A combination DNA and attenuated simian immunodeficiency virus vaccine strategy provides enhanced protection from simian/human immunodeficiency virus-induced disease

Abstract

Among the most effective vaccine candidates tested in the simian immunodeficiency virus (SIV)/macaque system, live attenuated viruses have been shown to provide the best protection from challenge. To investigate if preimmunization would increase the level of protection afforded by live attenuated SIVmac239Deltanef (Deltanef), macaques were given two priming immunizations of DNA encoding SIV Gag and Pol proteins, with control macaques receiving vector DNA immunizations. In macaques receiving the SIV DNA inoculation, SIV-specific cellular but not humoral responses were readily detectable 2 weeks after the second DNA inoculation. Following boosting with live attenuated virus, control of Deltanef replication was superior in SIV-DNA-primed macaques versus vector-DNA-primed macaques and was correlated with higher levels of CD8+/gamma-interferon-positive and/or interleukin-2-positive cells. Challenge with an intravenous inoculation of simian/human immunodeficiency virus (SHIV) strain SHIV89.6p resulted in infection of all animals. However, macaques receiving SIV DNA as the priming immunizations had statistically lower viral loads than control animals and did not develop signs of disease, whereas three of seven macaques receiving vector DNA showed severe CD4+ T-cell decline, with development of AIDS in one of these animals. No correlation of immune responses to protection from disease could be derived from our analyses. These results demonstrate that addition of a DNA prime to a live attenuated virus provided better protection from disease following challenge than live attenuated virus alone.

FIG. 1.

Timeline of study. The timeline of the study is presented in a line drawing beginning with the first DNA inoculation (week 0).

FIG. 2.

Proliferative responses of macaque PBMC to stimulation with SIV Gag protein. PBMC obtained from SIV-DNA-primed and vector-DNA-primed macaques at week 10 (2 weeks after the second DNA immunization) were stimulated in vitro with SIV Gag-Pol protein. After 6 days, [³H]thymidine was added to the cultures for 24 h. Uptake of ³H was measured, and raw data were converted to SIs based upon a negative control. SIs of >5 are considered positive.

FIG. 3.

Plasma viral loads and circulating CD4⁺ T cells in macaques. Following administration of Δnef at week 25, blood samples were obtained at the indicated time points for determining SIV RNA plasma viral loads and for determining the peripheral circulating CD4⁺ T-cell counts. (A) Group 1 macaques (vector DNA primed), viral load; (B) group 2 macaques (SIV DNA primed), viral load; (C) group 1 macaques, CD4⁺ T-cell counts; (D) group 2 macaques, CD4⁺ T-cell counts. Additionally, geometric mean viral loads were calculated for each group and plotted (E). The asterisk indicates a statistically significant difference in viral load setpoints between the SIV-DNA-primed group and the vector-DNA-primed group (total AUC) for weeks 28 and 61. The specific icons identifying the individual animals are consistent throughout all figures in the manuscript.

FIG. 4.

Analysis of Gag-specific CD8⁺ and CD4⁺ T cells following Δnef infection. PBMC obtained from immunized macaques were obtained and used for ICS to detect IFN-γ-secreting cells in response to stimulation with SIV Gag peptides, as described in Materials and Methods. Weeks (Wk) 27 and 49 (2 weeks and 24 weeks post-Δnef infection, respectively) were chosen for analysis of the immediate response (Wk 27) and the memory response (Wk 49). By use of flow cytometry, populations were separated into CD4⁺ and CD8⁺ fractions for analysis. The percentage of positive cells for each macaque is indicated on each graph. Median percentages of positive cells are calculated for each group and are represented by the bars. Asterisks indicate statistically significant differences between groups.

FIG. 5.

Plasma viral loads and circulating CD4⁺ T cells in macaques following challenge. Similar to results presented in Fig. 3, the plasma viral loads and CD4⁺ T-cell levels were analyzed with macaques at the indicated times following intravenous infection with SHIV89.6p. Challenge with SHIV89.6p occurred on week 65 of the study (indicated by the arrow on each graph). (A) Plasma viral load in vector-DNA-primed macaques; (B) plasma viral load in SIV-DNA-primed macaques; (C) plasma viral load in unimmunized control macaques; (D) CD4⁺ T cells in vector-DNA-primed macaques; (E) CD4⁺ T cells in SIV-DNA-primed macaques; (F) CD4⁺ T cells in unimmunized control macaques; (G) geometric mean viral loads in the three groups of macaques. Statistically significant differences in peak viral loads were observed between the vector-DNA-primed group and the control group, as well as between the SIV-DNA-primed group and the control group (indicated by the single asterisk). A statistically significant difference in viral load setpoints (total AUC, weeks 71 to 85) was observed between the SIV-DNA-primed group and the control group (indicated by the double asterisk).

FIG. 6.

Temporal analysis of SIV-Gag-specific CD4⁺ and CD8⁺ T cells following SHIV challenge. PBMC obtained from immunized macaques challenged with SHIV89.6p were used in ICS assays to detect IFN-γ-secreting cells in response to stimulation with SIV Gag peptides. Analyses were performed on the day of challenge (week 65 in the timeline), 2 weeks after challenge (week 67), and then 20 weeks after challenge (week 85). Populations of PBMC were gated on CD4⁺ or CD8⁺ cells for study. Percentages of positive cells are indicated for all macaques on the graphs (for vector-DNA-primed macaques, CD4⁺ [A] and CD8⁺ [D] cells; for SIV-DNA-primed macaques, CD4⁺ [B] and CD8⁺ [E] cells). The geometric mean percentages of positive cells were calculated for each group and are presented in panels C (CD4⁺ cells) and F (CD8⁺ cells). The asterisk indicates a statistically significant difference in mean percentages of IFN-γ⁺/CD8⁺ cells between the SIV-DNA-primed group and the vector-DNA-primed group at week 20. GMT, geometric mean titer.