of antibodies.
One mouse from each transgenic group showing high titer antibody was selected
for a final immunization with MCON6gp140CF. Mice were bled 4 days post-
immunization, and spleens were harvested to generate B-cell hybridomas. Fusion
efficiency of primary B-cells and Sp2/0 myeloma cells was low, between 10-15%,
yielding approximately 100-150 cell positive wells after HAT selection, for each
transgenic mouse. Hybridoma cells were seen to express huCD4 and huCCR5 after
fusion with transgenic mouse primary B-cells (Fig. 3-11A). Unfortunately, screening of
hybridoma supernatants against two HIV-1 envelope pseudotyped viruses yielded no
significant neutralizing activity in either of our transgenic lines (Fig. 3-11B), although
virus was consistently neutralized by control mAb b12.
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C
A B
Figure 3-11: B-cell hybridomas B-cell Figure 3-11:
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Figure 3-11: B-cell hybridomas (A) Flow cytometry analysis of human CD4
and CCR5 expression on the surface of mouse B-cell hybridomas. Splenocytes from wild
type and huCD4CCR5 mice immunized with MCON6gp140CF were fused with Sp2/0
myeloma cells and selected with HAT prior to FACS analysis. (B) Virus neutralization assay of select, individual hybridoma supernatants against HIV-1BAL and HIV-1DH12
envelope pseudotyped virus. Monoclonal antibody b12 (12.5 μg/ml) and virus alone
were used as controls for neutralizing activity. Infectivity was determined by measuring
luciferase activity of pseudotyped virus infection of TZM-bl cells. (C) Antibody binding
analysis of select transgenic mouse hybridoma supernatants to purified HIV-1BAL gp120,
MCON6gp140CF or to coating buffer alone, as determined by ELISA.
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Several hybridoma supernatants antibodies were tested for their ability to bind to HIV-1
envelope, even though it is unlikely that fusion intermediate antibodies would bind to
envelope in an unligated form. We found three hybridoma supernatants that bound to
MCON6gp140CF, two of which also bound to purified BAL gp120 (Fig. 3-11C). One of
these hybridoma supernatants also bound strongly to ELISA plates that had been coated
without any protein, suggesting that non-specific binding might account for this apparent
activity. None of the hybridoma supernatants seen to bind MCON6gp140CF showed any
neutralizing activity against Bal or DH12 Env pseudotyped viruses (data not shown).
Regardless of neutralizing antibody titers, we had hypothesized that the interaction of
envelope with receptors on B-cell surfaces would increase the likelihood of eliciting antibodies against rare epitopes. One such region is the gp41 MPER, bound by BR-Nabs
2F5, 4E10 and Z13. To determine whether transgenic mice might induce higher titer antibodies against gp41 MPER epitopes due to recruitment of envelope to naïve B-cells, we analyzed immunized mouse sera antibody binding to GST-gp41-fusion proteins -30, -
64 and -100, as described in detail in Chapter 2. All GST-fusion proteins, and a GST control, were coated at equivalent molar amounts (0.5 pmoles), and serum following each boost was used at a dilution factor of 300 (Fig. 3-12). Very poor antibody binding to
GST-gp41-30 was observed across all groups of immunized mice, which is not entirely unexpected given the rarity of antibodies against the MPER in chronically HIV-1 infected human patients (Braibant et al., 2006; Burton et al., 2004b; Wei et al., 2003; Zwick et al.). For all mouse groups, antibody responses against the larger GST-gp41-64 and-100 was far more pronounced. No reactivity was shown against the GST control protein alone
(data not shown), indicating specificity of binding antibodies to gp41 epitopes. Overall,
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Figure 3-12: Immunized mouse sera reactivity against gp41. ELISA analysis of
MCON6gp140CF immunized mouse serum antibody binding to GST-gp41-30, -64 and -
100 proteins (discussed in detail in Chapter 2). ELISA wells were coated with 0.5 pmol of each GST-gp41 fusion protein. Serum from individual wild type, huCD4 and huCD4CCR5 transgenic mice was diluted 300 fold.
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antibody responses against GST-gp41-64 and -100 were greater in the wild type mice
than in either of the transgenic mouse groups. This effect is likely a result of differences
between groups in overall antibody titers, as seen in reactivity against MCON6gp140CF
(Fig 3-10B), and may not necessarily represent a difference in the quality of antibodies
elicited against gp41 in different groups of mice. Interestingly, wild type mice wt-b) and
wt-c) only elicited antibodies against gp41 epitopes following the third immunization.
This is in contrast to the pattern observed in MCON6gp140CF antibody titer, where a
strong response was elicited after only two immunizations. Furthermore, wt-a) and wt-d)
appeared to have lower titer antibodies against gp41 after the third immunization than
after the second immunization, whereas all MCON6gp140CF specific antibodies
increased in titer following the third immunization. This may suggest that epitopes of
gp120 are highly immunogenic and may be immunodominant to gp41 epitopes,
especially so in the context of soluble, trimeric envelope where gp120-gp41 dissociation
does not occur (MCON6gp140CF lacks the gp120-gp41 cleavage site), thereby ensuring
that the highly immunogenic gp41 cluster I (residues 579-604 of HIV-1HXB2) epitopes
considered immunodominant remain occluded and are not exposed as gp41 stumps after
shedding (Moore et al., 2006).
Immunization Set D
The apparent reduced humoral response towards MCON6gp140CF observed in our transgenic mice was an unexpected result. We chose to repeat the above experiments in
order to determine if the lower antibody titers generated in our transgenic mice was spurious or reflected a compromised immune system intrinsic to these mice.
Furthermore, we wanted to assess whether the observed decreased humoral response was
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Immunization Set D.
A.
B. C.
D. E.
FIGURE 3-13
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Figure 3-13: Transgenic mice develop diminished humoral response against both
HIV-1 envelope protein and control protein lacking the ability to bind human CD4
or CCR5. (A) Four mice each from wild type, CD4 and CD4CCR5 transgenic groups
were immunized twice with 5μg HIV-1 MCON6gp140CF or 5 μg recombinant SARS-
Corona Virus Spike Protein cleaved at the transmembrane region (SARS-S(TM)), four weeks apart. Mice were immunized intraperitoneally with recombinant proteins and 5 μg
QS-21 as adjuvant. Serum was collected from blood through tail cuts, at week 6. Two
CD4CCR5 transgenic mice were selected for a third booster immunization with 5 μg
MCON6gp140CF at week 14. ELISA binding activity of immunized mouse serum
against 10ng/well MCON6gp140CF (B) or SARS S(TM) (C) protein. Preimmune, day
10, and week 6 sera from each mouse is pooled for each group, as indicated. Shown is
week 6 serum from each individual mouse. (D) Optical density (450 nm) of week 6
serum for each individual mouse immunized with recombinant proteins is averaged across each group and shown with standard deviation. (E) Two dual positive huCD4CCR5 mice received an additional boost with 5 g MCON6gp140CF + QS-21 at week 14. Two weeks post immunization, ELISA response against MCON6gp140CF was measured and compared to earlier responses in transgenic and wild type mice.
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specific to antigens involved in binding to huCD4 and/or huCCR5, or more generally
towards antigens that have no other interaction with B cells other than through binding to
B-cell receptors.
Four mice from each group were used in two immunization arms: one with 5 μg
MCON6gp140CF and one with 5 μg Severe Acute Respiratory Syndrome (SARS)-
Corona Virus (CoV) Spike(S) protein truncated at the transmembrane (TM) domain,
named SARS-CoV S(TM). Five micrograms of QS-21 was administered as adjuvant
upon each inoculation (Fig. 3-13A). Mice did not receive a recombinant vaccinia virus prime, as had been used in earlier experiments. Following the second immunization, one of the SARS-CoV S(TM) immunized wild type mice died before blood could be withdrawn.
After two immunizations, we observed a reduced humoral immune response in both huCD4 and huCD4CCR5 mice compared to wild type mice against both MCON6gp140 and SARS-CoV S(TM) antigens (Fig. 3-13B and C). The reduced capacity to elicit antibodies was far more pronounced in the dual-positive huCD4CCR5 mice. We did observe some variability in response among mice within the same group. A notable example was one of four huCD4 mice immunized with MCON6gp140CF that mounted a strong antibody response compared to the responses mounted by the other three. Serum from individual mice in each group were pooled together to compare the overall response against each antigen across the group, which further highlights the reduced capacity of our transgenic mice to mount antibodies against each antigen. The A450 value obtained from each individual mouse antiserum was averaged across each group, and represented in Figure 3-13D. Although there is large standard deviation within the huCD4 group immunized with MCON6gp140CF, there is a clear difference in humoral response
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between the wild type and huCD4CCR5 mice, for both antigens.
We chose to immunize huCD4CCR5 mice #1 and #2 with 5 μg MCON6gp140CF
at week 14 to boost antibody titers and stimulate B cells and to levels sufficient for hybridoma development. More mice were not immunized due to limitations in
availability to reagents. Nevertheless, results indicate increased antibody titers to
MCON6gp140CF with this subsequent boost, greater than that seen in wild type mice
after two immunizations (Fig. 3-13E). Neither mouse serum at 1:10 dilution nor
hybridoma supernatants screened showed any neutralizing activity against HIV-1 Bal,
DH12 or AD8.
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Discussion
Despite over 20 years of research, only a handful of BR-NAbs against HIV-1
envelope have been discovered. A major mechanism HIV-1 employs to avoid eliciting
such antibodies includes masking of conserved epitopes through complex structural
features and conformationally recessed binding sites. To promote elicitation of
neutralizing antibodies against conserved fusion intermediate epitopes, we generated two
transgenic mouse lines that express human CD4 or CD4 and CCR5 on the surface of B
cells. We hypothesized that HIV-1 envelope binding to receptors artificially expressed on naïve B-cells would substantially increase the likelihood of membrane-bound IgM antibodies encountering such epitopes. In our experiments, detection of human CD4 and
CCR5 by FACS analysis suggests that expression and transport of the proteins to the surface of mouse B cells occurred normally. Furthermore, the ability of CCR5 to bind
PSC-RANTES and mediate cell-to-cell fusion suggests that the proteins are structurally
intact and properly transported to the plasma membrane. The fact that these transgenic B
cells are permissive to HIV-1 envelope mediated fusion is essential, as presentation of
these epitopes to membrane bound immunoglobulins on naïve B-cells in the correct,
native setting, is integral in developing functional neutralizing antibodies (Finnegan et al.,
2001).
Both huCD4 and huCD4CCR5 transgenic mice were physiologically normal with
respect to their size, and ability to grow and to reproduce, and immunologically healthy
as unimmunized mice showed comparable concentrations of serum immunoglobulin
isotypes and subtypes compared with wild type mice. Furthermore, transgenic mice were
capable of mounting strong humoral immune responses against vaccinia viruses and were
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able to clear the virus infection similarly to wild type mice.
Mice immunized during several protocols using combinations of recombinant
vaccinia virus encoding for expression of HIV-1 envelope, whole AT-2 inactivated HIV-
1 virus particles and recombinant trimeric gp140, elicited HIV-1 specific antibody but
showed only limited breadth and potency of neutralizing antibody activity. Overall, AT-
2 inactivated HIV-1 particles possessed sub-optimal levels of envelope protein required
for vaccination, and included considerable protein contaminants that likely originated
from human cells and acted to divert mouse immune responses away from the HIV-1
envelope. Trimeric recombinant protein MCON6gp140CF proved to induce a greater
immune response in mice, but may have been an inappropriate antigen as it lacks the
gp120/gp41 cleavage site and the fusion peptide. Nevertheless, alternative fusion
intermediate structures and unique conformations may still be presented by
MCON6gp140 after receptor binding that are of significance to vaccine development.
In a limited attempt to generate hybridomas, we were unable to isolate any
monoclonal antibodies with neutralizing activity from these transgenic mice, although we
did identify hybridomas expressing antibodies capable of envelope binding.
Interestingly, transgenic mice appeared to mount lower titer vaccine antigen specific
antibodies than did wild type mice, irrespective of whether the antigen was capable of
binding HIV-1 receptors. It is apparent from our experiments that huCD4 and possibly
huCCR5 expression on mouse B-cells somehow decreases B cell function and
responsiveness to antigen, but with additional inoculations or continual antigen exposure, peak immune response is reached. Interestingly, HIV-1 envelope protein has been seen to induce apoptosis in human lymphocytes (Holm et al., 2004; Varbanov, Espert, and
Biard-Piechaczyk, 2006), initiated by gp120 binding and signaling events through both
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CD4 and coreceptor (Hivroz et al., 1993; Holm et al., 2004; Misse et al., 1999; Moutouh
et al., 1998). Transgenic mice likely signal through CD4 and CCR5 through identical
intracellular pathways (Oppermann et al., 1999; R&D_Systems). Such signaling events
could, in theory, play an important role in the observed diminished antibody response
seen in both transgenic mice compared to the wild type mice. Additionally, gp120 on
whole, AT-2 inactivated virions has also shown direct deleting activity on memory B
cells (Viau, Veas, and Zouali, 2007) depressing immune responsiveness and activation
(Cagigi et al., 2007; De Milito et al., 2001; Viau, Veas, and Zouali, 2007). Yet, the lower
titer of antibodies observed against SARS S (TM) in transgenic mice after two immunizations leads us to believe that this effect is irrespective of the antigen’s capability to bind to either CD4 or CCR5.
