Evasion of CD8+ T cells is critical for superinfection by cytomegalovirus

Abstract

Cytomegalovirus (CMV) can superinfect persistently infected hosts despite CMV-specific humoral and cellular immunity; however, how it does so remains undefined. We have demonstrated that superinfection of rhesus CMV-infected rhesus macaques (RM) requires evasion of CD8+ T cell immunity by virally encoded inhibitors of major histocompatibility complex class I (MHC-I) antigen presentation, particularly the homologs of human CMV US2, 3, 6, and 11. In contrast, MHC-I interference was dispensable for primary infection of RM, or for the establishment of a persistent secondary infection in CMV-infected RM transiently depleted of CD8+ lymphocytes. These findings demonstrate that US2-11 glycoproteins promote evasion of CD8+ T cells in vivo, thus supporting viral replication and dissemination during superinfection, a process that complicates the development of preventive CMV vaccines but that can be exploited for CMV-based vector development.

Fig. 1

Re-infection of RhCMV-positive animals is independent of viral dose. (A) At day 0, two cohorts of four RhCMV+ animals each were infected s.c. with 10² or 10⁴ PFU of RhCMV(gagL). The SIVgag-specific T cell responses in PBMC or in BAL were monitored by flow cytometric analysis of ICCS for CD69 and tumor necrosis factor (TNF)α (21) (fig. S2, S3). (B) Day of first detection of SIVgag-expressing virus in the urine or buccal swabs of each animal in the two cohorts shown in (A). Also included are results from a third cohort of eight RhCMV+ animals inoculated with 10⁷ PFU of RhCMV(gagL). Expression of SIVgag was determined by immunoblot using anti-SIVgag antibody from viral co-cultures (21). Each circle represents an individual animal. (C) Longterm secretion of SIVgag-expressing virus. Urine was isolated at the indicated days post-infection (PID) from each of the RhCMV(gagL)-infected RM and SIVgag-expression was detected from co-cultured virus by immunoblot. For control, a RhCMV-positive animal that did not receive RhCMV(gagL) was included.

Fig. 2

Interference with MHC-I assembly is not required for primary infection of CMV-naïve animals. Three cohorts of two RM each were inoculated s.c. with 10⁷ PFU of recombinant ΔUS2-11(gag), ΔVIHCEΔUS2-11(gag) or RhCMV(gag). (A) The RhCMV-specific T cell response in PBMC and the SIVgag-specific T cell response in PBMC and BAL were determined at the indicated days post infection using overlapping peptides to RhCMV immediate early genes IE1 and IE2 (IE) or SIVgag by flow cytometric analysis of ICCS for CD69, TNFα and IFNγ (21) (fig. S2, S3). (B) Immunoblot of RhCMV-IE2 or SIVgag expressed in co-cultures of urine samples obtained from animals infected with ΔUS2-11(gag) or ΔVIHCEΔUS2-11(gag). The IE2-blot confirms that the animals were negative for RhCMV prior to infection consistent with results from T cell assays (table S1B). (C) PCR-analysis of viral genomic DNA isolated from viral co-cultures at 428 days post infection. The presence or absence of indicated open reading frames (ORFs) was determined by PCR using specific primers (21). One of the animals infected with RhCMV(gag) served as control.

Fig. 3

US2-11 deleted RhCMV is unable to super-infect RhCMV+ rhesus macaques. (A) A cohort of four RhCMV+ RM was inoculated s.c. with 10⁷ PFU of ΔVIHCEΔUS2-11(gag) (ΔVΔU) at days 0 and 91. The CD4+ and CD8+ T cell response to SIVgag or RhCMV-IE was monitored by flow cytometric analysis of ICCS for CD69, TNFα and IFNγ in PBMC. The percentage of the responding, specific T cells within the overall memory subset is shown for each time point. At day 154 and again on day 224, the same cohort was inoculated with 10⁷ PFU of ΔUS2-11(gag) (ΔU) and RhCMV-IE and SIVgag-specific T cell responses were monitored bi-weekly. At day 737, the cohort was inoculated with ΔVIHCE(gag) (ΔV) and the T cell response was monitored as before. At day 989 the cohort was inoculated with ΔRh186-8(retanef) (ΔR). Besides SIVgag, a T cell response to SIVrev/nef /tat was detected by ICCS in all four animals (black lines) using corresponding overlapping peptides. Inset: A separate cohort of four animals was infected with wildtype RhCMV(gag) and the RhCMV-IE and SIVgag-specific CD4+ and CD8+ T cell response was monitored as described above at the indicated time points for 133 days. (B) The CD4+ and CD8+ T cell response to SIVgag in BAL was measured in parallel to the PBMC T cell responses shown in (A). (C) RhCMV secreted in the urine collected from the cohort infected with RhCMV(gag), or deletion viruses ΔVIHCEΔUS2-11(gag) or ΔUS2-11(gag), labeled ΔCMV. Virus was isolated at the indicated days by co-culture with telomerized rhesus fibroblasts (TRFs) and cell lysates were probed for expression of SIVgag by immunoblot. (D) Expression of RhCMV-IE2, SIVgag and SIVretanef by virus secreted in urine collected at the indicated days. Note that all animals were IE2-positive at the onset of the experiment confirming their RhCMV-positive T cell status (table S1D).

Fig. 4

CD8+ T cells protect rhesus macaques from infection by RhCMV lacking MHC-I inhibitors. (A) Four CMV-positive RM were treated at the indicated days with the anti-CD8 antibody CM-T807 prior to and after inoculation with 10⁷ PFU of ΔVIHCEΔUS2-11(gag) (2 animals, black line) or ΔUS2-11(gag) (two animals, red line). The absolute counts of CD8+ T cells in the blood of each animal are shown over time. (B) The presence of CD4+ and CD8+ T cell populations in PBMC of one representative animal is shown for the indicated days. (C) SIVgag-specific CD4+ and CD8+ T cell responses in PBMC and BAL of CD8+ T cell-depleted animals were monitored by ICCS for CD69, TNFα and IFNγ and are shown as a percentage of total memory CD4+ or CD8+ T cells. Note the delayed appearance of SIVgag-specific CD8+ T cells. (D) Expression of SIVgag or IE2 by RhCMV secreted in the urine of animals infected upon CD8+ depletion.