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1.  Immune-Correlates Analysis of an HIV-1 Vaccine Efficacy Trial Reveals an Association of Nonspecific Interferon-γ Secretion with Increased HIV-1 Infection Risk: A Cohort-Based Modeling Study 
PLoS ONE  2014;9(11):e108631.
Background
Elevated risk of HIV-1 infection among recipients of an adenovirus serotype 5 (Ad5)-vectored HIV-1 vaccine was previously reported in the Step HIV-1 vaccine efficacy trial. We assessed pre-infection cellular immune responses measured at 4 weeks after the second vaccination to determine their roles in HIV-1 infection susceptibility among Step study male participants.
Methods
We examined ex vivo interferon-γ (IFN-γ) secretion from peripheral blood mononuclear cells (PBMC) using an ELISpot assay in 112 HIV-infected and 962 uninfected participants. In addition, we performed flow cytometric assays to examine T-cell activation, and ex vivo IFN-γ and interleukin-2 secretion from CD4+ and CD8+ T cells. We accounted for the sub-sampling design in Cox proportional hazards models to estimate hazard ratios (HRs) of HIV-1 infection per 1-loge increase of the immune responses.
Findings
We found that HIV-specific immune responses were not associated with risk of HIV-1 infection. However, each 1-loge increase of mock responses measured by the ELISpot assay (i.e., IFN-γ secretion in the absence of antigen-specific stimulation) was associated with a 62% increase of HIV-1 infection risk among vaccine recipients (HR = 1.62, 95% CI: (1.28, 2.04), p<0.001). This association remains after accounting for CD4+ or CD8+ T-cell activation. We observed a moderate correlation between ELISpot mock responses and CD4+ T-cells secreting IFN-γ (ρ = 0.33, p = 0.007). In addition, the effect of the Step vaccine on infection risk appeared to vary with ELISpot mock response levels, especially among participants who had pre-existing anti-Ad5 antibodies (interaction p = 0.04).
Conclusions
The proportion of cells, likely CD4+ T-cells, producing IFN-γ without stimulation by exogenous antigen appears to carry information beyond T-cell activation and baseline characteristics that predict risk of HIV-1 infection. These results motivate additional investigation to understand the potential link between IFN-γ secretion and underlying causes of elevated HIV-1 infection risk among vaccine recipients in the Step study.
doi:10.1371/journal.pone.0108631
PMCID: PMC4219669  PMID: 25369172
2.  Vaccine-Induced Gag-Specific T Cells Are Associated With Reduced Viremia After HIV-1 Infection 
The Journal of Infectious Diseases  2013;208(8):1231-1239.
The contribution of host T-cell immunity and HLA class I alleles to the control of human immunodeficiency virus (HIV-1) replication in natural infection is widely recognized. We assessed whether vaccine-induced T-cell immunity, or expression of certain HLA alleles, impacted HIV-1 control after infection in the Step MRKAd5/HIV-1 gag/pol/nef study. Vaccine-induced T cells were associated with reduced plasma viremia, with subjects targeting ≥3 gag peptides presenting with half-log lower mean viral loads than subjects without Gag responses. This effect was stronger in participants infected proximal to vaccination and was independent of our observed association of HLA-B*27, –B*57 and –B*58:01 alleles with lower HIV-1 viremia. These findings support the ability of vaccine-induced T-cell responses to influence postinfection outcome and provide a rationale for the generation of T-cell responses by vaccination to reduce viremia if protection from acquisition is not achieved. Clinical trials identifier: NCT00095576.
doi:10.1093/infdis/jit322
PMCID: PMC3778967  PMID: 23878319
HIV-1 vaccine; Step study; Gag-specific T cells; HLA class I alleles
3.  Establishment and maintenance of a PBMC repository for functional cellular studies in support of clinical vaccine trials 
A large repository of cryopreserved peripheral blood mononuclear cells (PBMCs) samples was created to provide laboratories testing the specimens from human immunodeficiency virus-1 (HIV-1) vaccine clinical trials the material for assay development, optimization, and validation. One hundred thirty-one PBMC samples were collected using leukapheresis procedure between 2007 and 2013 by the Comprehensive T cell Vaccine Immune Monitoring Consortium core repository. The donors included 83 human immunodeficiency virus-1 (HIV-1) seronegative and 32 HIV-1 seropositive subjects. The samples were extensively characterized for the ability of T cell subsets to respond to recall viral antigens including cytomegalovirus, Epstein– Barr virus, influenza virus, and HIV-1 using Interferon-gamma (IFN-γ) enzyme linked immunospot (ELISpot) and IFN-γ/interleukin 2 (IL-2) intracellular cytokine staining (ICS) assays. A subset of samples was evaluated over time to determine the integrity of the cryopreserved samples in relation to recovery, viability, and functionality. The principal results of our study demonstrate that viable and functional cells were consistently recovered from the cryopreserved samples. Therefore, we determined that this repository of large size cryopreserved cellular samples constitutes a unique resource for laboratories that are involved in optimization and validation of assays to evaluate T, B, and NK cellular functions in the context of clinical trials.
doi:10.1016/j.jim.2014.04.005
PMCID: PMC4152919  PMID: 24787274
Peripheral blood mononuclear cells; Cryopreservation; Repository
4.  Optimization and qualification of a memory B-cell ELISpot for the detection of vaccine-induced memory responses in HIV vaccine trials 
Journal of immunological methods  2013;394(0):84-93.
