In this study, we analyzed the immunogenicity and protective efficacy of a mismatched Env vaccine delivered by heterologous prime-boost vaccination. We found that rLCMV could be used in alternative prime-boost combinations with plasmid DNA and adenoviral vaccine vectors. Importantly, these data provide the first evidence that unmatched Env alone is sufficient to confer protection against a heterologous SIV swarm in a repetitive mucosal NHP challenge. Vaccination with this vector combination stimulates multiple arms of the immune system, including antigen-specific CD4+ and CD8+ T cells in the blood, spleen, and gut-associated tissue, along with HIV-1 Env-specific antibodies. It was not unexpected that rLCMV would be effective in boosting antibody responses to Env in the NHP, since it stimulates dendritic cells to provide T-cell help, especially during the boost phase of the immune response.
Although rLCMV-based vaccine vectors have been only recently described, wild-type LCMV has been a broadly used tool to study T-cell immunology in mice (46
). As detailed here and published previously in regard to wild-type LCMV, the glycoprotein of this prototypic arenavirus efficiently targets antigen-presenting cells, especially DEC205+
dendritic cells, and activates them, resulting in the priming of B and T cells (41
). This particular targeting of DCs may explain why even low doses of rLCMV elicit a substantial immune response after boosting, whereas higher doses of Ad5 (up to several log units higher) are necessary to reach similar potency. rAd5 is known to infect cells ubiquitously, and only the rAd5 fiber shaft and penton base interact with the DC and mononuclear subsets necessary for T-cell priming (11
). Although the NHP prime-boost regimen confirms the mouse data, the effect of the boost on the cellular immune response was less than that observed in mice. This difference was likely due to the relatively lower LCMV vector dose per kilogram, a problem that may be remedied when large-scale production of the vector is achieved.
One of the major problems with rAd5 is the high seroprevalence of Ad5 in the human population, particularly in sub-Saharan Africa, where an HIV vaccine is urgently needed (42
). Recently, several groups have proposed simian, great ape, or human alternative serotype adenoviral vectors and that a combination with another potent vector would be ideal. While rAd28 or rAd26 are representatives from one such alternative serotype, it has become apparent that seropositivity to these vectors is markedly increased in Africa, where AIDS is endemic (10
). As an alternative, the simian or great ape adenoviruses, particularly the serotype C chimpanzee Ad3 vector, may represent an alternative vector with similar immunogenicity but low seropositivity that could be used in such settings (33
). In contrast, replication-competent LCMV can replicate in human cells but rarely infects immunocompetent individuals (15
), as evidenced by its low (<5%) seropositivity worldwide (3
). In addition, even if preexisting antibodies exist, they are rarely neutralizing (19
The immunogenicity of one of these vector combinations was confirmed in NHP and used to evaluate its ability to protect against a tier 2-like SIV strain, SIVsmE660, in a mucosal challenge model. This challenge model was established to recapitulate human mucosal infection by HIV-1. It has been shown previously that DNA/Ad5 (26
) or DNA/modified vaccinia virus Ankara (MVA) vaccines expressing Env in addition to three antigens, including Gag, Pol, and Nef or Tat, protected in this challenge model. In the case of DNA/MVA vaccines, protective efficacy required the coexpression of granulocyte-macrophage colony-stimulating factor (GM-CSF). In these studies, it is unclear whether other immunogens aside from Env contributed to the protection. A more recent study showed that the addition of Env to Gag and Pol in the vaccine was required to delay SIVmac251 infection (4
). While the protective effect of a vaccine regimen consisting of Env alone was not demonstrated in this study, antibody responses to Env as assayed by ELISA, neutralization activity against a tier 1 clone, and Env V2 peptide binding antibodies also correlated with protection. The difference in efficacy in these studies may be due to one of several factors, including the specific challenge virus, the dose used for challenge, the choice of vaccine vectors, or the specific vaccine antigen that was used to establish immunity. Importantly, Barouch et al. suggested that addition of Env improved vaccine-induced protection, consistent with our conclusion, but they did not demonstrate that Env alone prevents acquisition (4
). Although other multivalent vaccines have been previously shown to protect against challenges with a defined single SIV or SHIV strain (4
), we show here for the first time that a vaccine expressing only mismatched Env reduced the risk of infection by 82% and clearly demonstrate that Env alone is necessary and sufficient to protect against lentiviral infection by a heterologous quasispecies. The sequence distance between the vaccine Env (SIVmac239) and the Env of the challenge viral strain (SIVsmE660) is comparable to that between two sequence-divergent isolates of clade B viruses (28
). The timing of the challenge relative to the boost can affect efficacy and does vary among published studies in the literature. In this study, the 6-week interval was chosen to take advantage of the high level of immunity observed at that time. Since the immune correlates are measured both on the day of challenge and at the peak of immune response, unless the mechanism of protection changes with time, this difference in timing is unlikely to change the immune correlate. In support of this notion, we have previously shown that the correlates of protection with a DNA/Ad vaccine regimen with challenge 16 weeks after boost agree with the present study (28
A previous study showed that inactivated SIV grown in human T cells generated antibodies to human cell surface proteins (5
). Specifically, antibodies to human CD4 in monkeys conferred protection against SHIV infection. The vectors used in the present study were purified from human HEK 293-derived vector packaging cell lines and did not generate detectable anti-CD4 immune responses as determined by ELISA or cell surface staining (see Fig. S1 in the supplemental material). This result suggests that anti-CD4 antibodies or antibodies to the producer 293 cells were not responsible for the vaccine protective effect seen in previous studies (5
). In addition, because antibody responses to viral Env were shown to correlate with protection, it is likely that antibodies to HIV-1 Env mediate this effect. Further, a previous study indicated that antibody-dependent cell-mediated cytotoxicity (ADCC) activity did not correlate with protection (28
). Antibodies that neutralize the challenge virus swarm when tested in PBMC, but not in TZM-bl cells, appear to identify the correlate, highlighting the importance of subtleties in different neutralization assays for assessing biologically meaningful correlates. This study confirmed the neutralizing activity assayed on PBMC as the correlate of protection revealed by a previous study using a larger cohort, 89 animals (28
). The fact that we did not observe more than 50% neutralization in this assay suggests that the challenge swarm is more similar to that of a tier 2 virus when tested on human PBMC. These data together provide a cautionary note about differences in alternative methods for measuring antibody neutralization. In particular, differences in expression levels of CD4, CCR5, or other cofactors between PBMC and transformed cell lines may affect viral entry or replication and affect the sensitivity or specificity of virus neutralization, which may hinder the ability to detect a correlate of protection. The sensitivity of detection for neutralizing activity targeted to particular epitopes may differ in TZM-bl cells and PBMC, as documented in the case of antibodies to HIV gp41 (6
). It will be important to evaluate such alternative assay formats and natural viral isolates to define these correlates in future clinical efficacy trials.
In summary, this study suggests that immunization with a mismatched Env is necessary and sufficient to protect against a tier 2-like SIV challenge model, and this protection correlated with the ability to generate a neutralizing antibody response to the infecting viral swarm. Furthermore, we have identified two viral vectors that together elicit both humoral and cellular immunity. In clinical studies, it is possible that nonhuman rAd vectors such as chimpanzee Ad3 might substitute for rAd5. Because such a combination provides substantial protection against mucosal lentiviral challenge, this finding suggests that attention should be focused on this HIV gene product, an approach that would facilitate the development of an effective AIDS vaccine.