CHARACTERIZATION OF NEUTRALIZING ANTIBODY EPITOPES ON HIV-1 SUBTYPE C ENVELOPE GLYCOPROTEINS TO SUPPORT VACCINE DESIGN Elin Solomonovna Gray A thesis submitted to the Faculty of Medicine, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, 2007 ii DECLARATION I declare that this thesis is my own work. It is being submitted for the degree of Doctor of Philosophy at the University of the Witwatersrand, Johannesburg. It has not been submitted before for any degree or examination at this or any other University. ______________________ Elin Solomonovna Gray _____ day of ________________, ______ iii ABSTRACT Since its discovery as the etiological agent of AIDS in 1983, HIV-1 has been the focus of unrelenting research into an effective vaccine to control viral infection. Neutralizing antibodies constitute a correlate of immune protection for most available vaccines, but the induction of these antibodies against HIV-1 has become a major challenge. The HIV-1 envelope glycoprotein has evolved to evade neutralizing antibodies in an extraordinary way, yet a vaccine that can stimulate such antibodies remains the best hope to provide sterilizing immunity. The existence of a group of monoclonal antibodies, such as IgG1b12, 2G12, 2F5 and 4E10, capable of neutralizing a broad range of primary isolates signals vulnerable areas on the envelope glycoprotein. Furthermore, passive transfer of these antibodies can completely protect against viral challenge in animal models. The epitopes recognized by these antibodies are being intensely pursued as vaccine targets, in the hope of inducing such specificities. This thesis encompasses a series of studies on characterizing the epitopes recognized by these broadly cross-reactive monoclonal antibodies in the context of subtype C viruses. HIV-1 subtype C is responsible for the vast majority of infections worldwide, however, until recently, little research has been done on these viruses in contrast to the well characterized subtype B strains. Chapter Two describes the characterization of paediatric subtype C viruses for their sensitivity to IgG1b12, 2G12, 2F5 and 4E10. This study was done because of a planned clinical trial of some of these antibodies as post-exposure prophylaxis to prevent mother-to-child HIV-1 subtype C transmission. Only the MAb 4E10 was able to neutralize all the viruses tested, while IgG1b12 was only partially effective. 2F5 and 2G12 did not neutralize any of the viruses. The conclusion was that only 4E10 and IgG1b12 would be suitable for use as prophylactic agents in a population where HIV-1 subtype C is prevalent. Given that subtype C viruses were found to be largely insensitive to 2G12 neutralization, the commonly absent glycan at iv position 295 was introduced into envelope glycoproteins from this clade. The work presented in Chapter Three explores the requirements of the 2G12 epitope on the envelopes of subtype C viruses. However, this antibody binding site was not readily reconstituted, suggesting structural differences from other HIV-1 subtypes in which the 2G12 epitope is naturally expressed. Chapter Four describes the study of 4E10 resistant virus quasispecies isolated from a seven year old perinatally HIV-1 infected child, in whom anti-MPER antibodies were found. Determinants of 4E10 neutralization were mapped to the epitope of this antibody in the MPER, as well as to the cytoplasmic tail, in particular, to four amino acids in the LLP-2 region. The role of neutralizing antibodies in natural HIV-1 subtype C infection was examined in Chapter Five by following the development of autologous and heterologous neutralizing antibodies in 14 patients during the first year of infection. Potent but relatively strain-specific neutralizing antibody responses were detected within 3-12 months of infection. The magnitude of the responses was associated with shorter V1-to-V5 envelope length and fewer glycosylation sites, in particular in the V1-V2 region. Furthermore, anti-MPER and anti-CD4i neutralizing antibodies were detected in some individuals; however, they were not associated with neutralization breadth. Finally, in Chapter Six these results are analyzed collectively, in the context of the latest findings in the field, and suggestions for further research are discussed. v PUBLICATIONS FROM THIS THESIS Gray ES, Meyers T, Gray G, Montefiori DC, Morris L. Insensitivity of Paediatric HIV-1 Subtype C Viruses to Broadly Neutralising Monoclonal Antibodies Raised against Subtype B. PLoS Med. 2006 Jul;3(7):e255. PMID: 16834457 (Chapter Two) Gray ES*, Moore PL*, Choge IA, Decker JM, Bibollet-Ruche F, Li H, Leseka N, Treurnicht F, Mlisana K, Shaw GM, Abdool Karim SS, Williamson C, Morris L; CAPRISA 002 Study Team. Neutralizing Antibody Responses in Acute Human Immunodeficiency Virus Type 1 Subtype C Infection. J Virol. 2007 Jun;81(12):6187-96. PMID: 17409164 * Joint first authors (Chapter Five) Gray ES, Moore PL, Pantophlet RA, Morris L. N-Linked Glycan Modifications in gp120 Of Human Immunodeficiency Virus Type 1 Subtype C Render Partial Sensitivity to 2G12 Antibody Neutralization. J Virol. 2007 Oct;81(19):10769-76. PMID: 17634239 (Chapter Three) Gray ES, Moore PL, Bibollet-Ruche F, Li H, Decker JM, Meyers T, Shaw GM, Morris L. 4E10 Resistant Variants in an HIV-1 Subtype C Infected Individual with an Anti-MPER Neutralizing Antibody Response J Virol. 2008 Mar;82(5):2367-75. PMID: (Chapter Four) vi OTHER PUBLICATIONS Rademeyer C, Moore PL, Taylor N, Martin DP, Choge IA, Gray ES, Sheppard HW, Gray C, Morris L, Williamson C; HIVNET 028 study team. Genetic Characteristics of HIV-1 Subtype C Envelopes Inducing Cross-Neutralizing Antibodies. Virology. 2007 Nov 10;368(1):172-81. PMID: 17632196 Moore PL*, Gray ES*, Choge IA, Ranchobe, Mlisana K, Abdool Karim SS, Williamson C, Morris L; CAPRISA 002 Study Team. The C3-V4 Region is a Major Target of Autologous Neutralizing Antibodies in HIV-1 Subtype C Infection. J Virol. In press * Joint first authors Morris L, Coetzer M, Gray ES, Cilliers T, Alexandre KB, Moore PL, Binley JM. Entry Inhibition of HIV-1 Subtype C Isolates Chapter in Book: Entry Inhibitors in HIV Therapy -Edited by Jacqueline D. Reeves and Cynthia A. Derdeyn. Pages 119-131. vii PRESENTATIONS AT MEETINGS Gray ES, Morris L. Susceptibility of subtype C viruses to neutralizing monoclonal antibodies raised against subtype B South African AIDS Conference 2005, Durban, South Africa (Oral) Gray ES, Montefiori DC, Morris L. Susceptibility of subtype C viruses to neutralizing monoclonal antibodies raised against subtype B AIDS Vaccine 2005, Montreal, Canada (Poster) Gray ES, Moore PL, Pantophlet RA, Morris L. Restoration of glycan 295 in subtype C viruses renders partial sensitivity to 2G12 neutralization HIV Vaccine 2006, Keystone Symposia, Keystone, Colorado, USA (Poster) Gray ES, Moore P, Choge I, Meyers T and Morris L. Characterization of naturally occurring 4E10 resistant viruses in a subtype C HIV-1 infected child AIDS Vaccine 2006, Amsterdam, Holland (Poster) Gray ES, Moore PL, Choge IA, Decker JM, Bibollet-Ruche F, Li H, Leseka N, Treurnicht F, Mlisana K, Shaw GM, Abdool Karim SS, Williamson C, Morris L; CAPRISA 002 Study Team. Neutralizing antibody responses in acute HIV-1 subtype C infection HIV Vaccine 2007, Keystone Symposia, Whistler Resort, British Columbia, Canada (Poster) Gray ES, Taylor N, Moore PL, Choge IA, Cave E, Puren A, Shaw GM, Morris L. HIV-1 subtype C infected plasma samples with broad specificity contain anti-MPER antibodies AIDS Vaccine 2007, Seattle, Washington, USA (Poster) viii I dedicate this work To the wonderful beings from whom I came, Zoya and Suleiman, To the special creature that came from me, Anthony, and To Brian with whom I am. ix ACKNOWLEDGMENTS I would like to express my most sincere thanks to the following for their valuable contribution to this work. To my supervisor, Prof. Lynn Morris, for believing in me, for her guidance and patience every step of the way, To all my collaborators and co-authors, To all the volunteers for their altruistic participation and without whom this work would not have been possible. To my fellow colleagues, Penny Moore, with whom I share a lot of this work, for being a great teammate, for listening patiently to all my rhetorical talks, and for all her help from experiments to prose, Sarah Cohen for her amazing commitment to her work, without whom this lab will be in chaos, Natasha Taylor, who initiated me into the field of neutralization assays, Isaac Choge for all his help in the Post-PCR and sequencing lab, Sue Hermann for her constant smile despite my pushy attitude towards the ordering process. Sheila Doing for keeping me organized despite my natural inability to it, and for enduring my literature review, And all the friendly and helpful people of the AIDS Research Unit that supported me during this work. To Dr. David Montefiori for opening the doors of his lab to me and for introducing me to the world of neutralizing antibodies, To Dr. George Shaw for allowing me to work in his lab as one of them, and the people from his lab: Dr. Fred Bibollet, Julie Decker, Dr. Hui Li and Katie Davis. To the Fogarty Training program for sponsoring both training visit to these labs. To the dedicated clinicians that provided me with the samples, Dr. Tammy Meyers, Dr. Glenda Gray, Dr Salim Abdool Karim, Dr Koleka Mlisana and all the CAPRISA staff. To Prof. Shoub for the opportunity to study at the NICD. On a personal note, To Maria Paximadis for offering me her friendship from the very beginning and being there during the highs and lows. To my family; Brian, Lisa, Arlene and Ann, for bearing my absences and helping me balance science and motherhood. This work was funded by the South African Vaccine Initiative (SAAVI), Centre for the AIDS Program of Research in South Africa (CAPRISA) and Center for HIV-AIDS Vaccine Immunology (CHAVI). x TABLE OF CONTENTS DECLARATION...............................................................................................................ii ABSTRACT.....................................................................................................................iii PUBLICATIONS FROM THIS THESIS........................................................................... v OTHER PUBLICATIONS ...............................................................................................vi PRESENTATIONS AT MEETINGS...............................................................................vii ACKNOWLEDGMENTS ................................................................................................ ix TABLE OF CONTENTS................................................................................................... x LIST OF TABLES ..........................................................................................................xii LIST OF FIGURES........................................................................................................xiii ABREVIATIONS ........................................................................................................... xv CHAPTER ONE INTRODUCTION ............................................................................................................ 1 1.1 BACKGROUND ....................................................................................................... 2 1.2 HIV ENVELOPE GLYCOPROTEIN.............................................................................. 3 1.2.1 The gp120 molecule ....................................................................................... 4 1.2.1.1 Structural domains of gp120.................................................................... 6 1.2.1.2 Functional sites of gp120......................................................................... 9 1.2.1.2.1 CD4 binding site (CD4bs)................................................................. 9 1.2.1.2.2 Coreceptor binding site ................................................................... 11 1.2.2 The gp41 molecule....................................................................................... 11 1.2.2.1 gp41 ectodomain and the fusion process ................................................ 12 1.2.2.2 Cytoplasmic tail of gp41........................................................................ 14 1.3 NEUTRALIZING ANTIBODIES ................................................................................. 16 1.3.1 Neutralizing antibody responses in HIV-1 infected patients.......................... 16 1.3.2 Mechanisms of evasion from neutralizing antibodies.................................... 17 1.3.3 Broadly neutralizing antibodies .................................................................... 18 1.3.4 Neutralizing antibody epitopes ..................................................................... 19 1.3.4.1 CD4bs: b12 epitope ............................................................................... 19 xi 1.3.4.2 Silent face: 2G12 epitope....................................................................... 20 1.3.4.3 MPER: 2F5 and 4E10 epitopes .............................................................. 21 1.3.4.4 V3 loop ................................................................................................. 23 1.3.4.5 Coreceptor binding site and/or CD4 induced epitope (CD4i) ................. 24 1.4 IMMUNOGEN DESIGN FOR INDUCING NEUTRALIZING ANTIBODIES ........................... 25 1.4.1 Native trimeric envelope glycoproteins as immunogens ............................... 25 1.4.2 Exposure of cryptic epitopes (CD4i epitope) ................................................ 27 1.4.3 Epitopes of nMAbs as immunogens ............................................................. 27 1.4.4 Multivalent and centralized immunogens...................................................... 29 1.4.5 Other factors that contribute to vaccine design ............................................. 30 1.5 GENETIC SUBTYPES OF HIV-1 AND NEUTRALIZATION IMMUNOTYPES .................... 31 1.6 OBJECTIVES OF THIS STUDY.................................................................................. 32 CHAPTER TWO INSENSITIVITY OF PAEDIATRIC HIV-1 SUBTYPE C VIRUSES TO BROADLY NEUTRALISING MONOCLONAL ANTIBODIES RAISED AGAINST SUBTYPE B ....... 34 CHAPTER THREE N-LINKED GLYCAN MODIFICATIONS IN GP120 OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 SUBTYPE C RENDER PARTIAL SENSITIVITY TO 2G12 ANTIBODY NEUTRALIZATION .................................................................... 43 CHAPTER FOUR 4E10 RESISTANT VARIANTS IN AN HIV-1 SUBTYPE C INFECTED INDIVIDUAL WITH AN ANTI-MPER NEUTRALIZING ANTIBODY RESPONSE ............................... 52 CHAPTER FIVE NEUTRALIZING ANTIBODY RESPONSES IN ACUTE HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 SUBTYPE C INFECTION .............................. 82 CHAPTER SIX SUMMARIZING DISCUSSION AND CONCLUSIONS .................................................. 93 APPENDIX A Review table of subtype C viruses neutralization by nMAb................... 101 APPENDIX B Ethical Clearence .................................................................................. 102 GENERAL REFERENCES........................................................................................... 103 xii LIST OF TABLES Page Chapter 2 Table 1. Patient information and viral isolate characteristics for HIV-1 subtype C cloned envelope genes????????????? 37 Table 2. Sensitivity of HIV-1 subtype C pseudovirions to anti-HIV MAbs, sCD4, and plasma??????????????????? 37 Table 3. Amino acid sequences of MAbs epitopes in cloned subtype C envelope genes??????????????????.?? 40 Chapter 3 Table 1. Neutralization of wild-type virus and glycosylation mutants by MAbs, CD4-IgG2, and HIV-positive plasmas from subtype C- infected individuals?????????????????.?. 48 Chapter 5 Table 1. Clinical data for 14 acutely HIV-1 subtype C-infected individuals?????????????????????.... 85 Table 2. Detection of CD4i antibodies in two patients prior to and after HIV-1 infection???????????????????? 89 xiii LIST OF FIGURES Page Chapter 1 Figure 1.1. Organization of gp120 in linear and two-dimensional diagrams? 5 Figure 1.2. Crystal structures of gp120 core in unliganded and CD4-bound conformations. ???????????????????.... 6 Figure 1.3. Models of the envelope trimer in the unliganded and liganded states..?????????????????????.?..... 8 Figure 1.4. Electron tomography density maps of the SIV envelope spike..?. 9 Figure 1.5. CD4 and CCR5 binding surface on unliganded and liganded gp120..???????????????????????. 10 Figure 1.6. Schematic representation of the functional domains on gp41??. 12 Figure 1.7. Model of the envelope-mediated fusion process???????. 13 Figure 1.8. CD4 and b12 recognition of gp120. ??????????...? 19 Figure 1.9. Model of the nMAb 2G12 in contact with glycans on gp120??. 20 Figure 1.10. Models of the nMAbs 4E10 and 2F5 Fabs bound to their epitopes. ??????????????????????.. 22 Chapter 2 Figure 1. Neutralisation Dose-Response Curves of the MAbs 2G12, 2F5, IgG1b12, and 4E10, Alone and in Combination?????...?.. 39 Chapter 3 Figure 1. Amino acid sequence alignment of C2-to-C5 region of gp120s of the three HIV-1 subtype C clones used in this study?????... 46 Figure 2. Introduction of an N-glycan attachment site at position 295 in viruses Du151.2, COT9.6, and COT6.15 leads to an increase in molecular mass. ????????????????...??... 46 Figure 3. V295N mutation increases 2G12 antibody binding to subtype C gp120. ??????????????????????... 