Various aspects of the human immune system can be analyzed to determine the efficacy of a vaccine. We have developed a B-cell ELISpot to measure HIV-specific antibody-secreting B cells in the peripheral blood as a result of vaccination or natural infection. Our method includes stimulating peripheral blood mononuclear cells with interleukin-2 and a polyclonal activator, R848, to induce memory B cells to differentiate into antibody-secreting cells. Total immunoglobulin-secreting as well as antigen-specific B cells are then quantified. We have tested several HIV Env gp120 and gp140 proteins from different HIV subtypes, as well as a sensitive consensus group M Env gp140. Our findings indicate that the B-cell ELISpot provides a sensitive and specific tool to detect antigen-specific memory B-cell responses, and it is equally suited to detect antibody-secreting plasmablasts present in the circulation shortly after infection or vaccination.
doi:10.1016/j.jim.2013.05.007
PMCID: PMC3736720  PMID: 23707324
B-cell ELISpot; Antibody-secreting cells; Memory B-cell responses; Vaccine; HIV; Mucosal responses
5.  FCGR2C polymorphisms associate with HIV-1 vaccine protection in RV144 trial 
The Journal of Clinical Investigation  2014;124(9):3879-3890.
The phase III RV144 HIV-1 vaccine trial estimated vaccine efficacy (VE) to be 31.2%. This trial demonstrated that the presence of HIV-1–specific IgG-binding Abs to envelope (Env) V1V2 inversely correlated with infection risk, while the presence of Env-specific plasma IgA Abs directly correlated with risk of HIV-1 infection. Moreover, Ab-dependent cellular cytotoxicity responses inversely correlated with risk of infection in vaccine recipients with low IgA; therefore, we hypothesized that vaccine-induced Fc receptor–mediated (FcR-mediated) Ab function is indicative of vaccine protection. We sequenced exons and surrounding areas of FcR-encoding genes and found one FCGR2C tag SNP (rs114945036) that associated with VE against HIV-1 subtype CRF01_AE, with lysine at position 169 (169K) in the V2 loop (CRF01_AE 169K). Individuals carrying CC in this SNP had an estimated VE of 15%, while individuals carrying CT or TT exhibited a VE of 91%. Furthermore, the rs114945036 SNP was highly associated with 3 other FCGR2C SNPs (rs138747765, rs78603008, and rs373013207). Env-specific IgG and IgG3 Abs, IgG avidity, and neutralizing Abs inversely correlated with CRF01_AE 169K HIV-1 infection risk in the CT- or TT-carrying vaccine recipients only. These data suggest a potent role of Fc-γ receptors and Fc-mediated Ab function in conferring protection from transmission risk in the RV144 VE trial.
doi:10.1172/JCI75539
PMCID: PMC4151214  PMID: 25105367
6.  Vaccine-elicited human T cells recognizing conserved protein regions inhibit HIV-1 
Virus diversity and escape from immune responses are the biggest challenges to the development of an effective vaccine against HIV-1. We hypothesized that T-cell vaccines targeting the most conserved regions of the HIV-1 proteome, which are common to most variants and bear fitness costs when mutated, will generate effectors that efficiently recognize and kill virus-infected cells early enough after transmission to potentially impact on HIV-1 replication and will do so more efficiently than whole protein-based T-cell vaccines. Here, we describe the first-ever administration of conserved immunogen vaccines vectored using prime-boost regimens of DNA, simian adenovirus and modified vaccinia virus Ankara to uninfected UK volunteers. The vaccine induced high levels of effector T cells that recognized virus-infected autologous CD4+ cells and inhibited HIV-1 replication by up to 5.79 log10. The virus inhibition was mediated by both Gag- and Pol- specific effector CD8+ T cells targeting epitopes that are typically subdominant in natural infection. These results provide proof of concept for using a vaccine to target T cells at conserved epitopes, showing that these T cells can control HIV-1 replication in vitro.
doi:10.1038/mt.2013.248
PMCID: PMC3911893  PMID: 24166483
7.  Analysis of HLA A*02 Association with Vaccine Efficacy in the RV144 HIV-1 Vaccine Trial 
Journal of Virology  2014;88(15):8242-8255.
ABSTRACT
The RV144 HIV-1 vaccine trial demonstrated partial efficacy of 31% against HIV-1 infection. Studies into possible correlates of protection found that antibodies specific to the V1 and V2 (V1/V2) region of envelope correlated inversely with infection risk and that viruses isolated from trial participants contained genetic signatures of vaccine-induced pressure in the V1/V2 region. We explored the hypothesis that the genetic signatures in V1 and V2 could be partly attributed to selection by vaccine-primed T cells. We performed a T-cell-based sieve analysis of breakthrough viruses in the RV144 trial and found evidence of predicted HLA binding escape that was greater in vaccine versus placebo recipients. The predicted escape depended on class I HLA A*02- and A*11-restricted epitopes in the MN strain rgp120 vaccine immunogen. Though we hypothesized that this was indicative of postacquisition selection pressure, we also found that vaccine efficacy (VE) was greater in A*02-positive (A*02+) participants than in A*02− participants (VE = 54% versus 3%, P = 0.05). Vaccine efficacy against viruses with a lysine residue at site 169, important to antibody binding and implicated in vaccine-induced immune pressure, was also greater in A*02+ participants (VE = 74% versus 15%, P = 0.02). Additionally, a reanalysis of vaccine-induced immune responses that focused on those that were shown to correlate with infection risk suggested that the humoral responses may have differed in A*02+ participants. These exploratory and hypothesis-generating analyses indicate there may be an association between a class I HLA allele and vaccine efficacy, highlighting the importance of considering HLA alleles and host immune genetics in HIV vaccine trials.
