Faculty Bibliography

Genetic and immunologic analyses of epidemiologically-linked HIV transmission enable insights into the impact of immune responses on clinical outcomes. Human vaccine trials and animal studies of HIV-1 infection have suggested immune correlates of protection; however, their role in natural infection in terms of protection from disease progression is mostly unknown. Four HIV-1⁺ Cameroonian individuals, three of them epidemiologically-linked in a polygamous heterosexual relationship and one incidence-matched case, were studied over 15 years for heterologous and cross-neutralizing antibody responses, antibody binding, IgA/IgG levels, antibody-dependent cellular cytotoxicity (ADCC) against cells expressing wild-type or CD4-bound Env, viral evolution, Env epitopes, and host factors including HLA-I alleles. Despite viral infection with related strains, the members of the transmission cluster experienced contrasting clinical outcomes including cases of rapid progression and long-term non-progression in the absence of strongly protective HLA-I or CCR5Δ32 alleles. Slower progression and higher CD4/CD8 ratios were associated with enhanced IgG antibody binding to native Env and stronger V1V2 antibody binding responses in the presence of viruses with residue K169 in V2. ADCC against cells expressing Env in the CD4-bound conformation in combination with low Env-specific IgA/IgG ratios correlated with better clinical outcome. This data set highlights for the first time that V1V2-directed antibody responses and ADCC against cells expressing open, CD4-exposed Env, in the presence of low plasma IgA/IgG ratios, can correlate with clinical outcome in natural infection. These parameters are comparable to the major correlates of protection, identified post-hoc in the RV144 vaccine trial; thus, they may also modulate the rate of clinical progression once infected. The findings illustrate the potential of immune correlate analysis in natural infection to guide vaccine development.

Elucidating the structural basis of antibody (Ab) gene usage and affinity maturation of vaccine-induced Abs can inform the design of immunogens for inducing desired Ab responses in HIV vaccine development. Analyses of monoclonal Abs (mAbs) encoded by the same immunoglobulin genes in different stages of maturation can help to understand the maturation process. We have analyzed four human anti-V3 mAbs with the same VH1-3*01 and VL3-10*01 gene usage. Two mAbs, TA6 and TA7, were developed from a vaccinee in the HIV vaccine phase I trial DP6-001 with a polyvalent DNA prime - protein boost regimen, and two others, 311-11D and 1334, were developed from HIV-infected patients. The somatic hypermutation (SHM) rates in VH of vaccine-induced mAbs are lower than in chronic HIV infection-induced mAbs, while those in VL are comparable. Crystal structures of the antigen-binding fragments (Fabs) in complex with V3 peptides show that these mAbs bind the V3 epitope with a new cradle-binding mode, and that the V3 β-hairpin lies along the antigen-binding groove, which consists of residues from both heavy and light chains. Residues conserved from the germline sequences form specific binding pockets accommodating conserved structural elements of the V3 crown hairpin, predetermining the Ab gene selection, while somatically mutated residues create additional hydrogen bonds, electrostatic interactions, and van der Waals contacts, correlating with an increased binding affinity. Our data provide a unique example of germline sequences determining the primordial antigen-binding sites and SHMs correlating with affinity maturation of Abs induced by vaccine and natural HIV infection.IMPORTANCE Understanding the structural basis of gene usage and affinity maturation for anti-HIV-1 antibodies may help vaccine design and development. Antibodies targeting the highly immunogenic third variable loop (V3) of HIV-1 gp120 provide a unique opportunity for detailed structural investigations. By comparing the sequences and structures of four anti-V3 mAbs at different stages of affinity maturation but of the same V gene usage, two induced by vaccination and another two by chronic infection, we provide a fine example of how germline sequence determines the essential elements for epitope recognition and how affinity maturation improves the antibody's recognition of its epitope.

Major advances in donor identification, antigen probe design, and experimental methods to clone pathogen-specific antibodies have led to an exponential growth in the number of newly characterized broadly neutralizing antibodies (bnAbs) that recognize the HIV-1 envelope glycoprotein. Characterization of these bnAbs has defined new epitopes and novel modes of recognition that can result in potent neutralization of HIV-1. However, the translation of envelope recognition profiles in biophysical assays into an understanding of in vivo activity has lagged behind, and identification of subjects and mAbs with potent antiviral activity has remained reliant on empirical evaluation of neutralization potency and breadth. To begin to address this discrepancy between recombinant protein recognition and virus neutralization, we studied the fine epitope specificity of a panel of CD4-binding site (CD4bs) antibodies to define the molecular recognition features of functionally potent humoral responses targeting the HIV-1 envelope site bound by CD4. Whereas previous studies have used neutralization data and machine-learning methods to provide epitope maps, here, this approach was reversed, demonstrating that simple binding assays of fine epitope specificity can prospectively identify broadly neutralizing CD4bs-specific mAbs. Building on this result, we show that epitope mapping and prediction of neutralization breadth can also be accomplished in the assessment of polyclonal serum responses. Thus, this study identifies a set of CD4bs bnAb signature amino acid residues and demonstrates that sensitivity to mutations at signature positions is sufficient to predict neutralization breadth of polyclonal sera with a high degree of accuracy across cohorts and across clades.