47 Figure 4. Neutralization of wild-type and V295N mutant subtype C envelope-pseudotyped viruses by 2G12. ????????...?. 47 Figure 5. Impact of V295N and S448N mutations on 2G12 binding to monomeric and oligomeric gp120 from COT6.15?????...... 48 Figure 6. N-glycosylation at position 442 affects 2G12 and IgG1b12 binding to monomeric gp120. ??????????????. 49 Figure 7. Location of the Asn at position 442 relative to the location of the V3 loop region and other N-glycans involved in 2G12 binding?. 50 Chapter 4 Figure 1. Frequency analysis of substitutions in the MPER region of gp41 molecular clones obtained from the TM20 isolate??????.. 75 Figure 2. Neutralization of TM20 envelope clones.???????............ 76 Figure 3. Full length amino acid sequences of the functional envelope clones TM20.5, TM20.6 and TM20.13. ??????????.. 77 xiv Figure 4. Changes in positions 674 and 677 in the MPER affect 4E10 neutralization ????????????????????... 78 Figure 5. Changes in the cytoplasmic tail affect neutralization sensitivity??????????????????????. 79 Figure 6. Role of gp120 and the cytoplasmic tail on infectivity and envelope incorporation. ???????????????.?.. 80 Figure 7. Anti-MPER neutralization activity present in TM20 serum............ 81 Chapter 5 Figure 1. Comparison of autologous neutralization sensitivities of multiple envelope clones from individual patients. ?????????.. 85 Figure 2. Correlation between number of N-linked glycosylation sites, variable loop length, and autologous neutralization titer. ??.?.. 86 Figure 3. Heterologous neutralization of enrollment viruses by sera obtained at 6 and 12 months postinfection. ???????.?? 86 Figure 4. Neutralization of SF162, a neutralization-sensitive subtype B virus????????????????????????. 87 Figure 5. Neutralization sensitivities of early envelope clones from HIV-1 subtype C-infected patients. ??????????????? 87 Figure 6. Correlation between neutralization sensitivity and genetic characteristics of the envelope. ?????????????... 88 Figure 7. CD4i NAb responses in acute HIV-1 subtype C infection???.. 88 Figure 8. MPER NAb responses in acute HIV-1 subtype C infection??? 89 Figure 9. Mapping of anti-MPER neutralizing activity in two patients?.?. 90 Chapter 6 Figure 6.1. Percentage of viruses sensitive to neutralization by the broadly nMAbs IgG1b12, 2G12, 2F5 and 4E10. ?????????.... 96 xv ABREVIATIONS ?g microgram AIDS Acquired Immunodeficiency Syndrome CAPRISA Centre for the AIDS Program of Research in South Africa CCR1, 2b, 3, 5, 8 chemokine (C-C motif) receptor 1, 2b, 3, 5, 8 CD4bs CD4 binding site CD4, CD8 cluster differentiation 4, 8 CD4i CD4 induced CDR complementarity determining region CT cytoplasmic tail CTL cytotoxic T-lymphocyte CXCR4, CXCR6 chemokine (C-X-C motif) receptor 4, 6 DC-SIGN dendritic cell-specific intracellular adhesion molecule-3 grabbing nonintegrin DEAE-dextran diethylaminoethyl-dextran D-MEM Dulbecco?s Modified Eagle?s Media DNA deoxyribonucleic acid EDTA ethylenediamine tetra acetic acid ELISA enzyme linked immunoabsorbent assay ENF enfuvirtide env envelope gene Env envelope glycoprotein FACS fluorescence-activated cell sorting Gag group associated antigen protein GM-CSF Granulocyte Monocyte Colony Stimulating Factor GMT geometrical mean titer gp120, gp41 glycoprotein 120kda, 41kda GPR1, GPR15 G-protein receptor-1, -15 HAART highly active antiretroviral therapy HIV-1, HIV-2 Human Immunodeficiency Virus type-1, -2 HR-1, HR-2 first, second heptad repeat region HTLV-1 Human T-cell Lymphotropic Virus i.e. id est, that is IAVI International AIDS Vaccine Initiative IC50 50% inhibitory concentration ID50 50% inhibitory dilution IgG immunoglobulin LLP-1, LLP-2, LLP-3 lentivirus lytic peptide -1, -2, -3 LTR long terminal repeat MAb monoclonal antibody mg miligram MPER membrane proximal external region xvi MTCT Mother-to-child transmission MuLV Murine Leukaemia Virus NAb neutralizing antibodies nMAbs neutralizing monoclonal antibodies p24 gag protein 24kda PBMCs peripherals blood mononuclear cells PBS phosphate buffered saline PCR polymerase chain reaction PNGS potential N-linked glycosylation site RNA ribonucleic acid RT room temperature RT-PCR reverse transcriptase polymerase chain reaction SAAVI South African AIDS Vaccine Initiative sCD4 soluble CD4 SIV Simian Immunodeficiency Virus T-20 enfurvitide TBS tris buffered saline TCID50 50% tissue culture infectious doses TCLA T-cell line adapted UNAIDS United Nations programme on HIV/AIDS V1, V2, V3, V4, V5 variable regions 1-5 VLPs virus like particles AMINO ACID ABREVIATIONS Amino Acid Three- Letter code One-Letter code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamic acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V CHAPTER 1: INTRODUCTION 1 CHAPTER ONE INTRODUCTION CHAPTER 1: INTRODUCTION 2 1.1 BACKGROUND Human Immunodeficiency Virus type 1 (HIV-1) has infected more than 65 million people worldwide since 1983 when it was first described as the cause of the Acquired Immune Deficiency Syndrome (AIDS). In 2007, more than 33 million people were estimated to be living with HIV-1, 2.5 million became newly infected and around 2.1 million lost their lives to AIDS (UNAIDS 2007, www.unaids.org). Although antiretroviral therapies are highly effective in treating HIV infections, only a vaccine constitutes a practical and cost- effective intervention to control the spread of the HIV/AIDS pandemic. A major challenge for the development of an HIV vaccine has been to determine which immune responses should be elicited for protection. Currently licensed anti-viral vaccines are mostly effective against acute viral infections, conferring protection mainly through neutralizing antibodies. However, cell mediated immune responses are crucial in the control of established chronic virus infections such as cytomegalovirus (CMV), Epstein- Barr virus (EBV) and Herpes simplex virus (HSV) (Pantaleo and Koup, 2004) for which it has been more difficult to develop effective vaccines. In the case of HIV, the correlates of immune protection have not been clearly identified. Early studies have showed that immunization with the outer envelope protein (gp120) of HIV-1 induced antibodies that inhibited viral entry. However, subsequent clinical studies demonstrated that these antibodies did not protect against HIV-1 infection (Gilbert et al., 2005). This was later explained by their inability to neutralize primary HIV-1 isolates, despite having activity against neutralization sensitive T-cell line adapted strains (TCLA) that were used in the initial studies (Mascola and McNeil, 1995). These observations led many to question the relevance of this vaccine approach (Mammano et al., 1995, Moore et al., 1995, Schonning et al., 1998). Soon after, a new generation of vaccine candidates emerged which aimed to prime cellular immunity, specifically CD8+ cytotoxic T-lymphocytes (CTLs). Indeed, CHAPTER 1: INTRODUCTION 3 virus-specific CTLs are thought to be responsible for controlling viral replication in the acute phase of HIV infection (Koup et al., 1994). Furthermore, studies on simian immunodeficiency virus (SIV)-infected monkeys have shown that immunogens capable of inducing CTL responses can reduce viral set point and slow disease progression (Letvin, 2005). These observations suggested that vaccines capable of eliciting strong T-cell- mediated immune responses may be beneficial even if they do not induce sterilizing immunity. However, this type of response does not clear the virus reservoirs and resistance variants can emerge later in infection (Barouch et al., 2003). The failure of a recent ?test- of-concept? clinical trial of Merck?s candidate HIV-vaccine, which aimed to stimulate CTL responses, has further questioned this vaccine approach (http://www3. niaid.nih.gov/news/newsreleases/2007/step_statement.htm). There is renewed interest in neutralizing antibodies, prompted mainly by several observations that passive transfer of neutralizing antibodies are able to confer sterilizing immunity in animal studies (Mascola, 2002). In addition, it has been shown that although effector T cells can limit viral replication, they are not able to assist humoral immunity to prevent the establishment of initial infection (Mascola et al., 2003). As such, an enormous effort is currently being invested in the ?intelligent? design of immunogens capable of inducing broadly cross- reactive neutralizing antibodies against HIV-1 primary isolates. Such an endeavor requires not only an in-depth understanding of the viral envelope glycoprotein structure and function, but also the role of neutralizing antibodies in natural HIV-1 infection. 1.2 HIV ENVELOPE GLYCOPROTEIN The main targets of the anti-HIV neutralizing antibodies are the glycoprotein spikes on the virus envelope membrane. This glycoprotein complex interacts with the CD4 and coreceptor molecules present on the surface of target cell initiating the viral entry process. The functional envelope spike consists of a trimer of heterodimers formed by two CHAPTER 1: INTRODUCTION 4 glycoproteins, gp120 (the exterior envelope glycoprotein) and gp41 (the transmembrane glycoprotein). Three gp120 molecules interact non-covalently with three gp41 units forming an oligomer, where the trimeric structure is maintained by the interactions between the gp41 domains. In infected cells, the envelope glycoprotein is synthesized as a single polypeptide of approximately 845 to 870 amino acids in the rough endoplasmic reticulum (Allan et al., 1985). The extensive addition of N-linked high-mannose sugar chains forms the gp160 glycoproteins, which assemble into trimers (Earl et al., 1990). These oligomers are then transported to the Golgi apparatus, where accessible glycans are trimmed and modified to complex-oligosacharides. In the trans-Golgi, cellular proteases cleave the gp160 molecule into gp120 and gp41 (Decroly et al., 1997, Hallenberger et al., 1997). The mature spikes are then transported to the cell membrane, in particular to the detergent-insoluble membrane domains, known as lipid rafts (Rousso et al., 2000), where the virus assembly take place and the envelope spikes are incorporated into the budding virions. 1.2.1 The gp120 molecule The amino acid sequence of gp120 consists of five relatively conserved regions (C1-C5) interposed with five variable regions (V1-V5) which, with the exception of V5, are bracketed by cysteines forming disulfide bonds (Leonard et al., 1990) (Figure 1.1). Gp120 is a highly glycosylated protein with half of its mass being N-linked glycans (Lasky et al., 1986), far more glycosylated than the surface proteins from other retroviruses of similar size such as HTLV-1 and MuLV (Polonoff et al., 1982). Two types of N-linked glycosylations are found on the surface of gp120, mannose-rich and complex glycans. Structural modelling suggests that the high mannose glycans are clustered on one side of the surface while the complex glycans are localized within a distinct region of the gp120 (Zhu et al., 2000). CHAPTER 1: INTRODUCTION 5 Figure 1.1 Organization of gp120 in linear and two-dimensional diagrams. (a) Schematic representation of the envelope glycoprotein precursor. Digestion by cellular proteases, at the indicated cleavage sites (vertical arrows), releases the signal peptide, the gp120 and the gp41 molecules. The locations of the variable loops (V1-V5) in gp120 are indicated in red. (b) Gp120 two-dimensional diagram adapted from McCaffrey, et al., 2004. The small numbers indicate the amino acid positions in the HxB2 sequence. Arrows indicate the positions involved in the co-receptor binding site and asterisks indicate the residues involved in CD4 binding. In both diagrams the N-linked glycosylation sites are represented by the oligosaccharides structures expected in those positions: U-shaped branches for the complex glycans and tri-branched for the mannose rich glycans. a) b) CHAPTER 1: INTRODUCTION 6 1.2.1.1 Structural domains of gp120 Full-length gp120 has eluded structural analysis due to its lack of stability. To obtain crystal structures, HIV-1 and SIV gp120s have been deglycosylated and the N- and C- terminals, V1/V2 and V3 regions truncated, to generate what is commonly referred to as the ?gp120 core?. The first gp120 structure was obtained using the gp120 core of an HIV-1 virus stabilized with the D1D2 fragment of CD4 and a CD4 induced epitope-binding antibody (17b) (Kwong et al., 1998). Based on this structure, gp120 is organized into three regions: the inner domain, the outer domain and the bridging sheet (Figure 1.2b). The inner domain is formed mainly by the C1 and C5 regions and is largely devoid of glycans. It has long been suggested that this domain constitutes the major contact interface with the gp41 trans-membrane unit (Helseth et al., 1991, Moore et al., 1994). The outer domain is heavily glycosylated and modelling of the envelope oligomer suggests that these glycans cover the solvent-exposed part of the spike, protecting it from antibody recognition. In between the outer and inner domains is the bridging sheet region, formed by four anti- parallel ?-sheets: ?2 and ?3, which constitute the stem of the deleted V1/V2 loop; and the ?20 and ?21 of the C4 region. Figure 1.2: Crystal structures of gp120 core in unliganded and CD4-bound conformations. Ribbon diagram of (a) the unliganded SIV gp120 and (b) the HIV-1 liganded gp120 structures are depicted from the CD4 perspective. The structural domains are colored; green for the inner domain, blue for the outer domain and yellow for the bridging sheet. Variable loops stems and the ?-sheets that form the bridging sheet are also indicated. Figures were created in PyMol from the PDB data files 2BC4 (Chen et a., 2005) and 1GC1 (Kwong et al., 1998). a) b) CHAPTER 1: INTRODUCTION 7 More recently the gp120 of an SIV virus was resolved in the absence of CD4. This unliganded gp120 structure is presumed to represent the native state conformation (Chen et al., 2005). In contrast to the outer domain, which has a similar conformation as the liganded form, the inner domain and the bridging sheet revealed a very altered structure, suggesting that binding of CD4 induces radical conformational changes in these regions. The bridging sheet observed in the CD4 bound conformation appears segregated in the native structure (Figure 1.2a), with the ?2 and ?3 sheets, laying proximal to the inner domain, displaced 20-25 ? from the ?20 and ?21 loop. A third gp120 structure was resolved using an HIV-1 gp120 core that included the V3 loop, complexed to CD4 and the Fab X5 (Huang et al., 2005). In this structure the core resembles the liganded conformation. The V3 loop protrudes as an elongated structure from two anti-parallel ?-sheets in the outer domain. A disulphide bond between ?12 and ?13 stabilizes its base, while a long flexible stem extends away from the core ending in a conserved ?-turn tip. The trimeric model of this structure predicts the projection of the tip 30 ? towards the cell membrane, consistent with its positioning towards the coreceptor molecule (Huang et al., 2005) (Figure 1.3). However, it is not clear if V3 displays an extended structure in the context of the native oligomer. It has been suggested that the V3 is partially occluded by the V1/V2 loop of the adjacent protomer in the trimeric envelope glycoprotein, and it is only extended after CD4 interaction (Wyatt et al., 1998). The widely assumed trimeric structure of the envelope complex has recently been visualized using cryo-electron tomography microscopy by two separate groups (Zanetti et al., 2006, Zhu et al., 2006). Both studies have demonstrated similar dimensions and threefold symmetry for the envelope spike, but the actual map images displayed some distinct features (Figure 1.4). The Zhu et al. structure presents multiple lobes emanating from the core and tripod-like configuration of gp41, while the Zanetti et al. reconstruction CHAPTER 1: INTRODUCTION 8 t d y d j t j y F i g u r e 1 . 3 : M o d e l s o f t h e 1. 3: M od e l s o f t h e e nv el o p e tr i m e r in th e u n lig an de d an d li g an de d st at es . T h e st ru c t ur es a re d ep ic te d w i th th e v i ra l m em b r an e po si t io ne d at t h e to p of th e fi g ur e ab o v e th e sc h e m at i c r e pr e s en ta t io n o f g p4 1. (a ) T he u nl ig a n de d t ri m e r w as m od el ed f r om t h e SI V g p1 20 c or e at o m ic s t r u ct u r e b y C h e n, e t a l., 2 00 5. Ea ch o f t h e g p1 20 m o n om er s i s i llu str a t ed in a d iff e r e n t c ol o r , w i th N - li nk ed g ly c a ns re pr es en t e d by s t i c k m o d el s . (b ) T he tr i m e r ic m o d el o f t he C D 4- bo un d en v e l o pe g ly co pr ot e i n w as o bt ai n e d b y H ua ng e t a l. , 2 00 5 us in g th e V 3 - co nt an in g H I V -1 g p 1 20 c o r e st r uc tu re . T h e C D 4 re ce p t or i s de p i ct e d in y e l lo w . T he V 3 lo o p i s sh ow n in r e d, e xt en d i ng to w ar d s th e co -r e ce pt or m o l e c ul e on t h e ho s t m e m br a n e. CHAPTER 1: INTRODUCTION 9 constitutes a smooth structure with only three clear lobes and a single stem linking with the membrane (Roux and Taylor, 2007). These differences have been attributed to the techniques used for data collection and analysis (Subramaniam, 2006). Despite these limitations, this rapid evolving methodology promises to assist in solving the native structure of the envelope spike in more physiologically relevant environments, i.e. in the context of a viral or cell membrane. Figure 1.4: Electron tomography density maps of the SIV envelope spike. Side (a, c) and top (b, d) views of the 3D structure of SIV envelope glycoprotein as reported by Zhu et al., 2006 (a, b) and Zanetti et al., 2006 (c, d). Figure adapted from Roux and Taylor, 2007. 