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author:("Alam, S. muir")
1.  Cooperation of B Cell Lineages in Induction of HIV-1-Broadly Neutralizing Antibodies 
Cell  2014;158(3):481-491.
Summary
Development of strategies for induction of HIV-1 broadly neutralizing antibodies (bnAbs) by vaccines is a priority. Determining the steps of bnAb induction in HIV-1-infected individuals who make bnAbs is a key strategy for immunogen design. Here we study the B cell response in a bnAb-producing individual, and report cooperation between two B cell lineages to drive bnAb development. We isolated an autologous virus-neutralizing antibody lineage that targeted an envelope region (loop D) and selected virus escape mutants that resulted in both enhanced bnAb lineage envelope binding and escape mutant neutralization—traits associated with increased B cell antigen drive. Thus, in this individual, two B cell lineages cooperated to induce the development of bnAbs. Design of vaccine immunogens that simultaneously drive both autologous and broadly neutralizing B cell lineages may be important for vaccine-induced recapitulation of events that transpire during the maturation of neutralizing antibodies in HIV-1-infected individuals.
doi:10.1016/j.cell.2014.06.022
PMCID: PMC4150607  PMID: 25065977
2.  Progress in HIV-1 Vaccine Development 
The past two years have seen a number of basic and translational science advances in the quest for development of an effective HIV-1 vaccine. These advances include discovery of new envelope (Env) targets of potentially protective antibodies, demonstration that CD8+ T cells can control HIV-1 infection, development of immunogens to overcome HIV-1 T cell epitope diversity, identification of correlates of transmission risk in an HIV-1 efficacy trial, and mapping the co-evolution of HIV-1 founder Env mutants in infected individuals who develop bnAbs, thereby defining broad neutralizing antibody (bnAb) developmental pathways. Despite these advances, a promising HIV-1 vaccine efficacy trial published in 2013 failed to prevent infection, and the HIV-1 vaccine field is still years away from deployment of an effective vaccine. This review summarizes what some of the scientific advances have been, what roadblocks still remain, and what the most promising approaches are for progress in design of successful HIV-1 vaccine candidates.
doi:10.1016/j.jaci.2014.04.025
PMCID: PMC4133697  PMID: 25117798
HIV-1; vaccine; T cells; B cells; broadly neutralizing antibodies
3.  Polyreactivity and Autoreactivity among HIV-1 Antibodies 
Journal of Virology  2014;89(1):784-798.
ABSTRACT
It is generally acknowledged that human broadly neutralizing antibodies (bNAbs) capable of neutralizing multiple HIV-1 clades are often polyreactive or autoreactive. Whereas polyreactivity or autoreactivity has been proposed to be crucial for neutralization breadth, no systematic, quantitative study of self-reactivity among nonneutralizing HIV-1 Abs (nNAbs) has been performed to determine whether poly- or autoreactivity in bNAbs is a consequence of chronic antigen (Ag) exposure and/or inflammation or a fundamental property of neutralization. Here, we use protein microarrays to assess binding to >9,400 human proteins and find that as a class, bNAbs are significantly more poly- and autoreactive than nNAbs. The poly- and autoreactive property is therefore not due to the infection milieu but rather is associated with neutralization. Our observations are consistent with a role of heteroligation for HIV-1 neutralization and/or structural mimicry of host Ags by conserved HIV-1 neutralization sites. Although bNAbs are more mutated than nNAbs as a group, V(D)J mutation per se does not correlate with poly- and autoreactivity. Infrequent poly- or autoreactivity among nNAbs implies that their dominance in humoral responses is due to the absence of negative control by immune regulation. Interestingly, four of nine bNAbs specific for the HIV-1 CD4 binding site (CD4bs) (VRC01, VRC02, CH106, and CH103) bind human ubiquitin ligase E3A (UBE3A), and UBE3A protein competitively inhibits gp120 binding to the VRC01 bNAb. Among these four bNAbs, avidity for UBE3A was correlated with neutralization breadth. Identification of UBE3A as a self-antigen recognized by CD4bs bNAbs offers a mechanism for the rarity of this bNAb class.
IMPORTANCE Eliciting bNAbs is key for HIV-1 vaccines; most Abs elicited by HIV-1 infection or immunization, however, are strain specific or nonneutralizing, and unsuited for protection. Here, we compare the specificities of bNAbs and nNAbs to demonstrate that bNAbs are significantly more poly- and autoreactive than nNAbs. The strong association of poly- and autoreactivity with bNAbs, but not nNAbs from infected patients, indicates that the infection milieu, chronic inflammation and Ag exposure, CD4 T-cell depletion, etc., alone does not cause poly- and autoreactivity. Instead, these properties are fundamentally linked to neutralization breadth, either by the requirement for heteroligation or the consequence of host mimicry by HIV-1. Indeed, we show that human UBE3A shares an epitope(s) with HIV-1 envelope recognized by four CD4bs bNAbs. The poly- and autoreactivity of bNAbs surely contribute to the rarity of membrane-proximal external region (MPER) and CD4bs bNAbs and identify a roadblock that must be overcome to induce protective vaccines.
doi:10.1128/JVI.02378-14
PMCID: PMC4301171  PMID: 25355869
4.  Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-120 interface 
Nature  2014;515(7525):138-142.
