Dissecting Polyclonal Vaccine-Induced Humoral Immunity against HIV Using Systems Serology.

While antibody titers and neutralization are considered the gold standard for the selection of successful vaccines, these parameters are often inadequate predictors of protective immunity. As antibodies mediate an array of extra-neutralizing Fc functions, when neutralization fails to predict protection, investigating Fc-mediated activity may help identify immunological correlates and mechanism(s) of humoral protection. Here, we used an integrative approach termed Systems Serology to analyze relationships among humoral responses elicited in four HIV vaccine trials. Each vaccine regimen induced a unique humoral "Fc fingerprint." Moreover, analysis of case:control data from the first moderately protective HIV vaccine trial, RV144, pointed to mechanistic insights into immune complex composition that may underlie protective immunity to HIV. Thus, multi-dimensional relational comparisons of vaccine humoral fingerprints offer a unique approach for the evaluation and design of novel vaccines against pathogens for which correlates of protection remain elusive.

Gene signature discovery and systematic validation across diverse clinical cohorts for TB prognosis and response to treatment.

While blood gene signatures have shown promise in tuberculosis (TB) diagnosis and treatment monitoring, most signatures derived from a single cohort may be insufficient to capture TB heterogeneity in populations and individuals. Here we report a new generalized approach combining a network-based meta-analysis with machine-learning modeling to leverage the power of heterogeneity among studies. The transcriptome datasets from 57 studies (37 TB and 20 viral infections) across demographics and TB disease states were used for gene signature discovery and model training and validation. The network-based meta-analysis identified a common 45-gene signature specific to active TB disease across studies. Two optimized random forest regression models, using the full or partial 45-gene signature, were then established to model the continuum from Mycobacterium tuberculosis infection to disease and treatment response. In model validation, using pooled multi-cohort datasets to mimic the real-world setting, the model provides robust predictive performance for incipient to active TB risk over a 2.5-year period with an AUROC of 0.85, 74.2% sensitivity, and 78.3% specificity, which approximates the minimum criteria (>75% sensitivity and >75% specificity) within the WHO target product profile for prediction of progression to TB. Moreover, the model strongly discriminates active TB from viral infection (AUROC 0.93, 95% CI 0.91-0.94). For treatment monitoring, the TB scores generated by the model statistically correlate with treatment responses over time and were predictive, even before treatment initiation, of standard treatment clinical outcomes. We demonstrate an end-to-end gene signature model development scheme that considers heterogeneity for TB risk estimation and treatment monitoring.

Tuberculosis Treatment Monitoring and Outcome Measures: New Interest and New Strategies.

Despite the advent of new diagnostics, drugs and regimens, tuberculosis (TB) remains a global public health threat. A significant challenge for TB control efforts has been the monitoring of TB therapy and determination of TB treatment success. Current recommendations for TB treatment monitoring rely on sputum and culture conversion, which have low sensitivity and long turnaround times, present biohazard risk, and are prone to contamination, undermining their usefulness as clinical treatment monitoring tools and for drug development. We review the pipeline of molecular technologies and assays that serve as suitable substitutes for current culture-based readouts for treatment response and outcome with the potential to change TB therapy monitoring and accelerate drug development.

Sequence and vector shapes vaccine induced antibody effector functions in HIV vaccine trials.

Despite the advent of long-acting anti-retroviral therapy able to control and prevent infection, a preventative vaccine remains a global priority for the elimination of HIV. The moderately protective RV144 vaccine trial suggested functional IgG1 and IgG3 antibodies were a potential correlate of protection, but the RV144-inspired HVTN702 validation trial failed to demonstrate efficacy despite inducing targeted levels of IgG1/IgG3. Alterations in inserts, and antigens, adjuvant, and regimen also resulted in vaccine induced target quantitative levels of the immune correlates, but drove qualitative changes to the humoral immune response, pointing to the urgent need to define the influence of vaccine strategies on shaping antibody quality, not just quantity. Thus, defining how distinct prime/boost approaches tune long-lived functional antibodies represents an important goal in vaccine development. Here, we compared vaccine responses in Phase I and II studies in humans utilizing various combinations of DNA/vector, vector/vector and DNA/protein HIV vaccines. We found that adenoviral vector immunization, compared to pox-viral vectors, resulted in the most potent IgG1 and IgG3 responses, linked to highly functional antibody activity, including assisting NK cell related functions. Minimal differences were observed in the durability of the functional humoral immune response across vaccine regimens, except for antibody dependent phagocytic function, which persisted for longer periods in the DNA/rAd5 and rAd35/rAd5 regimen, likely driven by higher IgG1 levels. Collectively, these findings suggest adenoviral vectors drive superior antibody quality and durability that could inform future clinical vaccine studies. Trial registration: ClinicalTrials.gov NCT00801697, NCT00961883, NCT02207920, NCT00125970, NCT02852005).

