Depo-provera treatment does not abrogate protection from intravenous SIV challenge in female macaques immunized with an attenuated AIDS virus

Abstract

Background: In a previous study, progesterone treatment of female monkeys immunized with live, attenuated SHIV89.6 abrogated the generally consistent protection from vaginal simian immunodeficiency virus (SIV) challenge. The mechanisms responsible for the loss of protection remain to be defined. The objective of the present study was to determine whether Depo-Provera administration alters protection from intravenous SIV challenge in SHIV-immunized female macaques.

Methods and findings: Two groups of female macaques were immunized with attenuated SHIV89.6 and then challenged intravenously with SIVmac239. Four weeks before challenge, one animal group was treated with Depo-Provera, a commonly used injectable contraceptive progestin. As expected, SHIV-immunized monkeys had significantly lower peak and set-point plasma viral RNA levels compared to naïve controls, but in contrast to previously published findings with vaginal SIV challenge, the Depo-Provera SHIV-immunized animals controlled SIV replication to a similar, or even slightly greater, degree than did the untreated SHIV-immunized animals. Control of viral replication from week 4 to week 20 after challenge was more consistent in the progesterone-treated, SHIV-immunized animals than in untreated, SHIV-immunized animals. Although levels of interferon-gamma production were similar, the SIV-specific CD8(+) T cells of progesterone-treated animals expressed more functions than the anti-viral CD8(+) T cells from untreated animals.

Conclusions: Depo-Provera did not diminish the control of viral replication after intravenous SIV challenge in female macaques immunized with a live-attenuated lentivirus. This result contrasts with the previously reported effect of Depo-Provera(R) on protection from vaginal SIV challenge and strongly implies that the decreased protection from vaginal challenge is due to effects of progesterone on the genital tract rather than to systemic effects. Further, these results demonstrate that the effects of hormonal contraceptives on vaccine efficacy need to be considered in the context of testing and use of an AIDS vaccine.

Figure 1. Plasma vRNA levels after IV challenge with SIVmac239.

(A) unimmunized macaques, (B) SHIV-immunized macaques and (C) progesterone-treated, SHIV-immunized macaques. In panel (D), cumulative vRNA levels over the 20 weeks of observation were transformed into areas under the curve (AUC) and the median AUC of the 3 groups of animals were compared. The Kruskal-Wallis test and a pair-wise comparison between the 2 groups of immunized animals and the unimmunized group using Dunn's multiple comparisons test were performed.

Figure 2. Changes in peripheral CD4⁺ T cell populations after IV SIVmac239 challenge.

(A) Number of CD4⁺ T cells, and (B) the percentage of CD4⁺ CD95⁺ T cells in blood. (λ) Naïve control macaques (n = 7), (○) SHIV-immunized macaques (n = 5) and (5) Depo-Provera® SHIV-immunized macaques (n = 6). The number of CD4⁺ T cells (p = 0.0007) and the percentages of CD4⁺ CD95⁺ T cell (p<0.0001) were significantly higher (Friedman test) in the Depo-Provera®-treated, SHIV-immunized macaques than in the naïve control group.

Figure 3. SIV-specific T cell responses after IV SIVmac239 challenge.

(A) Mean number of SIV Gag p27 interferon (IFN)-γ secreting cells in PBMC before and after challenge. (B) Median area under the curve for the total IFN-γ secreting cells in each group from week 2 to 16 PC, the time-points for which samples were available. (C) Mean SIV-specific T cell proliferative responses in PBMC expressed as the stimulation index (SI) before and after SIV challenge. See Methods for details. (D) Median area under the curve for the cumulative SI in each group from week -4 to 20 PC. (λ) Naïve control macaques (n = 7), (○) SHIV-immunized macaques (n = 5) and (5) Depo-Provera® SHIV-immunized macaques (n = 6). P values of a T test are indicated.

Figure 4. Frequency and functional capacity of Gag CM9- and GY9-specific CD8⁺ T cells of SHIV-immunized animals after IV SIVmac239 challenge.

SIV-specific CD8⁺ T cell responses in cryopreserved PBMC from a Mamu-A*01 positive animal and a Mamu-A*02 positive animal in each vaccinated group are shown as pie charts. The number of positive SIV-specific T cells normalized to 10⁵ CD3⁺ T cells is shown for each response as the white number in the center of the pie chart. Each portion of a pie chart indicates the percentage of SIV-specific T cells that responded with one, two, or three functions; and the colored arcs around the pie show the cytokine or combination of cytokines comprising each response. ND not done, as samples were not available.

Figure 5. Frequencies of activated T cells after progesterone treatment and IV SIVmac239 challenge.

A) Percent of CD3⁺ CD4⁺ T cells expressing CD38 but not HLA-DR in PBMC. B) Percent of CD3⁺ CD8⁺ T cells expressing CD38 but not HLA-DR in PBMC. D) Percent of CD3⁺ CD8⁺ T cells expressing Ki-67⁺ in PBMC. E) Percent of CD3⁺ CD8⁺ T cells expressing caspase-3⁺ in PBMC. (○) SHIV-immunized macaques (n = 5) and (5) Depo-Provera®-treated, SHIV-immunized (n = 6).

Figure 6. Frequencies of regulatory CD4⁺ T cells after progesterone treatment and IV SIVmac239 challenge.

A) Percentages of CD4⁺ CD152⁺ T cells in PBMC. B) Percent of CD4⁺ FoxP3⁺ HLA-DR⁺ T cells after challenge in PBMC. C) Percentages of CD4⁺ CD152⁺ T cells in axillary LN of the vaccinated animals at 12 weeks post-challenge. D) Percentages of CD4⁺ FoxP3⁺ HLA-DR⁺ T cells in axillary LN of the vaccinated animals at 12 weeks post-challenge. (λ) Naïve control macaques (n = 7), (○) SHIV-immunized macaques (n = 5) and (5) Depo-Provera®-treated, SHIV-immunized macaques (n = 6).

Figure 7. Cytokine mRNA levels after IV SIVmac239 challenge.

Changes in gene expression are shown as the fold-change relative to the levels on the day of challenge for (A) TNF-α and (B) TGF-β. (C) Spearman rank correlation of TGF-β at 4 weeks PC, for all animals, with plasma viral RNA AUC between 1 and 20 weeks PC. (λ) Naïve control macaques (n = 7), (○) SHIV-immunized macaques (n = 5) and (5) Depo-Provera®-treated, SHIV-immunized macaques (n = 6).