IMPORTANCE The RV144 trial was the first to show efficacy against HIV-1 infection. Subsequently, much effort has been directed toward understanding the mechanisms of protection. Here, we conducted a T-cell-based sieve analysis, which compared the genetic sequences of viruses isolated from infected vaccine and placebo recipients. Though we hypothesized that the observed sieve effect indicated postacquisition T-cell selection, we also found that vaccine efficacy was greater for participants who expressed HLA A*02, an allele implicated in the sieve analysis. Though HLA alleles have been associated with disease progression and viral load in HIV-1 infection, these data are the first to suggest the association of a class I HLA allele and vaccine efficacy. While these statistical analyses do not provide mechanistic evidence of protection in RV144, they generate testable hypotheses for the HIV vaccine community and they highlight the importance of assessing the impact of host immune genetics in vaccine-induced immunity and protection. (This study has been registered at ClinicalTrials.gov under registration no. NCT00223080.)
doi:10.1128/JVI.01164-14
PMCID: PMC4135964  PMID: 24829343
8.  Increased Sequence Coverage through Combined Targeting of Variant and Conserved Epitopes Correlates with Control of HIV Replication 
Journal of Virology  2014;88(2):1354-1365.
A major challenge in the development of an HIV vaccine is that of contending with the extensive sequence variability found in circulating viruses. Induction of HIV-specific T-cell responses targeting conserved regions and induction of HIV-specific T-cell responses recognizing a high number of epitope variants have both been proposed as strategies to overcome this challenge. We addressed the ability of cytotoxic T lymphocytes from 30 untreated HIV-infected subjects with and without control of virus replication to recognize all clade B Gag sequence variants encoded by at least 5% of the sequences in the Los Alamos National Laboratory HIV database (1,300 peptides) using gamma interferon and interleukin-2 (IFN-γ/IL-2) FluoroSpot analysis. While targeting of conserved regions was similar in the two groups (P = 0.47), we found that subjects with control of virus replication demonstrated marginally lower recognition of Gag epitope variants than subjects with normal progression (P = 0.05). In viremic controllers and progressors, we found variant recognition to be associated with viral load (r = 0.62, P = 0.001). Interestingly, we show that increased overall sequence coverage, defined as the overall proportion of HIV database sequences targeted through the Gag-specific repertoire, is inversely associated with viral load (r = −0.38, P = 0.03). Furthermore, we found that sequence coverage, but not variant recognition, correlated with increased recognition of a panel of clade B HIV founder viruses (r = 0.50, P = 0.004). We propose sequence coverage by HIV Gag-specific immune responses as a possible correlate of protection that may contribute to control of virus replication. Additionally, sequence coverage serves as a valuable measure by which to evaluate the protective potential of future vaccination strategies.
doi:10.1128/JVI.02361-13
PMCID: PMC3911649  PMID: 24227851
9.  Efficacy Trial of a DNA/rAd5 HIV-1 Preventive Vaccine 
The New England journal of medicine  2013;369(22):2083-2092.
Background
A safe and effective vaccine for the prevention of human immunodeficiency virus type 1 (HIV-1) infection is a global priority. We tested the efficacy of a DNA prime–recombinant adenovirus type 5 boost (DNA/rAd5) vaccine regimen in persons at increased risk for HIV-1 infection in the United States.
Methods
At 21 sites, we randomly assigned 2504 men or transgender women who have sex with men to receive the DNA/rAd5 vaccine (1253 participants) or placebo (1251 participants). We assessed HIV-1 acquisition from week 28 through month 24 (termed week 28+ infection), viral-load set point (mean plasma HIV-1 RNA level 10 to 20 weeks after diagnosis), and safety. The 6-plasmid DNA vaccine (expressing clade B Gag, Pol, and Nef and Env proteins from clades A, B, and C) was administered at weeks 0, 4, and 8. The rAd5 vector boost (expressing clade B Gag-Pol fusion protein and Env glycoproteins from clades A, B, and C) was administered at week 24.
Results
In April 2013, the data and safety monitoring board recommended halting vaccinations for lack of efficacy. The primary analysis showed that week 28+ infection had been diagnosed in 27 participants in the vaccine group and 21 in the placebo group (vaccine efficacy, −25.0%; 95% confidence interval, −121.2 to 29.3; P = 0.44), with mean viral-load set points of 4.46 and 4.47 HIV-1 RNA log10 copies per milliliter, respectively. Analysis of all infections during the study period (41 in the vaccine group and 31 in the placebo group) also showed lack of vaccine efficacy (P = 0.28). The vaccine regimen had an acceptable side-effect profile.
Conclusions
The DNA/rAd5 vaccine regimen did not reduce either the rate of HIV-1 acquisition or the viral-load set point in the population studied. (Funded by the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT00865566.)
doi:10.1056/NEJMoa1310566
PMCID: PMC4030634  PMID: 24099601
10.  Characterization of Humoral and Cellular Immune Responses Elicited by a Recombinant Adenovirus Serotype 26 HIV-1 Env Vaccine in Healthy Adults (IPCAVD 001) 
The Journal of Infectious Diseases  2012;207(2):248-256.
Background. Adenovirus serotype 26 (Ad26) has been developed as a novel candidate vaccine vector for human immunodeficiency virus type 1 (HIV-1) and other pathogens. The primary safety and immunogenicity data from the Integrated Preclinical/Clinical AIDS Vaccine Development Program (IPCAVD) 001 trial, the first-in-human evaluation of a prototype Ad26 vector-based vaccine expressing clade A HIV-1 Env (Ad26.ENVA.01), are reported concurrently with this article. Here, we characterize in greater detail the humoral and cellular immune responses elicited by Ad26.ENVA.01 in humans.
Methods. Samples from the IPCAVD 001 trial were used for humoral and cellular immunogenicity assays.
