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In the Antibody Mediated Prevention (AMP) trials (HVTN 704/HPTN 085 and HVTN 703/HPTN 081), prevention efficacy (PE) of the monoclonal broadly neutralizing antibody (bnAb) VRC01 (vs. placebo) against HIV-1 acquisition diagnosis varied according to the HIV-1 Envelope (Env) neutralization sensitivity to VRC01, as measured by 80% inhibitory concentration (IC80). Here, we performed a genotypic sieve analysis, a complementary approach to gaining insight into correlates of protection that assesses how PE varies with HIV-1 sequence features. We analyzed HIV-1 Env amino acid (AA) sequences from the earliest available HIV-1 RNA-positive plasma samples from AMP participants diagnosed with HIV-1 and identified Env sequence features that associated with PE. The strongest Env AA sequence correlate in both trials was VRC01 epitope distance that quantifies the divergence of the VRC01 epitope in an acquired HIV-1 isolate from the VRC01 epitope of reference HIV-1 strains that were most sensitive to VRC01-mediated neutralization. In HVTN 704/HPTN 085, the Env sequence-based predicted probability that VRC01 IC80 against the acquired isolate exceeded 1 µg/mL also significantly associated with PE. In HVTN 703/HPTN 081, a physicochemical-weighted Hamming distance across 50 VRC01 binding-associated Env AA positions of the acquired isolate from the most VRC01-sensitive HIV-1 strain significantly associated with PE. These results suggest that incorporating mutation scoring by BLOSUM62 and weighting by the strength of interactions at AA positions in the epitope:VRC01 interface can optimize performance of an Env sequence-based biomarker of VRC01 prevention efficacy. Future work could determine whether these results extend to other bnAbs and bnAb combinations. [ABSTRACT FROM AUTHOR]

An objective in randomized clinical trials is the evaluation of "principal surrogates," which consists of analyzing how the treatment effect on a clinical endpoint varies over principal strata subgroups defined by an intermediate response outcome under both or one of the treatment assignments. The latter effect modification estimand has been termed the marginal causal effect predictiveness (mCEP) curve. This objective was addressed in two randomized placebo-controlled Phase 3 dengue vaccine trials for an antibody response biomarker whose sampling design rendered previously developed inferential methods highly inefficient due to a three-phase sampling design. In this design, the biomarker was measured in a case-cohort sample and a key baseline auxiliary strongly associated with the biomarker (the "baseline surrogate measure") was only measured in a further sub-sample. We propose a novel approach to estimation of the mCEP curve in such three-phase sampling designs that avoids the restrictive "placebo structural risk" modeling assumption common to past methods and that further improves robustness by the use of non-parametric kernel smoothing for biomarker density estimation. Additionally, we develop bootstrap-based procedures for pointwise and simultaneous confidence intervals and testing of four relevant hypotheses about the mCEP curve. We investigate the finite-sample properties of the proposed methods and compare them to those of an alternative method making the placebo structural risk assumption. Finally, we apply the novel and alternative procedures to the two dengue vaccine trial data sets. [ABSTRACT FROM AUTHOR]

The ability to produce a long-lasting, or durable, immune response is a crucial characteristic of many highly effective vaccines. A goal of early-phase vaccine trials is often to compare the immune response durability of multiple tested vaccine regimens. One parameter for measuring immune response durability is the area under the mean post-peak log immune response profile. In this paper, we compare immune response durability across vaccine regimens within and between two phase I trials of DNA-primed HIV vaccine regimens, HVTN 094 and HVTN 096. We compare four estimators of this durability parameter and the resulting statistical inferences for comparing vaccine regimens. Two of these estimators use the trapezoid rule as an empirical approximation of the area under the marginal log response curve, and the other two estimators are based on linear and nonlinear models for the marginal mean log response. We conduct a simulation study to compare the four estimators, provide guidance on estimator selection, and use the nonlinear marginal mean model to analyze immunogenicity data from the two HIV vaccine trials. [ABSTRACT FROM AUTHOR]