The isolation of human monoclonal antibodies (mAbs) is providing important insights regarding the specificities that underlie broad neutralization of HIV-1 (reviewed in1). Here we report a broad and extremely potent HIV-specific mAb, termed 35O22, which binds novel HIV-1 envelope glycoprotein (Env) epitope. 35O22 neutralized 62% of 181 pseudoviruses with an IC50<50 μg/ml. The median IC50 of neutralized viruses was 0.033 μg/ml, among the most potent thus far described. 35O22 did not bind monomeric forms of Env tested, but did bind the trimeric BG505 SOSIP.664. Mutagenesis and a reconstruction by negative-stain electron microscopy of the Fab in complex with trimer revealed it to bind a conserved epitope, which stretched across gp120 and gp41. The specificity of 35O22 represents a novel site of vulnerability on HIV Env, which serum analysis indicates to be commonly elicited by natural infection. Binding to this new site of vulnerability may thus be an important complement to current mAb-based approaches to immunotherapies, prophylaxis, and vaccine design.
doi:10.1038/nature13601
PMCID: PMC4224615  PMID: 25186731
5.  Enhanced Antibody Responses to an HIV-1 MPER Antigen in Mice Reconstituted with Cultured Lymphocytes 
We have shown that the protective HIV-1 antibody, 2F5, avidly reacts with a conserved mammalian self-antigen, kynureninase, and that the development of B cells specific for the 2F5 epitope is constrained by immunological tolerance. These observations suggest that the capacity to mount antibody responses to the 2F5 epitope is mitigated by tolerance, but such capacity may be latent in the pre-tolerance and/or anergic B cell pools. Here, we use B cell tetramer reagents to track the frequencies of B cells that recognize the HIV-1 2F5 epitope (SP62): in BL/6 mice, SP62-binding transitional B cells that bind this epitope are readily identified in bone marrow but are lost during subsequent development. Unsurprisingly then, immunization with SP62 immunogen does not elicit significant humoral responses in normal BL/6 mice. Reconstitution of Rag1null mice with normal congenic B cells that have matured in vitro, restores the capacity to mount significant serum Ab and germinal center responses to this HIV-1 epitope. These B cell cultures are permissive for the development of autoreactive B cells and support the development of SP62-specific B cell compartments normally lost in 2F5 antibody knockin mice. The recovery of humoral responses to the 2F5/SP62 epitope of HIV-1 by reconstitution with B cells containing forbidden, autoreactive clones provides direct evidence that normal BL/6 mice latently possess the capacity to generate humoral responses to a conserved, neutralizing HIV-1 epitope.
doi:10.4049/jimmunol.1302829
PMCID: PMC4003504  PMID: 24591365
6.  Modulation of Nonneutralizing HIV-1 gp41 Responses by an MHC-Restricted TH Epitope Overlapping Those of Membrane Proximal External Region Broadly Neutralizing Antibodies 
A goal of HIV-1 vaccine development is to elicit broadly neutralizing antibodies (BnAbs), but current immunization strategies fail to induce BnAbs, and for unknown reasons, often induce non-neutralizing Abs instead. To explore potential host genetic contributions controlling Ab responses to the HIV-1 Envelope (Env), we have used congenic strains to identify a critical role for MHC class II restriction in modulating Ab responses to the membrane proximal external region (MPER) of gp41, a key vaccine target. Immunized H-2d-congenic strains had more rapid, sustained, and elevated MPER+ Ab titers than those bearing other haplotypes, regardless of immunogen, adjuvant, or prime/boost regimen used, including formulations designed to provide T-cell help. H-2d restricted MPER+ serum Ab responses depended on CD4 TH interactions with Class II (as revealed in immunized intra-H-2d/b congenic or CD154-/- H-2d strains, and by selective abrogation of MPER re-stimulated, H-2d-restricted primed splenocytes by Class II-blocking Abs), and failed to neutralize HIV-1 in the TZM-b/l neutralization assay, coinciding with lack of specificity for an aspartate residue in the neutralization core of BnAb 2F5. Unexpectedly, H-2d restricted MPER+ responses functionally mapped to a core TH epitope partially overlapping the 2F5/z13/4E10 BnAb epitopes as well as non-neutralizing B-cell/Ab binding residues. We propose that Class II-restriction contributes to the general heterogeneity of non- neutralizing gp41 responses induced by Env. Moreover, the proximity of TH and B-cell epitopes in this restriction may have to be considered in re-designing minimal MPER immunogens aimed at exclusively binding BnAb epitopes and triggering MPER+ BnAbs.
doi:10.4049/jimmunol.1302511
PMCID: PMC3936991  PMID: 24465011
HIV-1; Env; MPER; BnAbs; 2F5; Immunogen
7.  Vaccine-Induced HIV-1 Envelope gp120 Constant Region 1-Specific Antibodies Expose a CD4-Inducible Epitope and Block the Interaction of HIV-1 gp140 with Galactosylceramide 
Journal of Virology  2014;88(16):9406-9417.