Safety and immune responses after a 12-month booster in healthy HIV-uninfected adults in HVTN 100 in South Africa: A randomized double-blind placebo-controlled trial of ALVAC-HIV (vCP2438) and bivalent subtype C gp120/MF59 vaccines.

BACKGROUND: HVTN 100 evaluated the safety and immunogenicity of an HIV subtype C pox-protein vaccine regimen, investigating a 12-month booster to extend vaccine-induced immune responses. METHODS AND FINDINGS: A phase 1-2 randomized double-blind placebo-controlled trial enrolled 252 participants (210 vaccine/42 placebo; median age 23 years; 43% female) between 9 February 2015 and 26 May 2015. Vaccine recipients received ALVAC-HIV (vCP2438) alone at months 0 and 1 and with bivalent subtype C gp120/MF59 at months 3, 6, and 12. Antibody (IgG, IgG3 binding, and neutralizing) and CD4+ T-cell (expressing interferon-gamma, interleukin-2, and CD40 ligand) responses were evaluated at month 6.5 for all participants and at months 12, 12.5, and 18 for a randomly selected subset. The primary analysis compared IgG binding antibody (bAb) responses and CD4+ T-cell responses to 3 vaccine-matched antigens at peak (month 6.5 versus 12.5) and durability (month 12 versus 18) timepoints; IgG responses to CaseA2_gp70_V1V2.B, a primary correlate of risk in RV144, were also compared at these same timepoints. Secondary and exploratory analyses compared IgG3 bAb responses, IgG bAb breadth scores, neutralizing antibody (nAb) responses, antibody-dependent cellular phagocytosis, CD4+ polyfunctionality responses, and CD4+ memory sub-population responses at the same timepoints. Vaccines were generally safe and well tolerated. During the study, there were 2 deaths (both in the vaccine group and both unrelated to study products). Ten participants became HIV-infected during the trial, 7% (3/42) of placebo recipients and 3% (7/210) of vaccine recipients. All 8 serious adverse events were unrelated to study products. Less waning of immune responses was seen after the fifth vaccination than after the fourth, with higher antibody and cellular response rates at month 18 than at month 12: IgG bAb response rates to 1086.C V1V2, 21.0% versus 9.7% (difference = 11.3%, 95% CI = 0.6%-22.0%, P = 0.039), and ZM96.C V1V2, 21.0% versus 6.5% (difference = 14.5%, 95% CI = 4.1%-24.9%, P = 0.004). IgG bAb response rates to all 4 primary V1V2 antigens were higher 2 weeks after the fifth vaccination than 2 weeks after the fourth vaccination: 87.7% versus 75.4% (difference = 12.3%, 95% CI = 1.7%-22.9%, P = 0.022) for 1086.C V1V2, 86.0% versus 63.2% (difference = 22.8%, 95% CI = 9.1%-36.5%, P = 0.001) for TV1c8.2.C V1V2, 67.7% versus 44.6% (difference = 23.1%, 95% CI = 10.4%-35.7%, P < 0.001) for ZM96.C V1V2, and 81.5% versus 60.0% (difference = 21.5%, 95% CI = 7.6%-35.5%, P = 0.002) for CaseA2_gp70_V1V2.B. IgG bAb response rates to the 3 primary vaccine-matched gp120 antigens were all above 90% at both peak timepoints, with no significant differences seen, except a higher response rate to ZM96.C gp120 at month 18 versus month 12: 64.5% versus 1.6% (difference = 62.9%, 95% CI = 49.3%-76.5%, P < 0.001). CD4+ T-cell response rates were higher at month 18 than month 12 for all 3 primary vaccine-matched antigens: 47.3% versus 29.1% (difference = 18.2%, 95% CI = 2.9%-33.4%, P = 0.021) for 1086.C, 61.8% versus 38.2% (difference = 23.6%, 95% CI = 9.5%-37.8%, P = 0.001) for TV1.C, and 63.6% versus 41.8% (difference = 21.8%, 95% CI = 5.1%-38.5%, P = 0.007) for ZM96.C, with no significant differences seen at the peak timepoints. Limitations were that higher doses of gp120 were not evaluated, this study was not designed to investigate HIV prevention efficacy, and the clinical significance of the observed immunological effects is uncertain. CONCLUSIONS: In this study, a 12-month booster of subtype C pox-protein vaccines restored immune responses, and slowed response decay compared to the 6-month vaccination. TRIAL REGISTRATION: ClinicalTrials.gov NCT02404311. South African National Clinical Trials Registry (SANCTR number: DOH--27-0215-4796).

Antibody and cellular responses to HIV vaccine regimens with DNA plasmid as compared with ALVAC priming: An analysis of two randomized controlled trials.

