Response rate and magnitude of response to Gag peptides or Ad5 empty vector at peak timepoint after three vaccine doses.

1<p>Bold denotes analytes with ≥50% response rate to either Gag or Ad5 with concentrations significantly 2-fold higher or lower than controls.</p>2<p>Wilcoxon sign-rank two-tailed test using responders when n ≥5 for each group.</p><p>Abbreviations: NA, not applicable.</p

Comparison of Gag- and Ad5-specific cellular responses in Ad5-seronegative and Ad5-seropositive vaccinees.

<p>PBMC from vaccinees collected at a peak timepoint (28 weeks) were tested by multiplex cytokine assay. Background-subtracted concentrations of a focus set of eleven analytes are shown for Ad5-seronegative (blue) and Ad5-seropositive (red) test groups stimulated by either (A) a Gag PTE peptide pool or (B) Ad5 empty vector. Only positive responders, as reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018526#pone-0018526-t002" target="_blank">Table 2</a>, were included in the analyses. Box and whisker plots indicate median and interquartile ranges, and groups were compared by Wilcoxon rank sums. Probability estimates are indicated when p≤0.05.</p

Peak vaccine response to Gag insert and Ad5 vector measured by <i>ex vivo</i> multiplex cytokine assay.

<p>Unfractionated PBMC from vaccinees collected at the peak response time point were assayed for 30 different cytokines and chemokines by multiplex bead array. Results are expressed as the log₁₀ fold change in concentration compared to negative control wells. (<b>A</b>) Overall response profile of Ad5-seronegative (n = 28, blue dots) and Ad5-seropositive (n = 28, red dots) vaccinees to Gag (top) and Ad5 (bottom) for all analytes assayed. Non-responders are shown in gray. Box plots indicate interquartile ranges and medians for responders only, and medians are connected by the green line for profile comparison. (<b>B</b>) Comparison of the magnitude of up-regulation of the analyte focus set (selection criteria described in the text) in response to either Gag or Ad5. Groups were compared by Wilcoxon sign-rank. Horizontal lines denote median values.</p

Ad5-specific neutralizing antibody titers before and after the MRKAd5 HIV-1 gag vaccine regimen.

<p>Serum samples collected at regular intervals during the vaccination schedule from Ad5-seronegative (n = 35, blue dots) and Ad5-seropositive (n = 34, red dots) vaccine recipients were assayed for Ad5 neutralizing antibody activity. Vaccination visits at 0, 4, and 26 weeks are indicated by green arrows. Bars indicate median neutralizing titers for each group at a given time point.</p

Analysis of antibodies against CD4 inducible (CD4i) epitopes.

<p>A) The presence of co-receptor binding site-directed antibodies was assayed by competition with the mAb, 17b. Competition titer indicates the serum dilution capable of outcompeting 50% of pseudoviral binding to 17b. B) Neutralizing antibody titers against HIV-1 JR-FL isolate without sCD4 treatment. C) Neutralizing antibody titers against HIV-1 JR-FL isolate with sCD4 treatment. D) Effect of V3 peptide treatment on neutralizing activity against sCD4-treated JR-FL.</p

Ability of vaccinee sera to mediate ADCC activity.

<p>CEMNK^r target cells were pulsed with gp120 prior to exposure of vaccine serum at a 1∶100 dilution. Target cell lysis indicates the ability of vaccinee serum to mediate cell killing by PBMC from a normal human donor. Dotted line indicates background cell lysis observed with a normal human sera control.</p

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

<div><p>Background</p><p>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.</p><p>Methods</p><p>We examined <i>ex vivo</i> 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 <i>ex vivo</i> 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-log_e increase of the immune responses.</p><p>Findings</p><p>We found that HIV-specific immune responses were not associated with risk of HIV-1 infection. However, each 1-log_e 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).</p><p>Conclusions</p><p>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.</p></div

Specificity of vaccine-induced antibody responses as determined through mAb competition.

<p>The ability of serially diluted human immune serum to outcompete binding of mAb to a JR-FL & VSV-G pseudotyped virus was measured. Competition titers indicate the serum dilution preventing 50% of pseudoviral binding to the mAb. A) Competition with carbohydrate-specific mAb, 2G12. B) Competition with V3 loop-specific mAb, 447-52D. C) Competition with CD4bs-specific mAb, b12.</p

Confirmation of neutralizing activities against representative HIV isolates.

<p>Neutralization antibody titers at 50% inhibition for each serum are shown against either SF162 (A) or SS1196.1 (B). Neutralizing activities against SC422661.8 (C) is shown as the fractions of individual sera from each trial either capable of achieving at least 50% inhibition of infection at a 1∶10 serum dilution (shaded portion) or unable to achieve 50% inhibition (open portion). All p values reaching significance (p<0.05) are presented in the figure. All other comparisons did not reach significant based upon Kruskal-Wallis and Dunn'significance tests.</p

Profiles of antibody responses elicited by three HIV vaccine regimens.

<p>Profiles of antibody responses elicited by three HIV vaccine regimens.</p