Results. We observed a dose-dependent expansion of the magnitude, breadth, and epitopic diversity of Env-specific binding antibody responses elicited by this vaccine. Antibody-dependent cell-mediated phagocytosis, virus inhibition, and degranulation functional activity were also observed. Env-specific cellular immune responses induced by the vaccine included multiple CD8+ and CD4+ T-lymphocyte memory subpopulations and cytokine secretion phenotypes, although cellular immune breadth was limited. Baseline vector-specific T-lymphocyte responses were common but did not impair Env-specific immune responses in this study.
Conclusion. Ad26.ENVA.01 elicited a broad diversity of humoral and cellular immune responses in humans. These data support the further clinical development of Ad26 as a candidate vaccine vector.
Clinical Trials Registration. NCT00618605.
doi:10.1093/infdis/jis671
PMCID: PMC3532832  PMID: 23125443
HIV-1; Vaccine; Adenovirus 26; Immunogenicity
11.  Optimization and Qualification of a Multiplex Bead Array to Assess Cytokine and Chemokine Production by Vaccine-specific Cells 
Journal of Immunological Methods  2012;382(1-2):117-128.
The magnitude and functional phenotype (e.g. proliferation, immune stimulation) of the vaccine-induced T-cell responses are likely to be critical in defining responses that can control pathogenic challenge. Current multi-parameter flow cytometric techniques may not be sufficient to measure all of these different functions, since characterizing T-cell responses by flow cytometry is presently limited to concurrent measurement of at most 10 cytokines/chemokines. Here, we describe extensive studies conducted using standardized GCLP procedures to optimize and qualitatively/quantitatively qualify a multiplex bead array (MBA) performed on supernatant collected from stimulated peripheral blood mononuclear cells (PBMC) to assess 12 cytokines and chemokines of interest. Our optimized MBA shows good precision (intra-assay, inter-day, inter-technician; coefficients of variation <30%) and linearity for most of the analytes studied. We also developed positivity criteria that allow us to define a response as positive or negative with a high degree of confidence. In conclusion, we provide a detailed description of the qualification of an MBA, which permits quantitative and qualitative evaluation of vaccine-induced immunogenicity and analysis of immune correlates of protection. This assay provides an excellent complement to the existing repertoire of assays for assessing immunogenicity in HIV vaccine clinical trials.
doi:10.1016/j.jim.2012.05.011
PMCID: PMC3406745  PMID: 22626638
Multiplex bead array; Vaccine; HIV; Cytokine; Chemokine
12.  HIV-1 Vaccine-Induced T-Cell Reponses Cluster in Epitope Hotspots that Differ from Those Induced in Natural Infection with HIV-1 
PLoS Pathogens  2013;9(6):e1003404.
Several recent large clinical trials evaluated HIV vaccine candidates that were based on recombinant adenovirus serotype 5 (rAd-5) vectors expressing HIV-derived antigens. These vaccines primarily elicited T-cell responses, which are known to be critical for controlling HIV infection. In the current study, we present a meta-analysis of epitope mapping data from 177 participants in three clinical trials that tested two different HIV vaccines: MRKAd-5 HIV and VRC-HIVAD014-00VP. We characterized the population-level epitope responses in these trials by generating population-based epitope maps, and also designed such maps using a large cohort of 372 naturally infected individuals. We used these maps to address several questions: (1) Are vaccine-induced responses randomly distributed across vaccine inserts, or do they cluster into immunodominant epitope hotspots? (2) Are the immunodominance patterns observed for these two vaccines in three vaccine trials different from one another? (3) Do vaccine-induced hotspots overlap with epitope hotspots induced by chronic natural infection with HIV-1? (4) Do immunodominant hotspots target evolutionarily conserved regions of the HIV genome? (5) Can epitope prediction methods be used to identify these hotspots? We found that vaccine responses clustered into epitope hotspots in all three vaccine trials and some of these hotspots were not observed in chronic natural infection. We also found significant differences between the immunodominance patterns generated in each trial, even comparing two trials that tested the same vaccine in different populations. Some of the vaccine-induced immunodominant hotspots were located in highly variable regions of the HIV genome, and this was more evident for the MRKAd-5 HIV vaccine. Finally, we found that epitope prediction methods can partially predict the location of vaccine-induced epitope hotspots. Our findings have implications for vaccine design and suggest a framework by which different vaccine candidates can be compared in early phases of evaluation.
Author Summary
The HIV epidemic is a major global health challenge leading to more than 1.8 million deaths annually, and despite significant efforts, the search for an efficacious and safe vaccine continues. Several candidate vaccines were designed to elicit CD8+ T-cell responses and were based on using recombinant Adenovirus serotype 5 (rAd-5) vector that expresses HIV-derived antigens. While none of these vaccines had protective effects, they provide an opportunity to study vaccine-induced T-cell responses on a population level. Here, we analyze data from the three largest epitope mapping studies performed in three clinical trials testing two rAd-5 vaccines. We find that vaccine-induced responses tend to cluster in “epitope hotspots” and that these hotspots are different for each vaccine and more surprisingly in two different vaccine trials testing the same vaccine. We also compared vaccine-induced hotspots to those elicited by natural infection and found that some of the vaccine-induced hotspots are not observed in natural infection. Finally, we show that epitope prediction methods can be useful for predicting vaccine induced hotspots based on participants HLA alleles.
doi:10.1371/journal.ppat.1003404
PMCID: PMC3688560  PMID: 23818843
13.  Uncommon Pathways of Immune Escape Attenuate HIV-1 Integrase Replication Capacity 
Journal of Virology  2012;86(12):6913-6923.