1.2.1.2 Functional sites of gp120 1.2.1.2.1 CD4 binding site (CD4bs) The CD4bs of gp120 constitutes a conformational region suggested to be only apparent in the context of the liganded structure of gp120. It is characterized by a hydrophobic pocket at the interface of the inner domain, the bridging sheet and outer domain, where the side CHAPTER 1: INTRODUCTION 10 chain of the Phe43 of CD4 is buried, otherwise known as the ?Phe43 cavity? (Figure 1.5). In the unliganded structure many of the residues involved in the interaction with CD4 are distributed around the interface of the inner and outer domain which is lined with hydrophobic residues (Chen et al., 2005). It has been proposed that the CD4 molecule first interacts with the internal face of the conformationally stable outer domains. However, this interaction is not energetically favorable, incurring a substantial drop in entropy (Kwong et al., 2002), and is only stable at the cell surface where the presence of multiple CD4 molecules, binding the trimer simultaneously, increases the avidity of this interaction (Zhou et al., 2007). The binding of CD4 induces large conformational changes in the inner domain, which leads to the formation of the bridging sheet and coreceptor binding site. Figure 1.5: CD4 and CCR5 binding surface on unliganded and liganded gp120. Molecular surface diagrams of the unliganded SIV and liganded HIV-1 gp120 core structures are shown from similar orientations. The residues interacting with the CD4 receptor, as documented by Zhou et al., 2007, are shown in red surrounding the Phe43 cavity. The amino acids involved in CCR5 binding, as determined by Rizzuto et al., 1998, are depicted in green in the vicinity of the V3 loop stem, shown in yellow. CHAPTER 1: INTRODUCTION 11 1.2.1.2.2 Coreceptor binding site In addition to CD4 receptors, HIV requires the presence of coreceptor molecules on the surface of the target cells. Most primary isolates use the ?-chemokine receptor CCR5 as an entry coreceptor, although some viruses undergo a coreceptor switch mainly to CXCR4 usage. Other minor coreceptors such as CCR1, CCR2b, CCR3, CCR8, CXCR6 (Bonzo/STRL33), Bob/GPR15 and GPR1, have also been shown to mediate virus entry in vitro, although their use in vivo is less certain (Moore et al., 2004). The V3 loop has been mapped as the major determinant of coreceptor switching, which demonstrates its involvement in the coreceptor-binding site. Other conserved structures of gp120 also form part of this functional region, such as the bridging sheet and the stem of the V3 loop (Rizzuto and Sodroski, 2000, Rizzuto et al., 1998). These residues are segregated in the unliganded gp120 structure and only converge after CD4 binding, to form a conserved pocket that harbors the sulfotyrosines on the N terminus of CCR5 (Figure 1.5). This interaction zips the flexible V3 stem into a rigid ?-hairpin (Huang et al., 2007). However, it is not clear if these changes occur before or after the tip of the V3 interacts with the second extracellular loop of CCR5 (Huang et al., 2007). 1.2.2 The gp41 molecule The gp41 molecule is a transmembrane glycoprotein, that is less variable and less glycosylated than gp120. It interacts non-covalently with gp120 and is responsible for maintaining the trimeric structure of the envelope glycoprotein, although its structure in the native conformation is unknown. Gp41 constitutes a class I fusion glycoprotein structurally homologous to the fusion proteins of other enveloped viruses such as orthomyxo-, paramyxo-, retro-, filo- and coronaviruses (Dimitrov, 2004). The linear amino acid sequence of gp41 can be divided CHAPTER 1: INTRODUCTION 12 into an ectodomain, a very conserved membrane spanning domain and a particularly long cytoplasmic tail (Figure 1.6). 1.2.2.1 gp41 ectodomain and the fusion process Several important features are present in the ectodomain of gp41 (Figure 1.6). The N- terminus hydrophobic glycine-rich peptide is essential for membrane fusion activity. This fusion peptide is linked, through a flexible polar segment, to a coiled-coil forming amphipathic ?-helix (Heptad repeat-1, HR1 or N-helix). A centrally located disulphide- bonded loop connects the HR1 to a second amphipathic ?-helix (HR2 or C-helix), which is followed by the membrane proximal external region (MPER). Figure 1.6: Schematic representation of the functional domains of gp41. (a) Functional motifs are indicated in a linear diagram of gp41. The membrane-spanning domain separates the ectodomain from the cytoplasmic tail. The disulphide loop and the glycans in the ectodomain are represented as S-S and Y, respectively. (b) Schematic diagram and (c) side and (d) top view of the atomic model of the six-helix bundle formed by the heptad repeats of gp41 shown in red and yellow (PDB code 1QBZ, Yang, et al.,1999). In free virions most of the gp41 ectodomain is buried by gp120, protecting this conserved fusion machinery from recognition by the immune system (Figure 1.7a). Only the MPER is CHAPTER 1: INTRODUCTION 13 thought to be exposed, as neutralizing antibodies against this region (2F5 and 4E10) are able to bind the native structure before receptor engagement (de Rosny et al., 2004, Zwick et al., 2001). The interface of gp41 with gp120 is not clearly discerned. However, cysteine scanning of the disulphide-bonded region of gp41, and the C1 and C5 regions in the inner domain of gp120 has identified residues that are able to covalently link these subunits (Binley et al., 2000a). Although the gp41 native structure in the prefusogenic state is unknown, experimental evidence suggests that this conformation may be stabilized through interactions with the inner domain ?-sandwich (Yang et al., 2003), which prevents the large interactive surfaces of HR1 and HR2 from collapsing into the highly stable six-helix bundle conformation (Weissenhorn et al., 1997). It has been suggested that in this prefusogenic state, the fusion peptide is in close proximity to the MPER (Lorizate et al., 2006) and the HR1 region does not form a coiled-coil structure (Mische et al., 2005). Figure 1.7: Model of the envelope-mediated fusion process. (a) Cartoon showing the envelope trimer before CD4 and co-receptor binding. The gp41 regions are depicted in the same color scheme as in Figure 1.6. The MPER region (green) may be exposed in the native conformation of the envelope. (b) After CD4 and coreceptor engagement, gp41 assumes an extended three-helix rod conformation inserting the fusion peptide (blue) into the host cell membrane. (c) The formation of the metastable six-helix bundle mediates the fusion of the virus membrane into the cell membrane. CHAPTER 1: INTRODUCTION 14 Upon CD4 and coreceptor binding the inner domain rearranges, destabilizing the structure of gp41 and inducing the pre-hairpin intermediate state. In this structure, the three gp41 molecules are associated in a trimeric coiled-coil in parallel orientation (Furuta et al., 1998, Jiang et al., 1993, Munoz-Barroso et al., 1998, Wild et al., 1994). The fusion peptide is released and inserted into the target cell membrane (Figure 1.7b). Although the existence of this structure is not clear, it has been extrapolated from the mechanism of the influenza virus hemagglutinin (HA) (Carr and Kim, 1993) and the ability of HR1 binding peptides, such as T-20 (Enfurvitide, DP-178), to trap this conformation and inhibit the fusion process (Furuta et al., 1998). The fusion-intermediate is brief, with the three HR2 helices rapidly folding into the hydrophobic grove on the surface of the HR1 coiled-coil trimer in an anti-parallel manner, creating a six-helical bundle structure that facilitates the fusion process by bringing the viral and cell membranes together (Figure 1.7c). This conformation is extremely stable, and it is believed that the free energy released during this step contributes substantially to overcome the energy barrier of the membrane fusion process (Chan and Kim, 1998). This post-fusion configuration of the ectodomain of gp41 has been structurally characterized (Weissenhorn et al., 1997), however it is not clear how the transition from the three-helix rod takes place (Chan et al., 1997). It has been suggested that the formation of the six- helical bundle is only completed once the fusion pore has formed (Markosyan et al., 2003). 1.2.2.2 Cytoplasmic tail of gp41 The membrane spanning domain (MSD) precedes an approximately 150 amino acids long segment in the intracellular compartment, known as the cytoplasmic tail (CT). The CT of HIV is, as for most lentiviruses, particularly long compared to other members of the Retroviridae family (Hunter and Swanstrom, 1990, Kalia et al., 2003). Multiple studies have demonstrated the critical role of the CT in virus assembly and CHAPTER 1: INTRODUCTION 15 envelope incorporation. Mutations or deletions in the matrix protein (p17) affect envelope incorporation, which can be reversed by compensatory mutations in the CT of gp41, demonstrating the interaction between these two proteins (Freed and Martin, 1996). Several lines of evidence suggest that Gag processing and the fusion machinery of the envelope glycoprotein are connected via the CT. The p55 precursor in the immature virion interacts with the CT of gp41 inhibiting the fusion process, as shown in protease defective viruses, while deletion of the CT disassociates these events (Murakami et al., 2004). This mechanism may avoid early fusion of immature particles that are not able to establish infection. The CT of gp41 contains a number of highly conserved functional motifs (Figure 1.6). The membrane proximal Tyr-based endocytosis (Yxx?) motif and the dileucine motif at the C- terminus mediate clathrin-dependent endocytosis, alter intracellular localization, and regulate envelope expression and incorporation in the virion (Byland et al., 2007, Day et al., 2004, LaBranche et al., 1995, West et al., 2002, Wyss et al., 2001, Ye et al., 2004). One or more palmitoylated cysteines (C764 and C837) are implicated in targeting envelope glycoprotein to lipid rafts and mutation of these cysteines to alanine decrease envelope incorporation and infectivity (Bhattacharya et al., 2004, Rousso et al., 2000). Three highly conserved amphipathic ?-helices, known as ?lentivirus lytic peptide? domains (LLP-1, LLP-2, and LLP-3) are implicated in interacting with the plasma membrane, decreasing bilayer stability, affecting envelope cell surface expression and incorporation into virus particles (Kalia et al., 2003, Piller et al., 2000, Wyss et al., 2005). They have also been described as calmodulin-binding domains that promote Fas-mediated apoptosis of infected cells (Micoli et al., 2006, Srinivas et al., 1993). CHAPTER 1: INTRODUCTION 16 1.3 NEUTRALIZING ANTIBODIES 1.3.1 Neutralizing antibody responses in HIV-1 infected patients In HIV-1 infection, antibodies capable of blocking virus infection in vitro develop in almost all individuals, although whether they are able to perform this function in vivo is less clear. Their absence during the acute phase of infection, when viral levels are brought under control, suggests that cellular immune responses may be more critical during this period (Moog et al., 1997, Koup et al., 1994). The earliest neutralizing antibodies can be detected after 3-12 months of infection, however, there is considerable variation in the kinetic, magnitude and breadth of this response (Kelly et al., 2005, Moog et al., 1997, Pellegrin et al., 1996, Richman et al., 2003, Wei et al., 2003). In general, the initial neutralization response is narrow, only effective against early autologous viruses (Li et al., 2006a, Moog et al., 1997, Richman et al., 2003) and some T-cell line adapted strains (Pilgrim et al., 1997). Nevertheless, the appearance of neutralization escape variants soon after the autologous response has developed, supports the notion that these antibodies exert immunological pressure on the virus (Wei et al., 2003, Richman et al., 2003). Antibodies capable of neutralizing heterologous viruses develop later in infection, with only a small percentage of chronically infected patients having broadly cross-reactive antibodies against multiple HIV-1 viruses (Braibant et al., 2006, Donners et al., 2002, Pilgrim et al., 1997). The nature of the antibodies in broadly cross-reactive sera, as well as why breadth develops so rarely, is not well understood. It is clear, however, that a threshold of viremia is necessary to induce neutralizing antibodies, demonstrated by their absence in individuals on highly active antiretroviral therapy (HAART) and in ?elite controllers? (Bailey et al., 2006, Binley et al., 2000b, Montefiori et al., 2001). In chronic infection, high viremia has been correlated with neutralization breadth (Deeks et al., 2006). On the other hand, rapidly progressing individuals, who lack control over viremia, usually display low neutralizing CHAPTER 1: INTRODUCTION 17 antibody titers, but this may be attributed to a general immune suppression (Cecilia et al., 1999, Pilgrim et al., 1997). A recent study has suggested that neutralizing antibodies might protect from HIV-1 superinfection, as this is more likely to occur during the early phase of infection when these antibodies are absent (Smith et al., 2006). Other studies have shown that maternal neutralizing antibodies can exert powerful protective and selective effects during perinatal HIV-1 transmission with resistant strains establishing infection in infants (Dickover et al., 2006, Wu et al., 2006). 1.3.2 Mechanisms of evasion from neutralizing antibodies HIV-1 has developed multiple escape mechanisms to avoid neutralization. The shedding of gp120 monomers diverts the immune system towards structures otherwise not found on the native trimer (Wyatt and Sodroski, 1998). The envelope spike is heavily glycosylated, with the poorly immunogenic glycans shielding antibody access to the peptidic structure (Johnson and Desrosiers, 2002). Furthermore, changes in glycan packing yield viruses resistant to the autologous neutralizing antibody response. This neutralization escape mechanism is referred as an ?evolving glycan shield? (Wei et al., 2003). The trimeric nature of the envelope glycoprotein shields conserved regions, while exposing relative amorphous highly glycosylated loop structures. These regions tolerate high levels of variation and therefore can easily escape from neutralizing antibodies (Wyatt et al., 1998). Multiple studies have suggested that the V1/V2 loops cover conserved epitopes involved in the coreceptor binding site of the neighboring protomer (Kwong et al., 2000), as deletion of these variable loops confers sensitivity to antibodies targeting this region (Sullivan et al., 1998, Wyatt et al., 1995). The coreceptor binding site is only transiently exposed after receptor engagement and thus out of antibody reach (Labrijn et al., 2003, Wu et al., 1996). The CD4bs on the other hand is exposed for functional reasons, however, a CHAPTER 1: INTRODUCTION 18 distinct type of camouflage, called ?entropic masking?, protects it. Binding to this epitope requires the fixation of the otherwise flexible gp120, imposing an entropic barrier for the high affinity antibody binding required for neutralization (Kwong et al., 2002). 1.3.3 Broadly neutralizing antibodies Despite all these defense mechanisms, a few rare broadly neutralizing monoclonal antibodies (nMAb) have been isolated from HIV-1 subtype B infected individuals. These MAb neutralize many primary isolates from different genetic subtypes, indicating some conserved structures on the envelope glycoproteins. Their epitopes include regions in gp41 (2F5 and 4E10), the CD4bs (b12), and part of the carbohydrate-masked ?silent face? of gp120 (2G12). Crystallographic analysis of these antibodies have revealed that they underwent remarkable structural adaptations to attain virus recognition (Burton et al., 2005). Passive immunization of primates challenged with chimeric simian?human immunodeficiency virus (SHIV) strains has shown that human nMAbs can protect against infection and are effective against intravenous (Baba et al., 2000, Mascola et al., 1999), oral (Hofmann-Lehmann et al., 2001) or intravaginal challenges (Mascola et al., 2000, Parren et al., 2001, Veazey et al., 2003). In cases where transmission occurs they can ameliorate disease by blunting the peak of viremia and lowering the viral set point (Ferrantelli et al., 2007). A recent study in humans showed that in some HIV-infected individuals these nMAbs can reduce the rate of viral rebound following a structured treatment interruption (Trkola et al., 2005). Furthermore, the existence of 2G12 escape variants in some of the treated patients demonstrated that this nMAb was indeed functional in vivo (Manrique et al., 2007, Nakowitsch et al., 2005). CHAPTER 1: INTRODUCTION 19 1.3.4 Neutralizing antibody epitopes 1.3.4.1 CD4bs: b12 epitope The neutralizing antibody b12 was obtained as a Fab through a phage display library strategy (Burton et al., 1991). The Fab b12 as well as the IgG1 recombinant MAb derived from it, IgG1b12, occlude the CD4bs on gp120 and prevents CD4 attachment (Burton et al., 1994, Roben et al., 1994). A key element of the CD4bs is a recess that forms a contact site for the Phe43 protruding from a loop of CD4 (Wyatt and Sodroski, 1998). The first crystal structure of IgG1b12 revealed that the protruding heavy chain complementarity determining region 3 (CDRH3) was unusually long, allowing it to access the CD4 binding pocket (Saphire et al., 2001). However, it was only the recent crystallized structure of the Fab b12 in complex with gp120 that clarified the mechanism behind this antibody neutralization. The b12 interactions with gp120 are mainly with residues in the structurally invariant outer domain (Figure 1.8). As a result, the b12 binding site, in contrast to the CD4bs, does not differ considerably from the pre- to post-attachment forms of gp120 (Zhou et al., 2007), explaining the previously shown low entropic cost of this interaction (Kwong et al., 2002). Figure 1.8: CD4 and b12 recognition of gp120. Ribbon diagram of gp120 bound to CD4 (yellow) and b12 (green-light blue) as obtained by Zhou, et al., 2007. The gp120 outer domain (red), inner domain (grey) and bridging sheet (dark blue) are indicated in both structures. CHAPTER 1: INTRODUCTION 20 1.3.4.2 Silent face: 2G12 epitope 2G12 recognizes a unique epitope on the surface of gp120 that is not directly associated with the receptor binding sites (Sanders et al., 2002a). Antibody mapping studies using monomeric gp120 showed that 2G12 forms a unique competition group in that no other MAb is able to prevent its binding to gp120 and vice versa (Moore and Sodroski, 1996). 