One hypothesis is that normal intracellular B cell signaling events involved in B cell activation may be disrupted. Normally, signaling is initiated through immunoglobulin/antigen complex internalization, leading to antigen presentation via
MHC class II to helper T cells, resulting in B cell activation. Disruption of signaling molecule binding to the BCR may occur through the presence of the huCD4 cytoplasmic tail on transgenic B cells. Tyrosine kinase p56lck is known to bind the CD4 cytoplasmic tail, and can phosphorylate the B cell receptor cytoplasmic tails upon cross-linking of B cell antigen receptors (Gold et al., 1994). Both p56lck and the p56lck binding-site on
CD4 is conserved between mouse and human CD4 (Salghetti, Mariani, and Skowronski,
1995). In transgenic mice, the cytoplasmic tails of the Ig- and Ig- components of the B cell receptor may compete with huCD4 for binding to p56lck, thus increasing the threshold required for B cell activation in transgenic mice. A similarly reduced humoral response was also seen in mice transgenic for human CD4 expression on T lymphocytes,
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especially evident by a delayed and weaker secondary humoral immune response compared to wild type mice (Seagal et al., 2001). This may be dependent on CD4 ligation and requires the cytoplasmic tail of CD4 (Marschner et al., 2002). CCR5 and
IgM also signal through related pathways involving PIP2, IP3, PKC and calcium release.
However, we cannot exclude the possibility that other signaling molecules such as Syc,
Lyn or ZAP-70 may be involved, and further study may be warranted. It may be advisable for future experiments to generate new lines of transgenic mice with minor modifications or deletions to the cytoplasmic tail of CD4 and CCR5 that still permit HIV-
1 binding and entry events yet fail to signal (Bedinger et al., 1988; Gosling et al., 1997;
Stantchev and Broder, 2000).
Further depression of B cell response may occur in our dual positive huCD4CCR5 mice by failure to recruit CD4 T cell help required for B-cell activation. Upon BCR triggering by antigen, B-cells rapidly induce expression of MIP-1 and MIP-1
(Krzysiek et al., 1999). Human CCR5 of the surface of mouse B cells, which is known to cross-react with mouse MIP-1/, may act to limit recruitment of CD4+ helper T-cells required for B cell activation.
Another interesting observation is that B-cells upregulate expression of DC-SIGN upon activation (Rappocciolo et al., 2006). Binding to DC-SIGN by HIV-1 gp120 or anti-DC-SIGN mAb promotes downregulation of genes encoding MHC class II (which likely decreases immune activation) and upregulation of MIP-1 and RANTES (which may play a role in induction of apoptosis following CD4 ligation) (Hodges et al., 2007).
SARS S protein also uses DC-SIGN as an alternative receptor to angiotensinogen- converting enzyme type 2 (ACE2) (Han, Lohani, and Cho, 2007; Yang et al., 2004) and
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may similarly act to downregulate MHC II molecules. There is still much work needed
to interpret the lower humoral response in our transgenic mice.
In these experiments we failed to induce broadly neutralizing antibodies. Several groups have suggested that antibody accessibility to conserved fusion intermediate
epitopes is excluded by steric limitations determined in part by the size of antibody in
relation to the space available for access to the epitope (Hamburger et al., 2005; Labrijn et al., 2003). For example, the antigen binding fragments of monoclonal antibodies 17b,
48D and X5 bind to a CD4i epitope (Moulard et al., 2002; Thali et al., 1993), but as the size of the molecule is increased from a scFv to a Fab to a full size IgG molecule, the neutralizing activity decrease correspondingly (Labrijn et al., 2003; Moulard et al., 2002;
Xiang et al., 2002). It is believed that the space between the target cell membrane and the
CD4-bound envelope may be insufficient to allow entire IgG molecule access to CD4i epitopes. Yet, we suggest that the close proximity of membrane bound IgM to receptor bound envelope on these unique transgenic B cells, combined with the inherent flexibility of both surface immunoglobulins and CD4 and the fluidity of plasma membrane may allow some accessibility of surface antibody to these conformational epitopes in our system.
The structural dimensions of membrane bound IgM are currently unknown.
However, for soluble IgG2a monoclonal antibody, the distance between the tip of the Fab and the farthest point of Fc is ~115Å (Harris et al., 1997; Saphire et al., 2001).
Membrane-bound IgM is anticipated to be similar in size accounting for the extension of
~40Å for an additional CH4 domain on IgM, less ~40-50Å for the hinge region it lacks.
Additionally, IgM exhibits greater Fab-Fab flexibility (56°) (Roux et al., 1998) than any
other immunoglobulin except IgD (77°) (Loset et al., 2004). To add, IgD is coexpressed
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with IgM on the surface of mature, naïve B cells, at a higher density than IgM
(Finkelman et al., 1995; Havran, DiGiusto, and Cambier, 1984), and has larger segmental
flexibility and longer distance between antigen binding sites. Importantly, IgD binding to
antigen can mediate B-cell activation in much the same way as IgM does, involving the
use of identical intracellular signal transduction machinery.
The crystal structure of monomeric, soluble CD4 (D1-D4) has been determined to
be approximately 25 x 25 x 125Å (Kwong et al., 1990). The distance between the cell
membrane and gp120 bound to CD4, as determined through solved crystal structures and
accounting for the considerable flexibility between the 2nd and 3rd extracellular domains of CD4, is estimated to be ~85Å (Labrijn et al., 2003; Wu, Kwong, and Hendrickson,
1997). Although this space may be smaller than the size of a full IgG molecule, the segmental flexibility of IgM and IgD combined with the fluidity of the B cell plasma membrane may permit interactions among B cell receptor and surface bound HIV-1 envelope at distances that even exceed the calculated minimums presented here.
However, an alternative approach to generate more space might be to extend the length of
CD4 by inserting a spacer (e.g. the (Gly4Ser)3 linker used in antibody scFv constructs) between the third and fourth flexible domains of CD4.
Although we did not detect broadly neutralizing antibodies in our immunized mice, these results may not stem from the failure to elicit antibodies against fusion intermediates, but rather from the inaccessibility of secreted antibodies to bind to short- lived CD4i epitopes (in the same manner as IgG-X5, -17b and -48D) (Labrijn et al.,
2003). Future experiments should take this difficulty in detection into account, and could consider cleavage of antibodies into smaller, neutralizing Fab molecules with the enzyme
papain prior to screening for neutralizing activity. Alternatively, by using RT-PCR on
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RNA from splenocytes and lymph nodes, recombinant ScFv antibodies can be produced
and cloned into a phage display vector, generating a phage antibody library (Embleton et
al., 1992). The scFv antibodies would be fused to the N-terminus of the phage g3p coat
protein. If presented the right target structure, such as complex, living cells undergoing envelope mediated fusion, these phages may be selected and enriched for binding to
specific biologically-active fusion intermediate structures (Hartley et al., 2003). Phages
displaying scFv may also be used in neutralization assays directly. Phage particles
consist essentially of flexible filaments of diameter 60-100 Å, and the g3p protein that
links the displayed antibody fragment to the tip of the phage particle contains two very
long naturally occurring glycine-rich linker domains (19 and 39 residues, respectively;
see Swiss-Prot accession number P69168). Since scFv are smaller and far more flexible
than full IgG antibodies when presented by phages, it is unlikely that phage-scFv
particles will sterically hinder interaction between phage gp3-linked antibody fragments
and target structures. Due to limited resources, we did not pursue this path in these
experiments.
An alternative approach might be to immunize members of camelidae (camels,
dromedaries, llamas, alpacas, guanacos and vicunas) as such animals are known to
generate single chain antibodies lacking light chains and CH1 constant heavy-chain
domains (Desmyter et al., 1996; Hamers-Casterman et al., 1993). These smaller antibody
fragments would have particular application in a project targeting fusion intermediate
epitopes. Although producing transgenic camels is highly unfeasible, steps have been
taken to generate transgenic mice expressing heavy-chain only antibodies derived from
dromedaries (Nguyen et al., 2003; Zou et al., 2005). It might be interesting to see if our
huCD4 and huCD4CCR5 mice crossed with these single-chain antibody mice might
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develop broad neutralizing activity against HIV-1 envelope fusion intermediates.
To give transgenic B cells more time to reorient membrane bound
immunoglobulins to the fusion intermediate epitopes, we could trap envelope in a
conformation post-coreceptor binding but prior to membrane fusion by treating mice with
the small molecule inhibitor enfuvirtide. Enfuvirtide, or T-20, halts fusion by binding to
the HR1 region of gp41 and blocking formation of the 6 helix-bundle involved in
promoting membrane fusion events (Kilby et al., 1998). Similarly, new mice could be
generated with decreased CCR5 expression levels by placing the CCR5 gene further
downstream of the B cell specific promoter. The rate of virus entry is strongly correlated
with coreceptor density (Lelievre et al., 2004; Lin et al., 2002). An alternative approach
would be to use slow-fusion envelope mutants that delay fusion kinetics and viral entry,
thereby prolonging exposure of CD4-CCR5-bound envelope to membrane-bound
antibody on the transgenic B-cell surface. Two such slow fusion envelopes have been
identified. Reeves et al.(Reeves et al., 2005; Reeves et al., 2004), while studying mutant viruses resistant to fusion inhibitor enfuvirtide (T20), found mutations in the HR1 domain of gp41 that substantially slowed fusion kinetics. In light of this, we have already placed
the responsible mutations of G36D and V38M in gp41 HR1 into the backbone of
MCON6p160 in our lab.
Given the low humoral response obtained against AT-2 HIV-1 particles in both
transgenic and wild type mice, it is desirable to ensure that if inactivated virions are used
as antigens in future studies, they express higher levels of trimeric envelope than is
typically found on HIV-1 particles (~14 spikes /virion) (Zhu et al., 2006). It may also be
useful to generate Virus Like Particles (VLPs) in non-human cell lines to prevent
elicitation of antibodies against human cellular proteins that may yield false-positive
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results in neutralization assays using human cellular targets. We experienced considerable difficulty in purifying HIV-1 VLPs in mouse NIH-3T3 cells that incorporated high level surface expression of envelope. Alternatively, we could immunize using whole, inactivated mouse lymphocytes expressing HIV-1 envelope. And as stated before, emulsion-based adjuvants ought to be avoided when choosing which adjuvant might be most effective when immunizing with trimeric gp160 bound to a lipid membrane.
Overall, our transgenic mice may require more antigen or an additional boost to obtain equivalent humoral response. Although we did not detect broadly neutralizing antibodies against HIV-1, as new fusion competent antigens are developed and enhanced methods for detection of virus neutralization emerge, these mice may yet prove useful as a tool to generate antibodies to short-lived envelope fusion intermediates.
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Acknowledgments
We are deeply indebted to Dr. Barton Haynes of Duke University Medical Center
Human Vaccine Institute for providing recombinant protein MCON6gp14CF, and vaccinia virus vvMCON6gp140CF, and to Dr. Jeff Lifson and Dr. Julian Bess for providing AT-2 inactivated HIV-1. This work would not have been possible without adjuvant QS-21 kindly provided by Antigenic Inc. Special thanks must be extended to
Dr. Scott Fulton for assistance in surgery during mouse intrasplenic immunization and to
Poki Wan and Dr. Man-Sun Sy for assistance in hybridoma development. The following reagents were obtained through the NIH AIDS Research and Reference Reagent
Program, Division of AIDS, NIAID, NIH: pNL4-3.Luc.R-E- from Dr. Nathaniel Landau;
TZM-bl from Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc. HIV-1Bal gp120 from Dr. Marvin Reitz.
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CHAPTER 4
Generating HIV-1 envelopes with antigenically diverse V3 loops:
Application for a polyvalent V3 vaccine.
Adam Penn-Nicholson1,3, and Michael W. Cho*1,2,3
Departments of Medicine1, Biochemistry2, Molecular Biology and Microbiology3
Case Western Reserve University School of Medicine, Cleveland, OH 44106
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Abstract
The extensive sequence diversity of Human Immunodeficiency Virus (HIV-1) poses
a major obstacle to the development of an effective global AIDS vaccine. It may be difficult
for any single immunogen to elicit protective neutralizing antibodies against multiple diverse viral isolates and prevent the rapid evolution of escape mutants. We hypothesize that polyclonal antibodies against multiple epitopes from diverse viral strains will exhibit greater breadth of neutralizing activity against HIV-1 isolates than any single immunogen, and may prevent the rapid evolution of escape mutants. The hypervariable V3 domain is crucial in virus-cell entry events and is considered the principal target for early immune responses, eliciting potent yet strain-specific neutralizing antibodies. Recent data suggest that gp120 V3 epitopes are well exposed, structurally semi-conserved, immunogenic and can elicit antibodies capable of neutralizing isolates from different viral clades. Herein, we establish a technique to generate considerable diversity within V3, representing the majority of circulating subtype B HIV-1 variants in the context of group M consensus gp120 or outer domain, either proportional or equivalent to the observed frequency of V3 amino acids of primary isolates. This diversity is extensive yet specific and can be easily manipulated. We propose our technique could be easily utilized to generate a large pool of diverse V3 loops for use in a polyvalent vaccine approach, characterizing immune responses or investigation of functional role of V3 in binding, entry and antibody resistance.