An attenuation of the HIV-1 replication capacity (RC) has been observed for immune-mediated escape mutations in Gag restricted by protective HLA alleles. However, the extent to which escape mutations affect other viral proteins during natural infection is not well understood. We generated recombinant viruses encoding plasma HIV-1 RNA integrase sequences from antiretroviral-naïve individuals with early (n = 88) and chronic (n = 304) infections and measured the in vitro RC of each. In contrast to data from previous studies of Gag, we observed little evidence that host HLA allele expression was associated with integrase RC. A modest negative correlation was observed between the number of HLA-B-associated integrase polymorphisms and RC in chronic infection (R = −0.2; P = 0.003); however, this effect was not driven by mutations restricted by protective HLA alleles. Notably, the integrase variants S119R, G163E, and I220L, which represent uncommon polymorphisms associated with HLA-C*05, -A*33, and -B*52, respectively, correlated with lower RC (all q < 0.2). We identified a novel C*05-restricted epitope (HTDNGSNF114–121) that likely contributes to the selection of the S119R variant, the polymorphism most significantly associated with lower RC in patient sequences. An NL4-3 mutant encoding the S119R polymorphism displayed a ∼35%-reduced function that was rescued by a single compensatory mutation of A91E. Together, these data indicate that substantial HLA-driven attenuation of integrase is not a general phenomenon during HIV-1 adaptation to host immunity. However, uncommon polymorphisms selected by HLA alleles that are not conventionally regarded to be protective may be associated with impaired protein function. Vulnerable epitopes in integrase might therefore be considered for future vaccine strategies.
doi:10.1128/JVI.07133-11
PMCID: PMC3393549  PMID: 22496233
14.  Immune-Correlates Analysis of an HIV-1 Vaccine Efficacy Trial 
The New England Journal of Medicine  2012;366(14):1275-1286.
BACKGROUND
In the RV144 trial, the estimated efficacy of a vaccine regimen against human immunodeficiency virus type 1 (HIV-1) was 31.2%. We performed a case–control analysis to identify antibody and cellular immune correlates of infection risk.
METHODS
In pilot studies conducted with RV144 blood samples, 17 antibody or cellular assays met prespecified criteria, of which 6 were chosen for primary analysis to determine the roles of T-cell, IgG antibody, and IgA antibody responses in the modulation of infection risk. Assays were performed on samples from 41 vaccinees who became infected and 205 uninfected vaccinees, obtained 2 weeks after final immunization, to evaluate whether immune-response variables predicted HIV-1 infection through 42 months of follow-up.
RESULTS
Of six primary variables, two correlated significantly with infection risk: the binding of IgG antibodies to variable regions 1 and 2 (V1V2) of HIV-1 envelope proteins (Env) correlated inversely with the rate of HIV-1 infection (estimated odds ratio, 0.57 per 1-SD increase; P = 0.02; q = 0.08), and the binding of plasma IgA antibodies to Env correlated directly with the rate of infection (estimated odds ratio, 1.54 per 1-SD increase; P = 0.03; q = 0.08). Neither low levels of V1V2 antibodies nor high levels of Env-specific IgA antibodies were associated with higher rates of infection than were found in the placebo group. Secondary analyses suggested that Env-specific IgA antibodies may mitigate the effects of potentially protective antibodies.
CONCLUSIONS
This immune-correlates study generated the hypotheses that V1V2 antibodies may have contributed to protection against HIV-1 infection, whereas high levels of Env-specific IgA antibodies may have mitigated the effects of protective antibodies. Vaccines that are designed to induce higher levels of V1V2 antibodies and lower levels of Env-specific IgA antibodies than are induced by the RV144 vaccine may have improved efficacy against HIV-1 infection.
doi:10.1056/NEJMoa1113425
PMCID: PMC3371689  PMID: 22475592
15.  MRKAd5 HIV-1 Gag/Pol/Nef Vaccine-Induced T-Cell Responses Inadequately Predict Distance of Breakthrough HIV-1 Sequences to the Vaccine or Viral Load 
PLoS ONE  2012;7(8):e43396.
Background
The sieve analysis for the Step trial found evidence that breakthrough HIV-1 sequences for MRKAd5/HIV-1 Gag/Pol/Nef vaccine recipients were more divergent from the vaccine insert than placebo sequences in regions with predicted epitopes. We linked the viral sequence data with immune response and acute viral load data to explore mechanisms for and consequences of the observed sieve effect.
Methods
Ninety-one male participants (37 placebo and 54 vaccine recipients) were included; viral sequences were obtained at the time of HIV-1 diagnosis. T-cell responses were measured 4 weeks post-second vaccination and at the first or second week post-diagnosis. Acute viral load was obtained at RNA-positive and antibody-negative visits.
Findings
Vaccine recipients had a greater magnitude of post-infection CD8+ T cell response than placebo recipients (median 1.68% vs 1.18%; p = 0·04) and greater breadth of post-infection response (median 4.5 vs 2; p = 0·06). Viral sequences for vaccine recipients were marginally more divergent from the insert than placebo sequences in regions of Nef targeted by pre-infection immune responses (p = 0·04; Pol p = 0·13; Gag p = 0·89). Magnitude and breadth of pre-infection responses did not correlate with distance of the viral sequence to the insert (p>0·50). Acute log viral load trended lower in vaccine versus placebo recipients (estimated mean 4·7 vs 5·1) but the difference was not significant (p = 0·27). Neither was acute viral load associated with distance of the viral sequence to the insert (p>0·30).
Interpretation
Despite evidence of anamnestic responses, the sieve effect was not well explained by available measures of T-cell immunogenicity. Sequence divergence from the vaccine was not significantly associated with acute viral load. While point estimates suggested weak vaccine suppression of viral load, the result was not significant and more viral load data would be needed to detect suppression.
doi:10.1371/journal.pone.0043396
PMCID: PMC3428369  PMID: 22952672
16.  Host Genetic Determinants of T Cell Responses to the MRKAd5 HIV-1 gag/pol/nef Vaccine in the Step Trial 
The Journal of Infectious Diseases  2011;203(6):773-779.