BACKGROUND: DNA plasmids promise a pragmatic alternative to viral vectors for prime-boost HIV-1 vaccines. We evaluated DNA plasmid versus canarypox virus (ALVAC) primes in 2 randomized, double-blind, placebo-controlled trials in southern Africa with harmonized trial designs. HIV Vaccine Trials Network (HVTN) 111 tested DNA plasmid prime by needle or needleless injection device (Biojector) and DNA plasmid plus gp120 protein plus MF59 adjuvant boost. HVTN 100 tested ALVAC prime and ALVAC plus gp120 protein plus MF59 adjuvant boost (same protein/adjuvant as HVTN 111) by needle. METHODS AND FINDINGS: The primary endpoints for this analysis were binding antibody (bAb) responses to HIV antigens (gp120 from strains ZM96, 1086, and TV1; variable 1 and 2 [V1V2] regions of gp120 from strains TV1, 1086, and B.CaseA, as 1086 V1V2 and B.CaseA were correlates of risk in the RV144 efficacy trial), neutralizing antibody (nAb) responses to pseudoviruses TV1c8.2 and MW925.26, and cellular responses to vaccine-matched antigens (envelope [Env] from strains ZM96, 1086, and TV1; and Gag from strains LAI and ZM96) at month 6.5, two weeks after the fourth vaccination. Per-protocol cohorts included vaccine recipients from HVTN 100 (n = 186, 60% male, median age 23 years) enrolled between February 9, 2015, and May 26, 2015 and from HVTN 111 (n = 56, 48% male, median age 24 years) enrolled between June 21, 2016, and July 13, 2017. IgG bAb response rates were 100% to 3 Env gp120 antigens in both trials. Response rates to V1V2 were lower and similar in both trials except to vaccine-matched 1086 V1V2, with rates significantly higher for the DNA-primed regimen than the ALVAC-primed regimen: 96.6% versus 72.7% (difference = 23.9%, 95% CI 15.6%-32.2%, p < 0.001). Among positive responders, bAb net mean fluorescence intensity (MFI) was significantly higher with the DNA-primed regimen than ALVAC-primed for 1086 V1V2 (geometric mean [GM] 2,833.3 versus 1,200.9; ratio = 2.36, 95% CI 1.42-3.92, p < 0.001) and B.CaseA V1V2 (GM 2314.0 versus 744.6, ratio = 3.11, 95% CI 1.51-6.38, p = 0.002). nAb response rates were >98% in both trials, with significantly higher 50% inhibitory dilution (ID50) among DNA-primed positive responders (n = 53) versus ALVAC-primed (n = 182) to tier 1A MW965.26 (GM 577.7 versus 265.7, ratio = 2.17, 95% CI 1.67-2.83, p < 0.001) and to TV1c8.2 (GM 187.3 versus 100.4, ratio = 1.87, 95% CI 1.48-2.35, p < 0.001). CD4+ T-cell response rates were significantly higher with DNA plasmid prime via Biojector than ALVAC prime (91.4% versus 52.8%, difference = 38.6%, 95% CI 20.5%-56.6%, p < 0.001 for ZM96.C; 88.0% versus 43.1%, difference = 44.9%, 95% CI 26.7%-63.1%, p < 0.001 for 1086.C; 55.5% versus 2.2%, difference = 53.3%, 95% CI 23.9%-82.7%, p < 0.001 for Gag LAI/ZM96). The study's main limitations include the nonrandomized comparison of vaccines from 2 different trials, the lack of data on immune responses to other non-vaccine-matched antigens, and the uncertain clinical significance of the observed immunological effects. CONCLUSIONS: In this study, we found that further investigation of DNA/protein regimens is warranted given enhanced immunogenicity to the V1V2 correlates of decreased HIV-1 acquisition risk identified in RV144, the only HIV vaccine trial to date to show any efficacy.

Distinct susceptibility of HIV vaccine vector-induced CD4 T cells to HIV infection.