ABSTRACT
Mucosal epithelial cell surface galactosylceramide (Galcer) has been postulated to be a receptor for HIV-1 envelope (Env) interactions with mucosal epithelial cells. Disruption of the HIV-1 Env interaction with such alternate receptors could be one strategy to prevent HIV-1 entry through the mucosal barrier. To study antibody modulation of HIV-1 Env-Galcer interactions, we used Galcer-containing liposomes to assess whether natural- and vaccine-induced monoclonal antibodies can block HIV-1 Env binding to Galcer. HIV-1 Env gp140 proteins bound to Galcer liposomes with Kds (dissociation constants) in the nanomolar range. Several HIV-1 ALVAC/AIDSVAX vaccinee-derived monoclonal antibodies (MAbs) specific for the gp120 first constant (C1) region blocked Galcer binding of a transmitted/founder HIV-1 Env gp140. Among the C1-specific MAbs that showed Galcer blocking, the antibody-dependent cellular cytotoxicity-mediating CH38 IgG and its natural IgA isotype were the most potent blocking antibodies. C1-specific IgG monoclonal antibodies that blocked Env binding to Galcer induced upregulation of the gp120 CD4-inducible (CD4i) epitope bound by MAb 17B, demonstrating that a conformational change in gp120 may be required for Galcer blocking. However, the MAb 17B itself did not block Env-Galcer binding, suggesting that the C1 antibody-induced gp120 conformational changes resulted in alteration in a Galcer binding site distant from the CD4i 17B MAb binding site.
IMPORTANCE Galactosyl ceramide, a glycosphingolipid, has been postulated to be a receptor for the HIV-1 envelope glycoprotein (Env) interaction with mucosal epithelial cells. Here, we have mimicked this interaction by using an artificial membrane containing synthetic Galcer and recombinant HIV-1 Env proteins to identify antibodies that would block the HIV-1 Env-Galcer interaction. Our study revealed that a class of vaccine-induced human antibodies potently blocks HIV-1 Env-Galcer binding by perturbing the HIV-1 Env conformation.
doi:10.1128/JVI.01031-14
PMCID: PMC4136246  PMID: 24920809
8.  Vaccine-Induced Env V1–V2 IgG3 Correlates with Lower HIV-1 Infection Risk and Declines Soon After Vaccination 
Science translational medicine  2014;6(228):228ra39.
HIV-1–specific immunoglobulin G (IgG) subclass antibodies bind to distinct cellular Fc receptors. Antibodies of the same epitope specificity but of a different subclass therefore can have different antibody effector functions. The study of IgG subclass profiles between different vaccine regimens used in clinical trials with divergent efficacy outcomes can provide information on the quality of the vaccine-induced B cell response. We show that HIV-1–specific IgG3 distinguished two HIV-1 vaccine efficacy studies (RV144 and VAX003 clinical trials) and correlated with decreased risk of HIV-1 infection in a blinded follow-up case-control study with the RV144 vaccine. HIV-1–specific IgG3 responses were not long-lived, which was consistent with the waning efficacy of the RV144 vaccine. These data suggest that specific vaccine-induced HIV-1 IgG3 should be tested in future studies of immune correlates in HIV-1 vaccine efficacy trials.
doi:10.1126/scitranslmed.3007730
PMCID: PMC4116665  PMID: 24648342
9.  Environmental Factors Influencing Epidemic Cholera 
Cholera outbreak following the earthquake of 2010 in Haiti has reaffirmed that the disease is a major public health threat. Vibrio cholerae is autochthonous to aquatic environment, hence, it cannot be eradicated but hydroclimatology-based prediction and prevention is an achievable goal. Using data from the 1800s, we describe uniqueness in seasonality and mechanism of occurrence of cholera in the epidemic regions of Asia and Latin America. Epidemic regions are located near regional rivers and are characterized by sporadic outbreaks, which are likely to be initiated during episodes of prevailing warm air temperature with low river flows, creating favorable environmental conditions for growth of cholera bacteria. Heavy rainfall, through inundation or breakdown of sanitary infrastructure, accelerates interaction between contaminated water and human activities, resulting in an epidemic. This causal mechanism is markedly different from endemic cholera where tidal intrusion of seawater carrying bacteria from estuary to inland regions, results in outbreaks.
doi:10.4269/ajtmh.12-0721
PMCID: PMC3771306  PMID: 23897993
10.  Chemical synthesis of highly congested gp120 V1V2 N-glycopeptide antigens for potential HIV-1–directed vaccines 
Journal of the American Chemical Society  2013;135(35):10.1021/ja405990z.