2G12 binds to high mannose and/or hybrid glycans, with mannose residues as essential components. Mutagenesis studies have implicated the glycans at positions 295, 332 and 392 in gp120 as being the most critical for 2G12 binding (Sanders et al., 2002a, Scanlan et al., 2002). The X-ray crystallography of this MAb in complex with mannose oligosaccharides, unveiled a unique antibody structure in which the two heavy chain variable domains (VH) interlock creating a swapped dimer arrangement (Figure 1.9). This creates an extended paratope that allows the recognition of a large oligomannose moiety. Figure 1.9: Model of the nMAb 2G12 in contact with glycans on gp120. The heavy chains (VH) of 2G12 (yellow and purple) interlock with the light chains (VL) (cyan), forming a unique domain-swapped antibody structure. The extended 2G12 idiotope has been modeled to interact with Man9GlcNAc2 groups (red), attached to the N332 and N392 residues of gp120 (grey) with its primary combining site. The glycan at N339 interacts with a secondary combining site formed by the VH/VH? interface. Figure was adapted from Calarese, et al., 2003. 2G12 MAb CHAPTER 1: INTRODUCTION 21 The authors concluded that 2G12 binds to the N-linked glycans at position 332 and 392 in the primary combining sites, with a potential interaction with glycn 339 at the VH/VH? interface of the antibody (Calarese et al., 2003). They also proposed that the glycan at position 295 plays an indirect role by preventing further processing of the glycan at 332 and maintaining its oligomannose structure as the one recognized by 2G12. 1.3.4.3 MPER: 2F5 and 4E10 epitopes 2F5 and 4E10 recognize two adjacent highly conserved epitopes in the extreme C-terminal of the gp41 ectodomain (Figure 1.10). This region is particularly attractive for vaccine design because it mediates the viral entry process and is highly conserved between viral strains. The 2F5 epitope has been mapped to the motif ELDKWA at the end of the HR2 region of gp41 (Muster et al., 1993), where the core residues D664, K665 and W666 are indispensable for antibody recognition (Zwick et al., 2005). Structural data of 2F5 MAb- epitope complexes shows that this region adopts an extended conformation with a type I ?- turn at the core of the epitope. Interestingly, the hydrophobic apex of the CDR H3 loop of 2F5 does not interact with the epitope directly (Ofek et al., 2004). It has been suggested that this region mediates interactions with the epitope-proximal viral membrane, explaining early evidence that 2F5 binding was enhanced in the presence of lipids (Grundner et al., 2002). The nMAb 4E10 recognizes a contiguous epitope at the C-terminus of the 2F5 binding region (Stiegler et al., 2001, Zwick et al., 2001). Mutagenesis experiments have demonstrated that the residues W672, F673 and W680 of the Trp-rich region of gp41 are indispensable for recognition by 4E10 (Zwick et al., 2005). The crystal structure of 4E10 bound to a 13-residue peptide revealed that this epitope assumes an unusual helical conformation. The hydrophobic face of this amphipathic helix is buried in the antibody CHAPTER 1: INTRODUCTION 22 combining site, where amino acids W672, F673, I675 and T676 are the key residues in this interaction (Cardoso et al., 2005). Further structural analysis of this epitope has extended it to the motif 672-WFx(I/L)(T/S)xx(L/I)W-680, where x does not play a major role in 4E10 binding (Cardoso et al., 2007). 4E10 2F5 Figure 1.10: Models of the nMAbs 4E10 and 2F5 Fabs bound to their epitopes. The structure of the 4E10 and 2F5 Fabs bound to their peptide epitopes, obtained by Cardoso, et al., 2005 and Ofek, et al., 2004, respectively, are shown in the context of the envelope spike (a) obtained by Zhu, et al., 2006, and (b) in the vicinity of the viral membrane. Figures were obtained from the IAVI report, 2006 (www.iavireport.org) and Burton, et al., 2005 a) b) CHAPTER 1: INTRODUCTION 23 1.3.4.4 V3 loop In addition to those described above, there are other epitopes able to induce neutralizing antibodies, but in a more limited way. This is the case for the V3 loop of gp120, which was previously considered the principal neutralizing determinant (Palker et al., 1988, Rusche et al., 1988). Later research demonstrated that this was only applicable to TCLA strains, where numerous passages in cell culture rendered these viruses highly sensitive to neutralization by anti-gp120 monoclonal antibodies, patients? sera, and soluble forms of CD4 (Wrin et al., 1995, Verrier et al., 2001, Follis et al., 1998). The mechanism by which sensitivity to neutralizing ligands is acquired is not clear and is manifested only in the context of the functional trimeric envelope spike. Monomeric gp120s derived from either a TCLA strain or a primary isolate exhibit similar affinities for sCD4 (Moore et al., 1991). By contrast the trimeric envelope glycoprotein of TCLA viruses bind the CD4 molecule more efficiently than the primary isolate (Moore et al., 1992, Kabat et al., 1994, Platt et al., 1997). The exposure of epitopes on TCLA viruses may reflect an optimization of the virus- cell interactions, particularly the CD4-gp120, in the absence of selective pressure provided by serum-neutralizing antibodies (Moore et al., 1995). The V3 loop is less important for primary isolate neutralization, presumably because this region is occluded in the trimeric structure prior to receptor binding. Furthermore, due to the variable nature of this region, most anti-V3 antibodies are isolate-specific. However, a group of these antibodies recognizes conformation-sensitive epitopes on V3 and they are able to neutralize a range of primary isolates. This is the case for the MAb 447-52D, which recognizes the GPGR motif at the tip of the V3 and main-chain atoms along the N-terminal side of the loop (Gorny et al., 1992, Huang et al., 2005, Stanfield et al., 2004). 447-52D neutralizes laboratory strains (Gorny et al., 1992, Gorny et al., 1993) and clinical isolates from various clades (Conley et al., 1994, Nyambi et al., 1998). Although, its activity is CHAPTER 1: INTRODUCTION 24 limited to viruses containing the GPGR sequence at the apex of V3 loop, the relatively broad neutralizing activity of 447-52D highlights the existence of conserved structures in the V3 loop and makes this region a potential vaccine target. 1.3.4.5 Coreceptor binding site and/or CD4 induced epitope (CD4i) The binding of CD4 to gp120 induces conformational changes that lead to the formation of the coreceptor binding site and enhanced binding of a group of antibodies, referred to as CD4i antibodies, such as: 17b, 21c, 23e, 48d, 49e (Xiang et al., 2002), X5 (Moulard et al., 2002), E51 (Xiang et al., 2003) and 412d (Xiang et al., 2005). Crystal structure and mutagenesis data have shown that the epitope recognized by these antibodies overlaps significantly with the highly conserved coreceptor binding site (Huang et al., 2005, Kwong et al., 1998, Xiang et al., 2002, Xiang et al., 2003). In many cases these antibodies mimic the coreceptor molecule by presenting sulfated tyrosine in their CDRH3 (Huang et al., 2007, Huang et al., 2004). CD4i antibodies are commonly found in HIV-infected individuals (Decker et al., 2005) suggesting that this epitope is highly immunogenic. Despite the extremely broad recognition of CD4i antibodies, neutralization is usually impaired. Several studies have shown that virus strains that do not require CD4 for entry are highly sensitive to neutralization by CD4i antibodies (Kolchinsky et al., 2001, Edwards et al., 2001). Moreover, CD4 dependence assures that this immunogenic and conserved region is only exposed after receptor engagement, where the close proximity of the viral and host membranes somewhat restricts the access to this region. This is supported by the fact that small forms of these antibodies, such as Fabs or single chains, display better neutralizing activity (Labrijn et al., 2003). Taken together, these observations preclude this epitope as a good target for vaccine design. CHAPTER 1: INTRODUCTION 25 1.4 IMMUNOGEN DESIGN FOR INDUCING NEUTRALIZING ANTIBODIES The lack of success of monomeric gp120 to induce neutralizing antibodies against primary isolates called for novel vaccine design strategies. Multiple approaches have been tested in the pursuit of an immunogen capable of inducing a broadly cross-reactive neutralization response some of which are described below. While the majority of these candidates have been able to induce neutralizing antibodies, they are usually only successful against viruses that are generally neutralization sensitive or closely related to the antigenic strain. 1.4.1 Native trimeric envelope glycoproteins as immunogens The observation that neutralization correlates with antibody-binding to the native trimeric envelope glycoprotein (Fouts et al., 1997, Sattentau and Moore, 1995) suggested the use of immunogens that resemble the oligomeric protein on the surface of the virus. Immunization with virus like particles (VLPs), pseudovirions and chemically inactivated viruses, constitute the most direct approaches to present the native trimer to the immune system (Buonaguro et al., 2005, Crooks et al., 2007, Grovit-Ferbas et al., 2000, McBurney et al., 2007, Quan et al., 2007, Race et al., 1995, Wagner et al., 1996). A key issue in these methodologies is the elicitation of antibodies to other cell membrane proteins incorporated in the VLPs, which could confound the interpretation of these results by showing neutralizing activity. Furthermore, the presence of gp120-gp41 monomers and gp41 stumps on these particles could induce non-neutralizing antibodies (Moore et al., 2006). To circumvent these issues it has been suggested that recombinant envelope trimers in proteoliposomes (EnvPL) be used as immunogens (Grundner et al., 2002). This approach was shown to be superior to monomeric gp120 in rabbit immunization experiments (Grundner et al., 2005). Another strategy to mimic the native envelope structure has been the use of soluble recombinant trimers as immunogens. This approach entails the production and purification CHAPTER 1: INTRODUCTION 26 of stable trimeric proteins. A widely followed method has been the use of soluble gp140 with a deleted cleavage site to avoid gp120 shedding (Barnett et al., 2001, Bower et al., 2004b, Srivastava et al., 2002, Yang et al., 2002, Yang et al., 2001, Zhang et al., 2001, Zhang et al., 2007). Further stabilization has been achieved by introducing heterologous trimerization domains such as the yeast transcription factor GCN4 (Yang et al., 2000). The cleavage defective soluble ectodomain construct YU2 gp140 (-/GCN4) has been shown to elicit neutralizing antibodies with some breadth compared to monomeric gp120 in mice, rabbits and guinea pigs (Grundner et al., 2005, Li et al., 2006c, Yang et al., 2001). However, observations that uncleaved forms of the envelope preferentially bind non- neutralizing antibodies suggest that they do not optimally resemble the native glycoprotein (Herrera et al., 2005, Pancera and Wyatt, 2005). As an alternative to preserving the cleavage site, other groups have stabilized the gp120-gp41 interaction by introducing inter- subunit disulphide bonds (SOS) (Binley et al., 2000a, Binley et al., 2002) with further trimerization improvement by introducing a helix-disturbing mutation in the HR1 of gp41 (SOSIP) (Beddows et al., 2007, Beddows et al., 2005, Sanders et al., 2002b). This approach once again rendered antigens superior to monomeric gp120, but they still failed to elicit broadly neutralizing antibodies (Beddows et al., 2007). It is possible that these manipulations do not achieve presentation of the native structure of the envelope glycoprotein. However, immunization experiments with non-stabilized gp140 oligomers were also limited in their ability to induce neutralizing antibodies (Kim et al., 2005). Strain-specific features may determine the stability of the trimeric structure as well as its capacity to induce broadly cross-reactive neutralizing antibodies (Rademeyer et al., 2007). A recent study showed that the R2 isolate-based uncleaved gp140 oligomer, derived from a patient with broadly cross-reactive neutralizing antibodies, elicited antibodies capable of neutralizing several heterologous viruses from multiple clades and constitutes the most CHAPTER 1: INTRODUCTION 27 successful immunogen reported to date (Zhang et al., 2007). 1.4.2 Exposure of cryptic epitopes (CD4i epitope) Other strategies have involved the exposure of cryptic epitopes, such as the conserved CD4i epitope, by deleting variable loops (Barnett et al., 2001, Gzyl et al., 2004, Kim et al., 2003, Lian et al., 2005, Srivastava et al., 2003) or glycosylation sites (Bolmstedt et al., 2001, Quinones-Kochs et al., 2002) (Nkosi et al. unpublished). Most of these modifications have been done in the context of soluble forms of the stabilized envelope trimer. An alternative to exposing the CD4i epitope has been to co-express gp120 with the CD4 subunit D1D2 (Fouts et al., 2002). This immunogen generated antibodies capable of inhibiting entry of several HIV-1 isolates, but concerns about the induction of anti-CD4 antibodies have been raised. To bypass this difficulty, mimetic forms of CD4 have been used, such as the gp120-M9 construct (Varadarajan et al., 2005); or gp120 liganded to the A32 antibody, which induces CD4i epitope exposure (Liao et al., 2004), but both these methods failed to elicit broadly neutralizing antibodies. 1.4.3 Epitopes of nMAbs as immunogens Another group of immunogens are based on the conserved epitopes recognized by the broadly nMAbs b12, 2G12, 2F5 and 4E10, aiming to recapitulate their antigenic site (Burton et al., 2004). The CD4bs is of particular interest as it is exposed on the surface of the virus for functional reasons. However, with the exception of IgG1b12, neutralizing antibodies to this region are not readily induced due to thermodynamic constraints (Kwong et al., 2002, Zhou et al., 2007). The stabilization of the gp120 monomer in a CD4-bound conformation has been proposed as a method to overcome this barrier. Rationalized structure manipulations, such as the introduction of cavity filling mutations and inter-domain disulfide bonds, have CHAPTER 1: INTRODUCTION 28 stabilized gp120 conformations that bind CD4 with minimal entropy (Dey et al., 2007, Zhou et al., 2007). Immunization with trimeric gp120-GCN4 with cavity filling mutations induced CD4i antibodies, but showed little improvement in eliciting neutralizing antibodies (Dey et al., 2007). Induction of anti-CD4bs antibodies, such as IgG1b12, has also been pursued through other strategies, such as glycan masking of non-neutralizing epitopes in the gp120 molecule, to focus the antibody response on the b12 epitope (Pantophlet et al., 2003, Pantophlet et al., 2004). While this approach succeeded in masking dominant non-neutralizing epitopes, it did not improve the neutralization response in immunized rabbits (Selvarajah et al., 2005). Assisted by new structural data on the b12 binding site and the biochemical properties of this interaction (Zhou et al., 2007), new strategies to induce b12-like antibodies are under development (Liu et al., 2007, Phogat et al., 2007). Attempts to reproduce the high mannose clustering recognized by 2G12 using synthetic glycosides and Man9 sugars conjugated to carrier molecules or synthetic scaffolds have been pursued (Dudkin et al., 2004, Geng et al., 2004, Lee et al., 2004, Li and Wang, 2004, Wang et al., 2004). While some of these synthetic oligosacarides inhibit the binding of 2G12 to gp120, their ability to induce neutralizing antibodies has not yet been reported. Another problem with these immunogens is their internal flexibility. Further strategies to enhance the immunogenicity to this epitope were discussed in a recent review by Scanlan et al. (Scanlan et al., 2007). They comprise the use of antigenic carriers and adjuvants that can break immunotolerance and the inclusion of antigenic carbohydrates such as rhamnose in these synthetic constructs. Structural data has shown that the nMAb 4E10 binds to a helical conformation of its epitope. On this basis, stabilization of a peptide containing the 4E10 epitope in a helical conformation has been pursued in the design of a better immunogen (Cardoso et al., 2007). CHAPTER 1: INTRODUCTION 29 Further manipulations to hide the non-binding site of this helix, taking membrane interactions into consideration, have also been proposed (Cardoso et al., 2007, Zwick, 2005). Other researchers have designed miniproteins containing the MPER in the context of a proteoliposome or the Hepatitis B surface antigen S1 protein (Ofek et al., 2004). Immunogenicity trials of these variants in a prime-boost regimen with native gp160 trimer have been suggested (Ofek et al., 2004). In a recent study, the MPER was engrafted into the V1/V2 region of gp120, but anti-MPER antibodies were not induced (Law et al., 2007). The knowledge obtained from the 2F5 and 4E10 antibody structures bound to their corresponding epitopes, is currently being exploited in the design of epitope-scaffold immunogens by in silico screening of potential carrier proteins (Ofek et al., 2007, Phogat et al., 2007). This inventive methodology promises to produce new candidates soon to be tested in pre-clinical studies. 