Understanding how human genetic variation impacts individual response to immunogens is fundamental for rational vaccine development. To explore host mechanisms involved in cellular immune responses to the MRKAd5 human immunodeficiency virus type 1 (HIV-1) gag/pol/nef vaccine tested in the Step trial, we performed a genome-wide association study of determinants of HIV-specific T cell responses, measured by interferon γ enzyme-linked immunospot assays. No human genetic variant reached genome-wide significance, but polymorphisms located in the major histocompatibility complex (MHC) region showed the strongest association with response to the HIV-1 Gag protein: HLA-B alleles known to be associated with differences in HIV-1 control were responsible for these associations. The implication of the same HLA alleles in vaccine-induced cellular immunity and in natural immune control is of relevance for vaccine design. Furthermore, our results demonstrate the importance of considering the host immunogenetic background in the analysis of immune responses to T cell vaccines.
doi:10.1093/infdis/jiq125
PMCID: PMC3071133  PMID: 21278214
17.  High-Functional-Avidity Cytotoxic T Lymphocyte Responses to HLA-B-Restricted Gag-Derived Epitopes Associated with Relative HIV Control ▿ 
Journal of Virology  2011;85(18):9334-9345.
Virus-specific cytotoxic T lymphocytes (CTL) with high levels of functional avidity have been associated with viral clearance in hepatitis C virus infection and with enhanced antiviral protective immunity in animal models. However, the role of functional avidity as a determinant of HIV-specific CTL efficacy remains to be assessed. Here we measured the functional avidities of HIV-specific CTL responses targeting 20 different, optimally defined CTL epitopes restricted by 13 different HLA class I alleles in a cohort comprising 44 HIV controllers and 68 HIV noncontrollers. Responses restricted by HLA-B alleles and responses targeting epitopes located in HIV Gag exhibited significantly higher functional avidities than responses restricted by HLA-A or HLA-C molecules (P = 0.0003) or responses targeting epitopes outside Gag (P < 0.0001). The functional avidities of Gag-specific and HLA-B-restricted responses were higher in HIV controllers than in noncontrollers (P = 0.014 and P = 0.018) and were not restored in HIV noncontrollers initiating antiretroviral therapy. T-cell receptor (TCR) analyses revealed narrower TCR repertoires in higher-avidity CTL populations, which were dominated by public TCR sequences in HIV controllers. Together, these data link the presence of high-avidity Gag-specific and HLA-B-restricted CTL responses with viral suppression in vivo and provide new insights into the immune parameters that mediate spontaneous control of HIV infection.
doi:10.1128/JVI.00460-11
PMCID: PMC3165743  PMID: 21752903
18.  Ets-1 maintains IL-7 receptor expression in peripheral T cells 
The expression of CD127, the IL-7 binding subunit of the IL-7 receptor, is tightly regulated during the development and activation of T cells and is reduced during chronic viral infection. However, the molecular mechanism regulating the dynamic expression of CD127 is still poorly understood. Here we report that the transcription factor Ets-1 is required for maintaining the expression of CD127 in murine peripheral T cells. Ets-1 binds to and activates the CD127 promoter, and its absence leads to reduced CD127 expression, attenuated IL-7 signaling, and impaired IL-7-dependent homeostatic proliferation of T cells. The expression of CD127 and Ets-1 is strongly correlated in human T cells. Both CD127 and Ets-1 expression are decreased in CD8+ T cells during HIV infection. In addition, HIV-associated loss of CD127 is only observed in Ets-1low effector memory and central memory but not in Ets-1high naïve CD8+ T cells. Taken together, our data identify Ets-1 as a critical regulator of CD127 expression in T cells.
doi:10.4049/jimmunol.1002099
PMCID: PMC3074256  PMID: 21148801
19.  Human adenovirus-specific T cells modulate HIV-specific T cell responses to an Ad5-vectored HIV-1 vaccine 
Recombinant viruses hold promise as vectors for vaccines to prevent infectious diseases with significant global health impacts. One of their major limitations is that preexisting anti-vector neutralizing antibodies can reduce T cell responses to the insert antigens; however, the impact of vector-specific cellular immunity on subsequent insert-specific T cell responses has not been assessed in humans. Here, we have identified and compared adenovirus-specific and HIV-specific T cell responses in subjects participating in two HIV-1 vaccine trials using a vaccine vectored by adenovirus serotype 5 (Ad5). Higher frequencies of pre-immunization adenovirus-specific CD4+ T cells were associated with substantially decreased magnitude of HIV-specific CD4+ T cell responses and decreased breadth of HIV-specific CD8+ T cell responses in vaccine recipients, independent of type-specific preexisting Ad5-specific neutralizing antibody titers. Further, epitopes recognized by adenovirus-specific T cells were commonly conserved across many adenovirus serotypes, suggesting that cross-reactivity of preexisting adenovirus-specific T cells can extend to adenovirus vectors derived from rare serotypes. These findings provide what we believe to be a new understanding of how preexisting viral immunity may impact the efficacy of vaccines under current evaluation for prevention of HIV, tuberculosis, and malaria.
doi:10.1172/JCI60202
PMCID: PMC3248307  PMID: 22201684
20.  Genetic impact of vaccination on breakthrough HIV-1 sequences from the Step trial 
Nature medicine  2011;17(3):366-371.