Two phase 3 placebo-controlled trials of the CYD-TDV vaccine, evaluated in children aged 2-14 y (CYD14) and 9-16 y (CYD15), demonstrated vaccine efficacy (VE) of 56.5% and 60.8%, respectively, against symptomatic virologically confirmed dengue (VCD). Sieve analyses were conducted to evaluate whether and how VE varied with amino acid sequence features of dengue viruses (DENVs). DENV premembrane/envelope amino acid sequences from VCD endpoint cases were aligned with the vaccine insert sequences, and extensions of the proportional hazards model were applied to assess variation in VE with amino acid mismatch proportion distances from vaccine strains, individual amino acid residues, and phylogenetic genotypes. In CYD14, VE against VCD of any serotype (DENV-Any) decreased significantly with increasing amino acid distance from the vaccine, whereas in CYD15, VE against DENV-Any was distance-invariant. Restricting to the common age range and amino acid distance range between the trials and accounting for differential VE by serotype, however, showed no evidence of VE variation with distance in either trial. In serotype-specific analyses, VE against DENV4 decreased significantly with increasing amino acid distance from the DENV4 vaccine insert and was significantly greater against residuematched DENV4 at eight signature positions. These effects were restricted to 2- to 8-y-olds, potentially because greater seropositivity of older children at baseline might facilitate a broader protective immune response. The relevance of an antigenicmatch between vaccine strains and circulating DENVs was also supported by greater estimated VE against serotypes and genotypes for which the circulating DENVs had shorter amino acid sequence distances from the vaccine. [ABSTRACT FROM AUTHOR]

Background: In the CYD14 and CYD15 Phase 3 trials of the CYD-TDV dengue vaccine, estimated vaccine efficacy (VE) against symptomatic, virologically confirmed dengue (VCD) occurring between months 13 and 25 was 56.5% and 60.8%, respectively.Methods: Neutralizing antibody titers to the 4 dengue serotypes in the CYD-TDV vaccine insert were measured at month 13 in a randomly sampled immunogenicity subcohort and in all VCD cases through month 25 (2848 vaccine, 1574 placebo) and studied for their association with VCD and with the level of VE to prevent VCD.Results: For each trial and serotype, vaccinees with higher month 13 titer to the serotype had significantly lower risk of VCD with that serotype (hazard ratios, 0.19-0.43 per 10-fold increase). Moreover, for each trial, vaccinees with higher month 13 average titer to the 4 serotypes had significantly higher VE against VCD of any serotype (P < .001).Conclusions: Neutralizing antibody titers postdose 3 correlate with CYD-TDV VE to prevent dengue. High titers associate with high VE for all serotypes, baseline serostatus groups, age groups, and both trials. However, lowest titers do not fully correspond to zero VE, indicating that other factors influence VE. [ABSTRACT FROM AUTHOR]

• This correlates analysis integrated host immune response & pathogen sequence data. • The nAb correlate of risk depends on dengue sequence in baseline seropositive participants. • Dependency of correlates on sequence-distance stronger in the vaccine (vs placebo) arm. • Infection- vs vaccine-induced nAbs may affect dengue risk differently. • nAb titer approximate surrogate endpoint of dengue disease in baseline-seropositive 9–14-year-olds. In the CYD14 trial of the CYD-TDV dengue vaccine in 2–14 year-olds, neutralizing antibody (nAb) titers to the vaccine-insert dengue strains correlated inversely with symptomatic, virologically-confirmed dengue (VCD). Also, vaccine efficacy against VCD was higher against dengue prM/E amino acid sequences closer to the vaccine inserts. We integrated the nAb and sequence data types by assessing nAb titers as a correlate of sequence-specific VCD separately in the vaccine arm and in the placebo arm. In both vaccine and placebo recipients the correlation of nAb titer with sequence-specific VCD was stronger for dengue nAb contact site sequences closer to the vaccine (p = 0.005 and p = 0.012, respectively). The risk of VCD in vaccine (placebo) recipients was 6.7- (1.80)-fold lower at the 90th vs 10th percentile of nAb for viruses perfectly matched to CYD-TDV, compared to 2.1- (0.78)-fold lower at the 90th vs 10th percentile for viruses with five amino acid mismatches. The evidence for a stronger sequence-distance dependent correlate of risk for the vaccine arm indicates departure from the Prentice criteria for a valid sequence-distance specific surrogate endpoint and suggests that the nAb marker may affect dengue risk differently depending on whether nAbs arise from infection or also by vaccination. However, when restricting to baseline-seropositive 9–14 year-olds, the correlation pattern became more similar between the vaccine and placebo arms, supporting nAb titers as an approximate surrogate endpoint in this population. No sequence-specific nAb titer correlates of VCD were seen in baseline-seronegative participants. Integrated immune response/pathogen sequence data correlates analyses could help increase knowledge of correlates of risk and surrogate endpoints for other vaccines against genetically diverse pathogens. Trial registration: EU Clinical Trials Register 2014-001708-24; registration date 2014-05-26. [ABSTRACT FROM AUTHOR]