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Introduction
The need for developing neutralizing antibodies through vaccination and during
natural infection of HIV-1 is widely understood as a requirement for protection, sterilizing
immunity and clearance of infection (Baba et al., 2000; Cho et al., 2001; Emini et al., 1992;
Mascola et al., 2000; Shibata et al., 1999; Trkola et al., 2005). During HIV-1 infection, patients elicit high-level virus specific antibodies, some of which are potently neutralizing, yet few of these antibodies, be they monoclonal or polyclonal patient sera, exhibit neutralizing activity against diverse HIV-1 isolates. Most of the antibodies elicited are either non-neutralizing, targeting irrelevant epitopes not involved in virus-host cell binding or entry events, or strain-specific, targeting regions of envelope showing significant variation among isolates (Parren et al., 1997). In over twenty years of AIDS research, only a handful of broadly reactive neutralizing antibodies (BR-NAbs) have been discovered, and these are rarely seen in HIV-1 infected patient sera (Burton et al., 1991; Gorny et al., 1992;
Muster et al., 1993; Roben et al., 1994; Stiegler et al., 2001; Trkola et al., 1996b; Zhang et al., 2006; Zwick et al., 2001b).
Elicitation of such desirable BR-NAbs is a challenging aspect of vaccine development, primarily due to several unusual strategies HIV-1 utilizes to avoid immune detection of critical residues. These include extensive masking of conserved residues by structures not involved in binding or entry events, lowered immunogenicity by an extensive “glycan shield”, and complex tertiary and quaternary structures that limit antibodies access, both temporally and spatially, to conserved regions of gp120 (Kwong et al., 2000; Wyatt et al.,
1998; Wyatt and Sodroski, 1998). Additionally, a remarkable degree of sequence variation among different virus isolates, particularly evident in five hypervariable regions of gp120
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(V1-V5), allows rapid evolution of mutants that escape neutralizing antibodies (Wei et al.,
2003).
The V3 loop is particularly well exposed and immunogenic, eliciting a broad range of neutralizing antibodies that tend to be strain-specific (Burton et al., 2004b; Gorny et al.,
2004; Gorny et al., 2002; Nyambi et al., 2000; Poignard et al., 2001). Yet despite extensive sequence variability, the V3 loop is structurally a semi-conserved region of 34-35 amino acids (Hartley et al., 2005; Huang et al., 2005) and subject to stringent constraints, partly regulated by its essential role in binding coreceptors CCR5 or CXCR4 (Chesebro et al.,
1992; De Jong et al., 1992; Fouchier et al., 1992; Shioda, Levy, and Cheng-Mayer, 1992).
This is well illustrated through studies that found structural similarities between the V3 loop and the natural chemokine ligands that bind to CCR5 or CXCR4 (Sharon et al., 2003).
Furthermore, deletion or single point mutations of V3 regularly result in non-functional virus (Cao et al., 1997; Ivanoff et al., 1992; Saunders et al., 2005). Despite sequence variability, there may be greater limitations on structural diversity of functional envelope and, as such, far fewer V3 immunotypes than subtypes (Nyambi et al., 2000; Zolla-Pazner et al., 1999). Furthermore, Deeks et al. (Deeks et al., 2006) propose that there may be limits to the capacity of the virus to evolve continuously to escape neutralizing antibodies, and an effective vaccine might drive virus evolution towards development of defective mutants.
Importantly, anti-V3 antibodies have shown the ability to protect against HIV-1 infection in animal models (Emini et al., 1992; Letvin et al., 2001) and can neutralize both intra- and inter-clade isolates (Binley et al., 2004; Conley et al., 1994; Gorny et al., 2004;
Gorny et al., 2002; Zolla-Pazner, 2004; Zolla-Pazner et al., 2004). Of particular interest is mAb 447-52D which binds to a highly conserved core sequence of GPGR in the V3 loop tip and shows considerable breadth of neutralizing activity (Binley et al., 2004; Gorny et al.,
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1992; Gorny et al., 2004; Gorny et al., 2002). Together, this suggests that V3 loop may be
an attractive target for vaccine development.
To develop a vaccine, considerable challenge lies in overcoming viral genetic
diversity. One such mechanism involves artificially generating a consensus or ancestral envelope sequence. (Gao et al., 2005; Gaschen et al., 2002; Haynes et al., 2006; Kothe et al.,
2007; Kothe et al., 2006; Liao et al., 2006; Nickle et al., 2003; Tian, Lan, and Chen, 2002)
Although such a sequence may help preserve structure of predominant circulating viral
isolates throughout a population, certain V3 amino acids exhibit considerable variation both
within their own clades and others, and as such, may have limited application in a V3 target
vaccine. An interesting study showed that a single V3 subtype B peptide that clustered
around the consensus subtype B sequence could neutralize, at best, 31% of subtype B
primary isolate tested (Haynes et al., 2006). Another such mechanism involves targeting multiple isolate epitopes through a polyvalent vaccine (see (Hurwitz et al., 2005; Hurwitz et al., 2008; McBurney and Ross, 2007; Slobod et al., 2005) for reviews). Such vaccine may consist of multiple epitopes stemming from different virus components (such as gag, pol, env, nef) (Brave et al., 2007; Fischer et al., 2007; Shinoda et al., 2004), or a single gene product from multiple different viral isolates or clades (Cho et al., 2001; Ljungberg et al.,
2002; Lockey et al., 2000; Pal et al., 2005; Rollman et al., 2004; Wang et al., 2006; Zolla-
Pazner et al., 1998), with some studies focused specifically on multiple V3 epitopes (Cruz et al., 2004; Haynes et al., 1995; Hewer and Meyer, 2003; Hewer and Meyer, 2005; Montero et al., 1997; Schilling et al., 2006). By inducing antibodies against the principal neutralizing determinant of V3 from most HIV-1 isolates endemic to a particular region, we hypothesize we could provide greater breadth of neutralization through a polyclonal antibody response than through any single immunogen. This concept stems from previous observation that
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animals immunized with gp120 or V3 peptides from multiple HIV-1 isolates developed
antibodies with greater breadth of neutralizing activity than animals immunized with a
single envelope protein (Cho et al., 2001; Haynes et al., 1995; Montero et al., 1997; Pal et al., 2005; Rollman et al., 2004; Shinoda et al., 2004; Wang et al., 2006). Support for the use
of multi-strain based vaccines stems from established effective vaccines against
antigenically diverse organisms such as poliovirus, influenza virus and Streptococcus
pneumoniae, and human papilloma virus (reviewed in (Hurwitz et al., 2005)).
Vaccines typically target either conserved structures common among virus isolate, or
diverse neutralizable domains from different isolates in a polyclonal antibody response. We
have undertaken a technique to generate antigens representing V3 loop sequences that
represent the majority of circulating subtype B HIV-1 variants (covering 85% of all amino
acids at each variable position). V3 sequences were represented as either proportional or
equivalent of their observed frequency of circulating subtype B variants in a naturally
infected HIV-1 patient population, derived from 1289 sequences from the Los Alamos
National Laboratory HIV-1 sequence database. The generated V3 variants were presented
in the context of HIV-1 group M consensus envelope (MCON6) gp120 or within gp120
outer domain (OD), lacking the immunodominant yet non-neutralizing inner domain. We
propose our technique could easily be utilized to generate a large pool of diverse V3 loops
for use in a polyvalent vaccine approach, characterizing immune responses or investigation
of functional role of V3 in binding and entry.
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Methods and Materials
Cloning
To facilitate cloning of the variant V3 loop sequences into our HIV-1 envelope
expression constructs, restriction enzyme cloning sites surrounding the V3 sequence of
codon-optimized pcDNA-MCON6gp160 were introduced by site directed mutagenesis. To
ensure the DNA mutations introduced were silent, MluI and EcoRV cloning sites were
chosen. However, the MluI and EcoRV cloning sites of the V3 loop were not unique in the
pcDNA-MCON6gp160 backbone. To remove the MluI and EcoRV sites in the plasmid
backbone, pcDNA-MCON6gp160 was digested with EcoRV and XhoI, blunt ended with
Klenow fragment, purified and ligated. After transformation of DH5 E. coli and analysis
of miniprep plasmid DNA to assure removal of the EcoRV site, the resultant plasmid was
digested with MluI and blunt ended with Klenow fragment, purified, ligated and
transformed into DH5 E. coli. Removal of MluI and EcoRV in the plasmid backbone was
confirmed by DNA sequencing and intact promoter sequence was confirmed through gp160
protein expression. This plasmid was renamed pcDNA*MCON6gp160. By using this
plasmid, we could easily introduce our desired V3 loop cloning sites. The MCON6gp160
envelope was also cloned into vector pTM-1 NdeI (Kleinman et al., 1988) under control of a
T7-Pol promoter, with BamHI and EcoRI, and cut with EcoRI and NdeI, blunt ended and
ligated. Site directed mutagenesis was undertaken to introduce restriction enzyme cloning
sites surrounding the V3 sequence of codon optimized pTM-MCON6gp160. Silent
mutations to generate MluI and EcoRV sites near the V3 loop cysteine residues responsible
for disulphide bond formation were introduced. For the MluI sites, sense primer 5’-
GAGATCAACTGCACGCGT-CCCAACAACAAC–3’, and antisense primer 5’-
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GTTGTTGTTGGGACGCGTGCAGTT-GATCTC–3’ were used with QuikChange XL
Site-Directed Mutagenesis Kit (Stratagene) according to manufacturers protocol. EcoRV
sites were introduced using sense primer 5’-CGAGATCATCGGCGATATCCGCCAGGC-
CCAC–3’ and antisense primer 5’-GTGGGCCTGGCGGATATCGCCGATGATCTCG–3’.
Underlined sequences signify restriction enzyme sites, and bold letters represent
mutagenesis sites. Restriction enzyme digestion and DNA sequencing confirmed correct
cloning. The plasmid is referred to as pTM-MCON6gp160-V3.
To generate secreted gp120, we used PCR cloning techniques to introduce a six-
Histidine (6-His) tag and a stop codon at the gp120-gp41 cleavage site. Sense primer 5'-
GACCATCACCCTGCCCTGCC-3' and antisense primer 5'-GGGCCCCTCGAGTTAATG-
GTGATGATGGTGATGGCGCTTCTCGCGCTCCACCACGCG-3' were used in a PCR reaction using pTM-MCON6gp160-V3 as a template. The XhoI restriction enzyme site is underlined. Purified PCR fragments and pTM-MCON6gp160-V3 were digested with XhoI and BsrGI, ligated and transformed into DH5 E. coli. The resultant clone DNA was confirmed by restriction enzyme analysis, DNA cloning and protein expression. The gp120H-V3 portion was also transferred into pcDNA*MCON6gp160. Generation of gp120 outer domain (OD) is discussed elsewhere (Kim et al., CROI 2007 poster 471, and see
Appendix section 2).
Generating polyvalent V3 loop sequences
To generate multiple V3 loop sequences representing amino acids representing no fewer than 85% of the 1289 subtype B isolate V3 regions sequenced in the LANL database, we utilized a nested PCR amplification technique. The inner set of primers consisted of 32 forward (primer 2F#1-#31) or 40 reverse primers (primer 2R#1-#40). The outer set of
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primers overlapped the inner set by 9 nucleotides, and consisted of 2 forward (primer 1F#1-
#2) and one reverse primer (1R#1). The outer set of primers contained Mlu and EcoRV
restriction enzyme sites in forward and reverse primers, respectively, to allow for easy
cloning into expression vectors. (For details on primer sequences, see Table 1). Pools of
primers were prepared by mixing primer amounts either equivalently, or proportionally
according to representation as seen in HIV-1 subtype B population sequenced by the LANL.
V3 Primers were added to 50ul PCR reaction mixtures at final concentration of 20 pmoles each of forward and reverse primer pools. PCR conditions were carried out in triplicate, using 2.5U per reaction High Fidelity DNA polymerase PfuUltraHF (Stratagene cat no.
600382-51) and consisted of 94°C hot-start for 3 min, 35 cycles of 94°C for 30 sec, 57°C for
1 min, 72°C for 1 min, followed by 72°C for 4 min. PCR products were collected and redistributed evenly amongst 15 PCR reaction mixtures containing the outer primer set.
Approximately 15 PCR reactions were run simultaneously to ensure complete representation of each V3 variant. Reaction conditions were identical to above. PCR products were purified and washed with QIAquick Gel Extraction Kit (QIAGEN) to remove primers and nucleotides. Cleaned PCR products and expression vectors (either pcDNA*- or pTM- based
MCON6gp120H-V3 or MON6OD-V3) were digested with restriction enzymes MluI and
EcoRV, purified through gel extraction, ligated and the entire ligation mix was transformed into DH5 E. coli cells. E. coli transformed with ligated CIP treated vector lacking digested
PCR products was used as a negative control to ensure appropriate selection by 100 g/ml
Ampicillin when spread on antibiotic selective LB plates. Plates were grown overnight at
37°C, and the next morning colonies were picked and plasmid DNA sequenced. All colonies were pooled, (approximately 10,000 to 15,000 colonies) and maxicultures used to
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prepare pooled polyV3 DNA. Additionally, E. coli pools were stored at -80°C in 15% glycerol.
Protein Expression
Protein expression was tested in both HeLa and 293T cells, by transfecting 90-95% confluent T25 flasks with 30 g DNA by standard calcium phosphate techniques for 16 h at
37°C, 5% CO2. Cell culture media was replaced with complete DMEM (10% FBS,
penicillin/streptomycin, glutamine) and incubated for 60-72 h at 37°C, 5% CO2.