1.4.4 Multivalent and centralized immunogens To overcome the genetic diversity of HIV, multivalent or centralized envelope immunogens have been used in preclinical and clinical trials. The first strategy combined the envelopes of multiple HIV-1 clades in one immunization protocol to increase the breadth of the immune responses (Cho et al., 2001, Pal et al., 2005, Rollman et al., 2004, Seaman et al., 2005, Wang et al., 2006). The second approach minimizes epitope diversity by artificially designing envelope genes representing consensus or ancestral sequences of those available in at the Los Alamos HIV Database. This strategy is based on the assumption that these sequences will be more closely related to circulating variants than the circulating variants will be to one another (Nickle et al., 2003). Six centralized immunogens have been generated to date; two consensus M, CON6 (Gao et al., 1996) and CON-S (Liao et al., 2006); subtype-specific consensus ConC (Kothe et al., 2006) and ConB (Kothe et al., 2007), and ancestral Anc1-EnvB (Doria-Rose et al., 2005) and AncC CHAPTER 1: INTRODUCTION 30 (Kothe et al., 2006). Of them, the newer consensus, CON-S, was shown to elicit greater neutralization breadth. This suggests that as more sequences are entered into the database, better coverage of envelope variability will be attained. 1.4.5 Other factors that contribute to vaccine design In addition to antigen design, progress has been made in other areas that contribute to the envelope glycoprotein immunogenicity. DNA prime and recombinant protein boost immunization regimens have been shown to improve the neutralizing antibody responses (Beddows et al., 2005, Law et al., 2007, Lian et al., 2005, Shu et al., 2007, Wang et al., 2005). Other combinations such as DNA prime-VLP boost (Buonaguro et al., 2007a) and DNA prime-viral vector boost (Gomez et al., 2007, Seaman et al., 2007, Shinoda et al., 2006) have also been promising. Novel adjuvant formulations have been tried in conjunction with newly designed immunogens. The GlaxoSmithKline adjuvants, AS01B, AS02A and AS03 have been shown to enhance the neutralizing titers induced by a soluble trimeric envelope in comparison to the more commonly used Ribi adjuvant (Li et al., 2006c, Zhang et al., 2007). The fusion of the innate activator C3d to an envelope construct proved to enhance immunogenicity (Bower et al., 2004a, Bower et al., 2006). Another formulation involving the co-delivery of GM-CSF DNA was shown to increase avidity of the antibody response (Robinson et al., 2006) and IL-21 and IL-15 gene delivery can increase the level of anti- HIV IgG and the longevity of immune responses (Bolesta et al., 2006). The use of CpG as a mucosal adjuvant was shown to enhance humoral and cellular immune responses in these compartments (Kang and Compans, 2003). Comparison between the numerous vaccine candidates and immunization strategies has been difficult as various neutralization assays and viral strains have been used in these different studies. This constitutes a major complication in the decision about which CHAPTER 1: INTRODUCTION 31 immunogens to advance into clinical trials. Mascola et al, suggested the use of cloned envelopes in a pseudovirion neutralization assay in combination with standardized viral panels to evaluate the neutralization responses elicited by candidate vaccines (Mascola et al., 2005). To date, two panels have been assembled, which include viruses from early subtype B and C infections (Li et al., 2005, Li et al., 2006b). Furthermore, a multitier system groups these viruses according to neutralization sensitivity, allowing comparisons of the potencies and breadths of the elicited antibodies (Mascola et al., 2005). 1.5 GENETIC SUBTYPES OF HIV-1 AND NEUTRALIZATION IMMUNOTYPES An important goal in the development of an effective HIV-1 vaccine is to overcome the extensive genetic heterogeneity of the virus. HIV-1 viruses have been divided in three groups based on their nucleotide sequences: group M (main), group O (outlier), and group N (non-M non-O) (McCutchan et al., 1996). Group M, the largest one, is further divided into genetic subtypes or clades: A, B, C, D, F1, F2, G, H, J and K, plus at least 20 circulating recombinant forms (Buonaguro et al., 2007b). Subtype C is the most prevalent in the world, being common in India and the southern African countries of Botswana, Zimbabwe, Malawi, Mozambique, and South Africa. Subtype B is dominant in North America and Western Europe and has been, until recently, the major focus for vaccine development. Although subtypes are a useful means to categorize HIV-1, the relevance of genetic subtype for vaccine design is uncertain. Genetic subtypes do not readily corresponds to neutralization serotypes and there is no compelling evidence to suggest that HIV-1 positive sera better neutralize viruses from the same clade than viruses from another clade (Kostrikis et al., 1996, Moore et al., 1996, Moore et al., 2001, Nyambi et al., 2000, Weber et al., 1996). However, some have reported stronger intraclade neutralization for subtype A/E (Mascola et al., 1996) or a regional clustering amongst subtype C viruses (Bures et al., 2002). The difficulties associated with finding neutralization immunotypes CHAPTER 1: INTRODUCTION 32 have been attributed to the variability of the neutralization titers and specificities in HIV-1 positive sera, as well as the variability of the neutralization sensitivity of the viruses and the high background-to-noise ratio of the methodologies used in these studies. A recent study, which used monoclonal antibodies in a highly reproducible single round neutralization assay, defined five neutralization immunotypes of HIV-1 somewhat associated with genetic clades (Binley et al., 2004). In this case, the absence or presence of some antibody epitopes could be defined in the envelope sequence and correlated with the observed phenotype. The fact that many of the epitopes recognized by these broadly cross- reactive nMAbs have been pursued as targets for vaccine design, underscores the importance of their study in the context of other HIV-1 subtypes. 1.6 OBJECTIVES OF THIS STUDY The aim of this work was to characterize the neutralizing antibodies epitopes on the HIV-1 subtype C envelope glycoprotein as well as the neutralization response that develops in HIV-1 subtype C infection, in particular the antibody specificities associated with this response. ? Firstly, the sensitivity of HIV-1 subtype C envelope glycoproteins to the broadly neutralizing monoclonal antibodies 2G12, IgG1b12, 4E10 and 2F5 were determined. The presence of the epitope recognized by these antibodies was assessed and compared to the neutralization phenotype. ? Given that subtype C viruses were found to be insensitive to 2G12 neutralization, envelope clones generated in the above study were used in an attempt to reconstitute the 2G12 epitope in subtype C envelopes. This information was further used to assess the antigenic conservation of subtype C envelopes in comparison with their subtype B counterparts. CHAPTER 1: INTRODUCTION 33 ? The 4E10 epitope is very conserved amongst HIV-1 viruses and an important target for vaccine design. The identification of an HIV-1 subtype C infected individual carrying 4E10 resistant variants allowed the study of the determinants of 4E10 sensitivity in the envelope gene of these viral quasispecies. ? Finally, the kinetics of the neutralization response during natural HIV-1 subtype C infection was analyzed by examining the development of autologous and heterologous neutralizing antibodies in 14 individuals during the first year of infection. In addition, the presence of antibody specificities, such anti-MPER and anti-CD4i neutralizing antibodies, was also assessed. CHAPTER 2: NEUTRALIZING MAb EPITOPES IN SUBTYPE C VIRUSES 34 CHAPTER TWO INSENSITIVITY OF PAEDIATRIC HIV-1 SUBTYPE C VIRUSES TO BROADLY NEUTRALISING MONOCLONAL ANTIBODIES RAISED AGAINST SUBTYPE B Published: PLoS Medicine 3(7):1023-1030 (2006) CHAPTER 2: NEUTRALIZING MAb EPITOPES IN SUBTYPE C VIRUSES 35 CHAPTER 2: NEUTRALIZING MAb EPITOPES IN SUBTYPE C VIRUSES 36 CHAPTER 2: NEUTRALIZING MAb EPITOPES IN SUBTYPE C VIRUSES 37 CHAPTER 2: NEUTRALIZING MAb EPITOPES IN SUBTYPE C VIRUSES 38 CHAPTER 2: NEUTRALIZING MAb EPITOPES IN SUBTYPE C VIRUSES 39 CHAPTER 2: NEUTRALIZING MAb EPITOPES IN SUBTYPE C VIRUSES 40 CHAPTER 2: NEUTRALIZING MAb EPITOPES IN SUBTYPE C VIRUSES 41 CHAPTER 2: NEUTRALIZING MAb EPITOPES IN SUBTYPE C VIRUSES 42 CHAPTER 3: 2G12 EPITOPE IN SUBTYPE C VIRUSES 43 CHAPTER THREE N-LINKED GLYCAN MODIFICATIONS IN GP120 OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 SUBTYPE C RENDER PARTIAL SENSITIVITY TO 2G12 ANTIBODY NEUTRALIZATION Published: Journal of Virology 81(19): 10769-10776 (2007) CHAPTER 3: 2G12 EPITOPE IN SUBTYPE C VIRUSES 44 CHAPTER 3: 2G12 EPITOPE IN SUBTYPE C VIRUSES 45 CHAPTER 3: 2G12 EPITOPE IN SUBTYPE C VIRUSES 46 CHAPTER 3: 2G12 EPITOPE IN SUBTYPE C VIRUSES 47 CHAPTER 3: 2G12 EPITOPE IN SUBTYPE C VIRUSES 48 CHAPTER 3: 2G12 EPITOPE IN SUBTYPE C VIRUSES 49 CHAPTER 3: 2G12 EPITOPE IN SUBTYPE C VIRUSES 50 CHAPTER 3: 2G12 EPITOPE IN SUBTYPE C VIRUSES 51 CHAPTER 4: 4E10 NEUTRALIZATION DETERMINANTS 52 CHAPTER FOUR 4E10 RESISTANT VARIANTS IN AN HIV-1 SUBTYPE C INFECTED INDIVIDUAL WITH AN ANTI-MPER NEUTRALIZING ANTIBODY RESPONSE Published: Journal of Virology, in press. CHAPTER 4: 4E10 NEUTRALIZATION DETERMINANTS 53 4E10 Resistant Variants in an HIV-1 Subtype C Infected Individual with an Anti- MPER Neutralizing Antibody Response Elin S. Gray1, Penny L. Moore1, Frederic Bibollet-Ruche3, Hui Li3, Julie M. Decker4, Tammy Meyers2, George M. Shaw3 and Lynn Morris1* 1 AIDS Virus Research Unit, National Institute for Communicable Diseases and 2 Harriet Shezi Clinic, Chris Hani Baragwanath Hospital, University of the Witwatersrand, Johannesburg, South Africa, 3 University of Alabama at Birmingham, Birmingham, AL 35294, USA. Number of words in abstract: 213 Number of words in main text: 4,638 Number of pages: 24 Number of figures: 7 Short Title: HIV-1 resistance to MAb 4E10 Keywords: MPER antibodies, 4E10 resistance, HIV-1 subtype C, cytoplasmic tail, LLP-2 *Corresponding author mailing address: Prof Lynn Morris, National Institute for Communicable Diseases, Johannesburg, Private Bag X4, Sandringham 2131, Johannesburg, South Africa. Phone: +27-11-386-6332. Fax: +2711-386-6333. E-mail: lynnm@nicd.ac.za. CHAPTER 4: 4E10 NEUTRALIZATION DETERMINANTS 54 Abstract: The broadly neutralizing monoclonal antibody (MAb) 4E10 recognizes a linear epitope in the C-terminus of the membrane proximal external region (MPER) of gp41. This epitope is particularly attractive for vaccine design because it is highly conserved amongst HIV-1 strains and neutralization escape in vivo has not been observed. Multiple env genes were cloned from an HIV-1 subtype C virus isolated from a 7 year old perinatally infected child who had anti-MPER neutralizing antibodies. One clone (TM20.13) was resistant to 4E10 neutralization as a result of an F673L substitution in the MPER. Frequency analysis showed that F673L was present in 33% of the viral variants and in all cases was linked to the presence of an intact 2F5 epitope. Two other envelope clones were sensitive to 4E10 neutralization, but TM20.5 was 10-fold less sensitive than TM20.6. Substitutions at 674 and 677 within the MPER rendered TM20.5 more sensitive to 4E10, but had no effect on TM20.6. Using chimeric and mutant constructs of these two variants, we further demonstrated that the LLP-2 domain in the cytoplasmic tail affected the accessibility of the 4E10 epitope, as well as virus infectivity. Collectively, these genetic changes in the face of a neutralizing antibody response to the MPER, strongly suggested immune escape from antibody responses targeting this region. CHAPTER 4: 4E10 NEUTRALIZATION DETERMINANTS 55 Introduction: The membrane proximal external region (MPER) of the HIV-1 envelope glycoprotein comprises the last 23 amino acids, from residues 660 to 683, of the extracellular domain of gp41 just before the transmembrane domain. This region has attracted a lot of attention in the field of HIV vaccinology due to some particular features: i) it is the target of two of the few broadly neutralizing monoclonal antibodies against HIV-1, namely 4E10 and 2F5, ii) it has been shown to be important in the fusion process and therefore in viral entry (11, 28), and iii) it is a highly conserved linear region among all HIV-1 subtypes. The MAb 4E10 recognizes an epitope containing the sequence NWF(D/N)IT (30, 38) in the tryptophan-rich region of gp41. Mutagenesis experiments have shown that residues W672, F673 and W680 are indispensable for 4E10 recognition (37). Crystal structures of the Fab 4E10 in complex with a peptide containing the epitope, illustrate that residues W672, F673, I675 and T676 are the key residues in this interaction (7). A more recent study extended the 4E10 epitope to the motif WFx(I/L)(T/S)xx(L/I)W (residues 672-680), where the amino acids marked with an x do not play a major role in 4E10 binding (6). The sequence ELDKWA (residues 663-667) immediately N-terminal to the 4E10 epitope, is the target of the 2F5 MAb (21). Mutagenesis studies had revealed that the amino acid motif DKW is required for recognition by this MAb (37), and structural studies have demonstrated that these three residues are deeply buried in the interface with 2F5 (25). While 4E10 neutralizes viruses from all HIV-1 subtypes, 2F5 fails to neutralize subtype C and some subtype D viruses and this can be directly correlated to changes in the antibody epitope (3, 14). Despite the high level of conservation of the MPER and its importance in the fusion process, multiple studies have demonstrated that mutations in this region do not necessarily impair viral infectivity (5, 37). It has been proposed that this region is not targeted by the host immune response and therefore is not under diversifying selection pressure (36). Recent studies have addressed the question of whether HIV-1 infection induces the production of neutralizing antibodies that target the MPER. The presence of such antibodies was assessed using a novel strategy where the HIV-1 MPER was engrafted onto an SIV (35) or HIV-2 envelope (F. Bibollet-Ruche et al., presented at the Keystone Symposium on HIV Vaccines, Keystone Resort, Keystone, CO, 2006). These studies CHAPTER 4: 4E10 NEUTRALIZATION DETERMINANTS 56 indicated that antibodies with specificities such as 4E10 and 2F5 are rarely produced (J. M. Decker et al., presented at the Keystone Symposium on HIV Vaccines, Keystone Resort, Keystone, CO, 2006) (35), however, other anti-MPER antibodies were detected in around a third of HIV-1 infected patients (F. Bibollet-Ruche et al., presented at the Keystone Symposium on HIV Vaccines, Keystone Resort, Keystone, CO, 2006). It remains unclear what the effect of such antibodies is on the viral population, as escape variants have not been described. In this study we characterized HIV-1 subtype C viral quasispecies with different sensitivities to the MAb 4E10. We explored the genetic determinants of these phenotypes as well as the anti-MPER antibody response that developed in the individual from whom this virus was isolated. CHAPTER 4: 4E10 NEUTRALIZATION DETERMINANTS 57 Methods: Cloning of envelope genes and production of pseudovirions: Proviral DNA extracted from in vitro infected PBMCs was used to amplify full-length envelope genes using the primers envA and envM (13). The 3Kb PCR fragments were cloned into an expression vector and used to generate Env-pseudotyped viruses as previously described (14). Envelope sequencing: gp41 was amplified from viral RNA from culture supernatant by nested RT-PCR using published primers (9, 13) and cloned using the TOPO TA Cloning? Kit (Invitrogen Corporation, Carlsbad, CA). The gp41 and gp160 clones were sequenced using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA) and