Induction of potent local cellular immunity with low dose X4 SHIV(SF33A) vaginal exposure

Abstract

Intravaginal inoculation of rhesus macaques with varying doses of the CXCR4 (X4)-tropic SHIV(SF33A) isolate revealed a threshold inoculum for establishment of systemic virus infection and a dose dependency in overall viral burden and CD4+ T cell depletion. While exposure to inoculum size of 1000 or greater 50% tissue infectious dose (TCID(50)) resulted in high viremia and precipitous CD4+ T cell loss, occult infection was observed in seven of eight macaques exposed to 500 TCID(50) of the same virus. The latter was characterized by intermittent detection of low level virus with no evidence of seroconversion or CD4+ T cell decline, but with signs of an ongoing antiviral T cell immune response. Upon vaginal re-challenge with the same limiting dose 11-12 weeks after the first, classic pathogenic X4 SHIV(SF33A) infection was established in four of the seven previously exposed seronegative macaques, implying enhanced susceptibility to systemic infection with prior exposure. Pre-existing peripheral SIV gag-specific CD4+ T cells were more readily demonstrable in macaques that became systemically infected following re-exposure than those that were not. In contrast, early presence of circulating polyfunctional cytokine secreting CD8+ T cells or strong virus-specific proliferative responses in draining lymph nodes and in the gut associated lymphoid tissue (GALT) following the first exposure was associated with protection from systemic re-infection. These studies identify the gut and lymphoid tissues proximal to the genital tract as sites of robust CD8 T lymphocyte responses that contribute to containment of virus spread following vaginal transmission.

Figure 1

(A) Viral load and absolute CD4+ T cells counts in macaques systemically infected following IVAG exposure to 500, 1000 and 3500 TCID₅₀ of pathogenic X4 SHIV_SF33A. The viral load detection threshold is 125 RNA copies/ml plasma. (B) Comparison of the kinetics and magnitude of virus replication and peripheral CD4+ T cell loss in macaques exposed IVAG to 1000 and 3500 TCID₅₀ of X4 SHIV_SF33A. Standard deviation of the means for macaques in the 1000 TCID₅₀ (n=4) and 3500 TCID₅₀ (n=3) are shown.

Figure 2

Plasma viremia (RNA copies/ml) and CD4:CD8 ratios in X4 SHIV_SF33A ES macaques. Macaques were exposed IVAG to 500 TCID₅₀ X4 SHIV_SF33A and monitored for virus load and CD4+ T cell loss.

Figure 3

Virus-specific CD4+ and CD8+ T cell proliferative responses in exposed seronegative (ES) macaques. PBMCs from the seven ES macaques were stimulated with peptides spanning SIV gag (●), and then stained for cell surface markers and Ki67. Cells from L041, the infected macaque (*), were also analyzed for comparison. Dashed line denotes cutoff point of three-fold above background (medium with DMSO alone), the upper limit of background proliferation induced by the peptides in four naïve control macaques analyzed. Baseline data are available for the group II macaques (BT29, DL34, T467, L041) and are shown. # indicates animals with viral blips or intermittent proviral SHIV PCR positivity.

Figure 4

Cytokine (IFN-γ and IL-2) production in response to antigen stimulation in CD8+ T cells of ES macaques. 4-color FACS analyses were performed on PBMCs stained for CD3, CD8, IFN-γ and IL-2. (A) Flow cytometry dot plots of cytokine production in response to superantigen SEB, Gag and HIV/SIV Reg peptide stimulation in macaque CA91 at wk1 post-exposure. (B) Percentage of CD8+ T cells that secrete IFN-γ in response to SIV Gag (■) or HIV/SIV Reg (), or both IFN-γ and IL-2 to SIV Gag () and HIV/SIV Reg (□) of all seven ES macaques. Dashed line denotes a 0.2% response level, the upper limit of cumulative responding cells to viral peptides in six naïve macaques analyzed. Baseline values available for two of the macaques (BT29 and T467) as well as data for L041 are also shown. * indicates systemic infection and # indicates animals with viral blips or intermittent proviral SHIV PCR positivity.

Figure 5

Antigen specific proliferative (A) and cytokine (B) responses in tissues of ES macaques. Surgery was performed in macaques BT29, DL34 and T467 at 3 weeks-post-exposure to 500 TCID₅₀ X4 SHIV_SF33A and tissue samples were collected. Cells prepared from various tissues were (A) labeled with CFSE and stimulated with either SEB (beige bars) or SIV gag peptides (other colored bars) for 6 days. Percentage of proliferating T cells is shown. (B) Tissue cells were stimulated with SHIV antigens and the percentage of CD4+ and CD8+ T cells that secrete IFN-γ in response to SIV Gag (■) or HIV/SIV Reg (), or both IFN-γ and IL-2 to SIV Gag () and HIV/SIV Reg (□) is shown. For both sets of data, the colors designate three broadly classified tissue sites: green, draining lymph nodes; red, gut associated lymphoid tissue; purple, distal or secondary lymphoid tissues. # indicates animals with viral blips or intermittent proviral SHIV PCR positivity, and − /+ indicates the absence or presence respectively of SHIV specific DNA in the various tissue compartments as determined by semi-nested env PCR.