Supernatant or cell lysate was collected and subjected to western blot analysis. To ensure strong expression levels and efficient representation of all possible V3 variants, the cells were transfected a minimum of 3 consecutive times. Approximately four T75 flasks containing 7.5x106 293T cells each (75% confluency) were transfected with 75 g of
pcDNA*MCon6gp120H-V3 or pcDNA*MCon6OD-V3 by standard calcium phosphate
transfection techniques. After overnight transfection and incubation at 37°C, 5% CO2, the
media was replaced with complete DMEM (10% FBS, penicillin/streptomycin, glutamine)
and incubated for a further 8-10 h. Cells were again transfected overnight with 75 g DNA
by calcium phosphate. In the morning, the media was replaced and cells were allowed to
recover for several days. After 3-4 days, flasks were plated at 50% confluency and
incubated for a further 24 h before a third transfection, as described above. Transfected cells
were selected with 500 g/ml neomycin for 2 weeks and 200 g/ml for 2 weeks. Cells were
tested for protein expression by western blot. During culture, cells were occasionally re-
transfected to ensure efficient representation of all possible V3 variants. To generate large
quantities of protein, approximately 40 culture dishes containing 2x107 cells were prepared.
The media was removed, cells were washed in serum free DMEM and cultured in DMEM
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containing 0.5% FBS, for 48 h. Culture media was collected, centrifuged at 3000 rpm in a
Heraeus/Sorvall swinging bucket rotor (75006445) for 10min. Supernatant was collected
and again centrifuged for 20-30 min to remove all contaminating cellular debris.
Supernatant was collected and stored at -70°C.
Western Blot Analysis
Cell media supernatant were subjected to SDS-PAGE and Western blot. Envelope proteins were detected with rabbit anti-gp160 polyclonal antibody at 1:1000-2000 dilution, followed by goat anti-rabbit IgG conjugated horseradish peroxidase (Pierce). Protein bands were visualized with SuperSignal chemiluminescent substrates (Pierce) according to the manufacturer’s protocol.
Protein Purification
6-His tagged proteins were purified using equilibrated Ni-NTA Superflow resin from
QIAGEN at a volume ratio of 1:100 with approximately 500-750 ml cell-free supernatant described above, under non-denaturing conditions. Binding of protein to resin was allowed to proceed overnight at 4°C before loading into a column. Flow through was collected, and reloaded to column twice to assure complete capture, and final flow through was collected and labeled as “unbound sample.” Captured resin was washed sequentially three times with
500 ml of Buffer A (20 mM Tris-HCl (pH7.4), with either 100-, 200-, or 500 mM NaCl).
Flow through was collected and labeled as “wash-1, -2, or -3.” Non-specifically bound proteins were removed by two sequential elution steps with 3 bed volumes of either 10- or
20 mM imidazole in buffer B (20 mM Tris-HCl (pH 7.4), 150 mM NaCl). Final 6-His tagged gp120 or OD proteins were eluted in 1 ml fractions with 250 mM imidazole in Buffer
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B, and labeled “Elution-1 or -2”. Proteins were dialyzed overnight with 20 mM HEPES, 50 mM NaCl. The eluted proteins were shown by western blot analysis and silver staining of a
10% SDS polyacrylamide gel. The purified proteins were stored at 80°C. Western blot analyses used rabbit anti-gp160 polyclonal antibody (Willey et al., 1991) at 1:1000-2000 dilution, followed by and goat anti-rabbit IgG conjugated horseradish peroxidase (Pierce).
Protein bands were visualized with SuperSignal chemiluminescent substrates (Pierce) according to the manufacturer’s protocol. Agarose bound Galanthus nivalas lectin (vector laboratories, Burlingame California) purification was obtained in similar methods described above, except purified proteins were eluted with 0.5M Methyl D-mannose pyronoside.
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Results
Although the V3 region is the principal neutralizing determinant, it is highly variable in sequence. We hypothesize that inclusion of multiple V3 regions as antigenic components of a polyvalent vaccine may elicit a broad and potent polyclonal humoral response that provides greater breadth of neutralization against diverse virus isolates than any single V3 sequence. In this study, multiple diverse V3 loops were selected by generating primers that include sequences incorporating the most commonly observed amino acids at the most variable residue positions of subtype B V3 sequences (Figure 4-1). The generation of multiple V3 sequences was achieved using a nested PCR amplification system using a large pool of primer pairs representative of 1289 circulating subtype B isolates, as determined by the Los Alamos National Laboratory HIV-1 Sequence database (Figure 4-2). The amino acids we have chosen for each position of V3, when combined, would represent about 85% of circulating subtype B virus isolates. Theoretically, about 2560 different V3 sequences are represented. At present, no vaccine antigen has provided protection against multiple forms of HIV-1. By presenting potently neutralizing epitopes of the vast majority of observed subtype B V3 sequences, we hypothesize a broader, more practical vaccine approach.
Generating V3 Diversity
The inner polyV3 primer pairs consisted of 32 different sense and 40 antisense primers, each sense and antisense primer designed with a nine nucleotide conserved overlapping sequence at the 3’ and 5’ ends, respectively. This allows polyV3 sense and antisense primers to bind to complementary sequences and use the opposite primer as
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Figure 4-1: Schematic figure detailing selection of V3 loop amino acid residues required for coverage of 85% of subtype B isolates, as based on 1289 sequences from the Los Alamos National Laboratory HIV database.
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Figure 4-1: Schematic figure detailing selection of V3 loop amino acid residues required for coverage of, at minimum, 85% of subtype B isolates, as based on 1289 sequences from the Los Alamos National Laboratory HIV database. HIV-1 envelope structure is expanded to show consensus subtype B V3 loop amino acid sequence on the top line (highlighted in yellow) where “consensus” means the most common amino acid found in each position among the sequences of the subtype. 1289 sequences were used to construct the subtype B consensus V3 sequence. Other observed amino acids in that position are presented directly below the consensus sequence, in descending order of prevalence. Subscripts record amino acid frequency with which each amino acid is observed at that position. Asterisk (*) subscript signifies less than 0.5% prevalence and a dash (-) indicates a gap inserted to maintain alignment. Amino acid residues selected to represent at least 85% of subtype B V3 loop diversity are highlighted in yellow .
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Figure 4-2: Schematic of methodology used to generate polyvalent V3 loop DNA sequences.
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Figure 4-2: Schematic of methodology used to generate polyvalent V3 loop DNA
sequences. Using a nested polymerase chain reaction (PCR) technique, employing 32
forward primers (F2) and 40 reverse primers (R2) for the inner PCR reaction, and 2 forward
(F1) and 1 reverse (R1) primer for the outer reaction, we are able to generate 2560 different
V3 loops, representing 85% of the amino acid observed at each V3 residue position.
Vertical black bars represent regions of nine-nucleotide primer sequence overlap. MCon6
Envelope amino acid positions are indicated with arrows above sequence. By encoding
unique restriction enzyme sites into the outer primer pairs, V3 loop DNA sequences can
easily be cloned into HIV-1 Group M Consensus gp120 or outer domain (OD) expression
vectors. V3 loop amino acids in a pool can be represented either equivalently, or in
proportion to their frequency as seen in circulating viral primary isolates.
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template for elongation. The inner PCR reaction yields 66 base pair nucleotide fragments
(Fig. 4-3B) that act as templates for a second PCR step using outer primer pairs. The inner
polyV3 primers consist of two sense and one antisense primer, incorporating MluI and
EcoRV restriction enzyme sites, respectively. Each outer sense and antisense primer has a
nine nucleotide conserved sequence that overlaps with and allows binding to either end of
the 66bp inner PCR product, which acts as template for the outer PCR reaction. The
resulting PCR products, representing 2560 different V3 sequences, is 99bp in length (Fig. 4-
3B) and contains unique MluI and EcoRV to allow for easy cloning. Restriction enzymes
sites MluI and EcoRV were introduced in to MCON6gp120H and MCON6-OD internal of
the disulphide bond forming the V3 loop region by site directed mutagenesis. These
nucleotide mutations remained silent (Fig. 4-3A).
Generating MCON6gp120 and Outer Domain
Recombinant proteins have great potential as vaccine candidates, and are shown to
induce strong humoral response. We chose to generate monomeric, recombinant gp120
from HIV-1 Group M Consensus sequence (MCON6gp160) using a simple PCR cloning
technique. By introducing a six-Histidine tag (6His) at the cleavage site of gp120 and gp41
of pcDNA-MCON6gp160, we allowed for efficient mammalian cell expression and easy
purification of recombinant protein through a Ni-NTA column. A similar technique was
undertaken to develop MCON6-OD, and the protein is described in Appendix section 2.
Our hypothesis is that OD acts to redirect antibody response against the PND of V3 by diminishing humoral response against the immunogenic yet classically non-neutralizing inner-domain. Studies described in Appendix section 2 show MCON6-OD is antigenically correct, and V3 of OD is recognized by V3-specific antibodies, including mAb 447-52D.
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Representation of V3 variants
PolyV3 primers were prepared as pools to represent V3 loop sequences either
proportionally or equivalently to their occurrence as circulating subtype B primary virus
isolates. For example, in our model the tip of the V3 crown can be either GPGR or GPGQ.
In the 1289 subtype B primary isolates sequence, arginine was observed at that position 76% of the time, whereas only 9% of sequences showed glutamine at that position. Together, these total 85% of all possible amino acids at that particular position. Proportional representation favors presentation of the most-common amino acid over the less-common amino acids (in this case, arginine over glutamine) in proportions relative to their occurrence in nature. Equivalent representation gives each amino acid equal probability of presentation
(i.e. 50/50). In Table 4-1, we show DNA sequences for each primer, and their relative percentage of representation within the pool in either model of proportional or equivalent representation.
PolyV3 PCR fragments were digested with restriction enzymes MluI and EcoRV and cloned into corresponding sites of MCON6gp120H-V3 and MCON6OD-V3 plasmids.
For each experiment, we generated a sufficient number of individual bacterial colonies
(approximately 10,000-15,000) to assure us that cloning, ligation and transformation of
DNA was not a limiting factor of our methodology. DNA was prepared from about 100 individual bacterial clones for each strategy. All remaining colonies were pooled together and amplified. DNA pools were used for protein expression (Fig. 4-3C).
Variant Analysis
From 99 proportionally- and 93 equivalently represented sequences randomly selected for DNA sequencing, we observed that our technique was successful in establishing
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Table 4-1: Poly V3 primer-encoded amino acid sequences.
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Table 4-1: Poly V3 primer-encoded amino acid sequences. (A.) MCON6 V3 loop nucleotide and corresponding amino acid sequence is shown, with clear identification of the most common amino acids identified at each position to cover at least 85% of all subtype B primary isolates. Nucleotides shown in blue italics identify sites targeted to introduce silent mutations that correspond to unique restriction enzyme sites MluI and EcoRV on the
MCON6 V3 sequence and corresponding primers. (B) Sequences from each individual primer set are shown. Colored amino acids identify mutant residues introduced in MCON6
V3 sequence to cover at least 85 % of all subtype B primary isolates, as based on 1289 subtype B sequenced isolates. Percentage of each primer added to proportional and equivalent polyV3 pool is indicated.
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Figure 4-3: (A) Schematic showing V3 loop sequence with silent mutations to introduce unique restriction enzyme sites internal of cysteine residues responsible for V3 loop disulphide bond formation. Amino acid positions selected for substitution to generate
MCON6 V3 variants representing 85% of the diversity of all amino acids for subtype B V3 at each position, are highlighted. (B) 1.5% Agarose gel showing DNA bands of nested PCR products from inner and outer reactions, at 66 and 99 base pairs, respectively. Outer primer band was purified, digested with EcoRV and MluI and cloned in to MCon6V3 to generate
V3 variants. (C) Western blot expression of pcDNA-MCon6gp120 and –polyV3gp120H proportional and equivalent transfected 293T cell supernatant, developed with rabbit anti- gp160 polyclonal antibody (Willey et al., 1991) at a 1:2000 dilution.
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vast diversity of V3 loop amino acid residues in MCON6-gp120 and -OD backgrounds
(Table 4-2). Each amino acid was represented at similar percentages as to what was
expected of our pool in the proportional representation experiment. We did see some
deviance from expectation of amino acid representation in the equivalently expressed pool, with E324R appearing strongly under-represented. This could be due to limited sampling size, primer design (limited choice in primers could result in self-annealing or primer- dimers), or errors in DNA sequencing of ‘CG’ rich regions. Additionally, we observed some incorporation of amino acids we did not encode for within our pool of primer. This result, although unexpected, encoded for residues that are similarly represented in the circulating population of subtype B isolates, and relative to their anticipated representation in our pool, do not appear to be of any significance. Furthermore, we did observe some variants
(approximately 2%) that included missense or nonsense mutations in the genetic code.
However, it is unlikely to have any effect on our antigen sample, as, without inclusion of a
6His tag, these mutants will not be purified.
We observed that most of our variants incorporated at least one mutation or non- consensus V3 amino acid (Fig. 4-4). As expected, the proportional pool had less variation from consensus V3 sequence that was seen in the equivalent pool. From a total 9 possible amino acid positions, the majority of proportionally represented V3 mutants included 1-3 residues that deviated from consensus, whereas the majority of equivalently represented V3 variants had 4 to 6 sequence variations. It was encouraging to find one V3 variant DNA sequence that included amino acid variations at all 9 possible positions, suggesting that our technique is efficient at generating vast diversity in a controlled manner.