resolved on an ABI 3100 automated genetic analyzer. The sequences were assembled and edited using Sequencher v.4.0 software (Genecodes, Ann Arbor, MI). Single cycle neutralization assay: Neutralization was measured as a reduction in luciferase gene expression after a single round infection of JC53bl-13 cells with Env-pseudotyped viruses (20). Briefly, 200 TCID50 of pseudoviruses in 50 ?l were incubated with 100 ?l of serially diluted MAbs, CD4-IgG2, plasma or T-20 (Enfurvirtide) in DMEM with 10% FBS in a 96-well plate, in triplicate for 1 h at 37?C. A 100 ?l of JC53bl-13 cells (1x104 cells/well) containing 75 ?g/ml DEAE dextran (Sigma-Aldrich, St. Louis, MO) was added and the cultures were incubated at 37?C in 5% CO2. Infection was determined 48 hours later using the Bright GloTM Reagent (Promega, Madison, WI). Luminescence was measured in a Wallac 1420 Victor Multilabel Counter (Perkin Elmer, Norwalk, CT). Titers were calculated as the inhibitory concentration (IC50) or reciprocal plasma dilution (ID50) causing 50% reduction of relative light unit (RLU) compared to the virus control (wells with no inhibitor) after subtracting the background (wells without virus infection). Virus infectivity and envelope incorporation: The amount of virus was normalized by the quantity of p24 pelleted through a 20% sucrose cushion for 2 hours at 20,000g. JC53bl-13 cells were infected with 10 ng of p24 in the presence of 30 ?g/ml DEAE dextran, and cultures were incubated at 37?C in 5% CO2 for 48 h. Infection was monitored by evaluating the luciferase activity. Envelope incorporation was estimated by Western blot. Viral proteins were resolved in a Criterion 4-15% Tris-HCl Gradient Gel (Bio-Rad Laboratories, Hercules, CA) and blotted onto a PVDF nitrocellulose membrane (GE Healthcare Life Science, Piscataway, NJ). The membranes were blocked overnight with 5% milk in TBS/0.05% Tween 20 and probed with the anti-gp120 (D7324), anti-gp41 CHAPTER 4: 4E10 NEUTRALIZATION DETERMINANTS 58 (7B2) or the anti-p24 (D7312) antibodies, and developed with an HRP-labeled secondary antibody (Sigma) using the enhanced chemiluminescence (ECL) detection system (GE Healthcare Life Science, Piscataway, NJ). To determine envelope expression levels, 293T cells were transfected with envelope constructs using the Fugene transfection reagent (Roche, Applied Science, Indianapolis, IN). The cells were harvested after 48 hours and lysated with the non-denaturing lysis buffer, 1% Triton X-100, 300 mM NaCl, 5 mM EDTA, 50 mM Tris-Cl, pH 7.4 with Complete protease inhibitor cocktail (Roche). The Western Blot was carried out as described above. Site directed mutagenesis: Specific amino acid changes in the HIV-1 and HIV-2 envelope glycoproteins were introduced using the QuikChange Site Directed Mutagenesis Kit (Stratagene, La Jolla, CA). The presence of mutations was confirmed by sequence analysis. Construction of chimeric envelope glycoproteins: To construct the gp120-gp41 chimeras, the TM20.5 and TM20.6 env plasmids were digested with NdeI and the interchanged fragments ligated with T4 DNA Ligase (Invitrogen Corporation). The correct orientation of the cloned fragments was determined by a colony PCR with primers T7 and EnvM. The ectodomain/cytoplasmic tail chimeras were constructing by overlapping PCR spanning the transmembrane domain using the primers: TM20CTfo: CAGATCCGTGAGATTAGTGAGCGGATTCTTAG and EnvM, TM20CTre: CTAAGAATCCGCTCACTAATCTCACGGATCTG and EnvA. The resultant amplicons was cloned into pcDNA3.1D (Invitrogen Corporation, Carlsbad, CA). Chimerism was confirmed by sequencing. Anti-MPER neutralization assay: This assay was adapted from that previously described by Decker et al. (J. M. Decker et al., presented at the Keystone Symposium on HIV Vaccines, Keystone Resort, Keystone, CO, 2006) using chimeric HIV-2/HIV-1 MPER constructs (F. Bibollet-Ruche et al., presented at the Keystone Symposium on HIV Vaccines, Keystone Resort, Keystone, CO, 2006) (15). Briefly, 2000 infectious units of virus pre-incubated with plasma/serum at a starting dilution of 1:20 and serially diluted 1:5 in the presence of normal human plasma/serum, was added to 40% confluent JC53bl-13 cells, seeded the day before. Infection was measured 48 hours later by evaluating the luciferase activity as described above. Nucleotide sequence accession numbers: The GeneBank database accession numbers for the three env clones described in this study are EU161643-EU161645. CHAPTER 4: 4E10 NEUTRALIZATION DETERMINANTS 59 Results: Discovery of a naturally-occurring 4E10 resistant virus: While characterizing functional envelope genes from HIV-1 subtype C viruses isolated from children (14) we identified an envelope clone, TM20.13 that was resistant to the MAb 4E10. This was the result of an F to L mutation at position 673, which is known from mutagenesis studies to confer resistance to 4E10 (37). However, this phenotype has not been previously described in patient samples and analysis of sequences in the Los Alamos HIV database suggested that this mutation is very rare in group M of HIV-1. This clone was derived from a virus isolated from a 7 year old female who was infected perinatally and at the time of blood collection had a CD4 count of 334 cells/?l and a viral load of 34,310 copies/ml. Although this patient had lymphocytic interstitial pneumonitis she was considered to be a slow progressor (8). The virus TM20 was isolated on donor PBMC and used the CCR5 coreceptor with a V3 sequence typical of subtype C viruses (8). Only a single sample from this patient was available. Attempts to obtain additional samples either from this individual or from her mother were unsuccessful. Frequency of the F673L mutation in the TM20 virus isolate: To determine the frequency of the F673L mutation in this cultured virus isolate, we analyzed the gp41 sequences of 43 molecular clones. The molecular clones were generated from 3 independent RT-PCR reactions from the viral RNA to ensure that the mutation was not introduced by PCR error. The mutation F673L was observed in 14 of the 43 (33%) molecular clones sequenced (Figure 1). In total 6 different quasispecies were identified. Interestingly the F673L mutation was always present in conjunction with the mutation S665K in the 2F5 epitope, as well as a group of associated mutations within the HR2 domain and the cytoplasmic tail of gp41 (Figure 1 and data not shown). Generation and characterization of functional TM20 envelope clones: Full-length envelope genes were amplified from the TM20 isolate and cloned into an expression vector to generate functional clones. Three envelope clones were pseudotyped and characterized for their sensitivity to the broadly neutralizing MAbs 4E10 and 2F5, and the entry inhibitor T-20 (Enfuvirtide, Fuzeon, DP178) (Figure 2A). As predicted from the sequence analysis, the clone TM20.13 was not neutralized by 4E10, but it was sensitive to 2F5. The other two clones were sensitive to 4E10 and resistant to 2F5, but TM20.6 (IC50=1.5 ?g/ml) showed at CHAPTER 4: 4E10 NEUTRALIZATION DETERMINANTS 60 least 10-fold higher sensitivity to 4E10 than the clone TM20.5 (IC50=17.5 ?g/ml). There were no differences in the neutralization sensitivity of the three clones to T-20, which binds to the HR1 region of gp41. In addition, all clones showed similar sensitivity to the HIV-1 positive plasma samples BB12 and BB107 (Figure 2B), suggesting that the increased sensitivity of the clone TM20.6 to 4E10 was not due to a generally neutralization sensitive phenotype. All three clones were resistant to the contemporaneous autologous serum (below 50% neutralization), which is typical in HIV-1 infection. Sequence analysis of functional clones from TM20: The full-length envelope genes of the three functional clones were sequenced (Figure 3). The genetic variation observed between the clones was consistent with the 7 year duration of infection; however the extent of variability from the C1 to C3 region of the gp120 was surprisingly low. Since these clones were amplified from in vitro grown virus, we cannot exclude the possibility that quasispecies bearing different sequences in this region of the envelope existed in vivo. The clone TM20.13 had a F673L mutation in the 4E10 epitope as well as the DKW motif intrinsic to the 2F5 epitope, which correlated with the observed phenotypes. TM20.5 and TM20.6 were identical in the gp41 ectodomain, except for two amino acids in the MPER at positions 674 and 677. Multiple other differences between the clones TM20.5 and TM20.6 were observed along the gp120 and the cytoplasmic tail of gp41. All three clones had conserved HR1 and V3 regions which are involved in T-20 sensitivity (10, 27), consistent with the observed equivalent sensitivity to this compound. Changes in the MPER are partially responsible for the differential sensitivity of TM20.5 and TM20.6 to 4E10: As noted above, TM20.5 was 10-fold less sensitive to 4E10 than TM20.6. These two clones differed in two amino acids in the 4E10 epitope (Figure 3). In order to determine if these residues were associated with 4E10 sensitivity we introduced the mutations D674N and K677N into TM20.6 by site-directed mutagenesis. These changes did not render the TM20.6 mutant more resistant to 4E10 neutralization (Figure 4). Conversely, when the mutations N674D and N677K were introduced in TM20.5 the 4E10 IC50 dropped 3-fold suggesting that in some contexts these positions may have an effect on antibody binding affinity. 4E10 neutralization of envelope chimeras: These apparently discordant results suggested CHAPTER 4: 4E10 NEUTRALIZATION DETERMINANTS 61 that regions outside the MPER might influence the sensitivity to 4E10. We therefore constructed chimeras in which large regions of the envelope gene were interchanged between the TM20.5 and TM20.6 clones (Figure 5). For both clones, the gp120 subunit had very little to no effect on the neutralization sensitivity indicating that the major determinants of 4E10 sensitivity were in the gp41 region. When the cytoplasmic tail from TM20.6 was combined with the ectodomain of TM20.5, which included gp120 and the external region of gp41, the resulting chimera Ecto5-CT6 was around 4-fold more sensitive than TM20.5. Conversely, Ecto6-CT5 was 3-fold less sensitive than TM20.6. Altogether these results suggest that while the major determinants of 4E10 sensitivity are in the MPER, the cytoplasmic tail can have a tangible impact on the exposure of this epitope. Mutations in the LLP-2 domain affect sensitivity to neutralization: The cytoplasmic tail sequence of clones TM20.5 and TM20.6 differed by ten amino acid residues (Figure 3), four in the lentivirus lytic peptide-2 domain (LLP-2). Frequency analysis showed that the LLP-2 sequence of TM20.6 was present in 17% of the quasispecies while 36% had the TM20.5 LLP-2 sequence. To determine if variations in LLP-2 were responsible for the changes in 4E10 sensitivity in the cytoplasmic tail chimeras, we exchanged the LLP-2 regions of TM20.5 and TM20.6 by introducing the mutations E783A, T784I, G789V and T792L in TM20.5 (TM20.5 LLP2-6) and the converse changes in TM20.6 (TM20.6 LLP2- 5). We tested all constructs for their sensitivity to neutralization by 4E10 and T-20 as well as IgG1b12 and CD4-IgG2 which bind to gp120 and block interactions with CD4. As observed for the chimera Ecto5-CT6, the mutant TM20.5 LLP2-6 became more sensitive to neutralization by 4E10 when compared to the parental TM20.5 virus (Figure 5). Similarly, these changes in the LLP-2 explained the decrease in sensitivity to 4E10 in the chimera Ecto6-CT5 when compared to the parental TM20.6. Thus, in both cases, the four amino acids in the LLP-2 domain accounted for the altered changes in sensitivity to 4E10 neutralization. These changes also had a minor effect on the neutralization sensitivity for IgG1b12 (Figure 5) and CD4-IgG2 (data not shown) but had no effect on T-20 neutralization. In order to look at the combined effects of changes in the MPER and LLP-2 we introduced these four mutations into the two MPER mutants to form TM20.5 MPER-6 LLP2-6 and CHAPTER 4: 4E10 NEUTRALIZATION DETERMINANTS 62 TM20.6 MPER-5 LLP2-5. For TM20.5 the resultant clone showed a 9-fold increase in neutralization sensitivity to 4E10 and the IC50 titer decreased to a similar level to that obtained for the TM20.6 clone (Figure 5). The opposite mutant, TM20.6 containing the MPER and LLP-2 of TM20.5, became 7-fold more resistant to neutralization, but the IC50 titer remained slightly lower than for TM20.5 (11.3 versus 17.5 ?g/ml), suggesting that perhaps other areas of the envelope may influence this phenotype. These double mutants showed similar sensitivities to IgG1b12 and CD4-IgG2 as the corresponding LLP-2 only mutants corroborating that the MPER plays no role in determining sensitivity to these reagents. Effect of the gp120 and cytoplasmic tail switching on envelope incorporation and viral infectivity: The LLP-2 domain is a highly conserved cationic amphipathic ?-helix with calmodulin-binding properties (29), that is involved in cell-to-cell fusion (17, 33), neutralization sensitivity (16), envelope cell surface expression (4), envelope incorporation into virions and virus infectivity (24). To explore the mechanism behind the effect of the cytoplasmic tail on 4E10 neutralization sensitivity, we compared the viral infectivity and envelope incorporation of the two TM20 clones and their chimeras. Chimeras bearing the cytoplasmic tail from TM20.6 (ie gp120(5)-gp41(6) and Ecto5-CT6) displayed low infectivity similar to the parental virus (Figure 6A). The TM20.5 cytoplasmic tail conferred an infectivity advantage to the variants carrying it (ie gp120(6)- gp41(5) and Ecto6-CT5). Furthermore, the LLP-2 mutants of TM20.5 displayed the same reduced infectivity as the Ecto5-CT6, while TM20.6 LLP-2 mutants, like Ecto6-CT5, were more infectious (data not shown). This suggested that the increased infectivity conferred by the TM20.5 cytoplasmic tail was largely determined by four amino acids in LLP-2. The levels of envelope incorporation into virus particles and envelope expression were determined by Western Blot. The virus input was standardized by using equal quantities of p24 antigen, which was confirmed by p24 detection on the blots. The amount of envelope incorporated into virions was assessed by staining with the anti-gp120 antibody D7324 and the anti-gp41 MAb 7B2 to detect gp160 (Figure 6B). In addition, envelope transfected cells were lysed and the levels of envelope expression were determined. Surprisingly, the less infectious TM20.6 clone showed greater envelope expression and incorporation into CHAPTER 4: 4E10 NEUTRALIZATION DETERMINANTS 63 virions than the clone TM20.5. This appeared to be determined by the gp120 as all chimeras containing TM20.6 gp120 showed higher levels of envelope irrespective of the gp41 sequence. Thus, the differential neutralization sensitivity between the clones could not be explained by the levels of envelope on virus particles. These results agree with other studies showing that the LLP-2 region can affect viral infectivity without changes in envelope incorporation (17, 24). Evaluation of anti-MPER neutralizing antibodies in the sera from TM20: The variability within the MPER among the quasispecies and the concomitant variations in 4E10 sensitivity, suggested the presence of immunological pressure targeting this region. To determine if this was the case, we tested the sera of this patient for antibodies to the MPER. We measured the neutralization activity of this serum against chimeric HIV-2 viruses engrafted with complete or partial HIV-1 MPER sequences (F. Bibollet-Ruche et al., presented at the Keystone Symposium on HIV Vaccines, Keystone Resort, Keystone, CO, 2006). Using this method we were able to detect anti-MPER antibodies in serum from patient TM20 (Figure 7). This serum was able to neutralize viruses that carried the full MPER sequence of either subtype B (7312A-C1), C (7312A-C1C) or a more similar match to the autologous MPER sequence where positions 671 and 676 were mutated to serine (7312A-C1Cm). This neutralization response was mapped to the second half of the MPER as evidenced by the neutralization of C4, C4GW and C8, but not C3 or C7. It has previously been shown that the 4E10 MAb neutralizes the chimeras, C1, C4 and C6 at similar antibody concentrations (J. M. Decker et al., presented at the Keystone Symposium on HIV Vaccines, Keystone Resort, Keystone, CO, 2006). Since TM20 sera failed to neutralize C6, this suggested the absence of 4E10?like antibodies. However, it partially neutralized the C4 chimera that in contrast to C6 includes the W680 residue which has been described to be important for 4E10 recognition (37). Taken together, our results suggest that the serum from this individual contained anti-MPER antibodies that recognize an epitope closely related, but not identical to the 4E10 epitope. Neutralization of C1C F673L by serum from patient TM20: The mutation F673L observed in the clone TM20.13 was introduced in the HIV-2/HIV-1 chimeric virus C1C by site- directed mutagenesis (C1C F/L). This single mutation conferred resistance to the 4E10 MAb relative to the sensitive C1C parental virus (data not shown). The TM20 serum was CHAPTER 4: 4E10 NEUTRALIZATION DETERMINANTS 64 unable to neutralize the C1C F/L mutant virus (Figure 7), suggesting that the F673L mutation observed in the clone TM20.13 could indeed be