Rhesus monkey model of repeated, low-dose SIV mucosal exposure.
A rhesus monkey model was established to study immune correlates of natural protection against infection following repeated, low-dose mucosal exposure to SIV. Viral challenge stocks were prepared from two genetically disparate SIV isolates, SIVmac251 and SIVsmE660. Two stocks were created to allow the confirmation of any findings by using different virus challenge systems. Monkeys were exposed to virus by intrarectal inoculation through a cluster of six weekly administrations. Using three monkeys/dose/virus stock, groups of animals were exposed to 6 × 107, 6 × 106, and 6 × 105 copies of virus with each administration. The cohorts of nine SIVmac251-exposed and nine SIVsmE660-exposed monkeys then were monitored for evidence of infection by assessing plasma samples obtained on a weekly schedule for SIV gag virus RNA. As shown in Fig. , all three monkeys exposed to 6 × 107 copies of SIVsmE660, two of three exposed to 6 × 106 copies of SIVsmE660, and one of three exposed to 6 × 105 copies of SIVsmE660 developed persistent viremia; further, all three monkeys exposed to 6 × 107 copies of SIVmac251, two of three exposed to 6 × 106 copies of SIVmac251, and none of the monkeys exposed to 6 × 105 copies of SIVmac251 developed persistent viremia. This created a cohort of seven monkeys that were exposed by a mucosal route to highly pathogenic SIV isolates but did not develop overt evidence of infection.
FIG. 1. Systemic infections following repeated rectal exposure of rhesus monkeys to SIVmac251 or SIVsmE660. Monkeys were exposed by intrarectal inoculation for 6 successive weeks to cell-free virus using three monkeys/dose/challenge stock and doses of 6 × (more ...) SIV-specific cellular immune responses in seven exposed, uninfected rhesus monkeys.
These seven exposed, uninfected rhesus monkeys then were evaluated for systemic SIV-specific cellular immunity to determine whether repeated mucosal exposure to virus can stimulate systemic immune responses that might confer protection against subsequent exposure to virus. Cellular immunity to SIV first was evaluated using a routine ELISPOT assay to assess PBMC IFN-γ responses following exposure to Env, Gag, Pol, and Nef SIVmac239 peptide pools. While PBMC of 7 of the animals in the initial cohort of 18 monkeys that became infected following mucosal exposure to virus demonstrated SIV-specific T-cell responses, PBMC of the seven exposed, uninfected monkeys demonstrated no T-cell responses (Fig. ). Recently it has been reported that the sensitivity of ELISPOT assays can be substantially enhanced by adding cytokines to the peptide-exposed PBMC and increasing the duration of exposure of the PBMC to the viral peptides (6
). PBMC of the exposed, uninfected monkeys evaluated using this highly sensitive assay also showed no evidence of virus-specific cellular immunity (data not shown).
FIG. 2. Exposed, uninfected monkeys developed no peripheral blood T-lymphocyte IFN-γ ELISPOT responses to SIV antigens. PBMC were isolated from exposed, uninfected monkeys, naïve control monkeys, and monkeys that became infected following intrarectal (more ...)
PBMC of these same monkeys also were assessed for SIV-specific cellular immunity by an intracellular cytokine staining (ICS) assay, evaluating both CD8+ and CD4+ T lymphocytes for evidence of IFN-γ, TNF-α, IL-2, and MIP-1β production following exposure to the same SIVmac239 peptide pools. Consistent with the findings of the ELISPOT study, these assays also demonstrated robust CD4+ and CD8+ T-lymphocyte responses by PBMC of the monkeys that developed overt infections, but no significant responses were detected in PBMC of the exposed, uninfected monkeys (Fig. and data not shown). These findings suggested that repeated mucosal exposure to virus did not elicit systemic cellular immune responses that could be detected by routine assays.
FIG. 3. Exposed, uninfected monkeys developed no peripheral blood T-lymphocyte ICS responses to SIV antigens. PBMC isolated from exposed, uninfected; naïve control; and SIV-infected monkeys were exposed to pools of Env, Gag, Pol, and Nef peptides and (more ...) Further exposures of uninfected rhesus monkeys to SIV.
It was possible that the initiation of a systemic infection in one of these monkeys was a stochastic event and that further mucosal exposures of these uninfected monkeys to virus would result in the initiation of additional infections. To examine that possibility, each of the seven uninfected monkeys received a second series of six weekly intrarectal exposures to the same quantity of the same SIV isolate it had received previously, and plasma viral RNA assays were monitored for evidence of the initiation of a systemic infection. These six further exposures to virus did not initiate additional infections.
Since all of the monkeys that received mucosal exposures to 6 × 107 copies of viral RNA became infected during the initial cluster of virus inocula, we explored the possibility that the seven multiply exposed but uninfected monkeys would become infected following exposure to this larger number of viral particles. These seven animals were subjected to a further cluster of six weekly intrarectal exposures to the same virus isolate to which they were previously exposed, but all exposures were to 6 × 107 copies of viral RNA. As shown in Fig. , only two of these seven monkeys developed a systemic infection. Therefore, five monkeys received 18 mucosal exposures to SIVsmE660 or SIVmac251 and did not acquire a systemic infection.
FIG. 4. Systemic infections following repeated rectal exposure of exposed, uninfected rhesus monkeys to large inocula of SIVmac251 or SIVsmE660. The monkeys that remained uninfected following 12 intrarectal exposures to SIV were exposed for 6 successive weeks (more ...) Evaluation of exposed, uninfected rhesus monkeys for cellular immunity by tetramer staining.
