Strong viremia control in vaccinated macaques does not prevent gradual Th17 cell loss from central memory

Abstract

It has been proposed that microbial translocation might play a role in chronic immune activation during HIV/SIV infection. Key roles in fighting bacterial and fungal infections have been attributed to Th17 and Tc17 cells. Th17 cells can be infected with HIV/SIV, however whether effective vaccination leads to their maintenance following viral challenge has not been addressed. Here we retrospectively investigated if a vaccine regimen that potently reduced viremia post-challenge preserved Th17 and Tc17 cells, thus adding benefit in the absence of sterilizing protection. Rhesus macaques were previously vaccinated with replication-competent Adenovirus recombinants expressing HIVtat and HIVenv followed by Tat and gp140 protein boosting. Upon SHIV(89.6P) challenge, the vaccines exhibited a significant 4 log reduction in chronic viremia compared to sham vaccinated controls which rapidly progressed to AIDS [39]. Plasma and cryopreserved PBMC samples were examined pre-challenge and during acute and chronic infection. Control macaques exhibited a rapid loss of CD4(+) cells, including Th17 cells. Tc17 cells tended to decline over the course of infection although significance was not reached. Immune activation, assessed by Ki-67 expression, was associated with elevated chronic viremia of the controls. Significantly increased plasma IFN-γ levels were also observed. No increase in plasma LPS levels were observed suggesting a lack of microbial translocation. In contrast, vaccinated macaques had no evidence of immune activation within the chronic phase and preserved both CD4(+) T-cells and Tc17 cells in PBMC. Nevertheless, they exhibited a gradual, significant loss of Th17 cells which concomitantly displayed significantly higher CCR6 expression over time. The gradual Th17 cell decline may reflect mucosal homing to inflammatory sites and/or slow depletion due to ongoing low levels of SHIV replication. Our results suggest that potent viremia reduction during chronic SHIV infection will delay but not prevent the loss of Th17 cells.

Fig. 1. Viral load and CD4 count after SHIV_89.6P challenge

(A) Geometric mean viral loads post challenge. A 4-log significant difference in median chronic viremia between control and vaccinated macaques over weeks 6 to 24 was previously reported [37]. With 6 additional controls, the 3.5 log difference remained significant (p<0.0001) (B) Mean CD4⁺ T-cell counts in blood over the course of the study. The dashed line indicates 100 cells/µl. The solid line marks the restoration of CD4 T cells in vaccinated macaques to pre-challenge levels. Post challenge, CD4⁺ T-cell counts declined to <10 cells/µl in controls. Error bar = standard error of the mean (sem).

Fig. 2. Flow Cytometry gating strategy

On the left hand side, singlets were further gated on the lymphocytic population followed by a live/dead gate and selection of CD3⁺ cells. CD3⁺ cells were gated on CD4 or CD8 with further gating for central memory (CM), effector memory (EM), and total memory (TM) using CD28 and CD95. As an example, CD4 CM cells and CD8 TM cells were gated as shown for IL-17 secretion.

Fig. 3. Loss of Th17 cells and up-regulation of CCR6 on Th17 cells during the course of infection in the vaccinated macaques

(A) Successive decline in the total population of Th17 cells (pre vs wk 4 and pre vs chronic, p = 0.027 for both) in vaccinated, protected macaques. (B) Up-regulation of CCR6 during the course of SHIV_89.6P infection. Significantly higher expression was seen in the total Th17 population (pre vs wk4 and pre vs chronic, p = 0.0003 for both). Plotted are mean values with sem.

Fig. 4. CCR6 expression on Tc17 cells and population dynamics over the course of infection

(A) Mean levels of CCR6 expression on Tc17 cells from the control animals (black bars) and vaccinated animals (open bars). Significantly increased expression on total memory cells (pre vs chronic and wk4 vs chronic, p = 0.0001 for both) was observed for control animals. (B) Tc17 memory population dynamics over the course of infection in control animals (black bars) and vaccinated animals (open bars). Error bar = sem.

Fig. 5. Lipopolysaccharide (LPS) levels in plasma, measured by LAL assay

Shown are LPS levels for paired samples before and after challenge (wk24, chronic phase).

Fig. 6. Chronic immune activation among CD8 and CD4 memory populations in control and vaccinated macaques

(A) Mean Ki-67 expression among CD8⁺ CM and (B) EM cells pre- and post-challenge (wk4 and wk24). Ki-67⁺ cells were elevated in controls compared to vaccinated macaques during chronic infection (wk24) for both CD8⁺ CM (p = 0.0043) and EM (p = 0.0006) cells. (C) Mean Ki-67 expression among CD4⁺ CM and (D) EM cells pre- and post-challenge (wk4 and wk24). Ki-67⁺ cells were elevated in controls compared to vaccinated macaques at wk4 and wk24 for CD4⁺ CM cells (p = 0.014 and p<0.0001, respectively) (C), whereas no differences were observed for EM cells (D). (E) Geometric mean viral loads illustrating increased viral burdens in control compared to vaccinated macaques post-challenge at wk4 and wk24 (p<0.0001 for both).

Fig. 7. Plasma IFN-γ levels post-challenge normalized to pre-challenge values and real time PCR analysis of MxA and IL-8 mRNA

(A) Plasma levels of IFN-γ as indicated over the course of infection. The mean of duplicate assays are plotted. The dotted line indicates the time of challenge. IFN-γ levels in control macaques compared to the vaccinated group were elevated at wk11 and wk20 post-challenge (p = 0.036 and 0.0036, respectively). (B) Mean fold increases of MxA and IL-8 mRNA at wk6 and wk20 post-challenge compared to pre values for the vaccinated and control groups are shown. Significant elevations in MxA mRNA are indicated as follows: A: p = 0.047; B: p = 0.0026; C: p = 0.0021. No significant differences between levels in control and vaccinated macaques were observed. Significant elevations in IL-8 mRNA are indicated as follows: D: p<0.0001; E: p = 0.047. IL-8 mRNA dropped significantly in the vaccinated and control groups between wk6 and wk20 (p = 0.0005 and p = 0.048, respectively). Error bar = sem.