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POPULATION BASED ON 1289 HIV-1 SUBTYPE B V3 SEQUENCES
PROPORTIONAL EQUIVALENT
population expected observed population expected observed N 84 90 80 N 81 50 49 S 91015 S 10 50 51
K 83 85 91 K 83 50 28 R 15 15 9 R 15 50 72
S 74 80 87 S 74 50 78 G 18 20 12 G 18 50 20 F 001 A 002
H 56 64 74 H 56 25 21 P 17 20 16 P 17 25 33 N 9104 N 92520 T 565 T 52525
I 69 79 60 I 69 50 57 L 18 21 40 L 18 50 42 F 1
R 76 89 82 R 76 50 29 Q 91118 Q 95071
F 73 83 92 F 73 50 46 W 15 17 7 W 15 50 51 I 201 L 502
T 56 58 66 T 56 50 52 A 41 42 34 A 41 50 48
E 35 40 39 E 35 20 27 D 25 28 23 D 25 20 23 Q 19 22 31 Q 19 20 31 R 562 R 5202 A 444 A 42017
based on app. 93 sequences based on app. 99 sequences
Table 4-2: Frequency of each amino acid in the pool of proportional and equivalent polyV3 sequences. Proportional and equivalent representation is based on DNA sequences of 93 and 99 clones, respectively. Results are inclusive of both MCon6 polyV3-gp120H and
MCon6 polyV3-OD clones. Observed frequencies are represented as a percentage of what was expected from the design of our primer pool, and frequencies found in circulating subtype B primary isolate population is used for comparison.
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Number of 0 1 2 3 4 5 6 7 8 9 mutations % observed 3 27 32 23 2 6 2 0 0 1
prop prop % expected 5.35 20.95 32.02 25.59 11.97 3.44 0.61 0.07 0.00 0.00
% observed 0 2 5 10 31 29 16 6 0 0
equiv % expected 0.04 0.55 3.20 10.39 20.78 26.80 22.42 11.80 3.55 0.47
Figure 4-4: Table and graph showing percentage of polyV3 variants in proportional and equivalent groups with amino acid mutations at each of nine possible residue positions of consensus subtype B V3 sequence targeted in our study.
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Protein Expression and Purification
PolyV3 DNA plasmid pools were bulk expressed in mammalian cell lines. Initially, we utilized HeLa cells, but found expression levels were lower compared to 293T cells (data not shown). Furthermore, 293T cells are an easily transfectable subclone of 293 cells. In order to maximize transfection efficiency and to ensure appropriate representation of all possible polyV3 variants during protein expression, we chose to use 293T cells for expression. Additionally, we performed large-scale transfection of cells with pcDNA*polyV3 plasmid DNA three times, in an effort to assure that all V3 variant plasmids would be transfected into cells in a manner that is representative of the DNA pool. The transfected 293T cells appeared to maintain gp120H and OD expression more than one month after transfection, allowing large volumes of protein to be collected and purified.
We observed considerable difficulty in purifying large quantities polyV3-gp120H and –OD to levels that would be suitable for a vaccination study. This was exacerbated by the presence of FBS in the media, so cells were cultured in low serum containing media
(0.5% FBS) in an attempt to circumvent this. In Figure 4-5, we show silver stained SDS-
PAGE gels of eluted fractions of polyV3 gp120H after two Ni-NTA purification steps, or polyV3 OD after one Ni-NTA purification and one Galanthus nivalis lectin purification.
Although purity of the samples is not appropriate for vaccination studies, our technique generates extensive diversity within V3 region that can be easily manipulated to specific requirements. Furthermore, polyV3 sequences may be better targeted by the immune system if combined with OD or fusion competent envelope constructs lacking V1/V2. We propose our technique could be easily utilized to generate a large pool of diverse V3 loops for use in a future polyvalent vaccine approach, characterizing immune responses or investigation of functional role of V3 in binding, entry and antibody resistance.
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Figure 4-5: Purification of polyV3 gp120H and OD. (A) Silver Stain analysis of polyV3 gp120H proportional and equivalent samples after two rounds of Ni-NTA purification. (B)
Silver Stain analysis of polyV3 OD proportional and equivalent samples after one round of
Ni-NTA purification and one round of Galanthus nivalis lectin purification U = Unbound fraction; E1, E2, E3 = Elution fraction one, two and three respectively; mwm = molecular weight marker.
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Discussion
Given the rapid emergence of HIV-1 neutralizing antibody escape mutants, it seems
unlikely that any single vaccine immunogen will have the desired effect of generating
broadly neutralizing monoclonal antibodies. We, and others, propose targeting the HIV-1
V3 region in a polyvalent vaccine approach to generate a polyclonal antibody response
against a region known as the principal neutralizing determinant of HIV-1.
Herein, we have established a practical method to generate diverse DNA sequences that span 85% of all amino acids at each position of the entire V3 region of subtype B primary isolates. This proof-of-concept study may be applied to other subtypes or geographically relevant viral strains. V3 diversity was obtained by targeting nine amino acid positions showing high variability within the V3 sequence, and substituting with two to five amino acids for each, thereby generating 2560 different V3 variants. By incorporating the most predominantly represented V3 residues in our approach, we hypothesized that we were increasingly likely to incorporate the vast majority of immunotypes of subtype B V3 epitopes in our antigen pool.
By expressing such polyvalent V3 loops in the backbone of an HIV-1 group M consensus envelope (MCON6) gp120 and outer domain, epitopes presented are likely to be closer to the native V3 conformation than would occur if V3 were presented as a linear peptide. Short peptides are highly flexible and may adopt unpredictable 3D structures that favor the exposure of some epitopes at the expense of others. Indeed, both monoclonal and polyclonal V3 specific neutralizing antibodies that have shown great breadth of activity recognize conformation specific epitopes of V3 (Conley et al., 1994; Gorny et al., 2004;
Gorny et al., 2002; Krachmarov et al., 2006). Furthermore, the vast majority of the V3
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monoclonal antibodies elicited with or selected with peptides are directed to structures that
are largely irrelevant in the context of primary isolate infectivity (Gorny and Zolla-Pazner,
2003). By using V3 in the context of native gp120 or OD that may be closer to native
conformation, we hope to circumvent some of these issues.
This technique appears particularly appealing given recent advances made in DNA
vaccine research. In the past, DNA immunization technology has been widely accepted as a
tool to induce cell-mediated immunity. Recently, strong humoral responses have been seen in a DNA-prime protein-boost immunization approach in HIV-1 and SIV vaccine studies on small animals and non-human primates. (Letvin et al., 1997; Lu et al., 1996; Pal et al., 2005;
Richmond et al., 1998; Wang et al., 2005; Wang et al., 2006)
We designed our V3 antigens with the intent of representing the majority of subtype
B primary isolates, however, it must be noted that variants are generated artificially from the
consensus V3 sequence. It is possible that viral isolates incorporating the least prevalent
amino acids at each position may not represent functional virus, and as such, will not be
represented in a natural infection setting. Upon BLAST protein alignment of reported
primary isolate V3 sequences, we identified several isolates with 91% (32/35) amino acid
identity to the least prevalent variant in our pool, yet the natural V3 sequences exhibited
94% (33/35) predicted physio-chemical conservation. Some isolates had >97% positive
physio-chemical conservation. This would suggest that, overall, there is good reason to
believe the polyV3 pool represents native virus V3 loops. On the other hand, some V3
variants may be poorly represented in the natural population, not as a factor of decreased
entry efficiency, but rather as a result of greater susceptibility to neutralizing antibodies, and
thus negative selection pressure against that variant. Nevertheless, by using a polyV3 pool
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based on proportional representation of isolates observed in a natural population, we will not
unnecessarily enhance targeting of rare structural motifs.
Overall, DNA sequencing analysis of variant clones developed through our
technique successfully demonstrates the generation of considerable diversity that closely
resembles anticipated results for both equivalent and proportional polyV3 pools. Three V3 residues in the equivalent pool showed somewhat deviant representation from anticipated results. Variant residues K309R, S310G and R317Q were each expected to occur at a ratio of 50/50, but were observed at a ratio of 28/72, 78/20 and 29/71, respectively (Table 2A).
However, an inverse relationship of each variant amino acid to the predominant consensus residue was observed in the polyV3 proportional pool for positions K309R and R317Q, close to anticipated results suggesting the small sample size may pose challenges to analysis.
In both the proportional and equivalent polyV3 pools, position E324D/Q/R/A showed under-representation of arginine, suggesting possible difficulty in primer binding or elongation. Additionally, we did observe unexpected substitutions of amino acids not specifically encoded for into select residue positions. Although the frequency of these substitutions is rare (less than 2%), it is unclear exactly why this may have occurred.
Taken as a whole, we have successfully established a rapid and easy technique to introduce considerable sequence diversity with a high degree of design precision into the V3 region of HIV-1 envelope. These polyvalent V3 sequences could easily be modified for cloning into any number of constructs and may serve to elicit conformation specific V3 antibodies. Furthermore, envelope constructs with individual V3 loops could provide a good model in which to explore structure/function, viral entry, tropism switching or resistance to chemokine inhibitors.
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It might be of great value to combine our polyvalent V3 loop vaccine with our functional envelope constructs with shortened or deleted V1 and V2 loops. By removing the highly immunogenic V1/V2 region, we could aid in refocusing the immune response against neutralizable epitopes of V3. As shown by Kim et al. (Kim et al., 2003), V1/V2 deletion enhances antibody responses against other regions of envelope protein, including a strong response against the V3 loop. In light of this, we have already taken steps to develop
MCON6 constructs with deletions in V1, V2, or both V1/V2 (see Appendix section 1 for methods and materials and results). When expressed as full-length gp160 on cell surfaces as trimers, several of these envelope constructs maintained the ability to induce membrane fusion with CD4 and CCR5 bearing cells. Ability to fuse assures us these constructs are still functional and present V3 in a conformationally relevant structure.
For future consideration in polyvalent V3 vaccine design, it may be more appropriate to include only V3 sequences associated with R5 tropic viruses, as all new infections transmitted sexually are caused by viruses using CCR5 as their coreceptor, or utilize only viruses from certain geographic regions.
Immunogen dilution in large pool in polyvalent vaccine approach is possible (as seen in an 8-valent study by Wang et al. (Wang et al., 2006)) and may result in antibody responses being directed away from the V3 loop towards other non-variable regions of our consensus envelope. Cruz et al. (Cruz et al., 2004) observed a reduced V3 specific immune response against a mixotope vaccine consisting of 5000 different V3 sequences in comparison to consensus V3 sequence peptide antigen. Interestingly, they observed strong antibody response against conserved regions of V3, which are better represented than variant regions in a polyvalent vaccine. This finding could support the hypothesis of preferentially eliciting and promoting maturation of antibodies that target conserved structural features shared
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among variant sequences, especially a conserved region such as the tetrameric GPGR-tip targeted by BR-Nab 447-52D.
Given the strong immunodominance of V3 region and the rapid appearance of V3 specific antibodies early in infection and after brief immunization protocols, we do not foresee poor immune response towards V3 as a whole.
We believe our technique could be easily utilized to generate a large pool of diverse V3 loops for use in a polyvalent vaccine approach, characterizing immune responses or investigation of functional role of V3 in binding, entry and antibody resistance.
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Acknowledgements
We are grateful to Drs. Beatrice Hahn providing codon optimized Group M Consensus
Envelope sequence plasmid pcDNA-MCON6gp160. We also thank Dr. Michael Lederman for invaluable support. Several steps of protein purification were assisted by Dr. Dong P.
Han and Dr. Soon J. Kim. Construction, expression and characterization of MCON6gp120-
OD were performed by Soon J. Kim.
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CHAPTER 5
CONCLUSIONS
AND
FUTURE PROSPECTS FOR HIV VACCINE RESEARCH
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CHAPTER 5
Conclusions and future prospects for HIV vaccine research.
Current clinical trials and what we now know: Even as several large-scale
clinical trials continue, hope is limited given the failure of the last two Phase III trials to
generate sterilizing immunity against HIV-1. At present, one other Phase III trial is being
conducted in Thailand, at enormous financial cost. It has suffered intense criticism from
several prominent scientists who say that while the original aims of the trial “remain
fundamentally worth addressing, [they] doubt whether these immunogens have any prospect of stimulating immune responses anywhere near adequate [to] prevent infection and/or lead to the immune control of HIV-1 replication post-infection.” (Burton et al., 2004a) The vaccine involves a prime-boost regimen consisting of Aventis Pasteur’s canarypox candidate
ALVAC vCP1521 followed by boosting with recombinant gp120 AIDSVAX from clades B and E. The canarypox vector is thought to be poorly immunogenic, as seen in Phase I and II trials, and the AIDSVAX gp120 component has shown no efficacy to date in two independent phase III trials.
Recently, the most promising HIV vaccine candidate in Phase IIb clinical trials failed to protect against infection. The Merck vaccine, MRKAd5, consisted of an adenovirus 5
(Ad5) vector to deliver HIV-1 gag/pol/nef genes. More shocking than the vaccine’s failure to protect, this T-cell vaccine may actually have led to enhancement of infection in individuals who had high preexisting levels of antibodies against the Ad5 viral vector. It is still not entirely clear why this may have occurred (Cohen, 2007).