We analyzed HIV-1 genome sequences from 68 newly-infected volunteers in the Step HIV-1 vaccine trial. To determine whether the vaccine exerted selective T-cell pressure on breakthrough viruses, we identified potential T-cell epitopes in the founder sequences and compared them to epitopes in the vaccine. We found greater distances for sequences from vaccine recipients than from placebo recipients (p-values ranging from < 0.0001 to 0.09). The most significant signature site distinguishing vaccine from placebo recipients was Gag-84, a site encompassed by several epitopes contained in the vaccine and restricted by HLA alleles common in the cohort. Moreover, the extended divergence was confined to the vaccine components of the virus (Gag, Pol, Nef) and not found in other HIV-1 proteins. These results represent the first evidence of selective pressure from vaccine-induced T-cell responses on HIV-1 infection.
doi:10.1038/nm.2316
PMCID: PMC3053571  PMID: 21358627
21.  Increased Breadth and Depth of Cytotoxic T Lymphocytes Responses against HIV-1-B Nef by Inclusion of Epitope Variant Sequences 
PLoS ONE  2011;6(3):e17969.
Different vaccine approaches cope with HIV-1 diversity, ranging from centralized1–4 to variability-encompassing5–7 antigens. For all these strategies, a concern remains: how does HIV-1 diversity impact epitope recognition by the immune system? We studied the relationship between HIV-1 diversity and CD8+ T Lymphocytes (CTL) targeting of HIV-1 subtype B Nef using 944 peptides (10-mers overlapping by nine amino acids (AA)) that corresponded to consensus peptides and their most common variants in the HIV-1-B virus population. IFN-γ ELISpot assays were performed using freshly isolated PBMC from 26 HIV-1-infected persons. Three hundred and fifty peptides elicited a response in at least one individual. Individuals targeted a median of 7 discrete regions. Overall, 33% of responses were directed against viral variants but not elicited against consensus-based test peptides. However, there was no significant relationship between the frequency of a 10-mer in the viral population and either its frequency of recognition (Spearman's correlation coefficient ρ = 0.24) or the magnitude of the responses (ρ = 0.16). We found that peptides with a single mutation compared to the consensus were likely to be recognized (especially if the change was conservative) and to elicit responses of similar magnitude as the consensus peptide. Our results indicate that cross-reactivity between rare and frequent variants is likely to play a role in the expansion of CTL responses, and that maximizing antigenic diversity in a vaccine may increase the breadth and depth of CTL responses. However, since there are few obvious preferred pathways to virologic escape, the diversity that may be required to block all potential escape pathways may be too large for a realistic vaccine to accommodate. Furthermore, since peptides were not recognized based on their frequency in the population, it remains unclear by which mechanisms variability-inclusive antigens (i.e., constructs enriched with frequent variants) expand CTL recognition.
doi:10.1371/journal.pone.0017969
PMCID: PMC3065451  PMID: 21464919
22.  Trivalent Adenovirus Type 5 HIV Recombinant Vaccine Primes for Modest Cytotoxic Capacity That Is Greatest in Humans with Protective HLA Class I Alleles 
PLoS Pathogens  2011;7(2):e1002002.
If future HIV vaccine design strategies are to succeed, improved understanding of the mechanisms underlying protection from infection or immune control over HIV replication remains essential. Increased cytotoxic capacity of HIV-specific CD8+ T-cells associated with efficient elimination of HIV-infected CD4+ T-cell targets has been shown to distinguish long-term nonprogressors (LTNP), patients with durable control over HIV replication, from those experiencing progressive disease. Here, measurements of granzyme B target cell activity and HIV-1-infected CD4+ T-cell elimination were applied for the first time to identify antiviral activities in recipients of a replication incompetent adenovirus serotype 5 (Ad5) HIV-1 recombinant vaccine and were compared with HIV-negative individuals and chronically infected patients, including a group of LTNP. We observed readily detectable HIV-specific CD8+ T-cell recall cytotoxic responses in vaccinees at a median of 331 days following the last immunization. The magnitude of these responses was not related to the number of vaccinations, nor did it correlate with the percentages of cytokine-secreting T-cells determined by ICS assays. Although the recall cytotoxic capacity of the CD8+ T-cells of the vaccinee group was significantly less than that of LTNP and overlapped with that of progressors, we observed significantly higher cytotoxic responses in vaccine recipients carrying the HLA class I alleles B*27, B*57 or B*58, which have been associated with immune control over HIV replication in chronic infection. These findings suggest protective HLA class I alleles might lead to better outcomes in both chronic infection and following immunization due to more efficient priming of HIV-specific CD8+ T-cell cytotoxic responses.
Author Summary
Unique HIV-infected individuals have remained healthy with stable CD4 counts and HIV RNA levels below the detection threshold in sensitive assays without antiretroviral therapy for 20 years. These nonprogressors have been intensively studied in order to identify mechanisms that could inform the design of an efficacious HIV/AIDS vaccine. In addition to strong associations with certain host genes like HLA B*57, nonprogressors are distinguished from progressors by the superior ability of their HIV-specific CD8+ T-cells to proliferate and to efficiently kill HIV-infected CD4+ T-cell targets via perforin and granzyme B, the major proteins contained within killing granules. Here, for the first time, we apply sensitive measurements of CD8+ T-cell proliferation and perforin expression, granzyme B target cell activity and infected CD4+ T-cell elimination to samples derived from recipients of the Merck adenovirus serotype 5-HIV vaccine. We demonstrate readily detectable CD8+ T-cell-mediated killing in these vaccinees. Although the killing responses were less than those of nonprogressors, vaccinees expressing the protective HLA alleles B*27, B*57 or B*58 exhibited greater killing than those not possessing these alleles. These findings suggest protective HLA alleles lead to better outcomes in both chronic infection and following immunization through early interactions that induce superior antiviral CD8+ T-cell killing responses.
doi:10.1371/journal.ppat.1002002
PMCID: PMC3044701  PMID: 21383976
23.  Impact of select immunologic and virologic biomarkers on CD4 decline in chronic HIV-1 subtype C infection: Results from Sinikithemba longitudinal population-based cohort, Durban, South Africa 
Background
The extent to which immunologic and clinical biomarkers influence HIV outcomes remains incompletely characterized, particularly for non-B subtypes. Based on data supporting in vitro HIV protein-specific CD8 T-lymphocyte responses as correlates of immune control in cross-sectional studies, we assessed the relationship of these responses, along with established HIV biomarkers, with rates of CD4 decline in subtype-C infection.