The concerns raised from adenovirus 5 (Ad5)-based HIV vaccine clinical trials, where excess HIV infections were observed in some vaccine recipients, have highlighted the importance of understanding host responses to vaccine vectors and the HIV susceptibility of vector-specific CD4 T cells in HIV vaccination. Our recent study reported that human Ad5-specific CD4 T cells induced by Ad5 vaccination (RV156A trial) are susceptible to HIV. Here we further investigated the HIV susceptibility of vector-specific CD4 T cells induced by ALVAC, a canarypox viral vector tested in the Thai trial RV144, as compared to Ad5 vector-specific CD4 T cells in the HVTN204 trial. We showed that while Ad5 vector-specific CD4 T cells were readily susceptible to HIV, ALVAC-specific CD4 T cells in RV144 PBMC were substantially less susceptible to both R5 and X4 HIV in vitro. The lower HIV susceptibility of ALVAC-specific CD4 T cells was associated with the reduced surface expression of HIV entry co-receptors CCR5 and CXCR4 on these cells. Phenotypic analyses identified that ALVAC-specific CD4 T cells displayed a strong Th1 phenotype, producing higher levels of IFN-γ and CCL4 (MIP-1ß) but little IL-17. Of interest, ALVAC and Ad5 vectors induced distinct profiles of vector-specific CD8 vs. CD4 T-cell proliferative responses in PBMC, with ALVAC preferentially inducing CD8 T-cell proliferation, while Ad5 vector induced CD4 T-cell proliferation. Depletion of ALVAC-, but not Ad5-, induced CD8 T cells in PBMC led to a modest increase in HIV infection of vector-specific CD4 T cells, suggesting a role of ALVAC-specific CD8 T cells in protecting ALVAC-specific CD4 T cells from HIV. Taken together, our data provide strong evidence for distinct HIV susceptibility of CD4 T cells induced by different vaccine vectors and highlight the importance of better evaluating anti-vector responses in HIV vaccination.

Evaluation of a mosaic HIV-1 vaccine in a multicentre, randomised, double-blind, placebo-controlled, phase 1/2a clinical trial (APPROACH) and in rhesus monkeys (NHP 13-19).

BACKGROUND: More than 1·8 million new cases of HIV-1 infection were diagnosed worldwide in 2016. No licensed prophylactic HIV-1 vaccine exists. A major limitation to date has been the lack of direct comparability between clinical trials and preclinical studies. We aimed to evaluate mosaic adenovirus serotype 26 (Ad26)-based HIV-1 vaccine candidates in parallel studies in humans and rhesus monkeys to define the optimal vaccine regimen to advance into clinical efficacy trials. METHODS: We conducted a multicentre, randomised, double-blind, placebo-controlled phase 1/2a trial (APPROACH). Participants were recruited from 12 clinics in east Africa, South Africa, Thailand, and the USA. We included healthy, HIV-1-uninfected participants (aged 18-50 years) who were considered at low risk for HIV-1 infection. We randomly assigned participants to one of eight study groups, stratified by region. Participants and investigators were blinded to the treatment allocation throughout the study. We primed participants at weeks 0 and 12 with Ad26.Mos.HIV (5 × 1010 viral particles per 0·5 mL) expressing mosaic HIV-1 envelope (Env)/Gag/Pol antigens and gave boosters at weeks 24 and 48 with Ad26.Mos.HIV or modified vaccinia Ankara (MVA; 108 plaque-forming units per 0·5 mL) vectors with or without high-dose (250 µg) or low-dose (50 µg) aluminium adjuvanted clade C Env gp140 protein. Those in the control group received 0·9% saline. All study interventions were administered intramuscularly. Primary endpoints were safety and tolerability of the vaccine regimens and Env-specific binding antibody responses at week 28. Safety and immunogenicity were also assessed at week 52. All participants who received at least one vaccine dose or placebo were included in the safety analysis; immunogenicity was analysed using the per-protocol population. We also did a parallel study in rhesus monkeys (NHP 13-19) to assess the immunogenicity and protective efficacy of these vaccine regimens against a series of six repetitive, heterologous, intrarectal challenges with a rhesus peripheral blood mononuclear cell-derived challenge stock of simian-human immunodeficiency virus (SHIV-SF162P3). The APPROACH trial is registered with ClinicalTrials.gov, number NCT02315703. FINDINGS: Between Feb 24, 2015, and Oct 16, 2015, we randomly assigned 393 participants to receive at least one dose of study vaccine or placebo in the APPROACH trial. All vaccine regimens demonstrated favourable safety and tolerability. The most commonly reported solicited local adverse event was mild-to-moderate pain at the injection site (varying from 69% to 88% between the different active groups vs 49% in the placebo group). Five (1%) of 393 participants reported at least one grade 3 adverse event considered related to the vaccines: abdominal pain and diarrhoea (in the same participant), increased aspartate aminotransferase, postural dizziness, back pain, and malaise. The mosaic Ad26/Ad26 plus high-dose gp140 boost vaccine was the most immunogenic in humans; it elicited Env-specific binding antibody responses (100%) and antibody-dependent cellular phagocytosis responses (80%) at week 52, and T-cell responses at week 50 (83%). We also randomly assigned 72 rhesus monkeys to receive one of five different vaccine regimens or placebo in the NHP 13-19 study. Ad26/Ad26 plus gp140 boost induced similar magnitude, durability, and phenotype of immune responses in rhesus monkeys as compared with humans and afforded 67% protection against acquisition of SHIV-SF162P3 infection (two-sided Fisher's exact test p=0·007). Env-specific ELISA and enzyme-linked immunospot assay responses were the principal immune correlates of protection against SHIV challenge in monkeys. INTERPRETATION: The mosaic Ad26/Ad26 plus gp140 HIV-1 vaccine induced comparable and robust immune responses in humans and rhesus monkeys, and it provided significant protection against repetitive heterologous SHIV challenges in rhesus monkeys. This vaccine concept is currently being evaluated in a phase 2b clinical efficacy study in sub-Saharan Africa (NCT03060629). FUNDING: Janssen Vaccines & Prevention BV, National Institutes of Health, Ragon Institute of MGH, MIT and Harvard, Henry M Jackson Foundation for the Advancement of Military Medicine, US Department of Defense, and International AIDS Vaccine Initiative.