an escape mutation from the anti- MPER response developed by this patient. CHAPTER 4: 4E10 NEUTRALIZATION DETERMINANTS 65 Discussion In the present study we describe three viral quasispecies from an HIV-1 subtype C infected child with different sensitivities to neutralization by the broadly cross-reactive MAb 4E10. Neutralization resistance was conferred by a rare mutation F673L in the 4E10 epitope. In addition, moderate changes in sensitivity between clones were modulated by secondary positions in this epitope and motifs in the cytoplasmic tail. The presence of anti-MPER neutralizing antibodies in this individual supports the hypothesis that escape from antibody-mediated immune pressure was driving these changes. The MAb 4E10 has been shown to be the most broadly cross-reactive antibody against HIV-1 (3), however it is not a particularly potent antibody, with >1 ?g/ml needed to neutralize most viruses at 50%. In this study, the clone TM20.13 was resistant to neutralization at up to 100 ?g/ml of 4E10 due to a mutation at F673L in the MPER. This is consistent with a report by Zwick and co-workers who showed that the introduction of the F673A mutation conferred resistance to 4E10 neutralization (37). The mutation F673L was present in a third of the quasispecies in this virus isolate. However, given that they were derived from an in vitro cultured virus, it is not clear if this mutation was present at higher or lower frequency in vivo, where neutralizing antibodies were present. While L673 is common amongst HIV-1 group O envelope sequences, this substitution is very rare among HIV-1 group M viruses in the Los Alamos HIV Sequence Database. The highly conserved nature of F673 suggests that this site either performs an important function or is not accessible to immune attack, as has been proposed (36). However, we found that virus infectivity was not obviously compromised by F673L (data not shown), similar to what was observed for the F673A mutant (37). This raises the question as to why this position is so highly conserved and under such strong negative selection pressure. Interestingly, in our study the F673L mutation was linked with the mutation S665K, which confers 2F5 sensitivity. All other clones were resistant to 2F5, due to the absence of a K at position 665, which is typical of subtype C viruses (3, 14). Furthermore, these mutations were accompanied in all cases by other mutations in gp41. HIV-1 is known to undergo considerable recombination during in vivo and in vitro proliferation (18), so the link between these changes may simply be the result of a recombination event between two divergent viral quasispecies. However, they could also represent compensatory changes associated with the mutation F673L that are required by the virus for survival in vivo. A CHAPTER 4: 4E10 NEUTRALIZATION DETERMINANTS 66 recent study on in vitro escape from 4E10 neutralization showed the appearance of F673L/V mutations in the resistant virus, but these viruses had impaired infectivity (19) which may explain why 4E10 escape variants were not observed in passive immunization studies with this antibody (22, 31). However, these viruses had no other genetic changes in this region, such as those shown here, that might have compensated for this loss of function (19). TM20.5 and TM20.6 showed a 10-fold difference in sensitivity to 4E10. These clones were identical in the ectodomain of gp41 except for two positions (674 and 677) within the 4E10 binding domain. Previous studies have shown that these two residues are dispensable for 4E10 recognition (7, 37). However, we observed that the introduction of mutations N674D/N677K into TM20.5 increased 4E10 sensitivity by 3-fold, suggesting that these positions can influence the presentation of the 4E10 epitope. On the other hand, the TM20.6 clone did not become more resistant to 4E10 when these two positions were changed. Therefore, TM20.6 D674N/K677N which had an identical MPER to TM20.5 showed a 5-fold difference in neutralization sensitivity to 4E10. This was also noted in another study where viruses with identical 4E10 epitopes had different IC50 values (3) suggesting that factors outside the MPER affect accessibility of this epitope. Indeed, substitutions in the HR1 region of gp41 as well as other factors that affect fusion kinetics have been shown to influence sensitivity to this MAb (26, 37). Thus, the observation that TM20.5 showed enhanced infectivity compared to TM20.6 may explain why only TM20.5 was sensitive to changes at positions 674 and 677 in the MPER possibly as a result of a limited window of opportunity for neutralization. We demonstrated that the cytoplasmic tail can modulate sensitivity to neutralization, in agreement with other studies (12, 16, 32, 34). Furthermore, we were able to identify four amino acids in the LLP-2 domain that affect both neutralization and infectivity. Such changes could impact on the amphipathicity of the LLP-2 ?-helix and therefore its membrane association, resulting in phenotypic differences, as observed here and by others (16, 24). It has been shown that deletion of the cytoplasmic tail or changes in this region, in particular the LLP-2, determines the fusogenicity of the envelope glycoprotein (1, 17, 24, 33) which could result in changes in sensitivity to some entry inhibitors (1) and MAbs (16). Abrahamyan and co-workers suggested that the cytoplasmic tail hinders the folding CHAPTER 4: 4E10 NEUTRALIZATION DETERMINANTS 67 of the trimeric coiled-coil into the six-helical bundle resulting in greater inhibition by peptides that target the fusion intermediates (1). The fact that we did not observe changes in sensitivity to T-20, which also targets this conformation, suggests that the mutations in the LLP-2 did not influence this stage of the fusion process. On the other hand, early events may have been affected, such as the differential exposure of neutralizing epitopes in the native structure or, more likely, a faster kinetic rate from the CD4 bound conformation to the trimeric coiled-coil. This may preferentially impact 4E10 neutralization as this antibody is capable of binding in the post-attachment stage (2). In agreement with this, we found that 4E10 neutralization was more affected by changes in the LLP-2 than neutralization by IgG1b12 and CD4-IgG2. Sensitivity to 4E10 neutralization was due to the combined effects of motifs in the MPER and the LLP-2. The two amino acid changes in the MPER and the four amino acid changes in the LLP-2 were sufficient to confer the 4E10 sensitive phenotype to TM20.5. However, engineering the more resistant phenotype appeared to require other sites in addition to these 6 amino acids. The fact that the TM20.6 MPER-5 LLP2-5 mutant showed the same sensitivity to 4E10 as the gp120(6)-gp41(5) construct, neither of which reached IC50 levels shown by TM20.5, suggested that perhaps sites in gp120 were also involved (Figure 5). Interestingly, the two mutations in the TM20.6 MPER, which failed to show any effect on their own, appeared to contribute to 4E10 resistance when co-expressed with the four LLP- 2 mutations that increased infectivity; again highlighting the role that infectivity plays in determining overall neutralization sensitivity. The presence of an anti-MPER neutralization response in this patient?s serum supports the hypothesis that the observed 4E10 resistant envelope quasispecies constituted escape variants. Though the antibodies elicited against the MPER in this patient were not clearly 4E10-like as evidenced by the inability to neutralize C6, their epitopes overlapped, and as for 4E10, the residues F673 and W680 were important for recognition. A recent study with the antibody Z13e1 showed that the mutation D674N, as observed in TM20.5, eliminates antibody binding (23). Therefore, it is also possible that antibodies similar to Z13, where the amino acid at position 674 constitutes a critical residue, might be involved in this anti- MPER neutralization response. CHAPTER 4: 4E10 NEUTRALIZATION DETERMINANTS 68 This study strongly suggests that the MPER is indeed immunogenic and accessible to antibodies that can drive the evolution of the virus toward escape variants. However, such an argument is only demonstrable in the context of a longitudinal study where the evolution of changes in the envelope glycoprotein can be tracked over time and in parallel to the development of the neutralization response. Considering the interest in the MPER as a vaccine target, it is important that the potential of in vivo escape from anti-MPER antibodies be clarified in such longitudinal studies. CHAPTER 4: 4E10 NEUTRALIZATION DETERMINANTS 69 Acknowledgements: We would like to thank Maria Salazar from UAB for her help with the sequences of the HIV-2 mutants and Isaac Choge for his technical support. We also thank James Robinson for providing MAb 7B2, and Dennis Burton and Ralph Pantophlet from the Neutralizing Antibody Consortium (NAC) of the International AIDS Vaccine Initiative (IAVI) for providing MAb 2F5, 4E10 and IgG1b12. Progenics Pharmaceuticals, Inc. and Roche Palo Alto kindly provided the CD4-IgG2 and Enfuvirtide respectively. 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CHAPTER 4: 4E10 NEUTRALIZATION DETERMINANTS 73 Figure legends: FIG 1: Frequency analysis of substitutions in the MPER region of 43 gp41 molecular clones obtained from the TM20 isolate. The substitutions K665S and F673L, associated with 2F5 and 4E10 resistance, respectively, are underlined and bolded. The functional envelope clones corresponding with some of these genotypes are indicated to the left of the sequences. FIG 2: Neutralization of TM20 envelope clones. The three functional clones were tested for neutralization by (A) the MAb 4E10, 2F5 and the entry inhibitor T-20 (Enfuvirtide), and (B) polyclonal antibodies from two broadly cross-reactive HIV-1 positive plasma and autologous contemporaneous sera. The dotted lines indicate 50% neutralization with only those values above the line considered positive FIG 3: Full length amino acid sequences of the functional envelope clones TM20.5, TM20.6 and TM20.13. Variable regions, heptad-repeat domains (HR-1 and HR-2), MPER, membrane spanning domain (MSD) and lentivirus lytic peptides (LLP-1 and LLP-2) are indicated. The mutation F673L in the MPER is underlined. The sensitivity of each of the clones to 2F5 and 4E10 neutralization is indicated with R denoting resistance and S denoting sensitivity. TM20.6 was extremely sensitive to 4E10 neutralization and is indicated with S++. FIG 4: Changes in positions 674 and 677 in the MPER affect 4E10 neutralization. TM20.6, TM20.5 and mutants TM20.6 D674N/K677N and TM20.5 N674D/N677K were tested for their sensitivity to neutralization by 4E10. Neutralization was scored as the antibody concentration required to reduce infectivity by 50% (IC50). The graph represents the mean of three independent experiments. P values are indicated when statistically significant differences between the means were observed in a Mann-Whitney nonparametric t-test analysis. FIG 5: Changes in the cytoplasmic tail affect neutralization sensitivity. Schematic representation of the chimeras, constructed by exchanging gp120 or cytoplasmic tail segments of TM20.5 and TM20.6, and LLP-2 and MPER mutants. All the constructs were tested for 4E10, IgG1b12 and T-20 neutralization. The mean IC50 of three independent experiments are indicated on the right. The IC50 values of the chimeras and mutants were CHAPTER 4: 4E10 NEUTRALIZATION DETERMINANTS 74 compared to the parental clones TM20.5 or TM20.6 and the IC50 ratio shown in each case. Statistically significant differences between means of the parental and chimeric/mutant IC50 values with P values <0.05 and <0.01 in a Mann-Whitney t-test are highlighted in light and dark gray respectively. FIG. 6: Role of gp120 and the cytoplasmic tail on infectivity and envelope incorporation. A) JC53bl-13 cells were infected with equal amounts of p24 (10 ng) of each parental and chimeric Env pseudotyped virus. Infectivity was determined by luciferase expression measured as Relative Light Units (RLU). The bars are colour coded according to the cytoplasmic tail carried by the construct, black for TM20.5 and white for TM20.6. B) Pelleted virions and C) env transfected cells, were lysed and subjected to SDS-PAGE, and visualized by Western blotting with anti-gp120 (D7312), anti-gp41 (7B2) or anti-p24 (D7312) antibodies. D) Schematic representation of the gp120, gp41 ectodomain and cytoplasmic tail encoded by the chimeric constructs. Regions derived from the clone TM20.5 are shaded grey and regions derived from TM20.6 are in white. FIG 7: Anti-MPER neutralization activity present in TM20 serum. The HIV-1 MPER sequences introduced into the 7321A HIV-2 chimeric or mutant viruses used in the neutralization assay are highlighted in grey. The bolded letters represent the sequence of the intact 2F5 and 4E10 epitope. The mutations N671S and T676S in C1Cm and F673L in C1CF/L are underlined. The IC50 titers are indicated on the right with those showing activity highlighted in grey. CHAPTER 4: 4E10 NEUTRALIZATION DETERMINANTS 75 Figure 1 CHAPTER 4: 4E10 NEUTRALIZATION DETERMINANTS 76 CHAPTER 4: 4E10 NEUTRALIZATION DETERMINANTS 77 Figure 3 TM20-5 MKVMGIQRNCPQWWIWGILGFWMLCNVVGNKDLWVTVYYGVPVWKEAKTTLFCASDAKGYEREVHNVWATHACVPTDPSP TM20-6 -------------------------S----------------------------------------------------N- TM20-13 ------------------------------GN----------------------------------------------N- ________V1__________ _________V2___ TM20-5 QEMVLENVTENFNMWNNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLKCSNITKINDTGEMKNCSFNTTTEVRDRKHNQ TM20-6 ---------------D----------V-----------------------R----V------------------------ TM20-13 --------------------------------------------------R----V------------------------ ______________________________ TM20-5 YALFYKLDIVPLSEKSNSSSSSESYRLINCNTSTITQACPKVSFDPIPIHYCTPAGYAILKCNNKTFNGTGPCNNVSTVQ TM20-6 ----------------------------------------------------A--------------------------- TM20-13 -------------------------------------------------------------------------------- _______________V3______________ TM20-5 CTHGIKPVVSTQLLLNGSLAEGEIIIRSKNLSDNAKTIIVHLNKSVPIVCTRPNNNTRTSTRIGPGQAFYATGDIIGDIR TM20-6 ---------------------E--V------------------------------------------------------- TM20-13 ---------------------E--V------------------------------------------------------- ____ ________________V4_______ TM20-5 QAHCNISREDWNKTLDMVERKLKEHFNKTIQFAPSSGGDLEITTHSFNCRGEFFYCNTSGLFNSISNETTTNGTTNGTIT TM20-6 --------------------------------------------------------S--R----T-----P-....---- TM20-13 -------------------------------------------------------------------------------- ___ ____V5___ TM20-5 IPCRIKQIINLWQEVGRAMYAPPIAGKITCNSSITGLLLVRDGGNEEN...DTEIFRPGGGDMRDNWRSELYKYKVVEIK TM20-6 --------------------------N------------S----HT--..KTE--------------------------- TM20-13 ----------M---------------N---K-N------SS---TDNSTKPE--T------------------------- gp120 <--> gp41 Ectodomain ___________________HR1_____ TM20-5 PLGIAPTEARRRVVEREKRAVGIGAVLLGFLGAAGSTMGAASITLTVQARQLLSGIVQQQSNLLKAIEAQQHMLQLTVWG TM20-6 ---------K---------------------------------------------------------------------- TM20-13 ---------K---------------------------------------------------------------------- _________ __________________HR2____ TM20-5 IKQLRARVLAIERYLKDQQLLGIWGCSGKLICTTNVRWNTTWSNRTRDDIWNNLTWMQWDKEIDNYTDTIYRLLEESQNQ TM20-6 -------------------------------------------------------------------------------- TM20-13 ------------------------------------P--D----K---E--------------N--------------I- ___ ___________MPER_________ ___ MSD ->gp41 Cytoplasmic tai l TM20-5 QEINEQELLALDSWKNLWSWFNISNWLWYIRIFIMIVGGLIGLRIIFAVLSIVNRVRQGYSPLSFQTLTPNPRGPDRPGG TM20-6 ---------------------D--K-------------------------------------------N--------L-E TM20-13 --R---------K-N-----LS--------K------------------------------------------------R __________LLP-2_________ TM20-5 IEEEGGEQDRDRSVRLVSGFLALFWDDLRSLCLFSYHRLRDFILVTARVVETLGQRGWETLKYLGSLGQYWGLELKKSAI TM20-6 ----------------------------------C---------------AI----V--L-----------V-------- TM20-13 -----------------------V-----N-------------------------------------------------- __________ LLP-1________ _ 2F5 4E10 TM20-5 SLLDTIAIVVAGGTDRVIEFIQRICRAIRNIPRRIRQGFETALL R S TM20-6 ----------------I--------------------------- R S++ TM20-13 -------------------------------------------- S R CHAPTER 4: 4E10 NEUTRALIZATION DETERMINANTS 78 Figure 4 4E10 0 5 10 15 20 p<0.01 TM20.6 TM20.6 D674N/K667N TM20.5 N674D/N667K TM20.5 CHAPTER 4: 4E10 NEUTRALIZATION DETERMINANTS 79 CHAPTER 4: 4E10 NEUTRALIZATION DETERMINANTS 80 Figure 6 CHAPTER 4: 4E10 NEUTRALIZATION DETERMINANTS 81 Figure 7 CHAPTER 5: DEVELOPMENT OF NEUTRALIZING ANTIBODIES IN HIV-1 INFECTION 82 CHAPTER FIVE NEUTRALIZING ANTIBODY RESPONSES IN ACUTE HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 SUBTYPE C INFECTION Published: Journal of Virology 81(12): 6187-6196 (2007) CHAPTER 5: DEVELOPMENT OF NEUTRALIZING ANTIBODIES IN HIV-1 INFECTION 83 CHAPTER 5: DEVELOPMENT OF NEUTRALIZING ANTIBODIES IN HIV-1 INFECTION 84 CHAPTER 5: DEVELOPMENT OF NEUTRALIZING ANTIBODIES IN HIV-1 INFECTION 85 CHAPTER 5: DEVELOPMENT OF NEUTRALIZING ANTIBODIES IN HIV-1 INFECTION 86 CHAPTER 5: DEVELOPMENT OF NEUTRALIZING ANTIBODIES IN HIV-1 INFECTION 87 CHAPTER 5: DEVELOPMENT OF NEUTRALIZING ANTIBODIES IN HIV-1 INFECTION 88 CHAPTER 5: DEVELOPMENT OF NEUTRALIZING ANTIBODIES IN HIV-1 INFECTION 89 CHAPTER 5: DEVELOPMENT OF NEUTRALIZING ANTIBODIES IN HIV-1 INFECTION 90 CHAPTER 5: DEVELOPMENT OF NEUTRALIZING ANTIBODIES IN HIV-1 INFECTION 91 CHAPTER 5: DEVELOPMENT OF NEUTRALIZING ANTIBODIES IN HIV-1 INFECTION 92 CHAPTER 6: SUMMARIZING DISCUSSION AND CONCLUSIONS 93 CHAPTER SIX SUMMARIZING DISCUSSION AND CONCLUSIONS CHAPTER 6: SUMMARIZING DISCUSSION AND CONCLUSIONS 94 The induction of neutralizing antibodies constitutes a major focus for the development of an HIV-1 vaccine, as it remains the only immune response that has been proven capable of completely blocking virus entry in animal models. Despite the fact that HIV-1 has evolved many strategies to avoid antibody recognition of the conserved regions in the envelope glycoprotein, the existence of a few broadly neutralizing MAbs, such as IgG1b12, 2G12, 2F5 and 4E10, highlights the presence of ?weak spots? that can be targeted for vaccine design. These nMAbs were isolated from subtype B infected patients and their epitopes have mainly been characterized in viruses from this subtype. Given that subtype C viruses are responsible for the majority of the HIV-1 infections worldwide, a growing number of researchers have expanded their interest to include these viruses. In this context, this work focuses on the study of the neutralizing antibody epitopes in the subtype C envelope glycoprotein. During the course of this study several subtype C viruses were characterized for their sensitivity to neutralization by the above mentioned broadly nMAbs. An overview of the neutralization efficacy of these antibodies for viruses from our laboratory is shown in Figure 6.1. It encompasses 26 subtype C envelope clones, including the paediatric viruses described in Chapter Two, the 14 acute infection viruses studied in Chapter Five and five other viruses isolated from chronically infected individuals. In summary, 4E10 constituted the most broadly reactive neutralizing antibody, followed by IgG1b12 that was able to neutralize around fifty percent of the tested viruses. 