Several challenges towards HIV vaccine research still exist, with some of the most pressing issues including: how to achieve induction of high-titer and potent broadly
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neutralizing antibodies and strong CTL responses; how to overcome poor immunogenicity of antigens presented in a native conformation; how to increase memory responses to allow long-lasting immunity; and how to overcome pre-immunity to vaccine vectors used in several trials.
There remain several candidates in clinical trial stages that have some potential for protecting against HIV-1 infection, or controlling viral replication and limiting transmission
(Table 5-1). Given the difficulty in inducing BR-NAbs against envelope, most of these vaccines aim primarily to induce strong cellular responses. T-cell vaccines recognize 9-11 amino acid peptides generated by proteolytic digestion of viral proteins and can be directed to the internal, more conserved capsid and enzymatic HIV-1 proteins. Such an approach may help overcome the significant sequence diversity observed among virus strains, but will likely fail to provide sterilizing immunity.
Of the vaccines currently in Phase I and Phase II clinical testing, several include the use of viral vectors such as Ad5 or Modified Vaccinia Ankara (MVA) which deliver multiple HIV-1 genes, including gag, nef, pol, and rev, and sometimes env which may act to stimulate a humoral response (for review, see (Robinson, 2007)). Furthermore, by including genes from multiple viral clades, the ability of escape mutants to arise may be reduced. Yet given the recent failure of the MRKAd5 vaccine, many clinical trials using recombinant Ad5 and other serotypes of adenovirus including Ad32 and Ad26, have been placed into a temporary holding pattern until more information can be obtained regarding their safety
(Kresge, 2007). Nevertheless, a few of these vaccines may be sufficiently different from
MRKAd5 to yield promising results, and proper analysis is still required. Differences include trials involving a prime/boost approach by priming with multiple DNA plasmids expressing HIV-1 genes and boosting with viral vectors, either recombinant Adenovirus or
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MVA. DNA-prime/viral-vector-boost strategies have shown considerable promise in
stimulating a strong cellular response in non-human primate models (Catanzaro et al., 2006;
Graham et al., 2006). Other Phase I clinical trials conducted by Wyeth Pharmaceuticals attempt to induce vaccine immunity by priming with subtype B specific DNA and boosting with multi-epitope CTL peptides.
These T-cell vaccines are unlikely to provide sterilizing immunity without the ability to induce potent neutralizing antibodies against HIV-1 envelope. As discussed above, recombinant gp120 from strain MN used in Phase III trials with ALVAC canarypox vector has previously failed to elicit protective antibodies against HIV-1 infection. The most promising candidate in this respect is in a current Phase I trial from Chiron involving a DNA prime of clade B gag and env, followed by a recombinant protein boost of a clade B oligomeric, V2-deleted gp140 (Srivastava et al., 2003a; Srivastava et al., 2003b). V2- deletion acts to better expose the conserved CD4 binding site, and trimerization ensures presentation of envelope in a more native setting. In preliminary studies, this V2-deleted gp140 partially protect macaques from infection with autologous virus, but has only limited effective neutralizing activity against other virus isolates (Xu et al., 2006). Most likely, the
V2 loops of most isolates exclude antibodies from binding to recessed conserved epitopes, no matter how prevalent such antibodies may be in serum.
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Table 5-1: A short list of candidate HIV vaccines currently in clinical trials. Taken from The International AIDS Vaccine Initiative (published Jan 2007). http://www.iavireport.org/specials/OngoingTrialsofPreventiveHIVVaccines.pdf
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Future prospects for an AIDS vaccine in the early stage of development.
There are a multitude of other antigens in the very early stages of development and
testing for efficacy and safety in animals, but until human trials are initiated, it is hard to
predict what the best antigens might be. To generate BR-NAbs against envelope, four
principal strategies are being explored. These include (1) mimicking envelope trimers, (2),
modifying envelope to expose conserved epitopes and remove/disguise variable and
immunodominant epitopes, (3) construction of stable fusion intermediate structures, (4)
rational structure–based mimotope design. These approaches have been discussed in detail
earlier and will only be briefly addressed here. Of greater interest are strategies being
undertaken to enhance antigen presentation and increase immunity (especially mucosal immunity) to novel antigens presenting conformationally relevant. A successful vaccine will likely induce both humoral and cellular immune responses that are potent, long-lived and can rapidly respond to a diverse number of HIV antigens to offer complete protection, or, at minimum, control of viral infection.
Trimeric envelope: Researchers are beginning to focus strongly on presenting
envelope as trimers as opposed to monomers (Beddows et al., 2006; Binley et al., 2000a;
Dey et al., 2007; Sanders et al., 2002; Yang et al., 2002). This helps prevent eliciting
antibodies against the gp120 inner domain, a highly immunogenic and conserved region that
is not accessible to antibodies on the function envelope trimer. Good trimer mimics bind
BR-NAbs well and bind non-neutralizing antibodies poorly. Several groups have shown
significantly higher neutralizing antibody titers elicited against trimeric envelope compared
to monomeric envelope, but none with appreciable breadth of neutralizing activity
(Beddows et al., 2005; Kim et al., 2005; Li et al., 2006). Other groups mimic mature trimers
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by expression of envelope on pseudoviruses or proteoliposomes (Grundner et al., 2005;
Sanders et al., 2002; Yang, Wyatt, and Sodroski, 2001). There is much promise in such an
approach, yet still much more to be learnt.
It is interesting that BR-NAbs 2F5, 4E10, b12 and several CD4i MAbs have
extended CDRH3 domains. This may facilitate in binding to envelope in a native, trimeric
structure and in the case of mAb b12, allows access to the deeply recessed CD4 binding
pocket. In this regard, assessment of the potential to elicit BR-NAbs antibodies in a mouse
model presents a substantial limitation, as mouse antibodies have notoriously shorter CDR3s than humans (Burton et al., 2004b). It may be more appropriate to immunize rabbits or a
humanized mouse model such as XenoMouse (Amgen/Abgenix), whereby their transgenic
mice produce fully functional human antibodies against antigens (Green, 1999).
Membrane bound envelope trimers: It is essential that antigens be presented in
native conformations without extensive structural rearrangements that may alter the manner
in which discontinuous epitopes are presented to the immune system. As such, membrane-
bound trimeric envelope antigens may be superior to soluble proteins in presenting native
conformation epitopes to immune cells. In this way, viral vector delivery of HIV-1 DNA
to targeted cells seems a reasonable approach to ensure viral envelope proteins are properly
glycosylated by host cellular machinery in a manner analogous to virus during natural
infection. Great effort has been placed in development of viral vector vaccines, and despite
mixed results, optimism persists. Another approach involves presenting trimeric envelop on
VLPs or pseudovirions (Devitt et al., 2007; Doan et al., 2005; Hammonds et al., 2005).
VLPs and pseudovirons have the advantage of being safer for immunization than many
common viral vectors, as they are normally replication-incompetent.
Viral vectors and pre-existing immunity: When using a viral vector vaccine
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regimen, it is important that any virally infected cell not induce either virally mediated cell lysis or stimulate an anti-viral vector CTL response against the infected cell, as this would diminish immune response against HIV-1 transgene proteins by removal of the transduced cell. For safety, several non-replicating viral vectors have been developed, and may also be used for gene therapy applications.
It is considered crucial that vaccine recipients not show high levels of preexisting immunity against viruses used as vectors, as this may blunt immune response to recombinant gene products (Mascola et al., 2005b). To overcome this problem, several strategies are being investigated to lower immune recall against viral vectors, including use of adenoviruses from uncommon serotypes; production of adenoviruses with alternate or chimeric fiber proteins; priming with DNA; prime-boost combinations of different viral vectors; and microencapsulation of viral vectors with inert polymers (Casimiro et al., 2004;
Sailaja et al., 2002; Santra et al., 2005). Likely, several non-human adenovirus vectors currently in development (bovine, porcine, canine, chimpanzee, ovine and fowl adenoviruses) will be further tested in human trials.
Given that preexisting immunity alters immunogenicity of recombinant viral vector vaccines, (prevalence of infection in a population and level of antibodies in each patient varies widely), in the future, it may be deemed necessary to prescreen vaccine subjects for vector immunity. Failure to identify subjects with strong vector immunity may result in high variability of protective efficacy and mixed interpretation of clinical trial results, or may, as seen in the MRKAd5 trial, even lead to increased risk of infection.
Specific cell targeting by viral vectors: An innovative approach has been employed in altering adenovirus to utilize alternative receptors for entry by replacing receptor-binding residues of adenovirus fiber protein with human cellular proteins that
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mimic ligands for specific cellular receptors (Roelvink et al., 1999). This allows vector targeting to specific tissue and cell types and improved delivery of genes.
Targeting conserved epitopes: An interesting concept is under development whereby engineered hyperglycosylated gp120 derivatives are produced as a means to preferentially target antibodies away from known variable domains and towards conserved epitopes. How this hyperglycosylation might affect overall structure is still unknown. A vast reduction of binding by non-neutralizing or weakly neutralizing antibodies was observed using envelopes showing 6-7 extra N-linked glycosylation sites, while maintaining the ability to bind BR-NAbs b12 and 2G12 (Pantophlet, Wilson, and Burton, 2003;
Pantophlet, Wilson, and Burton, 2004). Other Env modifications may involve removal of decoy epitopes to shift the balance of natural immunodominance away from non- neutralizing or strain-specific epitope to less immunodominant and conserved epitopes
(Garrity et al., 1997; Gzyl et al., 2004; Kim et al., 2003).
Stable Fusion Intermediates: Constructs that represent fusion intermediates or
CD4i epitopes have great potential, but obvious doubts exist as to whether antibodies elicited against such constructs will have access to the epitope on native virions during the fusion process (Xiao et al., 2003; Zipeto et al., 2006). However, additional antibodies against fusion intermediates would undoubtedly help decipher some of the poorly understood steps involved in the fusion process and may assist in identification of crucial residues involved. New antibodies may, too, result in the development of small molecule entry inhibitors. Nevertheless, mAb D5 targets a fusion intermediate structure on gp41 and does exhibit broad, albeit weak, neutralizing activity (Miller et al., 2005). New antigenic constructs, such as mimotopes encompassing D5 epitopes, may act to increase potency of antibodies against fusion intermediate and CD4i induces epitopes.
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Mimotopes: There is a good chance that future vaccines will hinge strongly on rational structural design of antigens known to bind HIV-1 neutralizing mAb. To this end, important efforts undertaken to solve the crystal structure of BR-NAbs in complex with viral envelope glycoproteins are underway (Brunel et al., 2006; Calarese et al., 2003; Cardoso et al., 2007; Cardoso et al., 2005; McGaughey et al., 2003; Ofek et al., 2004; Saphire et al.,
2001; Wilkinson et al., 2005). Hopefully, such studies will lead to development of immunogenic mimotopes (Dorgham et al., 2005; Saphire et al., 2007) with the potential to elicit BR-NAbs for use in future vaccine candidates.
Immunogen subtypes: HIV-1 clades B and E are considered antigenically distinct from other clades (Mascola et al., 1996), and there seems to be no particular preference for sera from patients infected with one virus to neutralize other viruses from the same clade.
Indeed several sera neutralize viruses from different clades (Mascola et al., 1996; Nyambi et al., 1996). All of the BR-NAbs known today have been obtained from Clade B infected individuals, mainly due to the nature of research and patient sample availability in developed European and North American nations being predominantly subtype B. Could other envelopes induce alternate BR-Nabs in patients? Could new antibodies be discovered in non-subtype B infected individuals? Researchers found distinct differences in the binding affinity of subtype B and subtype C trimeric envelopes to CD4 and mAb b12
(Srivastava et al., 2007). Significant strides are now being made to develop new immunogens from Subtype C envelopes (Lian et al., 2005; Srivastava et al., 2007). Given that the majority of infections caused worldwide are by viruses from clades other than subtype B, these studies may yield some promising new antigens.
Enhancing Immunity: Any effective AIDS vaccine may require both CTL and Ab mediated immunity. Difficulty in inducing potent antiviral immune responses that target
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diverse viral strains and prevent emergence of escape mutants has led to the development of
new vaccine strategies against HIV-1 (reviewed in (Berzofsky, Ahlers, and Belyakov,
2001)). Some of these strategies include (1) sequence modification to create enhanced
epitopes that bind with high affinity to MHC molecules; (2) targeted induction of mucosal
immunity; (3) use of synergistic combinations of cytokines, chemokines and costimulatory
molecules to enhance immune response; (4) relief of negative regulatory or suppressive
mechanisms that inhibit the immune response; (5) use of dendritic cells as vaccine vehicles;
and (7) formulation of the vaccine to incorporate agents inducing innate immunity.
Mucosal vaccines: Given that HIV-1 is primarily transmitted through vaginal and rectal intercourse, it is imperative that future vaccines aim to stimulate strong immunity at these mucosal barriers. The first cellular targets for HIV-1 infection are memory T-cells bearing CD4 and CCR5 found in high numbers in the mucosa of the gut-associated lymphoid tissue (GALT). Infection of GALT cells leads to rapid depletion of CD4 T-cells and is a major site of viral replication (Brenchley et al., 2004; Guadalupe et al., 2003; Li et
al., 2005b; Mehandru et al., 2007). Thus, novel vaccines, adjuvants and routes of delivery to
establish protective mucosal immunity at these sites should be a major objective of future
HIV vaccinology, and significant strides towards effective mucosal CTL responses are being
made. Novel VLPs have hopeful prospects of inducing strong mucosal immunity. A
particularly interesting approach included a bovine papillomavirus (BPV)-HIV-1 gp41
chimeric VLP, whereby the 2F5 ELDKWA sequence was fused to the BPV L1 capsid
protein, and used to generate VLPs (Zhang et al., 2004). A strong gp41 systemic and
mucosal antibody response was elicited after oral immunization of mice, and neutralizing
activity was detected in some mouse sera against HIV-1 strain MN, but not AD8 or DH12.