Critical to the search for an effective HIV-1 vaccine is the development of immunogens capable of inducing broadly neutralizing antibodies (BnAbs). A key first step in this process is to design immunogens that can be recognized by known BnAbs. The monoclonal antibody PG9 is a BnAb that neutralizes diverse strains of HIV-1 by targeting a conserved carbohydrate-protein epitope in the variable 1 and 2 (V1V2) region of the viral envelope. Important for recognition are two closely spaced N-glycans at Asn160 and Asn156. Glycopeptides containing this synthetically challenging bis-N-glycosylated motif were prepared by convergent assembly, and were shown to be antigenic for PG9. Synthetic glycopeptides such as these may be useful for the development of HIV-1 vaccines based on the envelope V1V2 BnAb epitope.
doi:10.1021/ja405990z
PMCID: PMC3826081  PMID: 23915436
11.  Toll-Like Receptor 7/8 (TLR7/8) and TLR9 Agonists Cooperate To Enhance HIV-1 Envelope Antibody Responses in Rhesus Macaques 
Journal of Virology  2014;88(6):3329-3339.
ABSTRACT
The development of a vaccine that can induce high titers of functional antibodies against HIV-1 remains a high priority. We have developed an adjuvant based on an oil-in-water emulsion that incorporates Toll-like receptor (TLR) ligands to test whether triggering multiple pathogen-associated molecular pattern receptors could enhance immunogenicity. Compared to single TLR agonists or other pairwise combinations, TLR7/8 and TLR9 agonists combined were able to elicit the highest titers of binding, neutralizing, and antibody-dependent cellular cytotoxicity-mediating antibodies against the protein immunogen, transmitted/founder HIV-1 envelope gp140 (B.63521). We further found that the combination of TLR7/8 and TLR9 agonists was associated with the release of CXCL10 (IP-10), suggesting that this adjuvant formulation may have optimally stimulated innate and adaptive immunity to elicit high titers of antibodies.
IMPORTANCE Combining TLR agonists in an adjuvant formulation resulted in higher antibody levels compared to an adjuvant without TLR agonists. Adjuvants that combine TLR agonists may be useful for enhancing antibody responses to HIV-1 vaccines.
doi:10.1128/JVI.03309-13
PMCID: PMC3957956  PMID: 24390332
12.  Induction of HIV-1 broad neutralizing antibodies in 2F5 knockin mice: selection against MPER-associated autoreactivity limits T-dependent responses 
Journal of immunology (Baltimore, Md. : 1950)  2013;191(5):10.4049/jimmunol.1300971.
A goal of HIV-1 vaccine development is to elicit broadly neutralizing antibodies (BnAbs). Using a knock-in (KI) model of 2F5, a human HIV-1 gp41 MPER-specific BnAb, we previously demonstrated that a key obstacle to BnAb induction is clonal deletion of BnAb-expressing B-cells. Here, in this model, we provide a proof-of-principle that robust serum neutralizing IgG responses can be induced from pre-existing, residual self-reactive BnAb-expressing B-cells in vivo, using a structurally compatible gp41 MPER immunogen. Furthermore, in CD40L-deficient 2F5 KI mice, we demonstrate that these BnAb responses are elicited via a type II T-independent pathway, coinciding with expansion and activation of transitional splenic B-cells specific for 2F5's nominal gp41 MPER-binding epitope (containing the 2F5 neutralization domain ELDKWA). In contrast, constitutive production of non-neutralizing serum IgGs in 2F5 KI mice is T-dependent, and originates from a subset of splenic mature B2-cells that have lost their ability to bind 2F5's gp41 MPER epitope. These results suggest that residual, mature B-cells expressing autoreactive BnAbs like 2F5 as BCR, may be limited in their ability to participate in T-dependent responses, by purifying selection that selectively eliminates reactivity for neutralization epitope-containing/mimicked host antigens.
doi:10.4049/jimmunol.1300971
PMCID: PMC3870053  PMID: 23918977
13.  Immunological and Virological Mechanisms of Vaccine-Mediated Protection Against SIV and HIV 
Nature  2013;505(7484):502-508.
Summary
A major challenge for the development of a highly effective AIDS vaccine is the identification of mechanisms of protective immunity. To address this question, we used a non-human primate challenge model with simian immunodeficiency virus (SIV). We show that antibodies to the SIV Envelope are necessary and sufficient to prevent infection. Moreover, sequencing of viruses from breakthrough infections revealed selective pressure against neutralization-sensitive viruses; we identified a two amino acid signature that alters antigenicity and confers neutralization resistance. A similar signature confers resistance of HIV-1 to neutralization by monoclonal antibodies against variable regions 1 and 2 (V1V2), suggesting that SIV and HIV share a fundamental mechanism of immune escape from vaccine- or naturally-elicited antibodies. These analyses provide insight into the limited efficacy seen in HIV vaccine trials.
doi:10.1038/nature12893
PMCID: PMC3946913  PMID: 24352234
14.  HIV-1 Vaccine-Induced C1 and V2 Env-Specific Antibodies Synergize for Increased Antiviral Activities 
Journal of Virology  2014;88(14):7715-7726.