Methods
Bi-variate and multivariate mixed effects models were used to assess the relationship of baseline CD4, plasma viral load (pVL), HLA class I alleles, and HIV protein-specific CD8 responses with rate of CD4 decline in a longitudinal population-based cohort of 300 therapy-naïve, chronically infected adults with baseline CD4>200 cells/mm3 and pVL>500 copies/ml, over a median 25 months follow-up.
Results
In bi-variate analyses, baseline CD4, pVL and possession of a protective HLA allele correlated significantly with rate of CD4 decline. No relationship was observed between HIV protein-specific CD8 responses and CD4 decline. Results from multivariate models, incorporating baseline CD4 (201–350 and >350), pVL (≤100,000 and >100,000), HLA (protective vs. not), yielded the ability to discriminate CD4 declines over a 10-fold range: the highest rate of decline was observed among individuals with CD4>350, pVL>100,000 with no protective HLA alleles (−59 cells/mm3/year), while the slowest decline was observed in individuals with CD4 201–350, pVL≤100,000 and a protective allele (−6 cells/mm3/year).
Conclusions
The combination of plasma viral load and HLA class I type, but not in vitro HIV protein-specific CD8 responses, differentiates rates of CD4 decline in chronic subtype-C infection better than either marker alone.
doi:10.1086/605503
PMCID: PMC2777678  PMID: 19663693
HIV subtype C; disease progression; CD4 decline; HLA class I; HIV-specific CTL responses
24.  Contribution of immunological and virological factors to extremely severe primary HIV-1 infection 
Background
During acute HIV infection, high viral loads and the induction of host immune responses typically coincide with the onset of clinical symptoms. However, clinically severe presentations during acute HIV-1 infection, including AIDS-defining symptoms, are unusual.
Methods
Virus isolates were tested for clade, drug susceptibility, coreceptor usage, and growth rate for two cases of clinically severe sexual transmission. HLA genotype was determined, and HIV-1-specific CTL responses to an overlapping peptide set spanning the entire HIV clade A and clade B proteome were assayed.
Results
The virus isolated from the two unrelated cases of severe primary HIV-1 infection showed R5/X4 dual/mixed tropism, belonged to clade B and CRF02-AG, and were highly replicative in peripheral blood mononuclear cell culture. Impaired humoral responses were paralleled by a profound absence of HIV-1-specific CTL responses to the entire viral proteome in the two study cases. One case for which the virus source was available, showed a remarkable HLA similarity between the transmission pair as all 4 HLA-A and -B alleles were HLA supertype-matched between the subjects involved in the transmission case.
Conclusions
The data suggest that concurrence of viral and host factors contribute to the clinical severity of primary HIV-1 infection and that subjects infected with highly replicative dual tropic viruses are more prone to develop AIDS-defining symptoms during acute infection if they are unable to mount humoral and cellular HIV-1-specific immune responses. Concordant HLA supertypes might facilitate the preferential transmission of HLA-adapted viral variants, further accelerating disease progression.
doi:10.1086/595704
PMCID: PMC2806184  PMID: 19093810
primary HIV-1 infection; HLA supertypes; CTL responses; R5/X4 dual tropism; rapid disease progression
25.  Induction of Robust Cellular and Humoral Virus-Specific Adaptive Immune Responses in Human Immunodeficiency Virus-Infected Humanized BLT Mice▿  
Journal of Virology  2009;83(14):7305-7321.
The generation of humanized BLT mice by the cotransplantation of human fetal thymus and liver tissues and CD34+ fetal liver cells into nonobese diabetic/severe combined immunodeficiency mice allows for the long-term reconstitution of a functional human immune system, with human T cells, B cells, dendritic cells, and monocytes/macrophages repopulating mouse tissues. Here, we show that humanized BLT mice sustained high-level disseminated human immunodeficiency virus (HIV) infection, resulting in CD4+ T-cell depletion and generalized immune activation. Following infection, HIV-specific humoral responses were present in all mice by 3 months, and HIV-specific CD4+ and CD8+ T-cell responses were detected in the majority of mice tested after 9 weeks of infection. Despite robust HIV-specific responses, however, viral loads remained elevated in infected BLT mice, raising the possibility that these responses are dysfunctional. The increased T-cell expression of the negative costimulator PD-1 recently has been postulated to contribute to T-cell dysfunction in chronic HIV infection. As seen in human infection, both CD4+ and CD8+ T cells demonstrated increased PD-1 expression in HIV-infected BLT mice, and PD-1 levels in these cells correlated positively with viral load and inversely with CD4+ cell levels. The ability of humanized BLT mice to generate both cellular and humoral immune responses to HIV will allow the further investigation of human HIV-specific immune responses in vivo and suggests that these mice are able to provide a platform to assess candidate HIV vaccines and other immunotherapeutic strategies.
doi:10.1128/JVI.02207-08
PMCID: PMC2704767  PMID: 19420076

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