While the multiply exposed but uninfected monkeys had no evidence of systemic antiviral immunity as determined by ELISPOT and ICS assays, it remained possible that a more sensitive assay for peripheral blood cellular immunity would detect virus-specific responses in these animals. Thirteen of the 18 rhesus monkeys employed in this study were Mamu-A*01+, and 4 of the 5 remaining uninfected monkeys were Mamu-A*01+. Therefore, there was no association between resistance to mucosal infection and expression of the Mamu-A*01 allele. We were, however, able to employ a Mamu-A*01/p11C tetramer to evaluate SIV Gag epitope-specific CD8+ T-lymphocyte responses in these monkeys. We first used this highly sensitive tetramer binding assay to assess PBMC of the exposed, uninfected monkeys for evidence of systemic antiviral cellular immunity (Fig. ). In fact, while PBMC of Mamu-A*01+ rhesus monkeys from the original cohort of 18 animals that developed systemic infections had readily demonstrable tetramer binding CD8+ peripheral blood T lymphocytes, PBMC of the 4 Mamu-A*01+-exposed, uninfected rhesus monkeys had no tetramer binding peripheral blood CD8+ T lymphocytes.
FIG. 5. Dominant epitope peptide/tetramer binding CD8+ T lymphocytes in the peripheral blood of the exposed, uninfected Mamu-A*01+ rhesus monkeys. PBMC were isolated from exposed, uninfected (EU) monkeys; naïve control monkeys; (more ...)
We also have shown that the sensitivity of this tetramer binding T-lymphocyte assay can be increased by a 1-week in vitro cultivation of PBMC with the optimal epitope peptide before the cells are evaluated in a tetramer binding assay (23
). This modified tetramer binding assay therefore was employed to evaluate PBMC of the exposed, uninfected Mamu-A*01+
rhesus monkeys (Fig. ). While tetramer binding CD8+
T lymphocytes were readily detected in epitope-peptide-stimulated PBMC from Mamu-A*01+
rhesus monkeys that had become infected, and while no tetramer binding CD8+
T lymphocytes were detected in similarly stimulated PBMC of naïve Mamu-A*01+
rhesus monkeys, no tetramer binding CD8+
T lymphocytes were detected in epitope-peptide-stimulated PBMC of the exposed, uninfected Mamu-A*01+
rhesus monkeys. Therefore, no evidence was found for systemic SIV-specific cellular immunity in the exposed, uninfected monkeys using highly sensitive tetramer binding assays.
Evaluation of rectal mucosal T lymphocytes for SIV-specific immunity.
Since it is possible that virus-specific T lymphocytes are in mucosal cell populations but not in the systemic circulation, we assessed lymphocytes sampled from the distal colonic mucosa of the exposed, uninfected monkeys for evidence of SIV-specific T lymphocytes (Fig. ). Lymphocytes were isolated from multiple colonic biopsy specimens obtained from the four Mamu-A*01+-exposed, uninfected monkeys and one Mamu-A*01+ SIV-infected monkey 22 weeks following the final mucosal exposure to virus, and these lymphocytes were assessed for Mamu-A*01/p11C tetramer binding CD8+ T cells. SIV Gag epitope-specific CD8+ T lymphocytes were demonstrated in this mucosal lymphocyte population of the SIV-infected rhesus monkey but not in the mucosal lymphocytes of the exposed, uninfected animals. Therefore, the multiply exposed, uninfected monkeys had no evidence of systemic or mucosal SIV-specific cellular immunity.
FIG. 6. Dominant epitope peptide/tetramer binding CD8+ T lymphocytes in the distal colonic mucosa of the exposed, uninfected Mamu-A*01+ rhesus monkeys. Lymphocytes were isolated from the distal colonic mucosa of the exposed, uninfected (more ...) Evaluation of rectal mucosal antibodies in exposed, uninfected monkeys.
Since protection against mucosal acquisition of an SIV infection could be antibody mediated, we assessed rectal mucosal secretions for anti-SIV antibodies after repeated rectal exposure of monkeys to SIV. A new cohort of six naïve rhesus monkeys was exposed to SIVsmE660 on a weekly schedule for 6 weeks, three monkeys to 6 × 107 RNA copies and three monkeys to 6 × 106 RNA copies. Only one of the six monkeys, CK8G, developed viremia. Low-titer anti-SIV IgA antibodies were detected in rectal sponge specimens obtained from this infected monkey and from two of the exposed, uninfected monkeys, CP11 and CP2C, 1 week following the final rectal exposure to virus (Table ). Therefore, repeated mucosal exposure to SIV elicited a local low-titer mucosal anti-SIV antibody response in some of these monkeys. However, the generation of this antibody response was not associated with protection from infection.
Levels of rectal anti-SIV IgA antibodies from multiply exposed rhesus monkeysa
Intravenous SIV challenge of exposed, uninfected monkeys.
We finally assessed whether there was any evidence of protective anti-SIV immunity in the 5 rhesus monkeys from the original cohort of 18 animals that had been exposed to SIV by intrarectal administration without becoming infected. These monkeys were inoculated by the intravenous route with 2 × 105 RNA copies of the same SIV isolate to which they had previously been exposed. All five monkeys became infected, and the kinetics of this viral replication during the period of primary infection was indistinguishable from the usual kinetics of SIV replication in naïve rhesus monkeys (Fig. ). These findings suggest that there was no potent systemic anti-SIV immunity in the exposed, uninfected animals.
FIG. 7. Systemic infections of exposed, uninfected rhesus monkeys following a single intravenous inoculation of SIVmac251 or SIVsmE660. The five monkeys that remained uninfected following 18 intrarectal exposures to SIV were inoculated by the intravenous route (more ...)