2G12 and 2F5 neutralized subtype C viruses poorly, in agreement with similar studies by other groups (Binley et al., 2004, Li et al., 2006b). The paucity of broadly nMAbs against some HIV-1 clades calls for further efforts in search of new antibodies. Furthermore, these reagents might point to new potential targets for immunogen design. Given that the existing nMAbs revealed vulnerable structures on the envelope glycoprotein, it is appropriate to speculate that other CHAPTER 6: SUMMARIZING DISCUSSION AND CONCLUSIONS 95 neutralizing antibodies that recognize similar epitopes, on non-subtype B envelopes, might also have broad activity, i.e antibodies that recognize a glycan arrangement or antibodies against the MPER. On the other hand, such antibodies may not be commonly found in HIV-1 infected individuals, as they have all shown extremely unusual features. These extraordinary structural adaptations (Burton et al., 2005) may depend on host genetic elements, such as the germline antibody repertoire or the case of the flexible hinge region of 2G12. Others have suggested that the capacity of the nMAbs 2F5 and 4E10 to recognize the lipid bilayer, as a mechanism to access the MPER, is associated with the polyreactivity of these antibodies (Haynes et al., 2005a). This suggests that B-cells carrying such autoreactive specificities normally would suffer clonal deletion, anergy or receptor editing (Haynes et al., 2005b). If this is indeed the case, it constitutes a major challenge to the field and a call for a better understanding of the B-cell populations where such unusual antibodies originate, as it may inform us how to manipulate the immune system to induce such specificities. However, a recent study showed that anti-HIV nMAbs are not exceptionally polyreactive, challenging the notion that autoantigen mimicry constitutes a mechanism of immunoevasion by HIV-1 (Scherer et al., 2007). In our study, resistance to the 2F5 MAb was explained, in most cases, by substitutions in the K665 residue to serine (S). Interestingly, the motif DKW of the intact 2F5 epitope was present in four viruses that were not sensitive to neutralization by this nMAb (Appendix A). However, all three viruses had a glutamine (Q) residue at position 667, which may explain their resistance to 2F5. Data from previous studies concur with this reasoning, as viruses where position 667 was substituted to Gln or Asp were found to be resistant despite the presence of an intact DKW motif (Binley et al., 2004, Li et al., 2005, Li et al., 2006b). These substitutions may affect the ?-turn conformation recognized by 2F5, as this residue is involved in the formation of one of the three intra-molecular hydrogen bonds formed in CHAPTER 6: SUMMARIZING DISCUSSION AND CONCLUSIONS 96 this epitope (Ofek et al., 2007). While the alanine residue at position 667 is relatively conserved in subtype B strains, this position is more polymorphic amongst subtype C viruses with common substitutions to K, Q, or D. Figure 6.1: Percentage of viruses sensitive to neutralization by the broadly nMAbs IgG1b12, 2G12, 2F5 and 4E10. A total of 26 subtype C viruses from our laboratory were tested against the nMAbs. Neutralization titers and specific antibody epitope sequences for each of the viruses are detailed in Appendix A. The common lack of reactivity of the nMAb 2G12 against subtype C viruses has been attributed to the absence of a glycosylation site at position 295, as reinforced in Figure 6.1 and Appendix A. The results presented in Chapter Three showed that the addition of a glycan at this position did not effectively reconstitute the 2G12 epitope in two subtype C viruses, even when other ancillary glycans were present, suggesting that this epitope may be displayed in a distinct conformation in the context of a subtype C core. Given that a subtype C gp120 structure has not been resolved, we speculate that it may differ from the well-characterized subtype B gp120 core. The following evidence supports this hypothesis; some positively selected sites found in subtype C envelopes are under negative selection in CHAPTER 6: SUMMARIZING DISCUSSION AND CONCLUSIONS 97 subtype B viruses (Travers et al., 2005), some of which map to the C3 region of gp120 (Choisy et al., 2004, Gaschen et al., 2002). This region includes the ?2-helix, which has been reported to have a very conserved amphipathic nature in subtype C viruses in comparison to its more hydrophobic subtype B counterpart (Gnanakaran et al., 2007). This suggests that this helix is more exposed in subtype C envelope glycoproteins. In contrast, the V3 loop of subtype C viruses is very conserved. A different pattern of glycosylation also distinguishes these subtypes (Zhang et al., 2004), such as the differential occurrence of glycosylation sites at positions 295 and 442. Taken together, these observations argue that the envelope glycoproteins of these subtypes may have distinct antigenic properties. This could have resulted from different selection pressures throughout their epidemic history, such as the mode of transmission, host genetics and presence of concomitant communicable diseases in these populations. Accordingly, differential glycan arrangements might affect viral tropism or affinity for molecules involved in virus binding and transport, such as DC-SIGN, mannose binding protein or syndecans (Gallay, 2004, Nguyen and Hildreth, 2003, Sanders et al., 2002a). In our study, the glycosylation mutants did not show apparent changes in viral entry; however, the effect on viral tropism or coreceptor affinity was not assessed. Furthermore, a recent study reported that deletion of a glycan increased macrophage tropism (Dunfee et al., 2007), while another study showed that in vitro generated 2G12 resistant viruses, became more sensitive to neutralization by several mannose-specific lectins (Huskens et al., 2007). Collectively, these observations support the necessity to explore whether the distinct glycosylation patterns and, in general, the differences between envelope glycoproteins of various subtypes could be the result of adaptations to different environmental conditions. All but one of the viruses tested here were sensitive to 4E10, supporting the cumulative evidence that this is the broadest neutralizing antibody available. The one exception was CHAPTER 6: SUMMARIZING DISCUSSION AND CONCLUSIONS 98 clone TM20.13, which was further characterized in the work described in Chapter Four. This study reinforced the importance of the MPER as a vaccine target, as it suggests that neutralizing antibodies to this region can indeed exert pressure on the virus, as evidenced by the appearance of escape variants. Due to the small scope of this study, with only one blood sample from this interesting case, it is not clear how escape from such antibodies occurred and how it affected disease progression. We explored the incidence of anti-MPER antibodies in an HIV-1 subtype C acute infection cohort. As presented in Chapter Five, four of the 14 studied individuals developed anti-MPER antibodies, however, their sera were not able to neutralize multiple heterologous viruses, suggesting that these antibodies were not conferring neutralization breadth. On the other hand, the role of these anti-MPER antibodies in autologous neutralization is unknown, and it will require further study. However, given the conservation of the MPER region, it is unlikely that these antibodies mediate type-specific recognition. The CAPRISA acute HIV-1 infection cohort gave us the opportunity to scrutinize the role of the neutralizing antibody response in natural infection. The work presented in Chapter Five is the beginning of a profound study of the evolution of neutralizing antibody responses in subtype C infection. In here, neutralization of the early autologous virus was detected only after 19 weeks post-infection, despite anti-HIV antibodies being detected as early as two weeks, anti-gp41 antibodies at 5-10 weeks and anti-V3 antibodies at 3-10 weeks post-infection (Alam et al., 2007, Moore et al., 2007). Furthermore, these autologous neutralization responses were highly type-specific with limited cross-reactivity even at two years post-infection (unpublished observations). The study of the specificities associated with this narrow neutralization response has been the subject of a recent study, which suggests that the C3-V4 region is a major target of type-specific autologous neutralizing antibodies (Moore et al., 2007). It is not clear if this finding can be extended CHAPTER 6: SUMMARIZING DISCUSSION AND CONCLUSIONS 99 to other HIV-1 subtypes, therefore, it is important to perform a similar study at least in subtype B infected individuals. Interestingly, in three patients, CD4i antibodies were detected very early in infection. Although the role of these antibodies is not clear, it has been proposed that CD4i antibodies may influence viral pathogenesis by constraining the virus to CD4 dependence. A comprehensive analysis of envelope sequences amplified from single genomes suggests that during acute infection there is no immune pressure on the virus as env diversity follows a Poisson distribution (Keele et al., 2007). Given that the inferred transmitted viruses showed CD4 dependence, no evidence is available to suggest that transmission of early virus replication favours CD4 independent variants. It is a conundrum for the field as to why there is a delay in the production of neutralizing antibodies to HIV-1 and particularly broadly cross-reactive antibodies. While in general the induction of neutralizing antibodies requires time to achieve affinity maturation, in early HIV-1 infection this period is prolonged because of the absence of an efficient T- helper response and a general non-specific immunoactivation (Brenchley et al., 2006). Furthermore, B-cells producing broadly cross-reactive neutralizing antibodies may have to compete with non-neutralizing antibodies to immunodominant epitopes and perhaps with non-neutralizing antibodies to the same epitope (Alam et al., 2007) or even type-specific neutralizing antibodies. While the initial vaccine response has to deal only with the latter, the rapid activation of memory B-cells upon infection is another stumbling block in HIV- vaccine development. Therefore, extensive research is needed into the understanding of the B-cell regulatory pathways that determine their activation, differentiation and establishment of long-lived plasma cells as well as memory B-cells. The continued monitoring of individuals enrolled in the CAPRISA study will allow us to the study the specificities of antibodies conferring cross-reactivity as well as provide useful CHAPTER 6: SUMMARIZING DISCUSSION AND CONCLUSIONS 100 samples from which new nMAbs could be obtained. Further studies will address the question of what constitutes neutralization breadth. While some studies suggest that neutralization breadth in HIV-1 infected patients is due to the presence of very few antibody specificities targeting vulnerable areas (Dhillon et al., 2007, Li et al., 2007) another possibility is that the accumulation of multiple ?type-specific? antibodies confers breadth. This is an important question to address as if the latter is found to be the case, then the induction of a broad neutralizing antibody response will constitute even more of a challenge that is currently appreciated. Furthermore, such data may inform additional strategies to be followed in the design of an HIV vaccine. APPENDICES 101 APPENDIX A Review table of subtype C viruses neutralization by nMAb Neutralization titers and amino acid sequences of the epitopes of broadly nMAb in cloned subtype C envelope gene s ENV clon e IgG1b1 2 <1 IC5 0 IC5 0 29 5 33 2 39 2 33 9 38 6 IC5 0 66 2 66 3 66 4 66 5 66 6 66 7 66 8 IC5 0 67 1 67 2 67 3 67 4 67 5 67 6 67 7 67 8 67 9 68 0 1- 2 Nx(S/T ) Nx(S/T ) Nx(S/T ) Nx(S/T ) Nx(S/T ) E L D K W A S N W F D I T N W L W 2-1 0 RP1.1 2 50. 0 50. 0 VC I NI S NG T NK T NT S 50. 0 A . . R . N N 13. 2 S . . S . . . . . . 10-2 0 RP6. 6 0. 9 50. 0 EC T NI S& NN S ND T NT T 50. 0 A . . N . N S 17. 1 . . . N . . . . . . 20-5 0 RP4. 3 11. 9 50. 0 VC T NI S NR T NN T DT S 50. 0 A . . S . N N 45. 8 . . . S . . K . . . >5 0 COT9. 6 50. 0 50. 0 VC T NI S NT S NR T NT S 50. 0 A . . S . K N 3. 0 S . . . . . K . . . COT6.1 5 3. 4 50. 0 VC T NI S NG T NK T NT S 50. 0 A . N S . Q N 35. 9 S . . S . . . . . . TM7. 9 0. 2 50. 0 VC T NI S NR R NK T NT S 50. 0 A . . S . K N 34. 5 . . . S . S . . . . TM3. 8 50. 0 50. 0 MC T NI S NS T NK T NT S 50. 0 A . . S . K N 21. 6 S . . N . S . . . . TM20.1 3 13. 4 50. 0 VC T NI S NT S NK T NS I 30. 9 A . . . . N N 50. 0 S . L S . S . . . SW7.1 4 50. 0 50. 0 VC T NI S NT S NK T NR T 50. 0 A . . S . N . 2. 1 S . . . . . S . . . Du174.1 5 0. 4 50. 0 VC T NI S NT S NK T NS M 50. 0 A . N . . Q N 9. 0 G . . S . . . . . . Du17 9 18. 3 50. 0 VC T NI S NT T NG T NS S& 50. 0 A . . . . Q N 5. 7 S . . S . . . . . . 525-2- 8 50. 0 50. 0 VC T NI S NT S LT T NS A 50. 0 A . . S . N N 7. 1 . . . G . . K . . . 536-10- 7 50. 0 50. 0 MC T NI S NT S ND T NN T 50. 0 A . . S . Q N 4. 4 S . . S . . . . . . CAP8 6 F 13. 7 50. 0 TC T NI S NT S TE T NE S 50. 0 A . . S . K N 7. 1 . . . . . . . . . . CAP45 G 3 0. 5 50. 0 VC R NI N NT T NR T KW S 50. 0 A . . S . N N 0. 7 . . . N . . . . . . CAP61 F1 0 50. 0 50. 0 EC V NI S NT S NK T NG T 50. 0 A . . . . Q N 18. 8 S . . . . . . . . . CAP63 A 9 24. 7 50. 0 VC A NI S NT S NT T NS T 50. 0 A . . . . Q N 2. 8 S . . N . S H . . . CAP84 3 2 50. 0 50. 0 VC T NI S NT T EK T NL N 50. 0 A . . S . N . 1. 1 . . . S . . K . . . CAP85 9 14. 4 50. 0 VC T NI S NT S NN T NS T 0. 3 A . . . . . N 0. 5 . . . . . . . . . . CAP88 B 5 19. 4 50. 0 VC I NI T NT S IA T NE N 50. 0 A . . S . N N 0. 6 . . . N . . Q . . . CAP206 8 50. 0 50. 0 VC T NL S NT T NT T NE N 50. 0 A . . S . K N 2. 3 . . . . . . K . . . CAP210 E 8 7. 8 50. 0 TC I NI S NT T NE T NS T 50. 0 A . . . . Q . 1. 0 S . . S . S S . . . CAP228 5 1 9. 9 50. 0 VC T NI S NT T NK T NE N 50. 0 A . . S . N N 9. 3 T . . N . S . . . . CAP239 G 3 50. 0 4. 6 NC T NI S NT S NE T NG T 50. 0 A . . S . N . 9. 2 . . . S . . . . . . CAP244 D 3 50. 0 50. 0 VC T NI D NT S NK T NN T 50. 0 A . N S . D . 0. 8 S . . S . . K . . . CAP255 1 6 50. 0 50. 0 NC I NI S NT S DK T NS T 50. 0 A . . S . N N 1. 0 T . . . . . . . . . CAP256 7 C 2. 4 10. 8 NC T NI S NT S EK T NG T 50. 0 A . . S . N . 5. 8 . . . S . S T . . . *Predicted N-linked glycosylation sites are bolded and italicise d **Residues crucial for 2F5 and 4E10 MAb activity are bolded and italicise d & The PNG site is moved one aa downstrea m 2F5 epitopes with intact DWK motif have been showe d 2G12 * 2F5* * 4E10* * APPENDICES 102 APPENDIX B Ethical Clearence GENERAL REFERENCES 103 GENERAL REFERENCES Alam, S. M., Searce, R. M., Parks, R., Plonk, K., Plonk, S. G., Sutherland, L. L., Gorny, M. K., Zolla-Pazner, S., Vanleeuwen, S., Moody, M. A., Xia, S. M., Montefiori, D., Tomaras, G. D., Weinhold, K. J., Karim, S. A., Hicks, C. B., Liao, H. X., Robinson, J., Shaw, G. M. & Haynes, B. (2007) HIV-1 gp41 antibodies that mask membrane proximal region epitopes: antibody binding kinetics, induction and potencial for regulation in acute infection. J. Virol, 82, 115-25. Allan, J. S., Coligan, J. E., Barin, F., McLane, M. F., Sodroski, J. G., Rosen, C. A., Haseltine, W. A., Lee, T. H. & Essex, M. 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