ABSTRACT
The RV144 ALVAC/AIDSVax HIV-1 vaccine clinical trial showed an estimated vaccine efficacy of 31.2%. Viral genetic analysis identified a vaccine-induced site of immune pressure in the HIV-1 envelope (Env) variable region 2 (V2) focused on residue 169, which is included in the epitope recognized by vaccinee-derived V2 monoclonal antibodies. The ALVAC/AIDSVax vaccine induced antibody-dependent cellular cytotoxicity (ADCC) against the Env V2 and constant 1 (C1) regions. In the presence of low IgA Env antibody levels, plasma levels of ADCC activity correlated with lower risk of infection. In this study, we demonstrate that C1 and V2 monoclonal antibodies isolated from RV144 vaccinees synergized for neutralization, infectious virus capture, and ADCC. Importantly, synergy increased the HIV-1 ADCC activity of V2 monoclonal antibody CH58 at concentrations similar to that observed in plasma of RV144 vaccinees. These findings raise the hypothesis that synergy among vaccine-induced antibodies with different epitope specificities contributes to HIV-1 antiviral antibody responses and is important to induce for reduction in the risk of HIV-1 transmission.
IMPORTANCE The Thai RV144 ALVAC/AIDSVax prime-boost vaccine efficacy trial represents the only example of HIV-1 vaccine efficacy in humans to date. Studies aimed at identifying immune correlates involved in the modest vaccine-mediated protection identified HIV-1 envelope (Env) variable region 2-binding antibodies as inversely correlated with infection risk, and genetic analysis identified a site of immune pressure within the region recognized by these antibodies. Despite this evidence, the antiviral mechanisms by which variable region 2-specific antibodies may have contributed to lower rates of infection remain unclear. In this study, we demonstrate that vaccine-induced HIV-1 envelope variable region 2 and constant region 1 antibodies synergize for recognition of virus-infected cells, infectious virion capture, virus neutralization, and antibody-dependent cellular cytotoxicity. This is a major step in understanding how these types of antibodies may have cooperatively contributed to reducing infection risk and should be considered in the context of prospective vaccine design.
doi:10.1128/JVI.00156-14
PMCID: PMC4097802  PMID: 24807721
15.  Structural basis for diverse N-glycan recognition by HIV-1–neutralizing V1–V2–directed antibody PG16 
HIV-1 uses a diverse N-linked-glycan shield to evade recognition by antibody. Select human antibodies, such as the clonally related PG9 and PG16, recognize glycopeptide epitopes in the HIV-1 V1–V2 region and penetrate this shield, but their ability to accommodate diverse glycans is unclear. Here we report the structure of antibody PG16 bound to a scaffolded V1–V2, showing an epitope comprising both high mannose–type and complex-type N-linked glycans. We combined structure, NMR and mutagenesis analyses to characterize glycan recognition by PG9 and PG16. Three PG16-specific residues, arginine, serine and histidine (RSH), were critical for binding sialic acid on complex-type glycans, and introduction of these residues into PG9 produced a chimeric antibody with enhanced HIV-1 neutralization. Although HIV-1–glycan diversity facilitates evasion, antibody somatic diversity can overcome this and can provide clues to guide the design of modified antibodies with enhanced neutralization.
doi:10.1038/nsmb.2600
PMCID: PMC4046252  PMID: 23708607
16.  TCR Affinity Associated with Functional Differences between Dominant and Subdominant SIV Epitope-Specific CD8+ T Cells in Mamu-A*01+ Rhesus Monkeys 
PLoS Pathogens  2014;10(4):e1004069.
Many of the factors that contribute to CD8+ T cell immunodominance hierarchies during viral infection are known. However, the functional differences that exist between dominant and subdominant epitope-specific CD8+ T cells remain poorly understood. In this study, we characterized the phenotypic and functional differences between dominant and subdominant simian immunodeficiency virus (SIV) epitope-specific CD8+ T cells restricted by the major histocompatibility complex (MHC) class I allele Mamu-A*01 during acute and chronic SIV infection. Whole genome expression analyses during acute infection revealed that dominant SIV epitope-specific CD8+ T cells had a gene expression profile consistent with greater maturity and higher cytotoxic potential than subdominant epitope-specific CD8+ T cells. Flow-cytometric measurements of protein expression and anti-viral functionality during chronic infection confirmed these phenotypic and functional differences. Expression analyses of exhaustion-associated genes indicated that LAG-3 and CTLA-4 were more highly expressed in the dominant epitope-specific cells during acute SIV infection. Interestingly, only LAG-3 expression remained high during chronic infection in dominant epitope-specific cells. We also explored the binding interaction between peptide:MHC (pMHC) complexes and their cognate TCRs to determine their role in the establishment of immunodominance hierarchies. We found that epitope dominance was associated with higher TCR:pMHC affinity. These studies demonstrate that significant functional differences exist between dominant and subdominant epitope-specific CD8+ T cells within MHC-restricted immunodominance hierarchies and suggest that TCR:pMHC affinity may play an important role in determining the frequency and functionality of these cell populations. These findings advance our understanding of the regulation of T cell immunodominance and will aid HIV vaccine design.