Background: Elucidating the structural basis of antibody (Ab) gene usage and affinity maturation of vaccine-induced Abs can inform the design of immunogens for inducing desired Ab responses in HIV vaccine development. Analyses of monoclonal Abs (mAbs) encoded by the same immunoglobulin genes in different stages of maturation can help to understand the maturation process.
Method(s): We have analyzed four human anti-V3 mAbs with the same VH1-3*01 and VL3-10*01 gene usage. Two mAbs, TA6 and TA7, were developed from a vaccinee in the HIV vaccine phase I trial DP6-001 with a polyvalent DNA prime-protein boost regimen, and two others, 311-11D and 1334, were developed from HIV-infected patients.
Result(s): The somatic hypermutation (SHM) rates in VH of the vaccine-induced mAbs are lower than that of the chronic HIV infection-induced mAbs, while those in VL are comparable. Crystal structures of the antigen-binding fragments (Fabs) in complex with V3 peptides show that these mAbs bind the V3 epitope with a new cradle-binding mode, and the V3 beta hairpin lies along the antigen-binding groove, which consists of residues of both heavy and light chains. Residues conserved from the germline sequences form specific binding pockets accommodating conserved structural elements of the V3 crown hairpin, predetermining the Ab gene selection, while somatically mutated residues create additional hydrogen bonds, electrostatic interactions, and van der Waals contacts, correlating with an increased binding affinity.
Conclusion(s): Our data provide a unique example of germline sequences determining the primordial antigen-binding sites and SHMs correlating with affinity maturation of Abs induced by vaccine and natural HIV infection

Background: HIV-1 infection of four individuals and subsequent superinfection of one of these individuals with related strains enables valuable insight into the impact of natural immune responses and host factors on clinical outcome from comparably "primed" individuals.
Method(s): Four HIV-1+ individuals, epidemiologically linked by their infecting viruses, were studied longitudinally for neutralization, antibody binding, IgA/IgG quantitation and antibody-dependent cellular cytotoxicity (ADCC). Immune responses and viral signatures were analyzed for correlates of protection and clinical parameters.
Result(s): Despite viral infection with related strains, the course of HIV-1+ infection varied greatly between each individual. For example, whereas the two slow progressors exhibited the highest neutralization, ADCC towards CD4-induced Env epitopes, SOSIP and/or V1V2 antibody binding, the two progressors had the highest envelope specific IgA/IgG ratios in plasma. One of the progressors additionally experienced superinfection with a virus that was traced back genetically to one of the slow progressors (linked superinfection). Of interest, superinfection induced immune responses comparable to the linked HIV-1+ individual including Envspecific IgA/IgG ratios, neutralization and antibody binding patterns. In the combined longitudinal data set, ADCC against cells expressing untriggered Env did not correlate with clinical parameters; however both heterologous neutralization and antibody binding against two different V1V2 scaffolded antigens were significantly associated with higher CD4/CD8 ratios.
Conclusion(s): Superinfection with a genetically linked HIV-1 strain induced comparable immune responses as observed in the related singly infected individual. Our longitudinal data set of four epidemiologically linked cases underline the importance of polyfunctional immune responses, including neutralization and V1V2 antibody binding, for beneficial clinical outcomes and possibly even protection of superinfection

Background: Reduced rates of HIV infection in humans and of SIV in macaques were correlated with antibodies (Abs) specific for the V2 region of the viruses' Envelope (Env) glycoprotein. To determine if a similar correlation exists after exposure to SHIV, we analyzed plasma samples from macaques immunized with a prime/boost vaccination strategy similar to RV144 to investigate immune correlates of viral control of SHIVBaL infection.
Method(s): Luminex assays were used to assess binding Abs in prechallenge plasma from 9 rhesus macaques co-immunized with envgag DNA (92TH023 gp160 and SHIV gag) and gp120 proteins (MN and A244) at weeks 0, 4, 12, & 20, followed by intrarectal challenge with SHIVBal at weeks 22 and 24. Antigens included those designed to identify V2 Abs specific for peptides (V2p), for quaternary epitopes (V2q) & for the alpha4beta7 integrin binding site (V2i).
Result(s): Abs from all 9 vaccinated macaques were cross-clade reactive with A, B and C gp120s. Reactivity occurred with a clade E cyclic V2 peptide and with unconstrained V1V2-scaffold proteins where the preferential structure of the V2 C-strand is an alpha-helix recognized by V2p Abs. There were no binding Abs vs. constrained V1V2-scaffold proteins which display the V2 C-strand as a beta-strand. All control monkeys exhibited high viremia, while 5 of 9 immunized animals (55%) displayed tight control of SHIVBaL infection postchallenge. There was a significant inverse correlation between Abs at the time of challenge that bound to unconstrained V1V21086-tags (where the V2 C-strand is preferentially an alpha-helix) and plasma virus load after challenge at weeks 24 ([r=-0.84, p=0.004]) and 35 ([r=-0.71, p=0.04). No other significant immune correlates were identified.
Conclusion(s): Control of SHIV infection correlated inversely with Ab binding to the alpha-helical V2 C-strand suggesting that non-neutralizing V2p Abs mediate tight control of SHIVBaL infection. These data are consistent with the correlates of protection for SIV in macaques and HIV in humans

