Suppression of adaptive immune responses during primary SIV infection of sabaeus African green monkeys delays partial containment of viremia but does not induce disease

Abstract

One of the most puzzling observations in HIV research is the lack of pathogenicity in most nonhuman primate species that are natural hosts of simian immunodeficiency virus (SIV) infection. Despite this, natural hosts experience a level of viremia similar to humans infected with HIV or macaques infected with SIV. To determine the role of adaptive immune responses in viral containment and lack of disease, we delayed the generation of cellular and humoral immune responses by administering anti-CD8- and anti-CD20 lymphocyte-depleting antibodies to sabaeus African green monkeys (Chlorocebus sabaeus) before challenge with SIV(sab9315BR). In vivo lymphocyte depletion during primary infection resulted in a brief elevation of viremia but not in disease. Based on the magnitude and timing of SIV-specific CD8(+) T-cell responses in the lymphocyte-depleted animals, CD8(+) T-cell responses appear to contribute to viral containment in natural hosts. We found no evidence for a contribution of humoral immune responses in viral containment. These studies indicate that natural hosts have developed mechanisms in addition to classic adaptive immune responses to cope with this lentiviral infection. Thus, adaptive immune responses in natural hosts appear to be less critical for viral containment than in HIV infection.

Figure 1

CD8⁺ T-cell and NK-cell depletion in SIV-infected AGMs. Absolute CD8⁺ T cells in peripheral blood (A-B) and lymph nodes (C-D) and peripheral blood NK-cell (E-F) counts in 12 sabaeus African green monkeys (AGMs) infected intravenously with SIV_sab9315BR. Six AGMs received 1 subcutaneous dose of 10 mg/kg of the anti-CD8α mAb cM-T807 on day 0 before simian immunodeficiency virus (SIV) infection and 2 intravenous doses of 5 mg/kg cM-T807 on days 3 and 7 after infection to deplete CD8⁺ lymphocytes (B,D,F). These 6 animals also received 50 mg/kg anti-CD20 rituximab antibody intravenously at days −7, 14, and 35 after infection to deplete B cells. The other 6 animals received 1 intravenous injection of 50 mg/kg IgIV 7 days before infection to serve as control animals (A,C,E). The ↓ in panels B, D, and F indicate the injection of the CD8⁺ lymphocyte-depleting mAb. CD8⁺ T cells were gated as CD8⁺ CD3⁺ lymphocytes and NK cells as CD8⁺ CD3⁻ lymphocytes. Absolute CD8⁺ T-cell and NK-cell numbers were calculated from white blood cell counts. p.i. indicates postinfection.

Figure 2

B-cell depletion in SIV-infected AGMs. Six sabaeus AGMs were depleted by mAb cM-T807 of CD8⁺ lymphocytes and by mAb rituximab of CD20⁺ lymphocytes (B,D), and 6 AGMs received purified control Ab IgIV (A,C). Absolute B-cell number in peripheral blood (A-B) and percentage of B cells in lymph node cell suspensions (C-D). The ↓ in panels B and D indicate the injection time points of the CD20⁺ lymphocyte-depleting mAb rituximab. p.i. indicates postinfection.

Figure 3

Temporally increased plasma SIV viremia in CD8⁺ and CD20⁺ lymphocyte-depleted SIV-infected AGMs. SIV_sab9315BR RNA copy numbers were determined by quantitative PCR in control Ab-treated AGMs (A) and CD8⁺ and CD20⁺ lymphocyte-depleted AGMs (B). Median viral RNA copy number for both groups (C). *Statistically significant difference between the control (solid line, ●) and CD8⁺ and CD20⁺ lymphocyte-depleted group (dashed line, □) at the indicated time points (Mann-Whitney test, P ≤ .05). p.i. indicates postinfection.

