Spectrum of Somatic Hypermutations and Implication on Antibody Function: Case of the anti HIV-1 antibody, b12 Mesfin Mulugeta Gewe A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2015 Reading Committee: Roland Strong, Chair Nancy Maizels Jessica A. Hamerman Program Authorized to Offer Degree: Molecular and Cellular Biology i ii ©Copyright 2015 Mesfin Mulugeta Gewe iii University of Washington Abstract Spectrum of Somatic Hypermutations and Implication on Antibody Function: Case of the anti HIV-1 antibody, b12 Mesfin Mulugeta Gewe Chair of the Supervisory Committee: Roland Strong, Full Member Fred Hutchinson Cancer Research Center Sequence diversity, ability to evade immune detection and establishment of human immunodeficiency virus type 1 (HIV-1) latent reservoirs present a formidable challenge to the development of an HIV-1 vaccine. Structure based vaccine design stenciled on infection elicited broadly neutralizing antibodies (bNAbs) is a promising approach, in some measure to circumvent existing challenges. Understanding the antibody maturation process and importance of the high frequency mutations observed in anti-HIV-1 broadly neutralizing antibodies are imperative to the success of structure based vaccine immunogen design. Here we report a biochemical and structural characterization for the affinity maturation of infection elicited neutralizing antibodies IgG1 b12 (b12). We investigated the importance of affinity maturation and mutations accumulated therein in overall antibody function and their potential implications to vaccine development. Using a panel of point iv reversions, we examined relevance of individual amino acid mutations acquired during the affinity maturation process to deduce the role of somatic hypermutation in antibody function. Biophysical characterizations of b12 point mutant interactions with gp120 monomers from two Clade B viruses (SF162 and QH0692) indicates importance of cooperative contributions by individual mutations accumulated due to the extensive maturation processes in attaining the observed broadly neutralizing properties of b12. However, effects of individual mutations on epitope binding do not correlate with their effect on virus neutralization potency. Establishment of viral latent reservoirs, rendering antibody-based immune responses ineffective as preventive treatments, precedes serum detections of infection elicited highly potent bNAbs. Bearing the goal of structure based vaccine immunogen design that recapitulate b12-like immune response, we also investigated the minimum mutations on germline antibody sequences required to garner b12 comparable epitope binding affinity and virus neutralization potency. Our progressive increase in mutations on germline b12 precursor antibody approach reveled the importance of mutations on both antibody complementarity determining (CDR) and framework regions (FWR) in epitope binding and virus neutralization. Here we report extensive mutations and prolonged affinity maturation are vital to the development of highly potent bNAbs. One potential mechanism in attaining increased affinity maturation following the antibody maturation process is conformational antibody rearrangement and rigidification of antibody variable domains. To determine the role of extensive antibody mutations in the overall antibody structural flexibility and conformational rearrangements, we examined the structures of germline precursor b12 light chain variable domains. Accordingly, our structural analyses reveal absence of apparent effect on global structure of antibody light chain variable domains following affinity maturation. However, local structural affects are observed where germline precursor antibody CDRs exhibited conformational samplings distinct from b12 light chain CDRs. While germline precursor antibody CDRs sample different conformations, the process of antibody maturation focused the epitope binding region to specific conformation enabling high affinity epitope binding. v These data suggest that structure based vaccine development that utilize b12-like bNAbs as templet need to develop a scheme that enables extensive affinity maturation observed in bNAbs. The bodies of work presented here, representing work on multiple aspects of bNAb development, potentially highlight key basic science research on HIV vaccinology that inform HIV vaccine development. vi Dedication To my wife Genet Tadesse and daughter Tinsae Mesfin for enduring the headwinds and to my parents Mulugeta Gewe and Fetlework Bekele for believing in me. vii Table of Content Abstract ........................................................................................................................................................ iv Dedication ................................................................................................................................................... vii Table of Content ........................................................................................................................................ viii List of Table .................................................................................................................................................. x List of Figures ............................................................................................................................................... x Chapter 1: ...................................................................................................................................................... 1 1.1 Introduction ................................................................................................................................................... 1 1.2 HIV-1 and antibody based immunity ............................................................................................................. 5 1.3 The antibody development paradigm.......................................................................................................... 10 1.4 The HIV vaccine design paradox ................................................................................................................ 13 1.5 Documents presented in this Thesis ........................................................................................................... 16 Chapter 2 ..................................................................................................................................................... 18 2.1 Overview ..................................................................................................................................................... 18 2.2 Results ........................................................................................................................................................ 21 2.2.1 Amino Acid Sequence Analysis of b12 Mature and Germline Precursor ................................................ 21 2.2.2 Characterization of b12 c/scFvs proteins ............................................................................................... 24 2.2.3 Epitope binding is affected by SHM on CDRs more than FWRs ............................................................ 28 2.2.4 Individual mSRs exhibit minimal effect on virus neutralization potency .................................................. 31 2.2.5 SHM on CDR micro satellites exhibit correlated effect on epitope binding and virus neutralization ....... 32 2.3 Discussion .................................................................................................................................................. 36 2.3.1 Structural implication of antibody light chain .......................................................................................... 39 2.4 Acknowledgment ........................................................................................................................................ 42 Chapter 3 ..................................................................................................................................................... 43 3.1 Overview ..................................................................................................................................................... 43 3.2 Results ........................................................................................................................................................ 45 3.2.1 gRBs for progressive SHM analysis ....................................................................................................... 45 3.2.2 b12 comparable binding kinetics necessitate extensive mutation of germline encoded precursor antibodies.............................................................................................................................................................. 49 3.2.3 Mature b12 comparable neutralization potency does not correlate with binding affinity. ........................ 51 3.3 Discussion .................................................................................................................................................. 56 3.4 Acknowledgment ........................................................................................................................................ 57 Chapter 4 ..................................................................................................................................................... 58 4.1 Overview ....................................................................................................................................................