Background: The antibody-mediated prevention (AMP) studies (HVTN 703/HPTN 081 and HVTN 704/HPTN 085) are harmonized phase 2b trials to assess HIV prevention efficacy and safety of intravenous infusion of anti-gp120 broadly neutralizing antibody VRC01. Antibodies for other indications can elicit infusion-related reactions (IRRs), often requiring premedication and limiting their application. We report on AMP study IRRs. Methods: From 2016 to 2018, 2699 HIV-uninfected, at-risk men and transgender adults in the Americas and Switzerland (704/085) and 1924 at-risk heterosexual women in sub-Saharan Africa (703/081) were randomized 1:1:1 to VRC01 10 mg/kg, 30 mg/kg, or placebo. Participants received infusions every 8 weeks (n = 10/participant) over 72 weeks, with 104 weeks of follow-up. Safety assessments were conducted before and after infusion and at noninfusion visits. A total of 40,674 infusions were administered. Results: Forty-seven participants (1.7%) experienced 49 IRRs in 704/085; 93 (4.8%) experienced 111 IRRs in 703/081 (P < 0.001). IRRs occurred more frequently in VRC01 than placebo recipients in 703/081 (P < 0.001). IRRs were associated with atopic history (P = 0.046) and with younger age (P = 0.023) in 703/081. Four clinical phenotypes of IRRs were observed: urticaria, dyspnea, dyspnea with rash, and "other." Urticaria was most prevalent, occurring in 25 (0.9%) participants in 704/085 and 41 (2.1%) participants in 703/081. Most IRRs occurred with the initial infusion and incidence diminished through the last infusion. All reactions were managed successfully without sequelae. Conclusions: IRRs in the AMP studies were uncommon, typically mild or moderate, successfully managed at the research clinic, and resolved without sequelae. Analysis is ongoing to explore potential IRR mechanisms. [ABSTRACT FROM AUTHOR]

Stepped wedge designs are increasingly commonplace and advantageous for cluster randomized trials when it is both unethical to assign placebo, and it is logistically difficult to allocate an intervention simultaneously to many clusters. We study marginal mean models fit with generalized estimating equations for assessing treatment effectiveness in stepped wedge cluster randomized trials. This approach has advantages over the more commonly used mixed models that (1) the population-average parameters have an important interpretation for public health applications and (2) they avoid untestable assumptions on latent variable distributions and avoid parametric assumptions about error distributions, therefore, providing more robust evidence on treatment effects. However, cluster randomized trials typically have a small number of clusters, rendering the standard generalized estimating equation sandwich variance estimator biased and highly variable and hence yielding incorrect inferences. We study the usual asymptotic generalized estimating equation inferences (i.e., using sandwich variance estimators and asymptotic normality) and four small-sample corrections to generalized estimating equation for stepped wedge cluster randomized trials and for parallel cluster randomized trials as a comparison. We show by simulation that the small-sample corrections provide improvement, with one correction appearing to provide at least nominal coverage even with only 10 clusters per group. These results demonstrate the viability of the marginal mean approach for both stepped wedge and parallel cluster randomized trials. We also study the comparative performance of the corrected methods for stepped wedge and parallel designs, and describe how the methods can accommodate interval censoring of individual failure times and incorporate semiparametric efficient estimators. [ABSTRACT FROM AUTHOR]