Such a novel approach has great potential to develop further.
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Dendritic cell antigen presentation: A novel approach to increase immune response
against HIV involves helping presentation of antigens to immune cells by employing
dendritic cells. Antigen-pulsed DCs have regularly been used to deliver antigen in vivo, but
such a patient-specific approach using autologous DCs is unfeasible in large-scale
vaccination programs in the developing world. By coupling antigen to a DC specific mAb,
both mice and humans showed enhanced immune responsiveness and kinetics when
compared to soluble antigen control (Bozzacco et al., 2007). Such an approach may help
stimulate stronger immunity in vaccines more applicable to developing nations.
Cytokine and chemokine adjuvants: Cytokine/chemokine codelivery with DNA encoding immunogens can increase the magnitude of immune response and improve vaccine
efficacy compared to delivery of DNA alone (Barouch et al., 2000). Codelivery of either
DNA or protein-based RANTES, granulocyte-macrophage colony stimulating factor (GM-
CSF) and interleukins IL-2 and IL-12 have shown particular appeal in inducing strong CTL
responses, and in some cases, strong humoral immunity (Belyakov et al., 2000; Kim et al.,
1997; Robinson et al., 2006; Xin et al., 1999). Such novel adjuvants will likely play an
important role in future DNA vaccine strategies. It might also be interesting to consider if
coimmunization of our huCD4CCR5 transgenic mice with antigen and RANTES or
RANTES expression plasmids might help recruit both transgene expressing B-cells and
mouse T-cells to the site of immunization and enhance a Th2 response.
Polyvalent vaccines: Polyvalent and multivalent vaccines, or vaccines that are
based on inclusion of multiple epitopes of different strains, thus broadening the specificities
of the antibodies produced (see (Hurwitz et al., 2005; Hurwitz et al., 2008; McBurney and
Ross, 2007; Slobod et al., 2005) for reviews), are currently being aggressively pursued.
Vaccine cocktail approaches may elicit immune responses with broad diversity and
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durability to prevent HIV-1 infection, stimulating both humoral and cellular immunity.
Polyvalent vaccines have the potential to stimulate a rapid secondary immune response
against a number of diverse antigens, and may capture some of the antigenic variability of
naturally evolving viruses, helping prevent initial infection and control virus replication in
infected individuals. Although proof-of-concept has been demonstrated, practicality of such
an approach is still questioned for two main reasons: (1) How many variant envelopes need
be included in a vaccine cocktail to confer complete protection, and (2) the laborious and
costly nature of preparing large pools of envelope. In Chapter 4, we address some of these
complex issues. Furthermore, the recent advances in DNA vaccine induced humoral and
cellular immunity provide an ideal mode of immunization due to the ease of manipulation
and preparation, and can efficiently be used in combination with viral vectors and soluble protein or peptide in prime-boost vaccine regimens.
Likely, future multivalent vaccines might include some form of consensus or ancestral HIV-1 DNA sequences for hypervariable regions, as was approached in Chapter 4.
The use of mosaic proteins from multiple sequences optimized by computational analysis
has already shown promising ability to represent diverse viral sequences (Fischer et al.,
2007). As results from several trials and studies are analyzed, compared and interpreted, it
should become clear how design of DNA constructs (e.g. promoter use, codon optimization
and CpG inclusion), regimen and route of delivery, vaccine formulations and genetic
adjuvants will best enhance desired immune response. This technology may become a
central strategy in vaccinating against infectious diseases in the future.
Partially protective vaccines: A polyvalent vaccine, as proposed in Chapter 4, may not provide complete protective immunity, but could provide protection against the most common variants in a particular geographic location. In Chapter 4, we developed antigens
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with the potential to elicit antibodies against 85% of subtype B isolates. At this stage of the
pandemic, some protection may be preferential to no protection at all. The concept of partial
vaccine efficacy can be defined in two ways. It can be used to describe a vaccine that only
protects some people in a population, but not others, possibly by protecting against only
certain virus strains. Or it can be used to describe a vaccine that does not completely protect
the recipient against infection, but may reduce the severity of disease. Most T-cell vaccine
candidates provide the second kind of coverage. Development of a polyvalent V3 vaccine
that could elicit neutralizing antibodies against the vast majority of subtype B primary
isolates, although providing only partial sterilizing immunity, could be used in concert with
other vaccines that induces strong cellular immunity, providing greater levels of protection.
Mathematical models have suggested that a vaccine with only 50% efficacy over 10 years given to 65% of adults could reduce HIV incidence by 25% to 60%. Even a vaccine with as little as 30% efficacy could have a significant impact on the HIV pandemic in
developing nations (Kahn, 2002). We can only hope that future vaccines will reach and
exceed this level of efficacy.
A partially protective vaccine capable of lowering peak viral loads, maintaining high
CD4 cell counts and promoting more rapid resolution of peak viremia in vaccine recipients
who do become infected is suggested to improve survival and delay the onset of AIDS.
However, these survival benefits seen in a population of vaccine recipients may lead to a greater prevalence of infection within this group and increased potential for virus transmission. The long-term consequence could involve a large population of HIV-1 infected individuals with neutralization resistant virus, leading to a loss of efficacy of the vaccine over time. Only if the antibody resistant mutations result in a decrease in fitness and transmission will such a partially protective vaccine be of any large-scale value.
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A modified course of infection with lowered transmission rates would indeed be welcome, but could partially protective vaccines with limited neutralizing activity against select R5-tropic strains drive virus evolution towards more rapid emergence of X4 tropic virus? Such consequences could be detrimental to patient health, as X4 tropic viruses are strongly associated with a fast decline in CD4 cells and the development of AIDS (Connor et al., 1997; Schuitemaker et al., 1992; Tersmette et al., 1988). However, there is no compelling evidence to suggest that antibody selection drives coreceptor switching from R5 to X4 tropic virus by selecting for specific virus mutants (Hoffman et al., 2002; Pastore,
Ramos, and Mosier, 2004). It appears that certain specific sets of mutations or insertions of charged residues (Arg, Lys) into precise positions (residues 310, 324 of MCON6 sequence) of the V3 loop are required for coreceptor switching (Hartley et al., 2005). Most likely, any vaccine that reduces virus load will be more effective in preventing coreceptor switching through diminished virus replication than through the specificity of antibody. As such, partially protective vaccines may have an important role to play in reducing AIDS- associated mortality.
The advantage HIV-1 gains over antibody neutralization through rapid diversification is a key mechanism of escape. Yet this advantage is lost at two key genetic bottlenecks: transmission to a new host, and during and immediately following coreceptor switching (Bunnik et al., 2007; Derdeyn et al., 2004). It may be that vaccines that target these two intervals of relative susceptibility are more likely to be effective than any vaccine that approaches all envelope sequences of the same importance in a vaccine. Given the diminished fitness and increased susceptibility of X4 tropic virus to neutralization compared to R5 tropic virus, it might be interesting to assess if an X4 tropic polyvalent vaccine would act to slow coreceptor switching and disease progression (See Figure 5-1).
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Figure 5-1. HIV-1 envelope evolution showing virus tropism and parallel changes in sensitivity of those viruses to neutralizing antibody. Figure adapted from (Mosier, 2005).
Recently transmitted R5 tropic virus and X4 tropic virus that arises at tropism switch are particularly susceptible to antibody-mediated neutralization.
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Conclusion
Some of the most pressing issues at hand today are how to achieve high-titer, broadly neutralizing antibodies and overcome poor immunogenicity and memory responses that
might allow a CTL response to control infection and limit evolution of escape mutants.
There is little doubt that enormous challenges lie ahead. Overcoming these challenges
requires a concerted commitment of scientific and economic resources, and strong yet
sensitive application in the developing world. The development of several internationally
collaborative organizations provides a promising framework in which to rapidly push
research forward to the forefront of clinical trails. These organizations include the
International AIDS Vaccine Initiative (IAVI), the WHO and UNAIDS, the Center for
HIV/AIDS Immunology (CHAVI), the HIV Vaccine Trial Network (HVTN) established by
National Institute of Allergy and Infectious Diseases (NIAID), the Neutralizing Antibody
Consortium (NAC), the European and Developing Countries Clinical Trials Partnership
(EDCTP), the French National AIDS Research Agency (ANRS), and the African AIDS
Vaccine Program (AAVP). It is also essential to increase funding for small HIV-1 vaccine
research projects and sponsor a large array of diverse, unconventional, high-risk-high-
reward projects. Current approaches towards effective AIDS vaccine development have not
worked, and sometimes the most startling and groundbreaking discoveries that shift entire
dogmas come from people who work from outside of the common field of research. We
might just find something by expanding the net, increasing the scope and integrating
scientists with diverse backgrounds of biomedical research who bring different perspectives
and skills to the table.
Additionally, establishing strong and trusting links with research institutions and
communities in developing countries is essential for future clinical trials. Historically,
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several unethically conducted drug study trials in Africa have left community members
unwilling to participate in future experimental research work (Grady, 2004). Given the
history of colonialism in some of the nations hardest hit by AIDS, care must be taken that developing nations do not feel exploited by these international research organizations. A balance must also be struck to ensure that participants enter into vaccine trials in a truly
voluntary nature, free from undue influence and coercion. Finally, it is imperative that
participants be fully informed of the risks and benefits of any vaccine trial, with full
understanding that an AIDS vaccine trail may not actually protect one from acquiring HIV
infection. The impact of failed vaccines on the acceptance of a community for further
vaccine efficacy testing is still somewhat unknown.
Despite the significant scientific and public health challenges, there is little doubt
that the development of a safe, effective and affordable HIV vaccine is a major priority for
HIV/AIDS vaccine researchers globally. We should not despair, but prepare ourselves by
promoting regional capacity to increase access to vaccines and therapy in developing nations
by engaging communities, training local scientists and nurses and building crucial
infrastructure.
It may be difficult to develop a single vaccine immunogen that includes all the
hypothesized requirements to develop broadly neutralizing antibodies. Likely, efficacy data
from each trial will stimulate and feed back into subsequent trials, resulting in the generation
of candidates with increased efficacy and safety, leading to a stepwise reduction in HIV-1
prevalence and AIDS-related morbidity. In the meantime, to stop the transmission of AIDS we should promote a healthy attitude towards sex and relationships by practicing three things we know already prevent acquisition of HIV. The ABCs of AIDS - abstain, be faithful and condomize, and not to forget the recently added recommendation, circumcise.
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Appendix
Section 1: Development of HIV-1 envelope constructs with shortened
V1/V2 loops that retain fusion competence.
Methods and Materials
V1 and V2 deletion from MCON6 envelope
To introduce unique restriction enzyme sites surround the V1 and V2 loops, which
would assist in partial deletion of these regions, we utilized a site-directed cloning technique
with QuikChange XL Site-Directed Mutagenesis Kit (Stratagene). Unique HpaI and
EcoRV restriction enzyme sites were introduced internal of pcDNA*MCon6gp160 V1 loop
using Hpa I primer pair 5’-GTGACCGTCAACTGCGTTAACGTGCGCAACGTGTCC-3’
(sense) and 5’-GGACACGTTGCGCACGTTAACGCAGTTCAGGGTCAC-3’ (antisense),
and EcoRV primer pair 5’-GAGACCGACAACGAGGATATCAAGAACTGCTCCTTC-3’
(sense) and 5’-GAAGGAGCAGTTCTTGATATCCTCGTTGTCGGTCTC-3’ (antisense).
Restriction enzyme sites are underlined and bold letters represent mutagenesis sites. Unique
SacI and BSTZ17 I restriction enzyme were introduced internal of the V2 loop using SacI primer pair 5’-CTTCAACATCACCACCGAGCTCCGCGACAAGAAGCAG-3’ (sense)
and 5’-CTGCTTCTTGTCGCGGAGCTCGGTGGTGATGTTGAAG-3’ (antisense), and with BstZ17 I primer pair 5’-CAAGAACTCCTCCGAGGTATACCGCCTGATCA-
ACTGC-3’ (sense) and 5’-GCAGTTGATCAGGCGGTATACCTCGGAGGAGTTCTTG-
3’ (antisense). Restriction enzyme sites are underlined and bold letters represent
mutagenesis sites. Correct cloning was confirmed by restriction enzyme digestion analysis
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and DNA sequencing. The resultant plasmids are referred to as pcDNA*MCon6gp160V1,
-V2 or –V1/V2. These plasmids were tested for expression and envelope function.
