The level of containment of viral replication observed in the present study suggests that vaccine-induced T-cell responses might indeed be more effective against the AIDS virus than we had previously considered. We, and others, have already shown that SIV vaccines based solely on inducing cellular immune responses (i.e., no Env in the vaccine) can reduce both acute and chronic phase viral replication of homologous challenge viruses (34
). Unfortunately, however, viral replication exceeded 1 million copies/ml during the acute phase in these earlier studies using homologous viral challenges, suggesting that it might be difficult to control acute-phase viral replication with vaccine-induced T-cell responses alone. Encouragingly, our new results indicate that vaccine-induced T-cell responses alone can control replication of a heterologous virus during both the acute and the chronic phases of infection even after a heterologous challenge.
Given the low levels of viral loads in vaccinee r00061, it is possible that this animal was not productively infected (Fig. ). Soon after the first challenge, we detected vRNA in plasma from this animal at days 9, 11, and 15. We also reamplified virus from frozen RNA extracted on these 3 days. Additionally, we observed a transient increase in 8 of 13 vaccine-induced CD8+ T-cell responses (see Fig. S4A in the supplemental material). However, two other independent laboratories were unable to amplify virus from shipped frozen samples from this animal. These two laboratories did amplify virus from a single time point (but not all time points) in shipped frozen plasma samples from vaccinee r02103, another animal with low acute-phase viral loads. This vaccinee was clearly infected given the expansion of 12 of 22 CD8+ and CD4+ vaccine-induced anamnestic T-cell responses at day 21 postinfection (Fig. ). The Mamu-A*02/Gag GY9-specific T-cell response expanded from 102 SFC/106 PBMCs 1 month prior to challenge to 2,525 SFC/106 PBMCs 21 days after infection in this vaccinee (Fig. ). We were also unable to isolate the virus from activated, CD8-depleted PBMCs from r00061 or r02103 on three different occasions. In contrast, we routinely cultured virus from PBMCs of two of the other vaccinees, r02114 and r01099, with higher acute-phase viral loads. Thus, we might have succeeded in productively infecting only seven of eight of our vaccinees. In contrast, we infected all of our naïve control animals.
Much of the attractiveness of vaccines that would elicit broadly reactive neutralizing antibodies relates to the ability of such antibodies to prevent infection or to limit viral replication during acute infection. This would diminish acute pathogenesis, reduce the generation of genetic diversity of the virus, and as an associated benefit, reduce the prospect of secondary transmission during acute infection, a time when higher levels of viremia are associated with increased transmission (21
). The present results suggest that vaccine-induced cellular immunity, in the absence of neutralizing antibody, may also be able to achieve these same important objectives. More than half of the vaccinated animals had peak viral loads of less than 80,000 vRNA copy eq/ml. The achievement of this level of control of a heterologous challenge virus during the acute phase represents an encouraging new benchmark in the evaluation of prophylactic AIDS vaccines.
The mucosal challenge model employed in the present study recapitulates the results of human mucosal exposures leading to clinical HIV-1 infections, and the vaccine regimen used provided dramatic protective effects against this challenge that were greater than those seen in other studies. However, as there has been less experience with this repeated mucosal heterologous challenge model than some other challenge systems, we considered whether the protection we observed was a function of superior vaccine efficacy or potentially a result of a less-rigorous challenge than that used in other studies. Unlike clonal SIV challenge stocks (e.g., SIVmac239), challenge stocks of virus swarms like SIVsmE660 can vary in their pathogenicity depending on how they are propagated. Indeed, in recently published studies, five of eight naïve animals and four of six naïve animals controlled SIVsmE660 replication to undetectable levels (32
). However, these do not represent typical results for SIVsmE660 challenge, since only 3 of 29 Indian rhesus macaques controlled replication of SIVsmE660 to undetectable levels in many previous studies (1
). We have previously used the same stock of SIVsmE660 used in the present study to intravenously challenge 10 naïve Indian rhesus macaques expressing a variety of MHC-I alleles, including six animals that expressed the “protective” alleles, Mamu-A*01
, and Mamu-B*17
) (see Fig. S5 in the supplemental material). All control animals became infected after one intravenous challenge with 100 TCID50
of this virus stock, and the mean peak plasma viremia during the acute phase was 5.1 × 106
vRNA copy eq/ml. All 10 of the naïve control animals had >100,000 vRNA copy eq/ml at 28 weeks postchallenge (see Fig. S5 in the supplemental material). Additionally, no “protective” effects were observed for any of the three MHC-I alleles after the SIVsmE660 challenge of these naïve control animals. Furthermore, in the current study, only 1 of 12 of our rectally challenged naïve macaques has controlled SIVsmE660 replication to undetectable levels (animal r96096) (see Fig. S2A in the supplemental material). Our stock of SIVsmE660, therefore, appears to be pathogenic and remarkably consistent from animal to animal after both intravenous and rectal challenge.