Figure 5

Antigen specific proliferative (A) and cytokine (B) responses in tissues of ES macaques. Surgery was performed in macaques BT29, DL34 and T467 at 3 weeks-post-exposure to 500 TCID₅₀ X4 SHIV_SF33A and tissue samples were collected. Cells prepared from various tissues were (A) labeled with CFSE and stimulated with either SEB (beige bars) or SIV gag peptides (other colored bars) for 6 days. Percentage of proliferating T cells is shown. (B) Tissue cells were stimulated with SHIV antigens and the percentage of CD4+ and CD8+ T cells that secrete IFN-γ in response to SIV Gag (■) or HIV/SIV Reg (), or both IFN-γ and IL-2 to SIV Gag () and HIV/SIV Reg (□) is shown. For both sets of data, the colors designate three broadly classified tissue sites: green, draining lymph nodes; red, gut associated lymphoid tissue; purple, distal or secondary lymphoid tissues. # indicates animals with viral blips or intermittent proviral SHIV PCR positivity, and − /+ indicates the absence or presence respectively of SHIV specific DNA in the various tissue compartments as determined by semi-nested env PCR.

Figure 6

Viremia (A), CD4:CD8 ratios (B) and virus-specific cytokine producing CD8+ T cells in peripheral blood of macaques susceptible (C) and resistant (D) to systemic infection following virus rechallenge. ES macaques received a second IVAG challenge 11-12 weeks after the first with the same inoculum dose (500 TCID₅₀) of X4-SHIV_SF33A. Viral load (SHIV RNA copies per ml plasma), peripheral CD4+ T cell number and the percentage of peripheral CD8+ T cells that secrete IFN-γ in response to SIV Gag (■) or HIV/SIV Reg (), or both IFN-γ and IL-2 to SIV Gag () and HIV/SIV Reg (□) were monitored at various time points post re-challenge. Virologic (A) and immunologic data (B, E) from L041, the macaque infected following a single exposure to 500 TCID50 of X4 SHIV_SF33A were included for comparison. +, indicates death due to euthanasia.

Figure 7

Antigen specific proliferative (A) and cytokine (B) responses in tissue compartments of rechallenged macaques. Surgery was performed in macaques BT29, DL34 and T467 at 3 weeks post-rechallenge and tissue samples were collected. Cells prepared from various tissues were (A) labeled with CFSE and stimulated with either SEB (beige bars) or SIV gag peptides (other colored bars) for 6 days. Percentage of CD8+ proliferating T cells is shown. (B) Tissue cells were stimulated with SHIV antigens and the percentage of CD4+ and CD8+ T cells that secrete IFN-γ in response to SIV Gag (■) or HIV/SIV Reg (), or both IFN-γ and IL-2 to SIV Gag () and HIV/SIV Reg (□) is shown. Color designations are the same as those outlined in Figure 4. The ratio of total CD4 to CD8 cytokine producing T cells in the various tissue compartments of each macaque was generated and shown for comparison. − /+ indicate the absence or presence respectively of SHIV specific DNA as determined by semi-nested env PCR. +, indicates death due to euthanasia. NA, not applicable.

Figure 7

Antigen specific proliferative (A) and cytokine (B) responses in tissue compartments of rechallenged macaques. Surgery was performed in macaques BT29, DL34 and T467 at 3 weeks post-rechallenge and tissue samples were collected. Cells prepared from various tissues were (A) labeled with CFSE and stimulated with either SEB (beige bars) or SIV gag peptides (other colored bars) for 6 days. Percentage of CD8+ proliferating T cells is shown. (B) Tissue cells were stimulated with SHIV antigens and the percentage of CD4+ and CD8+ T cells that secrete IFN-γ in response to SIV Gag (■) or HIV/SIV Reg (), or both IFN-γ and IL-2 to SIV Gag () and HIV/SIV Reg (□) is shown. Color designations are the same as those outlined in Figure 4. The ratio of total CD4 to CD8 cytokine producing T cells in the various tissue compartments of each macaque was generated and shown for comparison. − /+ indicate the absence or presence respectively of SHIV specific DNA as determined by semi-nested env PCR. +, indicates death due to euthanasia. NA, not applicable.