Effector memory T cell responses are associated with protection of rhesus monkeys from mucosal simian immunodeficiency virus challenge

Abstract

The rapid onset of massive, systemic viral replication during primary HIV or simian immunodeficiency virus (SIV) infection and the immune evasion capabilities of these viruses pose fundamental problems for vaccines that depend upon initial viral replication to stimulate effector T cell expansion and differentiation. We hypothesized that vaccines designed to maintain differentiated effector memory T cell (TEM cell) responses at viral entry sites might improve efficacy by impairing viral replication at its earliest stage, and we have therefore developed SIV protein-encoding vectors based on rhesus cytomegalovirus (RhCMV), the prototypical inducer of life-long TEM cell responses. RhCMV vectors expressing SIV Gag, Rev-Tat-Nef and Env persistently infected rhesus macaques, regardless of preexisting RhCMV immunity, and primed and maintained robust, SIV-specific CD4+ and CD8+ TEM cell responses (characterized by coordinate tumor necrosis factor, interferon-gamma and macrophage inflammatory protein-1beta expression, cytotoxic degranulation and accumulation at extralymphoid sites) in the absence of neutralizing antibodies. Compared to control rhesus macaques, these vaccinated rhesus macaques showed increased resistance to acquisition of progressive SIVmac239 infection upon repeated limiting-dose intrarectal challenge, including four macaques who controlled rectal mucosal infection without progressive systemic dissemination. These data suggest a new paradigm for AIDS vaccine development--vaccines capable of generating and maintaining HIV-specific TEM cells might decrease the incidence of HIV acquisition after sexual exposure.

Figure 1. RhCMV vectors engineered to express SIV proteins can re-infect RhCMV⁺ RM and initiate a de novo SIV-specific CD4⁺ and CD8⁺ T cell response

(a) Schematic of the SIV protein expression cassettes inserted into the RhCMV genome in the intergenic region between rh213 and Rh214 to create the RhCMV/Gag, RhCMV/Retanef and RhCMV/Env vectors. [See also Supplementary Figs. S1–S3] (b) Western blot analysis of telomerized rhesus fibroblasts co-cultured for 4 weeks with virus pelleted from urine collected at the designated intervals from initially RhCMV⁺ RM following their inoculation with RhCMV/Gag, RhCMV/Retanef and RhCMV/Env. (c) Flow cytometric intracellular cytokine analysis (FCICA) of peripheral blood T cells responding to wildtype RhCMV lysate, SIV Gag or Rev/Nef/Tat overlapping 15mer peptides in a typical, initially RhCMV⁺ RM following inoculation with the RhCMV/Gag and RhCMV/Retanef vectors. The values in the upper and lower right quadrants of the flow cytometric profiles indicate the net% (minus background) of the total CD4⁺ or CD8⁺ T cell population responding to the designated antigen with production of both TNF and IFN-γ or TNF alone, respectively.

Figure 2. RhCMV-vectored, SIV-specific T cell responses persist with a polarized T_EM phenotype and maintain high representation at extra-lymphoid effector sites

(a,b) Combined FCICA and surface phenotype analysis of CD4⁺ (a) and CD8⁺ (b) peripheral blood T cells responding to wildtype RhCMV lysate, SIV Gag or Rev/Nef/Tat overlapping 15mer peptides. The figure compares the CD28 vs. CCR7 phenotype of RhCMV and SIV antigen-responsive CD4⁺ or CD8⁺ T cells (CD69⁺; TNF⁺) in a representative, initially RhCMV⁺ RM that was inoculated 595 days and 330 days earlier with RhCMV/Retanef) and RhCMV/Gag, respectively (left and middle columns), and also compares the SIV Gag response of this RM to a representative RM that received a Gag protein prime and Ad5(Gag) boost (105 days after the boost; right column). [See also Supplementary Fig. S4] (c) FCICA of the response of peripheral blood vs. BAL T cells (CD4⁺ or CD8⁺) to SIV Rev/Nef/Tat overlapping 15mer peptides in an RM that received the RhCMV/Retanef vector 192 and 94 days earlier. The values in the profiles indicate the net% (minus background) of the total CD4⁺ or CD8⁺ T cell population responding to the Rev/Nef/Tat peptides with the designated cytokines. (d) Comparison of the mean frequencies (±SEM) of RhCMV-vectored SIV Gag-, Rev/nef/tat-, and Env-specific T cell responses in the peripheral blood memory compartment vs. the BAL (memory) T cell compartment in a group of 6 RM that are 192 days post initial inoculation and 94 days post a second inoculation with the RhCMV(Gag), RhCMV/Retanef and RhCMV/Env vectors.

Figure 3. RhCMV-vectored SIV-specific T cell responses maintain potent effector function

Representative FCICA of peripheral blood CD4⁺ and/or CD8⁺ T cells responding to SIV Gag or Rev/Nef/Tat overlapping peptides from RM that were inoculated with the corresponding RhCMV vector greater than 500 days earlier. The figures show coordinate analysis of a, TNF vs. IFN-γ vs. IL-2; b, TNF vs. IFN-γ vs. MIP-1β, and c, TNF vs. CD107 externalization (indicating degranulation of cytoplasmic cytotoxic granules). The left and middle profiles are gated on the overall CD4⁺ or CD8⁺ T cell populations with the percentage of the designated responding populations (CD69⁺, cytokine⁺ or CD107⁺) shown in each profile. The right profiles are Boolean gated on total responding T cells (those making any of the designated cytokines or expressing CD107, alone or in combination), with either the percentage of triple producers (a and b, colored blue), or the percentage of responding cells showing CD107 and TNF reactivity alone or in combination (c) designated in the figure [see also Supplementary Figs. S4 and S5].

Figure 4. RM inoculated with RhCMV vectors expressing Gag, Rev/Nef/tat, and Env are protected from progressive SIVmac239 infection following repeated, limiting dose intra-rectal challenge

(a) Vaccination and challenge protocol for efficacy assessment of RhCMV vectors. RhCMV/Gag, RhCMV/Retanef, and RhCMV/Env vectors were given individually at 133 day intervals in the following orders: 1) Gag/Retanef/Env, 2) Retanef/Env/Gag, and 3) Env/Gag/Retanef for 4 RM each. Half of each group was subsequently provided with a combined boost of all 3 vectors. The long-term anti-SIV T cell responses did not differ between these sub-groups, and all 12 of these RM were therefore combined into one vaccinated group for challenge (in comparison to 16 unvaccinated, but RhCMV⁺, control RM). (b) Mean pre-challenge frequencies (± SEM) of RhCMV-vectored, SIV Gag-, Rev/Nef/Tat-, and Env-specific responses among blood CD4⁺ and CD8⁺ memory T cells of the 12 vaccinated RM. (c) Plasma viral loads of the control (left panel) and vaccinated (right panel) RM cohorts over the course of, and subsequent to, limiting dose intra-rectal SIVmac239 challenge. Four of 12 vaccinated RM resisted progressive infection: these protected RM were treated with the humanized anti-CD8 mAb cMT807 at days 133, 136, 140 and 143 post-initial challenge (10, 5, 5, and 5 mg/kg doses, respectively) and were profoundly depleted of CD8⁺ T cells from blood (< 2.5% of baseline absolute counts) for 14 to 21 days. (d) FCICA of peripheral blood CD8⁺ T cells from the 4 protected vaccinees examining the response of these cells to SIV proteins that were (Rev/Nef/Tat) or were not (Pol and Vif) expressed by the administered RhCMV vectors, before (right panel) and 133 days after (left panel) initiation of SIVmac239 intra-rectal challenge protocol.