The recombinant, live, attenuated, tetravalent dengue vaccine CYD-TDV has shown efficacy against all four dengue serotypes. In this exploratory study (CYD59, NCT02827162), we evaluated potential associations of host human leukocyte antigen (HLA) alleles with dengue antibody responses, CYD-TDV vaccine efficacy, and virologically-confirmed dengue (VCD) cases. Children 4–11 years old, who previously completed a phase 2b efficacy study of CYD-TDV in a single center in Thailand, were included in the study. Genotyping of HLA class I and II loci was performed by next-generation sequencing from DNA obtained from 335 saliva samples. Dengue neutralizing antibody titers (NAb) were assessed as a correlate of risk and protection. Regression analyses were used to assess associations between HLA alleles and NAb responses, vaccine efficacy, and dengue outcomes. Month 13 NAb log geometric mean titers (GMTs) were associated with decreased risk of VCD. In the vaccine group, HLA-DRB1*11 was significantly associated with higher NAb log GMT levels (beta: 0.76; p = 0.002, q = 0.13). Additionally, in the absence of vaccination, HLA associations were observed between the presence of DPB1*03:01 and increased NAb log GMT levels (beta: 1.24; p = 0.005, q = 0.17), and between DPB1*05:01 and reduced NAb log GMT levels (beta: −1.1; p = 0.001, q = 0.07). This study suggests associations of HLA alleles with NAb titers in the context of dengue outcomes. This study was registered with clinicaltrials.gov: NCT02827162. [ABSTRACT FROM AUTHOR]

Multiple candidate vaccines to prevent COVID-19 have entered large-scale phase 3 placebo-controlled randomized clinical trials, and several have demonstrated substantial short-term efficacy. At some point after demonstration of substantial efficacy, placebo recipients should be offered the efficacious vaccine from their trial, which will occur before longer-term efficacy and safety are known. The absence of a placebo group could compromise assessment of longer-term vaccine effects. However, by continuing follow-up after vaccination of the placebo group, this study shows that placebo-controlled vaccine efficacy can be mathematically derived by assuming that the benefit of vaccination over time has the same profile for the original vaccine recipients and the original placebo recipients after their vaccination. Although this derivation provides less precise estimates than would be obtained by a standard trial where the placebo group remains unvaccinated, this proposed approach allows estimation of longer-term effect, including durability of vaccine efficacy and whether the vaccine eventually becomes harmful for some. Deferred vaccination, if done open-label, may lead to riskier behavior in the unblinded original vaccine group, confounding estimates of long-term vaccine efficacy. Hence, deferred vaccination via blinded crossover, where the vaccine group receives placebo and vice versa, would be the preferred way to assess vaccine durability and potential delayed harm. Deferred vaccination allows placebo recipients timely access to the vaccine when it would no longer be proper to maintain them on placebo, yet still allows important insights about immunologic and clinical effectiveness over time. [ABSTRACT FROM AUTHOR]