Despite the robust nature of SIVsmE660, it is formally possible that, like SHIV89.6P, SIVsmE660 may represent a less-than-stringent vaccine challenge virus, yielding misleadingly encouraging results in vaccine studies (5
). Vaccines designed to induce cellular immune responses only (i.e., using Gag/Pol and not Env) have had limited success at reducing acute-phase plasma viremia of SIVsmE660 below 1 × 106
vRNA copy eq/ml (16
). Furthermore, vaccination using attenuated SIVmac239, our best current vaccine, has shown limited ability to control heterologous SIVsmE660 replication during the acute phase (1
). These previous results are commensurate with our recent experiments using SIVmac239ΔNef. We vaccinated 10 Indian rhesus macaques with SIVmac239ΔNef and subsequently challenged them intravenously with SIVsmE660 using the stock of virus employed in the current study. Of these 10 SIVmac239ΔNef-vaccinated animals, only 4 showed some measure of control during the acute phase (see Fig. S6 in the supplemental material), and all 4 of these expressed the protective allele Mamu-B*08
. Thus, the robust pathogenic stock of SIVsmE660 used in the current experiments appears to represent a rigorous challenge for vaccine studies.
Nonhuman primate challenge models, with pathogenic SIVs used for the challenge, have been criticized for being too stringent. Challenges using the pathogenic viruses SIVmac239 and SIVmac251 often result in plasma viral concentrations of greater than 500,000 vRNA copy eq/ml in the chronic phase (2
). Our stock of SIVsmE660 given intravenously resulted in plasma viremia in excess of 100,000 vRNA copy eq/ml in the chronic phase (45
). Here, we have used a novel mucosal challenge regimen with a heterologous virus swarm in order to mimic the results of typical mucosal HIV exposure resulting in infection in humans. We have succeeded in infecting macaques with 1 to 4 variants, replicating the results that have been observed in HIV-infected humans. Mean peak viral loads of 2.5 × 106
vRNA copy eq/ml and 80,000 vRNA copy eq/ml in the chronic phase were observed in our control (naïve) animals. It has been suggested that our intrarectal challenge method might also have resulted in a less-stringent challenge. However, when we compared the viral loads in this study with those used by Reynolds et al., in which SIVsmE660 from the same stock was given intravenously, there was no statistical difference between the viral loads (45
) (see Fig. S7 in the supplemental material). These values are similar to average peak and chronic phase viral loads in humans (38
). Therefore, by several key measures we successfully mimicked HIV exposure in humans.
It will be important to calibrate the ability of other vaccine regimens to control the replication of SIVsmE660 after the challenge regimen we employed here. A benchmark of particular interest will be to formally assess whether the Merck Ad5 Gag, Pol, and Nef vaccine regimen, a vaccine approach that was not efficacious in the STEP study, can reduce viral replication after the type of mucosal heterologous SIVsmE660 challenge employed in the current study. Similarly, it will be critical to investigate whether SIVmac239ΔNef can control SIVsmE660 replication after a low-dose mucosal challenge in Indian rhesus macaques that do not express MHC-I alleles associated with the control of SIVmac239. Only moderate control of viral replication was observed in SIVmac239ΔNef-vaccinated monkeys after a high-dose intravenous challenge with this same stock of SIVsmE660 (45
). Interestingly, animals expressing the MHC-I alleles Mamu-B*08
showed the best control of this heterologous challenge during acute infection. It is important to note that the majority of SIVmac239ΔNef-vaccinated monkeys have shown complete control of standard highly pathogenic homologous challenges with SIVmac239 or SIVmac251, administered either intravenously or mucosally (14
To our knowledge, this is the first application of SGA-direct amplicon sequencing (29
) to the design and interpretation of an SIV vaccine trial. We combined a titration analysis of four naïve animals in this study, with nine additional naïve animals in another study (29a
), to estimate a SIVsmE660 inoculum size that would productively infect animals with less than five viruses, recapitulating the results that characterize the majority of human mucosal HIV exposures resulting in clinical infection. We then confirmed in the present study in 10 vaccinated or control animals that the numbers of transmitted viruses leading to productive infection were indeed between one and four (Table ). There were numerically lower numbers of transmitted viruses in vaccinated compared with control animals, but the difference was not statistically significant. An unexpected finding in the present study was a striking enrichment for G-to-A hypermutation observed in some (but not all) animals productively infected by a single virus. This was observed in vaccinated, control, and titration animals. While we have previously observed G-to-A hypermutation in HIV-1-infected humans (29
) and in SIVsmE660- and SIVmac251-infected Indian rhesus macaques (29a
), the extent of G-to-A hypermutation observed in some animals in the present study is unprecedented (e.g., animals 02103, 02114, and r92093) (Fig. and see Fig. S2E in the supplemental material). With low-dose virus exposure, infection by single viruses with altered Vif function could lead to correspondingly high levels of APOBEC-mediated hypermutation, thereby affecting virus replication efficiency in naïve and vaccinated animals. Such a result might not be apparent in animals productively infected by multiple viruses if one or more of these exhibited wild-type Vif function and better overall replication fitness.
The breadth and frequency of the vaccine-induced T-cell responses achieved in the present study may have been critical for the enhanced control of replication of this heterologous, pathogenic challenge virus. No other vaccine regimens to date have achieved these frequencies or induced the breadth of T-cell responses observed in the current experiments (13
). Whether the cellular immunity elicited by the replication-defective adenovirus HIV-1 vaccine in the STEP trial had comparable breadth and appropriate specificity such that one might have expected protection against the infecting strains in the reported cases is currently being investigated by using fine T-cell epitope mapping and viral sequencing. Those results will contribute to our understanding of the underlying reasons for the lack of vaccine efficacy in the STEP trial and help inform the next steps in HIV-1 vaccine research development.