Pre-existing adenovirus immunity modifies a complex mixed Th1 and Th2 cytokine response to an Ad5/HIV-1 vaccine candidate in humans

Abstract

The results of the recent Step Study highlight a need to clarify the effects of pre-existing natural immunity to a vaccine vector on vaccine-induced T-cell responses. To investigate this interaction, we examined the relationship between pre-existing Ad5 immunity and T-cell cytokine response profiles in healthy, HIV-uninfected recipients of MRKAd5 HIV-1 gag vaccine (HVTN 050, ClinicalTrials.gov #NCT00849732). Participants were grouped by baseline Ad5 neutralizing antibody titer as either Ad5-seronegative (titer ≤18; n = 36) or Ad5-seropositive (titer >200; n = 34). Samples from vaccine recipients were analyzed for immune responses to either HIV-1 Gag peptide pools or Ad5 empty vector using an ex vivo assay that measures thirty cytokines in the absence of long-term culture. The overall profiles of cytokine responses to Gag and Ad5 had similar combinations of induced Th1- and Th2-type cytokines, including IFN-γ, IL-2, TNF-α, IP-10, IL-13, and IL-10, although the Ad5-specific responses were uniformly higher than the Gag-specific responses (p<0.0001 for 9 out of 11 significantly expressed analytes). At the peak response time point, PBMC from Ad5-seronegative vaccinees secreted significantly more IP-10 in response to Gag (p = 0.008), and significantly more IP-10 (p = 0.0009), IL-2 (p = 0.006) and IL-10 (p = 0.05) in response to Ad5 empty vector than PBMC from Ad5-seropositive vaccinees. Additionally, similar responses to the Ad5 vector prior to vaccination were observed in almost all subjects, regardless of Ad5 neutralizing antibody status, and the levels of secreted IFN-γ, IL-10, IL-1Ra and GM-CSF were blunted following vaccination. The cytokine response profile of Gag-specific T cells mirrored the Ad5-specific response present in all subjects before vaccination, and included a number of Th1- and Th2-associated cytokines not routinely assessed in current vaccine trials, such as IP-10, IL-10, IL-13, and GM-CSF. Together, these results suggest that vector-specific humoral responses may reduce vaccine-induced T-cell responses by previously undetected mechanisms.

Figure 1. Peak vaccine response to Gag insert and Ad5 vector measured by ex vivo multiplex cytokine assay.

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. (A) 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) 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.

Figure 2. Comparison of Gag- and Ad5-specific cellular responses in Ad5-seronegative and Ad5-seropositive vaccinees.

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 Table 2, 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.

Figure 3. Frequency and magnitude of Ad5-stimulated cellular responses of vaccinees prior to vaccination.

PBMC collected from trial participants prior to vaccination (baseline) were tested by multiplex cytokine assay. Magnitudes of response for 30 analytes tested are expressed as log₁₀ fold change over background for Ad5-seronegative (n = 17, blue triangle) and Ad5-seropositive (n = 8, red triangle) volunteers. Non-responders are shown in gray. Box and whisker plots indicate median and interquartile ranges for responders only, while connecting line illustrates overall profile for comparison.

Figure 4. Ad5-specific neutralizing antibody titers before and after the MRKAd5 HIV-1 gag vaccine regimen.

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.