Modification of the Association Between T-Cell Immune Responses and Human Immunodeficiency Virus Type 1 Infection Risk by Vaccine-Induced Antibody Responses in the HVTN 505 Trial.

Background: HVTN 505 was a human immunodeficiency virus type 1 (HIV-1) preventive vaccine efficacy trial of a DNA/recombinant adenovirus serotype 5 (rAd5) vaccine regimen. We assessed antibody responses measured 1 month after final vaccination (month 7) as correlates of HIV-1 acquisition risk. Methods: Binding antibody responses were quantified in serum samples from 25 primary endpoint vaccine cases (diagnosed with HIV-1 infection between month 7 and month 24) and 125 randomly sampled frequency-matched vaccine controls (HIV-1 negative at month 24). We prespecified for a primary analysis tier 6 antibody response biomarkers that measure immunoglobulin G (IgG) and immunoglobulin A (IgA) binding to Env proteins and 2 previously assessed T-cell response biomarkers. Results: Envelope-specific IgG responses were significantly correlated with decreased HIV-1 risk. Moreover, the interaction of IgG responses and Env-specific CD8+ T-cell polyfunctionality score had a highly significant association with HIV-1 risk after adjustment for multiple comparisons. Conclusions: Vaccinees with higher levels of Env IgG have significantly decreased HIV-1 risk when CD8+ T-cell responses are low. Moreover, vaccinees with high CD8+ T-cell responses generally have low risk, and those with low CD8+ T-cell and low Env antibody responses have high risk. These findings suggest the critical importance of inducing a robust IgG Env response when the CD8+ T-cell response is low.

Higher T-Cell Responses Induced by DNA/rAd5 HIV-1 Preventive Vaccine Are Associated With Lower HIV-1 Infection Risk in an Efficacy Trial.

Background: It is important to identify vaccine-induced immune responses that predict the preventative efficacy of a human immunodeficiency virus (HIV)-1 vaccine. We assessed T-cell response markers as correlates of risk in the HIV Vaccine Trials Network (HVTN) 505 HIV-1 vaccine efficacy trial. Methods: 2504 participants were randomized to DNA/rAd5 vaccine or placebo, administered at weeks 0, 4, 8, and 24. Peripheral blood mononuclear cells were obtained at week 26 from all 25 primary endpoint vaccine cases and 125 matched vaccine controls, and stimulated with vaccine-insert-matched peptides. Primary variables were total HIV-1-specific CD4+ T-cell magnitude and Env-specific CD4+ polyfunctionality. Four secondary variables were also assessed. Immune responses were evaluated as predictors of HIV-1 infection among vaccinees using Cox proportional hazards models. Machine learning analyses identified immune response combinations best predicting HIV-1 infection. Results: We observed an unexpectedly strong inverse correlation between Env-specific CD8+ immune response magnitude and HIV-1 infection risk (hazard ratio [HR] = 0.18 per SD increment; P = .04) and between Env-specific CD8+ polyfunctionality and infection risk (HR = 0.34 per SD increment; P < .01). Conclusions: Further research is needed to determine if these immune responses are predictors of vaccine efficacy or markers of natural resistance to HIV-1 infection.

Identification of effective subdominant anti-HIV-1 CD8+ T cells within entire post-infection and post-vaccination immune responses.

Defining the components of an HIV immunogen that could induce effective CD8+ T cell responses is critical to vaccine development. We addressed this question by investigating the viral targets of CD8+ T cells that potently inhibit HIV replication in vitro, as this is highly predictive of virus control in vivo. We observed broad and potent ex vivo CD8+ T cell-mediated viral inhibitory activity against a panel of HIV isolates among viremic controllers (VC, viral loads <5000 copies/ml), in contrast to unselected HIV-infected HIV Vaccine trials Network (HVTN) participants. Viral inhibition of clade-matched HIV isolates was strongly correlated with the frequency of CD8+ T cells targeting vulnerable regions within Gag, Pol, Nef and Vif that had been identified in an independent study of nearly 1000 chronically infected individuals. These vulnerable and so-called "beneficial" regions were of low entropy overall, yet several were not predicted by stringent conservation algorithms. Consistent with this, stronger inhibition of clade-matched than mismatched viruses was observed in the majority of subjects, indicating better targeting of clade-specific than conserved epitopes. The magnitude of CD8+ T cell responses to beneficial regions, together with viral entropy and HLA class I genotype, explained up to 59% of the variation in viral inhibitory activity, with magnitude of the T cell response making the strongest unique contribution. However, beneficial regions were infrequently targeted by CD8+ T cells elicited by vaccines encoding full-length HIV proteins, when the latter were administered to healthy volunteers and HIV-positive ART-treated subjects, suggesting that immunodominance hierarchies undermine effective anti-HIV CD8+ T cell responses. Taken together, our data support HIV immunogen design that is based on systematic selection of empirically defined vulnerable regions within the viral proteome, with exclusion of immunodominant decoy epitopes that are irrelevant for HIV control.