Background: Experiments in rhesus macaques used DNA and protein co-immunizations with HIV-1 Envs mimicking the RV144 vaccine trial to study the protective effects of vaccine-elicited antibody (Ab) responses, particularly anti-V2 Abs.
Method(s): Nine rhesus macaques were co-immunized with gp160-92TH023 and SIV gag DNA via Gene Gun along with injections of gp120 A244 and gp120 MN proteins formulated in Adjuplex at 0, 4, 12 and 20 weeks. Intrarectal (IR) SHIV-BaL.P4 challenges began two weeks after immunization. SIV gag RNA was quantified in plasma samples, PBMCs and lymph nodes. Plasma Ab responses to HIV-1 envelope (Env) proteins and peptides were detected by ELISA and neutralization against SHIV-BaL was measured by the TZM-bl cell assay.
Result(s): Control animals were infected with peak plasma viral loads (PVL) of 106-107 copies/mL after two IR SHIV challenges. In contrast, PVL was delayed in four of nine immunized animals, peaking at 104-106 copies/mL and was undetectable in the remaining 5 macaques with low sporadic transient blips (controllers). Lymph nodes-and PBMC-associated VL, measured longitudinally, was undetectable suggesting a strong vaccine effect on the virus reservoir. Plasma Ab responses to heterologous gp120 developed in all animals with 50% titers of 104-105, with lower titers against heterologous V3 and V1V2 proteins. Correlates of immunity include an inverse correlation of plasma neutralization titers with SHIV infection (p=0.0357) and the level of anti-V2 Abs was significantly higher to some V2 antigens during immunization (weeks 0-22) in tight controllers compared to infected animals. In contrast to RV144 vaccine studies no IgG3 response was detected against gp120s and the ratio of IgA:IgG Abs titers was comparable between infected animals and controllers.
Conclusion(s): This study in macaques provides further support for the role of vaccine-elicited IgG Abs in control of viremia in blood and tissues with negligible, if any, contribution by plasma IgG3 or IgA Abs

Disease progression among HIV-1-infected individuals varies widely, but the mechanisms underlying this variability remains unknown. Distinct disease outcomes are the consequences of many factors working in concert, including innate and adaptive immune responses, cell-mediated and humoral immunity, and both genetic and phenotypic factors. Current data suggest that these multifaceted aspects in infected individuals should be considered as a whole, rather than as separate unique elements, and that analyses must be performed in greater detail in order to meet the requirements of personalized medicine and guide optimal vaccine design. However, the wide adoption of antiretroviral therapy (ART) influences the implementation of systematic analyses of the HIV-1-infected population. Consequently, fewer data will be available for acquisition in the future, preventing the comprehensive investigations required to elucidate the underpinnings of variability in disease outcome. This review seeks to recapitulate the distinct genotypic and phenotypic features of the immune system, focusing in particular on comparing the surface proteins of immune cells among individuals with different HIV infection outcomes.

The global intensification of ART can lead to increased rates of HIV drug resistance (HIVDR) mutations in treated, and also in ART naive patients. ART naive HIV-1 infected patients from Cameroon were subjected to a multimethod HIVDR analysis using Amplification Refractory Mutation System (ARMS)-PCR, Sanger sequencing, and longitudinal Next-Generation Sequencing (NGS), to determine their mutation profiles for K103N, Y181C, K65R, M184V, and T215F/Y. We processed 66 ART-naive HIV-1+ patients with highly diverse subtypes, underlining the predominance of CRF02_AG and the increasing rate of F2 and other recombinant forms in Cameroon. We compared three resistance testing methods for 5 major mutation sites. Using Sanger sequencing, the overall prevalence of HIVDR mutations was 7.6% (5/66) and included all studied mutations except K65R. Comparing ARMS-PCR with Sanger sequencing as a reference, we obtained a sensitivity of 100% (5/5) and a specificity of 95% (58/61), caused by three false positive calls with ARMS-PCR. For 32/66 samples we obtained NGS data and we observed two additional mismatches made up by minority variants (7%, 18%) that might not be clinically relevant. Longitudinal NGS analyses revealed changes in HIVDR mutations in all five positive subjects that could not be attributed to treatment. In one of these cases, superinfection led to the temporary masking of a resistant virus.HIVDR mutations can be sensitively detected by ARMS-PCR and Sequencing methods with comparable performance. Longitudinal changes in HIVDR mutations have to be considered even in the absence of treatment.