Background: HIV Vaccine Trials Network 703/HIV Prevention Trials Network 081 is a phase 2b randomized, double-blind, placebo-controlled trial to assess the safety and efficacy of passively infused monoclonal antibody VRC01 in preventing HIV acquisition in heterosexual women between the ages of 18 and 50 years at risk of HIV. Participants were enrolled at 20 sites in Botswana, Kenya, Malawi, Mozambique, South Africa, Tanzania, and Zimbabwe. It is one of the 2 Antibody Mediated Prevention efficacy trials, with HIV Vaccine Trials Network 704/ HIV Prevention Trials Network 085, evaluating VRC01 for HIV prevention. Methods: Intense community engagement was used to optimize participant recruitment and retention. Participants were randomly assigned to receive intravenous VRC01 10 mg/kg, VRC01 30 mg/kg, or placebo in a 1:1:1 ratio. Infusions were given every 8 weeks with a total of 10 infusions and 104 weeks of follow-up after the first infusion. Results: Between May 2016 and September 2018, 1924 women from sub-Saharan Africa were enrolled. The median age was 26 years (interquartile range: 22-30), and 98.9% were Black. Sexually transmitted infection prevalence at enrollment included chlamydia (16.9%), trichomonas (7.2%), gonorrhea (5.7%), and syphilis (2.2%). External condoms (83.2%) and injectable contraceptives (61.1%) were the methods of contraception most frequently used by participants. In total, through April 3, 2020, 38,490 clinic visits were completed with a retention rate of 96% and 16,807 infusions administered with an adherence rate of 98%. Conclusions: This proof-of-concept, large-scale monoclonal antibody study demonstrates the feasibility of conducting complex trials involving intravenous infusions in high incidence populations in sub-Saharan Africa. [ABSTRACT FROM AUTHOR]

Several vaccine candidates to protect against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or coronavirus disease 2019 (COVID-19) have entered or will soon enter large-scale, phase 3, placebo-controlled randomized clinical trials. To facilitate harmonized evaluation and comparison of the efficacy of these vaccines, a general set of clinical endpoints is proposed, along with considerations to guide the selection of the primary endpoints on the basis of clinical and statistical reasoning. The plausibility that vaccine protection against symptomatic COVID-19 could be accompanied by a shift toward more SARS-CoV-2 infections that are asymptomatic is highlighted, as well as the potential implications of such a shift. [ABSTRACT FROM AUTHOR]

We previously showed that Month 13 50% plaque reduction neutralization test (PRNT50) neutralizing antibody (nAb) titers against dengue virus (DENV) correlated with vaccine efficacy (VE) of CYD-TDV against symptomatic, virologically-confirmed dengue (VCD) in the CYD14 and CYD15 Phase 3 trials. While PRNT is the gold standard nAb assay, it is time-consuming and costly. We developed a next-generation high-throughput microneutralization (MN) assay and assessed its suitability for immune-correlates analyses and immuno-bridging applications. We analyzed MN and PRNT50 titers measured at baseline and Month 13 in a randomly sampled immunogenicity subset, and at Month 13 in nearly all VCD cases through Month 25. For each serotype, MN and PRNT50 titers showed high correlations, at both baseline and Month 13, with MN yielding a higher frequency of baseline-seronegatives. For both assays, Month 13 titer correlated inversely with VCD risk. Like PRNT50, high Month 13 MN titers were associated with high VE, and estimated VE increased with average Month 13 MN titer. We also studied each assay as a valid surrogate endpoint based on the Prentice criteria, which supported each assay as a valid surrogate for DENV-1 but only partially valid for DENV-2, -3, and -4. In addition, we applied Super-Learner to assess how well demographic, Month 13 MN, and/or Month 13 PRNT50 titers could predict Month 13–25 VCD outcome status; prediction was best when using demographic, MN, and PRNT50 information. We conclude that Month 13 MN titer performs comparably to Month 13 PRNT50 titer as a correlate of risk, correlate of vaccine efficacy, and surrogate endpoint. The MN assay could potentially be used to assess nAb titers in immunogenicity studies, immune-correlates studies, and immuno-bridging applications. Additional research would be needed for assessing the utility of MN titer in correlates analyses of other DENV endpoints and over longer follow-up periods. [ABSTRACT FROM AUTHOR]

