Samples were obtained from participants enrolled in the Step (21
) and HVTN 071 (a phase Ib open-label clinical trial using the same vaccine product) studies and from unvaccinated individuals enrolled in the study “Establishing Immunologic Assays for Determining HIV-1 Prevention and Control,” also referred to as SACs. Vaccine recipients received MRKAd5 HIV-1 gag/pol/nef
at weeks 0, 4, and 26 in the Step study, while HVTN 071 was halted based on the Step results before participants received the third vaccination; all participants received at least one vaccination, but none received all three. Ad5 nAb titers were measured at baseline as described previously (22
); titers of 18 or less defined seronegativity. Ad1 and Ad2 nAb titers were measured as described in Supplemental Methods.
Ad5 empty vector–specific immune responses.
Ad5-specific immune responses were assessed by intracellular cytokine staining using an empty Ad5 vector (ΔE1, ΔE3, ΔE4) provided by Gary Nabel at the Dale and Betty Bumper Vaccine Research Center, Bethesda, Maryland, USA (23
), as previously described (1
) (see Supplemental Methods). A study was performed in 17 individuals to compare the VRC vector to the empty Ad5 vector (ΔE1, WT E3, WT E4) provided by Merck (2
). Responses to both vectors were highly correlated (ρ = 0.9, P
< 0.0001 for both CD4+
T cells; data not shown), validating the use of the VRC vector for this study. Responses are reported as percentage of CD4+
T cells producing IFN-γ and/or IL-2. Positivity was established according to a previously published method (24
Ad5 and HIV peptides.
We synthesized 1,773 overlapping peptides for 11 Ad5 proteins/ORFs, including core, capsid, and non-structural proteins, covering about 40% of the Ad5 genome (5,134 amino acids; Supplemental Table 1). Ad5 peptides were synthesized as 15-mers overlapping by 11 amino acids (BioSynthesis). Lyophilized peptides were resuspended in DMSO and pooled as described in Supplemental Table 1.
HIV peptides covering the inserts in the studied vaccines were global potential T cell epitope (PTE-g) peptides representing Gag, Nef, and Pol that have been previously described (25
) (see Supplemental Methods).
Ad5 epitope mapping.
Epitope mapping, performed using IFN-γ ELISPOT, consisted of 4 peptide testing stages: master pool, sub-pool, matrix, and 15-mers. First, 12 Ad5 peptide master pools, consisting of 121–168 15-mer peptides each at a concentration of 1 μg/ml, were tested. Positive master pools were separated into 4 equal sub-pools and tested at 2 μg/ml. Positive sub-pools were tested in a 6 × 7 or 5 × 7 matrix at 2 μg/ml. Matrices were deconvoluted, and single 15-mers were tested at 2 μg/ml. In the first 3 rounds, the positive threshold was set at ≥2× background and ≥50 spot forming cells/million (SFC/M). At the final stage, the threshold was increased to ≥3× background. T cell responses to two consecutive 15-mer peptides were counted as a single response. Three consecutive 15-mers were counted as a single response if the overlapping sequence covered at least 8 amino acids; otherwise they were counted as two responses.
IFN-γ ELISPOT assays were performed using the Mabtech kit with 100,000 cells/well, as described in Supplemental Methods.
Group comparisons of treatment and serostatus were made for Ad5-specific CD4+
T cell response rates using multivariate logistic regression models. The covariates included to adjust for the potential imbalances and to increase the efficiency of the testing were region (North America or other), race (European descent or other), age (≤30 or >30), HSV-2 status, and circumcision status. A stepwise model selection procedure based on the Akaike information criterion (AIC) was used to reduce the number of predictors in the model (26
). If a main effect (treatment or serostatus) was not included in the final model, the P
value is reported as NS. For comparison of groups based on the magnitude of Ad5-specific CD4+
T cell responses, only samples with a positive response were included. The responses were log transformed to satisfy distributional assumptions of the subsequent linear regression modeling.
Associations between HIV-specific T cell responses and preexisting Ad5 immunity were tested using (a) multivariate linear regression for response magnitudes, (b) multivariate logistic regression for CD4+ T cell response rates, and (c) multivariate proportional odds logistic regression for CD8+ T cell response breadth. Since the CD8+ T cell response rate was high (32 of 34 vaccinees had detectable responses at peak), we assessed whether baseline Ad-specific responses influenced the breadth of the ensuing CD8+ T cell response (measured as the number of HIV proteins recognized, i.e., ranging from 0 to 3). First, univariate models were run on age (≤35, >35), sex, number of vaccinations, log10-transformed baseline Ad5 nAb titer, as well as baseline Ad-specific CD4+ and CD8+ T cell responses. These analyses suggested that number of vaccinations and sex could confound the relationship between the predictors of interest and the outcome, while age and Ad-specific CD8+ T cell responses were not predictive of HIV-specific cellular responses. Therefore, the final models included 4 baseline covariates: (a) sex, (b) number of vaccinations, (c) log10-transformed baseline Ad5 nAb titer, and (d) baseline Ad-specific CD4+ T cell response magnitude. A P value 0.05 or less was considered significant.
The institutional human subjects review committee at each clinical site approved all protocols prior to study initiation, and participants completed a thorough written informed consent process before study enrollment.
The following IRBs participated in the study: (a) HVTN sites: Fred Hutchinson Cancer Research Center, Seattle, Washington, USA; Emory University, Atlanta, Georgia, USA; University of Alabama at Birmingham, Birmingham, Alabama, USA; Brigham & Women’s Hospital, Boston, Massachusetts, USA; New York Blood Center, New York, New York, USA; Columbia University Medical Center, New York, New York, USA; University of Illinois at Chicago, Chicago, Illinois, USA; Fenway Community Health, Boston, Massachusetts, USA; AIDS Research Alliance, Los Angeles, California, USA; University of Pennsylvania, Philadelphia, Pennsylvania, USA; Saint Louis University New Hope Comprehensive Care Clinic, Saint Louis, Missouri, USA; Vanderbilt University Health Science Committee, Nashville, Tennessee, USA; San Francisco General Hospital (SFGH), San Francisco, California, USA; University of Rochester, Rochester, New York, USA; Asociación Civil Impacta Salud y Educación, Lima, Peru; Hospital Universitário Clementino Fraga Filho, Rio de Janeiro, Brazil; Centro de Referência e Treinamento, São Paulo, Brazil; University of Puerto Rico Medical Sciences, San Juan, Puerto Rico, USA; GHESKIO Centers, Port-au-Prince, Haiti; University of the West Indies, Kingston, Jamaica; Instituto Dermatológico y Cirugía de Piel, Santo Domingo, Dominican Republic; (b) Merck sites: North Jersey Community Research Initiative, Newark, New Jersey, USA; University of Colorado Health Sciences Center, Denver, Colorado, USA; New York University School of Medicine AIDS Clinical Trials Unit, New York, New York, USA; Center for Clinical Studies — Houston, Houston, Texas, USA; Center for Clinical Studies — Webster, Webster, Texas, USA; Care Resource, Miami, Florida, USA; AIDS Research Alliance, West Hollywood, California, USA; HIV Clinical Trials Unit IBAC, Sydney, New South Wales, Australia; Cohort St-Luc, Infectious Diseases, Montreal, Quebec, Canada; Canadian Immunodeficiency Research Collaborative Inc., Toronto, Ontario, Canada; Saint Paul’s Hospital — Immunodeficiency Clinic, Vancouver, British Columbia, Canada; Instituto Dominicano de Estudios Virológicos (IDEV), Santo Domingo, Dominican Republic; Asociación Vía Libre, Lima, Peru.