We used a DNA prime/rMVA boost regimen to induce HTL and a strong, multispecific CTL response. These CTL responses were detected in both systemic and mucosal tissues. By delivering two rMVA boosts via different routes, we were able to elicit the highest SIV epitope-specific CTL responses reported thus far for the DNA/rMVA strategy. In vaccinees, an average of 12% of their CD3+
lymphocytes were Mamu-A*01/CM9 tetramer-positive 1 week after an i.v. boost with rMVA. These vaccine-induced Gag181-189
CM9-specific tetramer levels were up to 2-fold higher than those achieved in a similar study using a DNA prime/rMVA boost strategy (6
), >10-fold higher than tetramer levels in a cytokine-augmented DNA vaccination regimen (8
), and approximately half of those induced by a DNA/CRL1005 prime/adenovirus boost regimen (50
). Moreover, the i.v. boost induced long-lived mucosal CTL, as shown by the expression of mucosal homing and retention markers on PBMC and detection of Gag181-189
CM9-specific CTLs in the sigmoid colon for up to 10 weeks postboost.
Our vaccine strategy differed from those reported previously in that we used the second i.v. rMVA boost to target CTL to the mucosa. Targeting vaccine-induced immune responses to mucosal surfaces may be important for several reasons. Mucosal surfaces provide the first line of defense against sexually transmitted HIV, and locating virus-specific CTL in the mucosa may facilitate a rapid response after exposure. In a murine study, virus-specific memory T cells derived from tertiary lymphoid tissues, including the intestinal mucosa, are more directly lytic than their splenic counterparts. Therefore, vaccine-induced mucosal CTL may be more effective at limiting early HIV/SIV replication. Since the vast majority of HIV infections occur across mucosal barriers, it may be crucial for successful HIV vaccines to elicit both systemic and mucosal responses. Our vaccine regimen successfully elicited α4β7+ and αEβ7+ CTL and tetramer-positive CD8+ cells in mucosal tissues.
After i.r. challenge with a high dose of SIVmac239, a strong anamnestic CTL response significantly reduced peak viremia in the vaccinated animals (P = 0.005). After peak viremia, the difference in the viral loads between the vaccinees and the controls lost statistical significance and none of the vaccinated animals were able to control viral replication in the chronic phase. Neutralizing antibodies to SIVmac239 were not detected in any of the animals at 6 months postchallenge. It is possible that with an increased number of animals in the study a statistically significant difference in the viral loads may have been maintained by the vaccinees into the chronic phase of infection. However, even with a small sample size, the present study suggests that multispecific CTLs are capable of providing a degree of protection against acute viral replication but, without neutralizing antibodies, they are unable to control chronic viral replication after a high-dose mucosal challenge of pathogenic SIV.
Taken together, our data suggest that even very vigorous CTL responses, targeted both systemically and mucosally, cannot alone control a pathogenic SIV challenge. In contrast, some recent reports have suggested that CTL-based vaccines can ameliorate the course of immunodeficiency diseases (6
). These studies used the chimeric virus SHIV-89.6P as the challenge, and the discrepancy between our results and those of other groups can likely be attributed to fundamental differences between SHIV-89.6P and SIVmac239. First, SHIV-89.6P and SIVmac239 exhibit different cell tropisms. HIV, SIV, and SHIV can be phenotyped based on the coreceptor used for cell attachment (10
). The major coreceptors for HIV and SIV are the chemokine receptors CCR5 and CXCR4 (11
). SIVmac239 is a CCR5-utilizing (R5) virus, and infected macaques typically show a gradual loss of peripheral CD4+
T cells; this loss is analogous to that seen in the course of most HIV infections (18
).SHIV-89.6P, meanwhile, expresses an env
gene derived from a dualtropic virus that could use CCR5 or CXCR4 for entry (R5X4) (42
). Macaques infected with SHIV-89.6P show a rapid and irreversible loss of CD4+
T lymphocytes in the peripheral lymphoid tissues that is similar to that seen in infections with CXCR4-using (X4) strains of HIV (17
). Moreover, SHIV-89.6P, unlike most primary strains of HIV, is sensitive to neutralizing antibodies (35
), whereas our data and those of others show that SIVmac239 is difficult to neutralize (16
). A previous study linked the ability of macaques to mount an antibody response to SHIV-89.6P to longer survival (30
), suggesting that antibodies play a significant role in the observed protection from disease progression. Thus, in recent vaccine studies with SHIV-89.6P as the challenge virus (6
), protection of CD4+
T cells from rapid depletion may have been the key to the vaccinees' long-term survival. If preserved, CD4+
T cells could provide adequate help to B cells, enabling them to mount an effective antibody response against neutralization-susceptible SHIV-89.6P. Furthermore, in at least three of these previous vaccine studies the viral envelope was used as an immunogen and, as a result, cross-reactive neutralizing antibodies may have developed rapidly after challenge. It is difficult to understand the rationale for using SHIV-89.6P in vaccine studies designed to test the efficacy of CTL-based vaccines when several SIV strains (such as SIVmac251, SIVmac239, and SIVmacE660) are readily available. In contrast to SHIV-89.6P, SIVmac239 resembles HIV in that it is very difficult to neutralize with antibodies (23
Despite strong CTL responses, including mucosally located responses, vaccinated macaques lost control of SIVmac239 by the chronic phase. This failure to control virus replication may be the result of several factors. Both the vaccinees and the controls were infected with a single, high dose of SIVmac239. Although several factors play a role in HIV transmission, recent studies have suggested that the viral load of the infected individual is the chief predictor of heterosexual transmission (20
). These studies show that the probability of transmission increases with rising virus levels. In addition, an empirical model of heterosexual HIV-1 transmission predicts that, when seminal levels in plasma are high (>100,000 copies/ml), transmission occurs in 1 of 100 sexual encounters, whereas the probability of transmission declines rapidly with decreasing seminal viral loads (15
). Thus, it may be that the single dose of SIVmac239 used to infect macaques is unnaturally high and does not accurately reflect the transmission of HIV in humans. In a challenge more closely imitating physiological conditions, vaccine-induced CTL, such as those we observed, may be able to control viral replication. However, the development of virus-specific CTL alone may not be sufficient to limit viral replication. Additional help from neutralizing antibodies and the innate immune system may be necessary to control HIV replication. Moreover, the induction of particularly strong immunodominant CTL responses, like those against Gag181-189
CM9 and Tat28-35
SL8, may hinder the stimulation of subdominant CTL responses during infection. The lack of activation of subdominant CTL responses may impede the immune system's ability to mount an effective response.
In conclusion, the DNA prime/rMVA boost vaccination regimen generated HTL and long-lived, multispecific systemic and mucosal CTL. After mucosal challenge with the highly pathogenic SIVmac239, a massive anamnestic CTL response was observed in the limited number of vaccinees, and these animals controlled peak viral replication (P = 0.005). In contrast to similar studies with SHIV-89.6P as the challenge virus, our vaccinated animals were unable to control viral replication in the chronic phase of infection. The present study suggests that multispecific CTL, in the absence of neutralizing antibodies, can achieve a modicum of control over early viral replication but are unable to control chronic viral replication after a high dose mucosal challenge with a pathogenic SIV.