Critical role for Env as well as Gag-Pol in control of a simian-human immunodeficiency virus 89.6P challenge by a DNA prime/recombinant modified vaccinia virus Ankara vaccine

Abstract

Cellular immune responses against epitopes in conserved Gag and Pol sequences of human immunodeficiency virus type 1 have become popular targets for candidate AIDS vaccines. Recently, we used a simian-human immunodeficiency virus model (SHIV 89.6P) with macaques to demonstrate the control of a pathogenic mucosal challenge by priming with Gag-Pol-Env-expressing DNA and boosting with Gag-Pol-Env-expressing recombinant modified vaccinia virus Ankara (rMVA). Here we tested Gag-Pol DNA priming and Gag-Pol rMVA boosting to evaluate the contribution of anti-Env immune responses to viral control. The Gag-Pol vaccine raised frequencies of Gag-specific T cells similar to those raised by the Gag-Pol-Env vaccine. Following challenge, these rapidly expanded to counter the challenge infection. Despite this, the control of the SHIV 89.6P challenge was delayed and inconsistent in the Gag-Pol-vaccinated group and all of the animals underwent severe and, in most cases, sustained loss of CD4(+) cells. Interestingly, most of the CD4(+) cells that were lost in the Gag-Pol-vaccinated group were uninfected cells. We suggest that the rapid appearance of binding antibody for Env in Gag-Pol-Env-vaccinated animals helped protect uninfected CD4(+) cells from Env-induced apoptosis. Our results highlight the importance of immune responses to Env, as well as to Gag-Pol, in the control of immunodeficiency virus challenges and the protection of CD4(+) cells.

FIG. 1.

Temporal frequencies of Gag-specific T cells. (A) Gag-specific CD8⁺ T-cell responses raised by DNA priming and rMVA booster immunizations. The schematic presents the geometric mean of Gag-CM9 tetramer binding for the A*01 animals in each group. (B) Gag-specific IFN-γ ELISPOTs in Gag-Pol-Env-vaccinated (solid bars) and Gag-Pol-vaccinated (open bars) macaques at various times prechallenge and at 2 weeks postchallenge. Three pools of 10 to 13 Gag peptides (22-mers overlapping by 12 amino acids) were used for the analyses. The values above the bars are the geometric means of the ELISPOTs of the groups. The numbers above the graphs designate individual animals. #, data not available; ∗, <20 ELISPOTs per 10⁶ PBMC. Data for the Gag-Pol-Env-vaccinated groups are reprinted from reference with permission of the publisher. Wk, week.

FIG. 2.

Intracellular cytokine analyses with Gag peptide pools in high-dose Gag-Pol-vaccinated animals at 1 week after administration of the rMVA booster. PBMC were stimulated with four Gag pools (22-mers overlapping by 12 amino acids) for 6 h, fixed, and stained for CD3, CD8, and IFN-γ. Cells were gated on lymphocytes, followed by CD3, and analyzed for CD8 and IFN-γ. Cells in the left quadrants represent CD4⁺ (CD3⁺ CD8⁻). The frequencies in the upper quadrants are IFN-γ-producing cells as percentages of the total number of CD4⁺ (left quadrants) or CD8⁺ (right quadrants) cells.

FIG. 3.

Gag-specific T-cell responses at 1 week after administration of the rMVA booster. Gag-specific CD4 and CD8 responses were determined for both high-dose and low-dose DNA-primed animals at 1 week after administration of the rMVA booster by intracellular cytokine analyses. The sum of responses for the four Gag pools that represent the total Gag response was used for the comparison. Each symbol represents an individual animal. Symbols for animals are the same as in Fig. 4. Horizontal bars represent the geometric means for each group.

FIG. 4.

Temporal viral loads and CD4⁺ cell counts postchallenge of vaccinated and control animals. Panels: A, geometric mean viral loads; B, geometric mean CD4⁺ cell counts; C, viral loads; D, CD4⁺ cell counts of individual animals in the vaccine and control groups. The key to animal numbers is presented in panel D. Assays for the first 12 weeks for the Gag-Pol-Env-immunized groups had a background of 1,000 copies of RNA per ml of plasma. Animals with loads of less than 1,000 copies of RNA per ml of plasma were scored with a load of 500. For all other assays, the background for detection was 300 copies of RNA per ml of plasma and animals with levels of virus below 300 copies of RNA per ml of plasma up to 12 weeks were scored at 500 copies of RNA per ml of plasma and the rest were scored at 300 copies of RNA per ml of plasma. The symbol † represents the death of an animal. Data points that are not connected reflect missing data. Data for the Gag-Pol-Env-immunized groups are reproduced in part from reference with permission of the publisher.

FIG. 5.

Temporal antibody responses. Amounts (micrograms) of total SIV239 Gag or 89.6 Env antibody were determined by using ELISAs. Neutralization titers are the reciprocal of the serum dilution giving 50% neutralization of the indicated viruses grown in human PBMC. Symbols for animals are the same as in Fig. 4. Data points that are not connected reflect missing data. Data for the Gag-Pol-Env-immunized groups are, in part, reproduced from reference . Neuts, neutralizing antibody; GMT, geometric mean titer.

FIG. 6.

Viral loads, infected cells, and CD4⁺ cell loss in the peripheral blood at 2 weeks postchallenge. (A) Intracellular p27 staining. PBMC were fixed and stained for intracellular Gag, CD3, and CD8. Cells were gated on lymphocytes, followed by CD3, and analyzed for CD8 and Gag. Cells in the left quadrants represent CD4⁺ cells (CD3 positive, CD8 negative). The frequencies in the upper left quadrant are Gag-positive cells as the percentages of the total number of CD4⁺ cells (left quadrants). Data are shown for animal 3 prechallenge and animals 3, 37, and 26 postchallenge. Animals were chosen to represent the range of the observed values. Slight differences in the gates in the different panels represent different photomultiplier tube settings for assays run at different times. (B) Comparison of viral loads, numbers of infected cells, and CD4⁺ cell losses at 2 weeks postchallenge. Geometric means of viral RNA copies and percent infected CD4⁺ cells and arithmetic means of percent CD4⁺ cell loss are represented as horizontal bars on the respective graphs. Arrows indicate symbols for the animals whose results are shown in panel A. Cont., control.