Author Summary
MHC-restricted CD8+ T cell populations that bind viral proteins are often present at different frequencies. It is thought that those virus-specific CD8+ T cells that are present at the highest frequency are predominantly responsible for eliciting control of viral infections. While the number of virus-specific CD8+ T cells is undoubtedly important, the functionality of these cells may also play an anti-viral role. It is not known if high-frequency virus-specific CD8+ T cells are more functionally effective against viral infection than those present at low frequencies. In this study, we characterized the functional differences between the SIV-specific cells present at high versus low frequencies in rhesus monkeys infected with simian immunodeficiency virus (SIV). We found that the high- and low-frequency SIV-specific cells had different functional capacities during acute and chronic SIV infection. We also found that the affinity with which a cell interacts with viral proteins may contribute to these functional differences. These findings further our understanding of anti-viral immune responses and may help to inform HIV vaccine development.
doi:10.1371/journal.ppat.1004069
PMCID: PMC3990730  PMID: 24743648
17.  IGHV1-69 B Cell Chronic Lymphocytic Leukemia Antibodies Cross-React with HIV-1 and Hepatitis C Virus Antigens as Well as Intestinal Commensal Bacteria 
PLoS ONE  2014;9(3):e90725.
B-cell chronic lymphocytic leukemia (B-CLL) patients expressing unmutated immunoglobulin heavy variable regions (IGHVs) use the IGHV1-69 B cell receptor (BCR) in 25% of cases. Since HIV-1 envelope gp41 antibodies also frequently use IGHV1-69 gene segments, we hypothesized that IGHV1-69 B-CLL precursors may contribute to the gp41 B cell response during HIV-1 infection. To test this hypothesis, we rescued 5 IGHV1-69 unmutated antibodies as heterohybridoma IgM paraproteins and as recombinant IgG1 antibodies from B-CLL patients, determined their antigenic specificities and analyzed BCR sequences. IGHV1-69 B-CLL antibodies were enriched for reactivity with HIV-1 envelope gp41, influenza, hepatitis C virus E2 protein and intestinal commensal bacteria. These IGHV1-69 B-CLL antibodies preferentially used IGHD3 and IGHJ6 gene segments and had long heavy chain complementary determining region 3s (HCDR3s) (≥21 aa). IGHV1-69 B-CLL BCRs exhibited a phenylalanine at position 54 (F54) of the HCDR2 as do rare HIV-1 gp41 and influenza hemagglutinin stem neutralizing antibodies, while IGHV1-69 gp41 antibodies induced by HIV-1 infection predominantly used leucine (L54) allelic variants. These results demonstrate that the B-CLL cell population is an expansion of members of the innate polyreactive B cell repertoire with reactivity to a number of infectious agent antigens including intestinal commensal bacteria. The B-CLL IGHV1-69 B cell usage of F54 allelic variants strongly suggests that IGHV1-69 B-CLL gp41 antibodies derive from a restricted B cell pool that also produces rare HIV-1 gp41 and influenza hemagglutinin stem antibodies.
doi:10.1371/journal.pone.0090725
PMCID: PMC3948690  PMID: 24614505
18.  Characterization of Host-Cell Line Specific Glycosylation Profiles of Early Transmitted/Founder HIV-1 gp120 Envelope Proteins 
Journal of proteome research  2013;12(3):1223-1234.
Glycosylation plays an essential role in regulating protein function by modulating biological, structural, and therapeutic properties. However, due to its inherent heterogeneity and diversity, the comprehensive analysis of protein glycosylation remains a challenge. As part of our continuing effort in the analysis of glycosylation profiles of recombinant HIV-1 envelope-based immunogens, we evaluated and compared the host-cell specific glycosylation pattern of recombinant HIV-1 surface glycoprotein, gp120, derived from clade C transmitted/founder virus 1086.C expressed in Chinese hamster ovary (CHO) and human embryonic kidney containing T antigen (293T) cell lines. We used an integrated glycopeptide-based mass mapping workflow that includes a partial deglycosylation step described in our previous study1 with the inclusion of the fragmentation technique, electron transfer dissociation (ETD), to complement collision induced dissociation (CID). The inclusion of ETD facilitated the analysis by providing additional validation for glycopeptide identification and expanding the identified glycopeptides to include coverage of O-linked glycosylation. The site-specific glycosylation analysis shows that the transmitted/founder 1086.C gp120 expressed in CHO and 293T displayed distinct similarities and differences. For N-linked glycosylation, two sites (N386 and N392), in the V4 region were populated with high mannose glycans in the CHO cell-derived 1086.C gp120, while these sites had a mixture of high mannose and processed glycans in the 293T cell-derived 1086.C gp120. Compositional analysis of O-linked glycans revealed that 293T cell-derived 1086.C gp120 consisted of cores 1, 2, and 4 type O-linked glycans while CHO cell-derived 1086.C exclusively consisted of core 1 type O-linked glycans. Overall, glycosylation site occupancy of the CHO and 293T cell-derived 1086.C gp120 show high degree of similarity except for one site at N88 in the C1 region. This site was partially occupied in 293T-gp120 but fully occupied in CHO-gp120. Site-specific glycopeptide analysis of transmitted/founder 1086.C gp120 expressed in CHO cells revealed the presence of phosphorylated glycans while 293T cell produced 1086.C gp120 glycans were not phosphorylated. While the influence of phosphorylated glycans on immunogenicity is unclear, distinguishing host-cell specific variations in glycosylation profiles provides insights into the similarity (or difference) in recombinant vaccine products. While these differences had minimal effect on envelope antigenicity, they may be important in considering immunogenicity and functional capacities of recombinant envelope proteins produced in different expression systems.
doi:10.1021/pr300870t
PMCID: PMC3674872  PMID: 23339644
19.  Estimating the Probability of Polyreactive Antibodies 4E10 and 2F5 Disabling a gp41 Trimer after T Cell-HIV Adhesion 
PLoS Computational Biology  2014;10(1):e1003431.