Background: High hemagglutination inhibition (HAI) and neuraminidase inhibition (NAI) titers are generally associated with reduced influenza risk. While repeated influenza vaccination reduces seroresponse, vaccine effectiveness is not always reduced.Methods: During the 2007-2008 influenza season, a randomized, placebo-controlled trial (FLUVACS) evaluated the efficacies of live-attenuated (LAIV) and inactivated influenza vaccines (IIV) among healthy adults aged 18-49 in Michigan; IIV vaccine efficacy (VE) and LAIV VE against influenza disease were estimated at 68% and 36%. Using the principal stratification/VE moderation framework, we analyzed data from this trial to assess how each VE varied by HAI or NAI responses to vaccination observed for vaccinated individuals and predicted counterfactually for placebo recipients. We also assessed how each VE varied with pre-vaccination/baseline variables including HAI titer, NAI titer, and vaccination history.Results: IIV VE appeared to increase with Day 30 post-vaccination HAI titer, albeit not significantly (p=0.20 and estimated VE 14.4%, 70.5%, and 85.5% at titer below the assay lower quantification limit, 512, and 4096 (maximum)). Moreover, IIV VE increased significantly with Day 30 post-vaccination NAI titer (p=0.040), with estimated VE zero at titer 10 and 92.2% at highest titer 640. There was no evidence that fold-change in post-vaccination HAI or NAI titer associated with IIV VE (p=0.76, 0.38). For LAIV, there was no evidence that VE associated with post-vaccination or fold-rise HAI or NAI titers (p-values >0.40). For IIV, VE increased with increasing baseline NAI titer in those previously vaccinated, but VE decreased with increasing baseline NAI titer in those previously unvaccinated. In contrast, for LAIV, VE did not depend on previous vaccination or baseline HAI or NAI titer.Conclusions: Future efficacy trials should measure baseline and post-vaccination antibody titers in both vaccine and control/placebo recipients, enabling analyses to better elucidate correlates of vaccine- and natural-protection.Trial Registration: ClinicalTrials.gov NCT00538512. October 1, 2007. [ABSTRACT FROM AUTHOR]

The broadly neutralizing antibody (bnAb) VRC01 is being evaluated for its efficacy to prevent HIV-1 infection in the Antibody Mediated Prevention (AMP) trials. A secondary objective of AMP utilizes sieve analysis to investigate how VRC01 prevention efficacy (PE) varies with HIV-1 envelope (Env) amino acid (AA) sequence features. An exhaustive analysis that tests how PE depends on every AA feature with sufficient variation would have low statistical power. To design an adequately powered primary sieve analysis for AMP, we modeled VRC01 neutralization as a function of Env AA sequence features of 611 HIV-1 gp160 pseudoviruses from the CATNAP database, with objectives: (1) to develop models that best predict the neutralization readouts; and (2) to rank AA features by their predictive importance with classification and regression methods. The dataset was split in half, and machine learning algorithms were applied to each half, each analyzed separately using cross-validation and hold-out validation. We selected Super Learner, a nonparametric ensemble-based cross-validated learning method, for advancement to the primary sieve analysis. This method predicted the dichotomous resistance outcome of whether the IC50 neutralization titer of VRC01 for a given Env pseudovirus is right-censored (indicating resistance) with an average validated AUC of 0.868 across the two hold-out datasets. Quantitative log IC50 was predicted with an average validated R2 of 0.355. Features predicting neutralization sensitivity or resistance included 26 surface-accessible residues in the VRC01 and CD4 binding footprints, the length of gp120, the length of Env, the number of cysteines in gp120, the number of cysteines in Env, and 4 potential N-linked glycosylation sites; the top features will be advanced to the primary sieve analysis. This modeling framework may also inform the study of VRC01 in the treatment of HIV-infected persons. [ABSTRACT FROM AUTHOR]

Although the HVTN 505 DNA/recombinant adenovirus type 5 vector HIV-1 vaccine trial showed no overall efficacy, analysis of breakthrough HIV-1 sequences in participants can help determine whether vaccine-induced immune responses impacted viruses that caused infection. We analyzed 480 HIV-1 genomes sampled from 27 vaccine and 20 placebo recipients and found that intra-host HIV-1 diversity was significantly lower in vaccine recipients (P ≤ 0.04, Q-values ≤ 0.09) in Gag, Pol, Vif and envelope glycoprotein gp120 (Env-gp120). Furthermore, Env-gp120 sequences from vaccine recipients were significantly more distant from the subtype B vaccine insert than sequences from placebo recipients (P = 0.01, Q-value = 0.12). These vaccine effects were associated with signatures mapping to CD4 binding site and CD4-induced monoclonal antibody footprints. These results suggest either (i) no vaccine efficacy to block acquisition of any viral genotype but vaccine-accelerated Env evolution post-acquisition; or (ii) vaccine efficacy against HIV-1s with Env sequences closest to the vaccine insert combined with increased acquisition due to other factors, potentially including the vaccine vector. [ABSTRACT FROM AUTHOR]