T Cell Responses against Mycobacterial Lipids and Proteins Are Poorly Correlated in South African Adolescents.

Human T cells are activated by both peptide and nonpeptide Ags produced by Mycobacterium tuberculosis. T cells recognize cell wall lipids bound to CD1 molecules, but effector functions of CD1-reactive T cells have not been systematically assessed in M. tuberculosis-infected humans. It is also not known how these features correlate with T cell responses to secreted protein Ags. We developed a flow cytometric assay to profile CD1-restricted T cells ex vivo and assessed T cell responses to five cell wall lipid Ags in a cross-sectional study of 19 M. tuberculosis-infected and 22 M. tuberculosis-uninfected South African adolescents. We analyzed six T cell functions using a recently developed computational approach for flow cytometry data in high dimensions. We compared these data with T cell responses to five protein Ags in the same cohort. We show that CD1b-restricted T cells producing antimycobacterial cytokines IFN-γ and TNF-α are detectable ex vivo in CD4(+), CD8(+), and CD4(-)CD8(-) T cell subsets. Glucose monomycolate was immunodominant among lipid Ags tested, and polyfunctional CD4 T cells specific for this lipid simultaneously expressed CD40L, IFN-γ, IL-2, and TNF-α. Lipid-reactive CD4(+) T cells were detectable at frequencies of 0.001-0.01%, and this did not differ by M. tuberculosis infection status. Finally, CD4 T cell responses to lipids were poorly correlated with CD4 T cell responses to proteins (Spearman rank correlation -0.01; p = 0.95). These results highlight the functional diversity of CD1-restricted T cells circulating in peripheral blood as well as the complementary nature of T cell responses to mycobacterial lipids and proteins. Our approach enables further population-based studies of lipid-specific T cell responses during natural infection and vaccination.

Assessment of the Safety and Immunogenicity of 2 Novel Vaccine Platforms for HIV-1 Prevention: A Randomized Trial.

BACKGROUND: A prophylactic HIV-1 vaccine is a global health priority. OBJECTIVE: To assess a novel vaccine platform as a prophylactic HIV-1 regimen. DESIGN: Randomized, double-blind, placebo-controlled trial. Both participants and study personnel were blinded to treatment allocation. (ClinicalTrials.gov: NCT01215149). SETTING: United States, East Africa, and South Africa. PATIENTS: Healthy adults without HIV infection. INTERVENTION: 2 HIV-1 vaccines (adenovirus serotype 26 with an HIV-1 envelope A insert [Ad26.EnvA] and adenovirus serotype 35 with an HIV-1 envelope A insert [Ad35.Env], both administered at a dose of 5 × 1010 viral particles) in homologous and heterologous combinations. MEASUREMENTS: Safety and immunogenicity and the effect of baseline vector immunity. RESULTS: 217 participants received at least 1 vaccination, and 210 (>96%) completed follow-up. No vaccine-associated serious adverse events occurred. All regimens were generally well-tolerated. All regimens elicited humoral and cellular immune responses in nearly all participants. Preexisting Ad26- or Ad35-neutralizing antibody titers had no effect on vaccine safety and little effect on immunogenicity. In both homologous and heterologous regimens, the second vaccination significantly increased EnvA antibody titers (approximately 20-fold from the median enzyme-linked immunosorbent assay titers of 30-300 to 3000). The heterologous regimen of Ad26-Ad35 elicited significantly higher EnvA antibody titers than Ad35-Ad26. T-cell responses were modest and lower in East Africa than in South Africa and the United States. LIMITATIONS: Because the 2 envelope inserts were not identical, the boosting responses were complex to interpret. Durability of the immune responses elicited beyond 1 year is unknown. CONCLUSION: Both vaccines elicited significant immune responses in all populations. Baseline vector immunity did not significantly affect responses. Second vaccinations in all regimens significantly boosted EnvA antibody titers, although vaccine order in the heterologous regimen had a modest effect on the immune response. PRIMARY FUNDING SOURCE: International AIDS Vaccine Initiative, National Institutes of Health, Ragon Institute, Crucell Holland.