A few broadly neutralizing antibodies, isolated from HIV-1 infected individuals, recognize epitopes in the membrane proximal external region (MPER) of gp41 that are transiently exposed during viral entry. The best characterized, 4E10 and 2F5, are polyreactive, binding to the viral membrane and their epitopes in the MPER. We present a model to calculate, for any antibody concentration, the probability that during the pre-hairpin intermediate, the transient period when the epitopes are first exposed, a bound antibody will disable a trivalent gp41 before fusion is complete. When 4E10 or 2F5 bind to the MPER, a conformational change is induced that results in a stably bound complex. The model predicts that for these antibodies to be effective at neutralization, the time to disable an epitope must be shorter than the time the antibody remains bound in this conformation, about five minutes or less for 4E10 and 2F5. We investigate the role of avidity in neutralization and show that 2F5 IgG, but not 4E10, is much more effective at neutralization than its Fab fragment. We attribute this to 2F5 interacting more stably than 4E10 with the viral membrane. We use the model to elucidate the parameters that determine the ability of these antibodies to disable epitopes and propose an extension of the model to analyze neutralization data. The extended model predicts the dependencies of for neutralization on the rate constants that characterize antibody binding, the rate of fusion of gp41, and the number of gp41 bridging the virus and target cell at the start of the pre-hairpin intermediate. Analysis of neutralization experiments indicate that only a small number of gp41 bridges must be disabled to prevent fusion. However, the model cannot determine the exact number from neutralization experiments alone.
Author Summary
Most people who become infected with HIV generate a strong antibody response to the infecting virus population. Unfortunately, the protection offered by the antibody is short lived as the virus rapidly mutates and renders the antibodies impotent in preventing further infection. There are a few antibodies, however, that have been isolated from infected individuals that can block infection by many different viral strains. Among these are several that target sites on the HIV that are exposed only after the virus has attached to a cell. These antibodies have a brief window of time to prevent fusion of the virus and cell. They are special in that they bind both to the viral membrane and to sequences on the gp41 protein that lie along the viral surface. Here, we present a model that predicts the concentrations at which these antibodies effectively neutralize the virus. The model tells us what properties of antibody binding are key in determining efficient neutralization and what properties have little influence. A prediction of the model is that in a standard neutralization assay there are only a small number of attachments between virus and cell and disabling these is sufficient to prevent infection.
doi:10.1371/journal.pcbi.1003431
PMCID: PMC3907291  PMID: 24499928
20.  Vaccine Induction of Antibodies Against a Structurally Heterogeneous Site of Immune Pressure within HIV-1 Envelope Protein Variable Regions 1 and 2 
Immunity  2013;38(1):176-186.
Summary
The RV144 HIV-1 trial of the canary pox vector (ALVAC-HIV) plus the gp120 AIDSVAX B/E vaccine demonstrated an estimated efficacy of 31%, that correlated directly with antibodies to HIV-1 envelope variable regions 1 and 2 (V1–V2). Genetic analysis of trial viruses revealed increased vaccine efficacy against viruses matching the vaccine strain at V2 residue 169. Here, we isolated four V2 monoclonal antibodies from RV144 vaccinees that recognize residue 169, neutralize laboratory-adapted HIV-1, and mediate killing of field isolate HIV-1-infected CD4+ T cells. Crystal structures of two of the V2 antibodies demonstrated residue 169 can exist within divergent helical and loop conformations, which contrasted dramatically with the beta strand conformation previously observed with a broadly neutralizing antibody PG9. Thus, RV144 vaccine-induced immune pressure appears to target a region that may be both sequence variable and structurally polymorphic. Variation may signal sites of HIV-1 envelope vulnerability, providing vaccine designers with new options.
