This study demonstrates that the DNA priming-rAd5 boosting immunization platform can elicit robust HIV-1-specific anti-Env antibody responses in nonhuman primates. Prior reports have described HIV-1-specific immune responses after DNA-rAd5 immunization of monkeys, but these have focused on cellular immunity. Our pilot study of four animals suggested that the antibody response generated by DNA-rAd5 immunization was larger than the response observed with sequential rAd5 immunizations. Since other studies have reported similar conclusions (5
), we did not pursue the immunization strategy with rAd5 alone for this study. Our study focused on the kinetics, potency, and breadth of the neutralizing antibody response elicited by DNA-rAd5 immunization. Using the neutralization-sensitive SF162 virus, we observed low but detectable levels of neutralizing antibodies after three DNA inoculations and a rapid increase in neutralizing antibodies within 1 week of the rAd5 boost. The vaccine-induced neutralizing antibody response persisted for 12 weeks after the rAd5 boost, which was the time of SHIV89.6P challenge.
The neutralization of heterologous primary HIV-1 isolates was tested with plasma samples from 2 weeks after the rAd5 boost. Data for all 12 animals that received Env immunogens are shown in Fig. . Immune plasma samples were tested at a dilution of 1:5 and were compared directly to the corresponding preimmune plasma samples in each assay. The bars in Fig. show means ± SEM for two to four independent neutralization assays for each plasma sample measured against each virus. Thus, the neutralization shown was reproducible.
Regarding the precision of neutralization, our single-round flow cytometric assay directly enumerates the number of HIV-1-infected CD4 T cells (14
). Since we counted 50,000 events by flow cytometry and since the infection rate in preimmune plasma is typically about 1 to 2% of the T cells in culture, this assay can readily distinguish a 50% decrease in the number of infected target cells. The fact that immune plasma demonstrated the neutralization of some viruses, but not others, strongly supports our conclusion of antibody-mediated virus neutralization. For example, HxB2/BaL immune plasma neutralized HIV-1 BaL more strongly than 89.6P immune plasma, while the reverse was true for the neutralization of HIV89.6. In addition, some viruses were not neutralized at all by immune plasma, and we included a MuLV control to test for nonspecific plasma effects on virus entry. Lastly, we purified IgG from two immune plasma samples and confirmed that the modest levels of neutralization observed (50 to 75% against HIV-1 BaL and JRCSF) were mediated by IgG. Thus, we are confident that the Env immunogens encoded by the DNA and rAd5 vectors elicited HIV-1-specific neutralizing antibodies.
Overall, the potency and breadth of neutralization elicited by our Env immunogens were limited. Even at a 1:5 dilution, immune plasma displayed ≥80% neutralization against only a few virus isolates. This is generally considered to be a low or modest level of antibody, but it may be possible that moderate levels of virus-neutralizing antibodies act in concert with anti-HIV-1 cellular responses to affect the acquisition of HIV-1 in humans or the disease course after infection (13
). Perhaps more important is the restriction in the breadth of virus neutralization. More than one-half of the viruses tested were not neutralized by any of the immune plasma samples. Our immunization strategy included a single clade B Env immunogen (either 89.6P or HxB2/BaL), and we do not know if a single Env immunogen presenting more conserved epitopes or a combination of Env immunogens would elicit a broader response. This is an active area of investigation in several laboratories.
An effective secondary antibody response might act during the early phase of viremia to limit viral spread. To assess if Env immunization primed animals for an anamnestic virus-neutralizing antibody response, we evaluated plasma neutralizing activity after a SHIV89.6P challenge. Our prior data suggested that the postchallenge neutralizing antibody response against the SHIV89.6P challenge virus was more rapid in 89.6P Env-immunized animals than in mock Env-immunized animals, but this difference was not statistically significant (12
). In this study, we extended these observations by evaluating the kinetics of the neutralizing antibody response to BaL and by assessing the breadth of the neutralizing response against a panel of eight viruses. While the mock Env-immunized animals (those that received only SIV Gag-Pol-Nef) developed neutralizing antibodies to SHIV89.6P within about 6 weeks postchallenge, these mock Env-immunized animals did not develop neutralizing antibodies to BaL (Fig. ) or the other heterologous viruses tested (Fig. ). Thus, without Env priming, the neutralizing antibody response was highly strain specific. In contrast, the HxB2/BaL and 89.6P Env-immunized animals developed anamnestic neutralizing antibody responses to several heterologous viruses (Fig. ). Since this secondary antibody response may potentially impact the early stages of viral spread, it is important that the breadth of the response seen at week 6 postchallenge was limited to those viruses that were initially neutralized by prechallenge plasma. These data suggest that the breadth of immunity elicited by the original vaccine immunogen will be the limiting factor, even during the secondary antibody response. There is an additional question of the impact of Env immunization on the long-term neutralizing antibody response, including the neutralization of autologous plasma viruses. We are currently investigating this question.
In summary, this report provides the first detailed description of the anti-HIV-1 neutralizing antibody response in nonhuman primates elicited by DNA-rAd5 immunization. In addition to eliciting potent HIV-1-specific cellular immunity, this immunization platform generates robust HIV-1-specific antibody responses that allow for quantitative measurements of HIV-1 neutralization. The HxB2/BaL and 89.6P Env immunogens tested elicited modest levels of plasma antibodies that neutralized several heterologous HIV-1 strains. Ongoing research is focusing on strategies to improve both the potency and breadth of Env immunization. Future approaches with novel Env immunogens can be tested with this immunization platform.