Priming immunization with DNA augments immunogenicity of recombinant adenoviral vectors for both HIV-1 specific antibody and T-cell responses

Abstract

Background: Induction of HIV-1-specific T-cell responses relevant to diverse subtypes is a major goal of HIV vaccine development. Prime-boost regimens using heterologous gene-based vaccine vectors have induced potent, polyfunctional T cell responses in preclinical studies.

Methods: The first opportunity to evaluate the immunogenicity of DNA priming followed by recombinant adenovirus serotype 5 (rAd5) boosting was as open-label rollover trials in subjects who had been enrolled in prior studies of HIV-1 specific DNA vaccines. All subjects underwent apheresis before and after rAd5 boosting to characterize in depth the T cell and antibody response induced by the heterologous DNA/rAd5 prime-boost combination.

Results: rAd5 boosting was well-tolerated with no serious adverse events. Compared to DNA or rAd5 vaccine alone, sequential DNA/rAd5 administration induced 7-fold higher magnitude Env-biased HIV-1-specific CD8(+) T-cell responses and 100-fold greater antibody titers measured by ELISA. There was no significant neutralizing antibody activity against primary isolates. Vaccine-elicited CD4(+) and CD8(+) T-cells expressed multiple functions and were predominantly long-term (CD127(+)) central or effector memory T cells and that persisted in blood for >6 months. Epitopes mapped in Gag and Env demonstrated partial cross-clade recognition.

Conclusion: Heterologous prime-boost using vector-based gene delivery of vaccine antigens is a potent immunization strategy for inducing both antibody and T-cell responses.

Trial registration: ClinicalTrials.gov NCT00102089, NCT00108654.

Figure 1. T cell responses to DNA are boosted by rAd immunization.

(A) IFN-γ ELISpot responses in subjects 4 weeks after third dose of DNA or after a single dose of rAd5 vector only compared to peak response at 4–6 weeks following rAd5 vector boosting. The scale indicates spot-forming cells per million peripheral blood mononuclear cells. Individual subject responses are shown for recipients of 4-plasmid (VRC 004) or 6-plasmid (VRC 007) vaccines (left panel), the rAd5 vector (VRC 006) vaccine (center panel), and the combined DNA/rAd5 prime-boost (right panel). Results for peptide pool stimulation are shown for each peptide pool representing a single gene product in the vaccine shown on the x-axis. Boxplots represent the median and IQR. Longitudinal T cell responses to EnvA peptides were measured by IFN-γ ELISpot (B), CD4⁺ (C) and CD8⁺ (D) ICS and data are shown for all 14 subjects. The line graph shows the mean response at each time point. Some clinical time points were aggregated and are shown here as the mean value of the measurements for the grouped time points: The data following DNA injections #2 and #3 are the means of results from 2 and 4 weeks after the immunization time point; 6 month data post-DNA are the means of results from 16, 24, and 30 weeks after DNA injection #3; and the <6 week post-rAd5 time point data are means of results from 2, 4, and 6 weeks after rAd5 vector immunization.

Figure 2. Phenotype of vaccine-induced T cells.

(A) Flow cytometric analysis of antigen-specific T cells. The top row of graphs shows the progressive gating to identify live CD3⁺CD4⁺ or CD3⁺CD8⁺ lymphocytes. For either CD4 or CD8 T cells, antigen-responsive cells (following in vitro stimulation) are identified by the production of IL2, IFN-γ, or TNF. This example shows EnvA-stimulated cells from one of the highest responders in the study; the phenotyping graphs illustrate the distribution of cells that make any cytokine (blue) overlaid on the total CD4 or CD8 population (grey). (B) The distribution of expression of a variety of cell surface markers on antigen-specific CD4 or CD8 T cells following vaccination. The colored bars represent the IQR for the distribution at different time points: 4 weeks post-DNA (blue); 6–18 months post DNA (red); 4 weeks post rAd5 boost (green); and 6 months post-boost (orange). (C) Two different phenotyping schemas, based on either CD27 or CCR7 expression in combination with CD45RO, were used to characterize antigen-specific T cells. The bar charts show the individual data points and IQR for the four time points following immunization. The pie charts summarize these distributions, showing the average proportion of the CD4 or CD8 vaccine-specific T cell response that is T_CM (light grey), T_EM (medium grey), or T_EF (black). Asterisks indicate distributions that are different from the earliest time point (4 weeks post DNA) at p<0.05 (Student's T test).

Figure 3. Function of vaccine-induced T cells.

The quality of the vaccine-elicited CD4 or CD8 T cell response is characterized by the proportion of cells making every possible combination of the three measured cytokines. The bar charts show the individual data points and IQR for four time points following immunization. Pie charts show the average proportion of the CD4 or CD8 vaccine-specific T cell response that is polyfunctional (black), producing two functions (medium grey), or is monofunctional (light grey). The memory time point had a significantly different quality of CD8 T cell response than earlier time points (SPICE permutation analysis).

Figure 4. The quality of the response within T_CM, T_EM, or T_EF cells.

Data for each subject is averaged over time points (there was no statistically significant change in quality over time within each subset). T_CM cells have significantly more polyfunctional T cells than T_EM or T_EF cells.

Figure 5. Clade coverage of epitope-specific responses.

Seven responses (A through G) to five minimum CD8⁺ T cell epitopes as defined in Table 2 are shown for four VRC 009 volunteers. Volunteer identifiers are shown in far right panels. Left panel shows the minimum epitope and sequence variants that were tested. Left center panel shows the frequency of each variant as it occurs within HIV clades A, B, C, and D. ELISpot responses expressed as SFCs per million PBMC at multiple peptide dilutions (µg/ml) to the epitope variants are shown from 4 weeks after the third dose of DNA (right center panel) and 4 weeks after rAd boost (far right panel). *  =  only the major variant was tested due to cell limitations. ND  =  not done due to absence of cells for any peptide titrations.