Activation of a dendritic cell-T cell axis by Ad5 immune complexes creates an improved environment for replication of HIV in T cells

Abstract

The STEP HIV vaccine trial, which evaluated a replication-defective adenovirus type 5 (Ad5) vector vaccine, was recently stopped. The reasons for this included lack of efficacy of the vaccine and a twofold increase in the incidence of HIV acquisition among vaccinated recipients with increased Ad5-neutralizing antibody titers compared with placebo recipients. To model the events that might be occurring in vivo, the effect on dendritic cells (DCs) of Ad5 vector alone or treated with neutralizing antiserum (Ad5 immune complexes [IC]) was compared. Ad5 IC induced more notable DC maturation, as indicated by increased CD86 expression, decreased endocytosis, and production of tumor necrosis factor and type I interferons. We found that DC stimulation by Ad5 IC was mediated by the Fcgamma receptor IIa and Toll-like receptor 9 interactions. DCs treated with Ad5 IC also induced significantly higher stimulation of Ad5-specific CD8 T cells equipped with cytolytic machinery. In contrast to Ad5 vectors alone, Ad5 IC caused significantly enhanced HIV infection in DC-T cell cocultures. The present results indicate that Ad5 IC activates a DC-T cell axis that, together with the possible persistence of the Ad5 vaccine in seropositive individuals, may set up a permissive environment for HIV-1 infection, which could account for the increased acquisition of HIV-1 infection among Ad5 seropositive vaccine recipients.

Figure 1.

DCs activation and maturation. (a) Flow cytometry analysis of CD86 expression after treatment of DCs with Ad5 vector alone (filled square), Ad5 IC (formed with a fixed Ad5 dose and increasing serum volumes [Ad5 NAb titer of 512]; filled diamonds), neutralizing serum (NS) alone (open triangles) and mock-treated DCs (open circle). (b) Untreated DCs (open circle) treated with a range of Ad5 doses (filled squares) or with Ad5 IC (filled diamonds) formed with 20 μl of sera (Ad5 NS titer of 512) and various Ad5 doses and with NS alone (open triangle). (c) Flow cytometry profile of CD40 and CD86 expression in DCs treated with Ad5 vector, Ad5 vector plus NNS, or Ad5 IC. (d) Cumulative data (five DC donors and eight different sera) of CD86 expression. (e) Ag uptake after incubation of DCs with Ad5 vector, Ad5 IC, LPS, or mock treated. The assays were repeated three times with similar results.

Figure 2.

Secretion of proinflammatory cytokines from DCs. (a) DCs were treated with Ad5 IC (TNF-α, filled diamonds; IFN-I, open diamonds) formed with a fixed dose of Ad5 vector and a range of serum volumes (Ad5 NS titer was 512), NS alone (TNF-α, filled triangles; IFN-I, open triangles). Culture supernatants were collected and analyzed for the presence of TNF-α by ELISA and IFN-I. (b) DCs were also treated with Ad5 vector alone (TNF-α and IFN-I, filled and open squares, respectively) or with Ad5 IC (TNF-α and IFN-I, filled and open diamonds, respectively) formed with 20 μl of serum (Ad5 NAb titer was 512) and untreated DCs (IFN-I and TNF-α, open and filled circles, respectively). Representative data of one out of nine subjects are shown. (c and d) Correlation between levels of TNF-α and IFN-I secretion and Ad5 NAb titers. Each point on the line results from the mean value of several pooled sera analyzed. At least 28 sera were analyzed for TNF-α or IFN-I in duplicate. Statistical analysis was performed using Spearman's rank correlation.

Figure 3.

Ad5 IC deliver the activation signal to DCs through FcγRs and TLR9. (a) DCs were treated with Ad5 IC in the presence of anti-FcγRI, IIa, and III blocking Abs. Culture supernatants were analyzed for the secretion of TNF-α and IFN-I. Results are expressed as the percent of the TNF-α or IFN-I secretion levels in the cultures stimulated with Ad5 IC in the presence or absence of FcγRs blocking Abs. (b) DCs were treated with Ad5 vector, helper-dependent vector, Ad5 IC, or HD IC . Supernatants were analyzed for the presence of TNF-α and IFN-I. Results are expressed as the percent of the TNF-α and IFN-I secretion levels versus controls (treatment of DCs with Ad5 IC). (c) DCs were stimulated with Ad5 IC (NAb titer of 512) and LPS (TLR4 agonist) or CpG in the presence or absence of TLR9 inhibitor. At the end of the stimulation period, supernatants were analyzed for the presence of TNF-α and IFN-I. Results are expressed as the percent of the cytokine levels measured in culture treated with TLR9 inhibitor versus those found in control cultures (absence of inhibitors). All of the experiments in Fig. 3 were performed twice in triplicate. Error bars represent SEM.

Figure 4.

Ad5 vector and Ad5 IC stimulate Ad5-specific CD4 and CD8 T cells. (a) Cumulative data on the percentage of the total cytokine response (IL-2 + TNF-α + IFN-γ) of Ad5-specific CD4 and CD8 T cells after stimulation with Ad5 vector or Ad5 IC–treated DCs. The data were generated from five Ad5 seropositive subjects with Ad5 Ab titers >1,000. Blood mononuclear cells were stimulated with DCs treated with either autologous or heterologous neutralizing sera. (b) Flow cytometry profiles of CD8 T cells secreting IL-2, TNF-α, and IFN-γ after stimulation with Ad5 vector, Ad5 + NNS, or Ad5 IC–treated DCs from a representative subject. All the possible combinations of the responses are shown on the x axis, and the functionally distinct cell populations within the responding CD8 T cell populations are shown on the y axis. Responses are grouped and color-coded on the basis of the number of functions. In the pie chart, each slice corresponds to the fraction of CD8 T cells with a given number of functions within the responding CD8 T cell populations. Bars correspond to the fractions of functionally distinct T cell populations within the total CD8 T cells. (c) Perforin and granzyme B expression in proliferating CD8 T cells. Analysis of perforin and granzyme B expression was determined on gated CFSE-low proliferating CD3⁺CD8⁺ T cells after stimulation with DCs treated with Ad5 or Ad5 IC. Cumulative data are also shown.

Figure 5.

Ad5 IC stimulates HIV infection in vitro. Mean p24 levels at day 0, 4, and 7 in blood mononuclear cells from donors were cultured with Ad5 or Ad5 IC–treated autologous DCs. Uninfected cultures and HIV infection in cultures in which blood mononuclear cells were mixed with untreated DCs were used as controls. Four donors were studied. Data represent the mean of triplicate determinations.