To delete V1 and/or V2 loops from pcDNA*MCon6gp160V1 and -V2, we used a
PCR cloning technique using each of these plasmids as template. Final plasmids containing deletions of both V1 and V2 were subcloned from these single variable loop deleted plasmids. Five variants of V1 deletion were generated, four of these using four different sense primers spanning the V1 region, and a single common antisense primer downstream of
V1, incorporating a convenient and unique BsrGI cloning site. The common antisense primer sequence was 5'-CCTTGTACTTGTACAGCTCGG-3', with BsrGI restriction
enzyme site underlined. This common antisense primer was use independently with each of
the following four sense primers. V1a): 5’-ATTAATGTTAACGTCAGGAATGTC-
TCCGATATCAAGAACTGCTCC-3’; V1b): 5’-ATTAATGTTAACGTCAGGTCGAA-
TGGGACGGATATCAAGAACTGCTCC-3’; V1c): 5’-ATTAATGTTAACGTCAGGGA-
GACCGACAACGAGGATATCAAG-3’; V1d): 5’-ATTAATGTTAACGTCAGGGA-
GGATATCAAGAACTGCTCC-3’. Underlined and double underlined sequences represent
HpaI and EcoRV restriction enzyme cloning sites of pcDNA*MCon6gp160V1. PCR products were purified and washed with QIAquick Gel Extraction Kit (QIAGEN) to remove primers and nucleotides. Cleaned PCR products and expression vector pcDNA*MCon6gp160V1 were digested with restriction enzymes HpaI and BsrGI and ligated together resulting in plasmid pcDNA*MCon6gp160-V1a) through -V1d). To generate the fifth V1 deletion variant with a Glycine-Alanine-Glycine replacement linker, we used sense primer 5'-CCTGTGGGTGACCGTGTACTACG-3' (BstE II site is
underlined), and antisense primer 5’-CGTACGGATATCGCCGGCTCCCAC-
GTTAACGCAGTTCAGGG-3’ (HpaI and EcoRV sites are underlined and double
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underlined, respectively, and GAG coding sequence is in bold). A single V2 deletion
variant with a Glycine-Alanine-Glycine replacement linker was made using sense primer 5’-
CGTACGGAGCTCGGCGCCGGAGTATACCGCCTGATCAACTGC-3’ (SacI and
BstZ17I restriction enzyme sites are underlined and double underlined respectively, and
GAG coding sequence is in bold), and antisense primer 5’-GCAGCTTCTTGGCCAC-
CTGC-3’ (MscI restriction enzyme site is underlined). PCR products and vector pTM-
MCon6gp160V2 were digested with SacI and MscI and ligated together. Resultant clone was named pTM-MCon6gp160-V2. Plasmids with deletions of both V1 and V2 loop were generated by digesting V1 deleted plasmids with Sac I and MscI and cloning into corresponding sites of pTM-MCon6gp160-V2. All plasmids were confirmed by digestion
analysis and DNA sequencing, and tested for both expression and envelope functionality.
Envelope insert was later cloned into pcDNA* backbone by digesting pTM insert with
HincII and BstEII and pcDNA* vector with Hind III, klenow to blunt end, and digestion
with BstEII. Variable loop deleted envelope inserts were cloned into pcDNA*MCon6gp160
backbone.
Cell-cell fusion assay
Approximately 1x105 HeLa cells in an 80% confluent 24 well culture plate were transfected in quadruplicate with 3 g either pcDNA, pcDNA-MCon6gp160 (control) or variable loop mutant envelope plasmid DNA, and with 0.75 g pCMV-Tat per well, by standard calcium phosphate techniques. Cells were incubated for 16h at 37°C + 5% CO2 in complete DMEM, whereupon media was removed and replaced with fresh complete DMEM and incubated at 37°C for a further 8h. After removal of medium and washing adherent cells three times with serum free DMEM, approximately 1x105 lightly trypsinzed and
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washed TZM-bl cells were added to each well in 750ul serum free DMEM. Cell mixtures were incubated at 37°C for 1h before addition of 750ul of DMEM containing 20% FBS.
Cells were incubated for a further 36h before determination of fusion by luciferase activity detection. Experiments were repeated a minimum of four times. For luciferase assay detection, cells were lysed and virus infectivity was determined using -Glo luminescence assay as per manufacturer’s protocol (Promega). Relative luminescence units (RLU) were measured using a luminometer (Bio-Rad). Cells transfected with pCMV-Tat and control plasmid pcDNA were used to determine background luminescence and mean background was subtracted from all readings. Fusion competence of each mutant envelope was determined as a percentage of pCMV-Tat and wild type pcDNA-MCon6gp160 cotransfected controls.
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RESULTS
V1/V2 deletion
To allow for easy deletion of V1 and V2 loops from HIV-1 group M consensus envelope sequence (MCON6), we introduced novel restriction enzyme cloning sites internal to the cysteine residues defining the base of each variable loop. MCon6 V1 sequence was mutated to introduce HpaI and EcoRV sites internal of cysteine residues involved in disulphide bonding and loop formation. Neither V1 mutations was silent, resulting in
T131V and E147D substitution in plasmid pcDNA*MCon6gp160V1, but was not deemed significant given the diversity of V1. Restriction enzyme sites SacI and BstZ17I were introduced internal of V2 cysteine residues resulting in one silent mutation and Y194V substitution in plasmid pcDNA*MCon6gp160V2. A plasmid with both V1 and V2 restriction enzyme sites was generated, named pcDNA-MCON6gp160V1/V2. As expected given the great diversity observed in variable loop regions, none of the amino acid substitutions appeared to have a significant effect on protein expression (data not shown).
We hypothesize partial deletions of V1 and V2 that still yielded a fusion competent virus would help generate a better gp120 antigen through a two-fold mechanism. Firstly, by redirecting immune responses of V1/V2 against neutralizable V3 and outer domain epitopes
(Gzyl et al., 2004; Kang et al., 2005; Kim et al., 2003), and secondly, by better exposing conserved neutralizing epitopes masked by these large, hypervariable regions (Barnett et al.,
2001; Kim et al., 2003; Lu et al., 1998; Xu et al., 2006). Indeed, studies using variable loop deleted envelopes show better binding of antibodies to CD4bs (Wyatt et al., 1993) and coreceptor-binding site (Cao et al., 1997; Wyatt et al., 1995). Several studies have shown higher titer antibodies against gp120 with variable loop deleted envelope (Kim et al., 2003;
243
Lu et al., 1998), and some studies have found elicitation of higher titer Nab upon variable
loop deleted envelope immunization, and broad, albeit weak, neutralization of hetrologous
virus strains (Barnett et al., 2001). Furthermore, only two variable loop deleted envelopes
that remain fusion competent have thus far been discovered (Cao et al., 1997; Stamatatos,
Wiskerchen, and Cheng-Mayer, 1998) and generation of a group M consensus sequence variable loop deleted yet fusion competent variant would be a particularly useful HIV-1 vaccine reagent.
In this regard, not knowing how tolerant MCon6 envelope would be of large deletions of variable loops, we generated several deletion variants and tested them for entry
functionality. As shown in Figure Appendix-1, we generated five V1 and one V2 deleted mutants through PCR cloning techniques utilizing our envelope constructs with unique
restriction enzyme cloning sites internal of the variable loop cysteine residues. We chose to
partially delete variable loops (maintaining stem flanking residues or tip regions) or delete
whole loops by replacing with Glycine-Asparagine-Glycine linkers, as had been previously
reported (Stamatatos, Wiskerchen, and Cheng-Mayer, 1998; Wyatt et al., 1995).
244
A.
B. V1 LOOP DELETION MUTANTS
NCVNVRNVSSNGTETDNEDIKNCSFN V1GAG: NCVNV-----GAG-----DIKNCSFN V1a): NCVNVRNVS------DIKNCSFN V1b): NCVNVR---SNGT-----DIKNCSFN V1c): NCVNVR------ETDNEDIKNCSFN V1d): NCVNVR------EDIKNCSFN
V2 LOOP DELETION MUTANTS
NCSFNITTELRDKKQKVYALFYRLDVVPIDDKNSSEISGKNSSEYYRLINCNTS V2: NCSFNITTEL------GAG------VYRLINCNTS
Figure Appendix-1: V1/V2 deletion. (A) Schematic diagram of V1 and V2 deletion mutants. Amino acid substitutions and novel restriction enzyme cloning sites highlighted.
N-linked glycosylation sites are indicated. (B) HIV-1 MCon6 V1 and V2 loop DNA sequences, and generated deletion mutants. V1 loops span cysteine residues (shown in bold) involved in disulphide bond formation. Dashes represent deleted amino acids. Underlined sequences represent location of introduced unique restriction enzyme cloning sites.
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V1 and V2 deleted envelopes are fusion competent
Fusion competence of MCON6gp160-V1, -V2 and V1/V2 deleted loop variants
(MCON6gp160-V1, V2, V1V2, respectively) was tested in a cell-cell fusion assay measuring luciferase reporter activity of TZM-bl cells as percentage of wild type
MCON6gp160 fusion (Figure Appendix-2A). To ensure that each envelope mutant construct was expressed at levels comparable to wild type MCON6gp160, a western blot of transfected HeLa cells used in cell-cell fusion assays is shown (Figure Appendix-2C). Only one construct, V1a)V2, showed any deviation in envelope expression level from wild type, being slightly enhanced. However, binding of antibody nonspecifically to human cellular proteins (Figure Appendix-2C lower panel) shows that this is not a result of increased expression level of the mutant envelope construct, but rather human error in gel loading. Similarly, the lane containing negative control pcDNA was also overloaded.
Nevertheless, we observed a marginal increase in fusion competence of V1/V2 (Figure
Appendix-2B), showing that substitutions of amino acids internal of V1 and V2 have little effect on entry function of envelope. Likewise, we saw no effect on functionality of V3 gp160. The most obvious reduction in fusion competence was observed in V1a) gp160, yet this reduction was not apparent when combined with V2 deletion. Interestingly, V1d) mutants, which has the largest deletion of V1 loop, showed only a 35% reduction in fusion efficiency. These results lead us to believe there is likely substantial interaction of each variable loop with other envelope structures, and these interactions are necessary for maintaining the complex structure of trimeric envelope to allow receptor binding and entry.
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Figure Appendix-2: HIV-1 MCon6gp160 V1 and V2 loop deletion mutant fusion competence.
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Figure Appendix-2: HIV-1 MCon6gp160 V1 and V2 loop deletion mutant fusion competence: (A) HeLa cells expressing HIV-1 MCon6 envelope constructs and Tat were mixed with HeLa derived reporter TZM-bl cells expressing CD4 and CCR5. Upon cell-cell fusion, b-galactosidase and luciferase genes under control of HIV-1 promoter are activated by Tat binding to the Long Terminal Repeat (LTR). Fusion is measured as a percentage of wild type envelope MCon6gp160 luciferase activity. (B) Western blot analysis of MCon6
Envelope construct expression used in cell-cell fusion experiments, and unrelated protein.
(C) Western Blot analysis of HeLa cell expression of MCon6gp160 mutant constructs.
Rabbit anti-gp160 antibody was used at 1:1000 dilution. An overdeveloped blot to show non-specific binding to human cellular proteins is shown in panel II.
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Section 2: Outer Domain
2007 Conference on Retroviruses and Opportunistic Infections poster abstract by Kim et al.
Poster #471. (http://www.retroconference.org/2007/Abstracts/30284.htm)
Construction and Characterization of Antigenic Properties of gp120 Outer Domain
Soon-Jeung Kim*, D Han, A Penn-Nicholson, Y Qin, and M Cho
ABSTRACT
Background: Eliciting neutralizing antibodies that are broadly reactive remains a critical
roadblock to developing a protective HIV-1 vaccine. Presently, only a few of such
neutralizing antibodies have been identified, all of which were isolated from patients. Of these antibodies, 3 target the outer domain of gp120 (gp120OD), namely b12, 2G12, and
447-52D. The outer domain is largely immunosilent due to extensive glycosylation.
Although the inner domain is highly immunogenic, antibodies against this region fail to
neutralize the virus. We hypothesize that the removal of the inner domain will divert antibody responses toward the outer domain of the protein, which would increase the likelihood of eliciting broadly reactive neutralizing antibodies. The objective of this study is to generate a structurally intact gp120OD that is antigenically correct. Methods: For
constructing gp120OD, we used codon-optimized M group consensus envelope (MCON6).
We used known crystal structures of gp120 to select suitable cleavage sites for separating
the inner and outer domains. 6xHis tag was attached to the C-terminal end of the protein to
facilitate protein purification. The protein was expressed by transient transfection of a
249
plasmid encoding the protein into HeLa or 293T cells. Antigenic properties of the proteins
were evaluated by immunoprecipitation and/or by ELISA using a panel of monoclonal
antibodies, including b12, 2G12 and 447-52D, as well as HIV-Ig and antisera from
individual HIV-infected patients. Results: MCON6 gp120OD was efficiently expressed and secreted into cell culture medium. It was quite homogeneous in size with an apparent mass of about 50 kD. The protein is glycosylated; deglycosylation with PNGaseF resulted in an expected size of about 26 kD. The protein could be immunoprecipitated by b12, 2G12,
447-52D, and 654-30D, but not by 48d or 17b. More importantly, gp120OD could co- immunoprecipitate CD4 demonstrating that our protein was able to bind CD4. Together, these results indicated that our gp120OD is structurally intact and antigenically correct.
HIV-Ig was able to immunoprecipitate full-length gp120, but not gp120OD, indicating that the OD is indeed poorly immunogenic. However, evaluation of antisera from individual patients indicated that there is a significant variation amongst patients in antibody responses against gp120OD. Conclusions: MCON6 gp120OD we generated is antigenically correct and has great potential to elicit broadly reactive neutralizing antibodies.
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Figure Appendix-3: Structure of gp120 and gp120 outer domain. (A) Crystal structure of gp120 showing the inner (blue) and outer (yellow) domains, and the V3 loop (red). (B) CD4 binding site of outer domain is highlighted in green.
251
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