Baseline Ad5 serostatus does not predict Ad5-HIV vaccine-induced expansion of Ad-specific CD4+ T-cells

Post-hoc analysis of the Merck STEP trial showed vaccination with an Ad5 vector-based HIV-1 vaccine was associated with increased HIV-1 acquisition rates in volunteers with baseline Ad5 neutralizing antibody (nAb) titers > 200¹. It was proposed that vaccination of Ad5 seropositive subjects caused activation and expansion of pre-existing Ad5-specific CD4⁺ T-cells, potentially serving as targets for HIV infection². However, neither the prevalence of Ad5-specific CD4⁺ T-cells in humans, nor their relationship with Ad5 nAb titer has been characterized. Moreover, it is unknown to what degree Ad5 vector administration stimulates pre-existing Ad5-specific CD4⁺ T-cells. Adequately addressing this from the STEP trial is impossible, as peripheral blood mononuclear cell (PBMC) samples were only obtained after vaccination (weeks 8, 30, 52, 104)³.

To characterize the relationship between Ad5 nAb titers and Ad5-specific CD4⁺ T-cell responses, we analyzed samples from 40 subjects with varying Ad5 nAb titers by intracellular cytokine staining using replication-defective Ad5 particles for stimulation^4–7. (Supplementary Methods online, Supplementary Fig. 1, Supplementary Table 1). Of these subjects, 15 (five seronegative weeks 0–4, five seronegative weeks 0–78, and five seropositive weeks 0–78) were enrolled in the Merck 016 phase I HIV-1 vaccine safety trial and received Ad5 vectors used in the STEP trial at weeks 0, 4, and 26⁸ (Supplementary Table 2). We detected similar frequencies of Ad5-specific CD4⁺ T-cells in > 80% of Ad5 seropositive and Ad5 seronegative subjects at baseline (Fig. 1a). Within Ad5 seropositive subjects, Ad5-specific CD4⁺ T-cell frequencies did not correlate with Ad5 nAb titers⁹ (Fig. 1b).

Four weeks after the first Ad5-HIV-1 vector administration in the 15 vaccinated subjects (Supplementary Table 2), Ad5 nAb titers in baseline seronegative subjects (n = ten) increased (P < 0.05), becoming comparable to those seen in baseline Ad5 seropositive subjects (n = five) in all but one individual (Fig. 1c) who seroconverted by week 8 (Fig. 1d). Ad5-specific CD4⁺ T-cells increased in both groups (P < 0.002, baseline seropositive; P < 0.03, baseline seronegative) after the initial vector dose (Fig. 1e, Supplemental Fig. 2). Successive vaccinations further expanded Ad5-specific T-cells in some subjects, but these responses were transient in most individuals (Fig. 1e, Supplemental Fig. 3). At no point was there a statistical difference between the serogroups.

We next examined the relationship between Ad5 serostatus and potential functional differences in Ad5-specific CD4⁺ T-cells before and after vaccination. Ad5- specific CD4⁺ T-cells that produced IFN-γ, IL-2, MIP-1α, TNF- α, and/or perforin were present at baseline in most individuals at similar frequency regardless of Ad5 serostatus (Fig. 2a). There was no correlation between Ad5 nAb titer and % Ad5-specific CD4⁺ T-cells that produced any one or more functions (data not shown). IFN-γ dominated the response in both serogroups, but accounted for only ~50% of the total response (Fig. 2b).

After the first vaccination Ad5-specific CD4⁺IFN-γ⁺ T-cells increased in both groups (P < 0.05), with the fold change in CD4⁺IFN-γ⁺ T-cells independent of serostatus (Fig. 2c, Supplemental Fig. 2–4). The frequency of Ad5-specific MIP-1 α⁺ CD4⁺ T-cells increased above baseline after vaccination (P<0.03 seropositives, P < 0.005 seronegatives), whereas IL-2 (P < 0.03) and TNF–α (P < 0.001) increased in seropositive subjects only and accounted for a higher proportion of the total response (P < 0.05) compared with seronegative subjects (Fig. 2c–d, Supplemental Fig. 2–3). Following three vector doses, perforin and MIP-1α were higher in seronegative subjects above baseline, but little differences in other functions were found in either serogroup after the second vaccine dose (Fig. 2d, Supplemental Fig. 3). Despite these transient increases in CD4⁺ T-cell functions within serogroups, there was never a significant difference between the groups for the percentage of Ad5-specific CD4⁺ T-cells producing IFN-γ, IL- 2, MIP-1α, TNF-α, or perforin. The degree of polyfunctionality of Ad5-specific CD4⁺ T-cells remained comparable between baseline Ad5 seronegative and seropositive subjects (Supplemental Fig. 4).

No difference was found in Ki-67 expression for total (data not shown) or Ad5- specific CD4⁺ T-cells (Fig. 2e, Supplemental Fig. 5) between the serogroups or compared to baseline. Expression of the mucosal trafficking-associated markers α4 and β7 did not differ significantly from baseline within either serogroup on total memory (T_M: all CD45RO⁺ and CCR7⁻CD45RO⁻) and effector memory (T_EM: CCR7⁻CD45RO⁺) CD4⁺ T-cells (Fig. 2f, Supplemental Fig. 5) or Ad5-specific T_M or T_EM CD4⁺ T-cells (Fig. 2g) after vaccination. Moreover, Ad5-specific CD4⁺ T_M and T_EM cells represented a small fraction of total circulating α4⁺β7⁺CD4⁺ T-cells, and did not change significantly after vaccination (Fig. 2h). Thus, while transient changes in the phenotype and magnitude of Ad5-specific CD4⁺ T-cell responses were detected within groups after vaccination, no significant differences between groups were observed.

Our results indicate that Ad5-specific CD4⁺ T-cells were unlikely to play a role in the possible increased susceptibility to HIV-1 infection observed in the STEP trial. Three findings support this conclusion: First, baseline Ad5 nAb titers are not predictive of Ad5-specific CD4⁺ T-cell responses. Second, Ad5-specific CD4⁺ T-cells within both baseline Ad5 seronegative and seropositive subjects expand in response to Ad vector administration. While slightly higher responses were observed early in baseline Ad5 seropositives, these differences were extremely transient, and would not account for increased acquisition primarily observed at later times (post-week 20) in STEP. Thus, vaccination does not appear to preferentially increase the pool of potentially infectable circulating Ad5-specific CD4⁺ T-cells in Ad5 seropositive individuals. Finally, Ad5 nAb seronegative subjects uniformly became Ad5 seropositive after a single vaccination, yet no enhanced susceptibility was noted in baseline Ad5 seronegative STEP participants after the first vaccine administration. Taken together, these data suggest that any linkage between Ad5 serostatus and Ad5-specific T-cell-related increase in HIV-1 acquisition should only have been observed early after the first vaccine dose, for afterwards Ad5-specific T-cell responses in baseline Ad5 seronegative subjects appear immunologically equivalent to baseline Ad5 seropositive subjects. With the caveat that our analyses are restricted to circulating CD4⁺ T-cells and do not address potential differences in activated Ad5-specific CD4⁺ T-cells within mucosal tissues after vaccination, our results do not support the hypothesis that Ad5-specific CD4⁺ T-cells contributed to the potential increased HIV-1 acquisition in the STEP trial.