doi:10.1016/j.immuni.2012.11.011
PMCID: PMC3569735  PMID: 23313589
21.  Reconstructing a B-Cell Clonal Lineage. II. Mutation, Selection, and Affinity Maturation 
Affinity maturation of the antibody response is a fundamental process in adaptive immunity during which B-cells activated by infection or vaccination undergo rapid proliferation accompanied by the acquisition of point mutations in their rearranged immunoglobulin (Ig) genes and selection for increased affinity for the eliciting antigen. The rate of somatic hypermutation at any position within an Ig gene is known to depend strongly on the local DNA sequence, and Ig genes have region-specific codon biases that influence the local mutation rate within the gene resulting in increased differential mutability in the regions that encode the antigen-binding domains. We have isolated a set of clonally related natural Ig heavy chain–light chain pairs from an experimentally infected influenza patient, inferred the unmutated ancestral rearrangements and the maturation intermediates, and synthesized all the antibodies using recombinant methods. The lineage exhibits a remarkably uniform rate of improvement of the effective affinity to influenza hemagglutinin (HA) over evolutionary time, increasing 1000-fold overall from the unmutated ancestor to the best of the observed antibodies. Furthermore, analysis of selection reveals that selection and mutation bias were concordant even at the level of maturation to a single antigen. Substantial improvement in affinity to HA occurred along mutationally preferred paths in sequence space and was thus strongly facilitated by the underlying local codon biases.
doi:10.3389/fimmu.2014.00170
PMCID: PMC4001017  PMID: 24795717
somatic hypermutation; experimental influenza infection; antibody selection; antibody affinity maturation; phylogenetics
22.  In Response 
doi:10.4269/ajtmh.13-0499b
PMCID: PMC3854909  PMID: 24306034
23.  HIV-1 antibodies from infection and vaccination: insights for guiding vaccine design 
Trends in microbiology  2012;20(11):532-539.
Attempts to formulate a protective HIV-1 vaccine through classic vaccine design strategies have not been successful. Elicitation of HIV-1-specific broadly neutralizing antibodies (bnAbs) at high titers that are present before exposure might be required to achieve protection. Recently, the application of new technologies has facilitated the study of clonal lineages of HIV-1 envelope (Env) antibodies, which have provided insights into HIV-1 antibody development during infection and upon vaccination. Strategies are being developed for the analysis of infection and vaccine candidate-induced antibodies, their gene usage, and their maturation pathways such that this information can be used to attempt to guide rational vaccine design.
doi:10.1016/j.tim.2012.08.011
PMCID: PMC3757512  PMID: 22981828
HIV-1 vaccine; broadly neutralizing antibodies; immunogen design
24.  Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus 
Nature  2013;496(7446):469-476.
Current HIV-1 vaccines elicit strain-specific neutralizing antibodies. However, cross-reactive neutralizing antibodies arise in ~20% of HIV-1-infected individuals, and details of their generation could provide a roadmap for effective vaccination. Here we report the isolation, evolution and structure of a broadly neutralizing antibody from an African donor followed from time of infection. The mature antibody, CH103, neutralized ~55% of HIV-1 isolates, and its co-crystal structure with gp120 revealed a novel loop-based mechanism of CD4-binding site recognition. Virus and antibody gene sequencing revealed concomitant virus evolution and antibody maturation. Notably, the CH103-lineage unmutated common ancestor avidly bound the transmitted/founder HIV-1 envelope glycoprotein, and evolution of antibody neutralization breadth was preceded by extensive viral diversification in and near the CH103 epitope. These data elucidate the viral and antibody evolution leading to induction of a lineage of HIV-1 broadly neutralizing antibodies and provide insights into strategies to elicit similar antibodies via vaccination.
doi:10.1038/nature12053
PMCID: PMC3637846  PMID: 23552890
25.  Antigenicity and Immunogenicity of Transmitted/Founder, Consensus, and Chronic Envelope Glycoproteins of Human Immunodeficiency Virus Type 1 
Journal of Virology  2013;87(8):4185-4201.
Human immunodeficiency virus type 1 (HIV-1) vaccine development requires selection of appropriate envelope (Env) immunogens. Twenty HIV-1 Env glycoproteins were examined for their ability to bind human anti-HIV-1 monoclonal antibodies (MAbs) and then used as immunogens in guinea pigs to identify promising immunogens. These included five Envs derived from chronically infected individuals, each representing one of five common clades and eight consensus Envs based on these five clades, as well as the consensus of the entire HIV-1 M group, and seven transmitted/founder (T/F) Envs from clades B and C. Sera from immunized guinea pigs were tested for neutralizing activity using 36 HIV-1 Env-pseudotyped viruses. All Envs bound to CD4 binding site, membrane-proximal, and V1/V2 MAbs with similar apparent affinities, although the T/F Envs bound with higher affinity to the MAb 17b, a CCR5 coreceptor binding site antibody. However, the various Envs differed in their ability to induce neutralizing antibodies. Consensus Envs elicited the most potent responses, but neutralized only a subset of viruses, including mostly easy-to-neutralize tier 1 and some more-difficult-to-neutralize tier 2 viruses. T/F Envs elicited fewer potent neutralizing antibodies but exhibited greater breadth than chronic or consensus Envs. Finally, chronic Envs elicited the lowest level and most limited breadth of neutralizing antibodies overall. Thus, each group of Env immunogens elicited a different antibody response profile. The complementary benefits of consensus and T/F Env immunogens raise the possibility that vaccines utilizing a combination of consensus and T/F Envs may be able to induce neutralizing responses with greater breadth and potency than single Env immunogens.
doi:10.1128/JVI.02297-12
PMCID: PMC3624376  PMID: 23365441

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