Lower Viral Loads and Slower CD4+ T-Cell Count Decline in MRKAd5 HIV-1 Vaccinees Expressing Disease-Susceptible HLA-B*58:02.

BACKGROUND: HLA strongly influences human immunodeficiency virus type 1 (HIV-1) disease progression. A major contributory mechanism is via the particular HLA-presented HIV-1 epitopes that are recognized by CD8(+) T-cells. Different populations vary considerably in the HLA alleles expressed. We investigated the HLA-specific impact of the MRKAd5 HIV-1 Gag/Pol/Nef vaccine in a subset of the infected Phambili cohort in whom the disease-susceptible HLA-B*58:02 is highly prevalent. METHODS: Viral loads, CD4(+) T-cell counts, and enzyme-linked immunospot assay-determined anti-HIV-1 CD8(+) T-cell responses for a subset of infected antiretroviral-naive Phambili participants, selected according to sample availability, were analyzed. RESULTS: Among those expressing disease-susceptible HLA-B*58:02, vaccinees had a lower chronic viral set point than placebo recipients (median, 7240 vs 122 500 copies/mL; P = .01), a 0.76 log10 lower longitudinal viremia level (P = .01), and slower progression to a CD4(+) T-cell count of <350 cells/mm(3) (P = .02). These differences were accompanied by a higher Gag-specific breadth (4.5 vs 1 responses; P = .04) and magnitude (2300 vs 70 spot-forming cells/10(6) peripheral blood mononuclear cells; P = .06) in vaccinees versus placebo recipients. CONCLUSIONS: In addition to the known enhancement of HIV-1 acquisition resulting from the MRKAd5 HIV-1 vaccine, these findings in a nonrandomized subset of enrollees show an HLA-specific vaccine effect on the time to CD4(+) T-cell count decline and viremia level after infection and the potential for vaccines to differentially alter disease outcome according to population HLA composition. CLINICAL TRIALS REGISTRATION: NCT00413725, DOH-27-0207-1539.

Vaccination With Heterologous HIV-1 Envelope Sequences and Heterologous Adenovirus Vectors Increases T-Cell Responses to Conserved Regions: HVTN 083.

BACKGROUND: Increasing the breadth of human immunodeficiency virus type 1 (HIV-1) vaccine-elicited immune responses or targeting conserved regions may improve coverage of circulating strains. HIV Vaccine Trials Network 083 tested whether cellular immune responses with these features are induced by prime-boost strategies, using heterologous vectors, heterologous inserts, or a combination of both. METHODS: A total of 180 participants were randomly assigned to receive combinations of adenovirus vectors (Ad5 or Ad35) and HIV-1 envelope (Env) gene inserts (clade A or B) in a prime-boost regimen. RESULTS: T-cell responses to heterologous and homologous insert regimens targeted a similar number of epitopes (ratio of means, 1.0; 95% confidence interval [CI], .6-1.6; P = .91), but heterologous insert regimens induced significantly more epitopes that were shared between EnvA and EnvB than homologous insert regimens (ratio of means, 2.7; 95% CI, 1.2-5.7; P = .01). Participants in the heterologous versus homologous insert groups had T-cell responses that targeted epitopes with greater evolutionary conservation (mean entropy [±SD], 0.32 ± 0.1 bits; P = .003), and epitopes recognized by responders provided higher coverage (49%; P = .035). Heterologous vector regimens had higher numbers of total, EnvA, and EnvB epitopes than homologous vector regimens (P = .02, .044, and .045, respectively). CONCLUSIONS: These data demonstrate that vaccination with heterologous insert prime boosting increased T-cell responses to shared epitopes, while heterologous vector prime boosting increased the number of T-cell epitopes recognized. CLINICAL TRIALS REGISTRATION: NCT01095224.

Efficacy trial of a DNA/rAd5 HIV-1 preventive vaccine.

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.).

Fitness-Balanced Escape Determines Resolution of Dynamic Founder Virus Escape Processes in HIV-1 Infection.