Measurement error is well known to cause bias in estimated regression coefficients and a loss of power for detecting associations. Methods commonly used to correct for bias often require auxiliary data. We develop a solution for investigating associations between the change in an imprecisely measured outcome and precisely measured predictors, adjusting for the baseline value of the outcome when auxiliary data are not available. We require the specification of ranges for the reliability or the measurement error variance. The solution allows one to investigate the associations for change and to assess the impact of the measurement error. [ABSTRACT FROM AUTHOR]

The RV144 clinical trial showed the partial efficacy of a vaccine regimen with an estimated vaccine efficacy (VE) of 31% for protecting low-risk Thai volunteers against acquisition of HIV-1. The impact of vaccine-induced immune responses can be investigated through sieve analysis of HIV-1 breakthrough infections (infected vaccine and placebo recipients). A V1/V2-targeted comparison of the genomes of HIV-1 breakthrough viruses identified two V2 amino acid sites that differed between the vaccine and placebo groups. Here we extended the V1/V2 analysis to the entire HIV-1 genome using an array of methods based on individual sites, k-mers and genes/proteins. We identified 56 amino acid sites or “signatures” and 119 k-mers that differed between the vaccine and placebo groups. Of those, 19 sites and 38 k-mers were located in the regions comprising the RV144 vaccine (Env-gp120, Gag, and Pro). The nine signature sites in Env-gp120 were significantly enriched for known antibody-associated sites (p = 0.0021). In particular, site 317 in the third variable loop (V3) overlapped with a hotspot of antibody recognition, and sites 369 and 424 were linked to CD4 binding site neutralization. The identified signature sites significantly covaried with other sites across the genome (mean = 32.1) more than did non-signature sites (mean = 0.9) (p < 0.0001), suggesting functional and/or structural relevance of the signature sites. Since signature sites were not preferentially restricted to the vaccine immunogens and because most of the associations were insignificant following correction for multiple testing, we predict that few of the genetic differences are strongly linked to the RV144 vaccine-induced immune pressure. In addition to presenting results of the first complete-genome analysis of the breakthrough infections in the RV144 trial, this work describes a set of statistical methods and tools applicable to analysis of breakthrough infection genomes in general vaccine efficacy trials for diverse pathogens. [ABSTRACT FROM AUTHOR]

The RV144 trial demonstrated 31% vaccine efficacy at preventing human immunodeficiency virus (HIV)-1 infection. Antibodies against the HIV-1 envelope variable loops 1 and 2 (Env V1 and V2) correlated inversely with infection risk. We proposed that vaccine-induced immune responses against V1/V2 would have a selective effect against, or sieve, HIV-1 breakthrough viruses. A total of 936 HIV-1 genome sequences from 44 vaccine and 66 placebo recipients were examined. We show that vaccine-induced immune responses were associated with two signatures in V2 at amino acid positions 169 and 181. Vaccine efficacy against viruses matching the vaccine at position 169 was 48% (confidence interval 18% to 66%; P = 0.0036), whereas vaccine efficacy against viruses mismatching the vaccine at position 181 was 78% (confidence interval 35% to 93%; P = 0.0028). Residue 169 is in a cationic glycosylated region recognized by broadly neutralizing and RV144-derived antibodies. The predicted distance between the two signature sites (21?±?7?Å) and their match/mismatch dichotomy indicate that multiple factors may be involved in the protection observed in RV144. Genetic signatures of RV144 vaccination in V2 complement the finding of an association between high V1/V2-binding antibodies and reduced risk of HIV-1 acquisition, and provide evidence that vaccine-induced V2 responses plausibly had a role in the partial protection conferred by the RV144 regimen. [ABSTRACT FROM AUTHOR]