Figure 4

Increased IFN-γ immune responses in CD8⁺ and CD20⁺ lymphocyte-depleted SIV-infected AGMs. SIV_sab IFN-γ cytokine release of PBMCs obtained from 12 SIV-infected sabaeus AGMs. PBMCs from 6 control Ab-treated AGMs (A,C) and 6 CD8⁺ and CD20⁺ lymphocyte-depleted AGMs (B,D) were stimulated with SIV_sab Gag and Env 15-mer peptide pools for 18 hours at 37°C. The values given represent the number of IFN-γ SFCs in 10⁶ PBMCs in response to stimulation with the Gag (A-B) or Env peptide pool (C-D). Responses were considered positive if their value was greater than 2 times the SD of the mean of the media control wells. All values were background corrected using the media controls. p.i. indicates postinfection.

Figure 5

Increased SIV-specific expression of IFN-γ, IL-2, and TNF-α cytokine responses in CD8⁺ T cells from CD8⁺ and CD20⁺ lymphocyte-depleted SIV-infected AGMs. Intracellular expression of IFN-γ (A,D,G), IL-2 (B,E,H), and TNF-α (C,F,I) in SIV_sab Gag and Env peptide pool-stimulated CD8⁺ T cells. Freshly isolated PBMCs were stimulated for 9 hours at 37°C in the presence of either Env or Gag peptide pools. The intracellular staining in CD8⁺ T cells stimulated with Gag or Env peptide pools is shown for control Ab-treated AGMs in black and for CD8⁺ and CD20⁺ lymphocyte-depleted AGMs in red (A-F). (G-I) Median responses of the 6 control AGMs in green and of the 6 CD8⁺ and CD20⁺ lymphocyte-depleted AGMs in blue. *Statistically significant difference between the control and CD8⁺ and CD20⁺ lymphocyte-depleted group at the indicated time points (Mann-Whitney test, P ≤ .05). Responses were considered positive if their value was greater than 2 times the SD of the mean of the media control wells. All values were background corrected using the media controls. Responses and background were determined separately for the different cytokines. p.i. indicates postinfection.

Figure 6

Delayed appearance of SIV-specific humoral immune responses has no effect on set-point viremia of SIV-infected AGMs. SIV_sab9315BR NAb titers in control Ab-treated AGMs (A) and CD8⁺ and CD20⁺ lymphocyte-depleted AGMs (B). The median NAb titer was calculated for both groups (C). *Statistically significant difference between the control (solid line, ●) and CD8⁺ and CD20⁺ lymphocyte-depleted group (dashed line, □) at the indicated time points (Mann-Whitney test, P ≤ .05). The 2 animals with the most efficient/longest B-cell depletion, no. 365 (D) and no. 366 (E), in the group of antibody-treated and SIV-infected sabaeus AGMs are shown. The appearance of NAbs (red lines) coincided with the reappearance of peripheral blood B cells (blue lines) but had no effect on set-point viremia. p.i. indicates postinfection.

Figure 7

CD8⁺ and CD20⁺ lymphocyte depletion in SIV-infected AGMs did marginally affect the number of memory CD4⁺ T cells and Ki-67⁺ memory CD4⁺ T cells in peripheral blood. CD4⁺ T cells of control Ab-treated AGMs (A-D) and CD8⁺ and CD20⁺ lymphocyte-depleted AGMs (B-E) were stained with anti-CD28 and anti-CD95 to determine naive (CD95⁻) and memory (CD95⁺) CD4⁺ T-cell subsets. The fold change in memory CD4⁺ T-cell numbers (A-C) and the fold change in Ki-67⁺ memory CD4⁺ T-cell numbers (D-E) were calculated for each time point compared with the median of 3 preantibody injection time points. (C) The median fold change in memory CD4⁺ T cells from the 6 control Ab-treated AGMs (solid line, filled symbols) and the 6 CD8⁺ and CD20⁺ lymphocyte-depleted AGMs (solid line, open symbols) are shown. (F) The median fold change in Ki-67⁺ memory CD4⁺ T cells from the 6 control Ab-treated AGMs (solid line, filled symbols) and the 6 CD8⁺ and CD20⁺ lymphocyte-depleted AGMs (solid line, open symbols) are shown. *Single time point with a significant difference (Mann-Whitney test) between the control group and the antibody-treated group of animals. No significant differences were detected between the control and antibody-treated groups at any of the other time points. p.i. indicates postinfection.