UNLABELLED: To understand the interplay between host cytotoxic T-lymphocyte (CTL) responses and the mechanisms by which HIV-1 evades them, we studied viral evolutionary patterns associated with host CTL responses in six linked transmission pairs. HIV-1 sequences corresponding to full-length p17 and p24 gag were generated by 454 pyrosequencing for all pairs near the time of transmission, and seroconverting partners were followed for a median of 847 days postinfection. T-cell responses were screened by gamma interferon/interleukin-2 (IFN-γ/IL-2) FluoroSpot using autologous peptide sets reflecting any Gag variant present in at least 5% of sequence reads in the individual's viral population. While we found little evidence for the occurrence of CTL reversions, CTL escape processes were found to be highly dynamic, with multiple epitope variants emerging simultaneously. We found a correlation between epitope entropy and the number of epitope variants per response (r = 0.43; P = 0.05). In cases in which multiple escape mutations developed within a targeted epitope, a variant with no fitness cost became fixed in the viral population. When multiple mutations within an epitope achieved fitness-balanced escape, these escape mutants were each maintained in the viral population. Additional mutations found to confer escape but undetected in viral populations incurred high fitness costs, suggesting that functional constraints limit the available sites tolerable to escape mutations. These results further our understanding of the impact of CTL escape and reversion from the founder virus in HIV infection and contribute to the identification of immunogenic Gag regions most vulnerable to a targeted T-cell attack. IMPORTANCE: Rapid diversification of the viral population is a hallmark of HIV-1 infection, and understanding the selective forces driving the emergence of viral variants can provide critical insight into the interplay between host immune responses and viral evolution. We used deep sequencing to comprehensively follow viral evolution over time in six linked HIV transmission pairs. We then mapped T-cell responses to explore if mutations arose due to adaption to the host and found that escape processes were often highly dynamic, with multiple mutations arising within targeted epitopes. When we explored the impact of these mutations on replicative capacity, we found that dynamic escape processes only resolve with the selection of mutations that conferred escape with no fitness cost to the virus. These results provide further understanding of the complicated viral-host interactions that occur during early HIV-1 infection and may help inform the design of future vaccine immunogens.

Safety and Immunogenicity of Novel Adenovirus Type 26- and Modified Vaccinia Ankara-Vectored Ebola Vaccines: A Randomized Clinical Trial.

IMPORTANCE: Developing effective vaccines against Ebola virus is a global priority. OBJECTIVE: To evaluate an adenovirus type 26 vector vaccine encoding Ebola glycoprotein (Ad26.ZEBOV) and a modified vaccinia Ankara vector vaccine, encoding glycoproteins from Ebola virus, Sudan virus, Marburg virus, and Tai Forest virus nucleoprotein (MVA-BN-Filo). DESIGN, SETTING, AND PARTICIPANTS: Single-center, randomized, placebo-controlled, observer-blind, phase 1 trial performed in Oxford, United Kingdom, enrolling healthy 18- to 50-year-olds from December 2014; 8-month follow-up was completed October 2015. INTERVENTIONS: Participants were randomized into 4 groups, within which they were simultaneously randomized 5:1 to receive study vaccines or placebo. Those receiving active vaccines were primed with Ad26.ZEBOV (5 × 10(10) viral particles) or MVA-BN-Filo (1 × 10(8) median tissue culture infective dose) and boosted with the alternative vaccine 28 or 56 days later. A fifth, open-label group received Ad26.ZEBOV boosted by MVA-BN-Filo 14 days later. MAIN OUTCOMES AND MEASURES: The primary outcomes were safety and tolerability. All adverse events were recorded until 21 days after each immunization; serious adverse events were recorded throughout the trial. Secondary outcomes were humoral and cellular immune responses to immunization, as assessed by enzyme-linked immunosorbent assay and enzyme-linked immunospot performed at baseline and from 7 days after each immunization until 8 months after priming immunizations. RESULTS: Among 87 study participants (median age, 38.5 years; 66.7% female), 72 were randomized into 4 groups of 18, and 15 were included in the open-label group. Four participants did not receive a booster dose; 67 of 75 study vaccine recipients were followed up at 8 months. No vaccine-related serious adverse events occurred. No participant became febrile after MVA-BN-Filo, compared with 3 of 60 participants (5%; 95% CI, 1%-14%) receiving Ad26.ZEBOV in the randomized groups. In the open-label group, 4 of 15 Ad26.ZEBOV recipients (27%; 95% CI, 8%-55%) experienced fever. In the randomized groups, 28 of 29 Ad26.ZEBOV recipients (97%; 95% CI, 82%- 99.9%) and 7 of 30 MVA-BN-Filo recipients (23%; 95% CI, 10%-42%) had detectable Ebola glycoprotein-specific IgG 28 days after primary immunization. All vaccine recipients had specific IgG detectable 21 days postboost and at 8-month follow-up. Within randomized groups, at 7 days postboost, at least 86% of vaccine recipients showed Ebola-specific T-cell responses. CONCLUSIONS AND RELEVANCE: In this phase 1 study of healthy volunteers, immunization with Ad26.ZEBOV or MVA-BN-Filo did not result in any vaccine-related serious adverse events. An immune response was observed after primary immunization with Ad26.ZEBOV; boosting by MVA-BN-Filo resulted in sustained elevation of specific immunity. These vaccines are being further assessed in phase 2 and 3 studies. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT02313077.

Immune-correlates analysis of an HIV-1 vaccine efficacy trial.

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.

Increased sequence coverage through combined targeting of variant and conserved epitopes correlates with control of HIV replication.

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.