In the experiments described herein, the adaptive immune system was effectively bypassed by the use of gene transfer technology to generate long-lived, protective anti-SIV biologic activity in the sera of otherwise naïve monkeys. Six of nine immunized monkeys were protected against infection by the SIV challenge, and all nine were protected from AIDS, whereas all six of the controls became infected and two-thirds (four of six) died over the course of the experiment.
Much of the success in these experiments can be attributed to AAV gene transfer technology. AAV vectors have an established record of high efficiency gene transfer in a variety of model systems 32,33
. When delivered to post-mitotic organs like muscle, brain, or liver, AAV vector genomes take on the form of intranuclear high molecular weight episomal concatamers that direct transgene expression for extended periods of time 34-39
. Our previous work demonstrated the feasibility of this approach in mice for in vivo
HIV neutralization 20
, and other investigators have adapted AAV (and other vector systems) for antibody gene delivery for a variety of purposes 40-50
. In this experiment, we showed that in monkeys, a single intramuscular injection of an AAV vector could direct long-term (> 1 year) continuous expression of a biologically active protein. In addition, serum immunoadhesin levels achieved using the self-complementary (double-stranded DNA) AAV vectors (4L6 and 5L7) were far superior to those observed with the traditional single-stranded DNA vector (N4).
Three important caveats emerged from our data. First, transgene immunogenicity appeared to be an important correlate of protection that must be better understood. We tested three different immunoadhesins and observed anti-immunoadhesin responses that ran the gamut from undetectable (4L6) to almost completely incapacitating (5L7). It is tempting to speculate, but remains unproven, whether immunoadhesins expressed by in vivo
transduction might behave like exogenously administered recombinant DNA-derived proteins (e.g., monoclonal antibodies or chimeric molecules like etanercept) whose safety profiles can be readily established. Moreover, it should be possible to reduce the potential for immunogenicity by specific residue modifications 51,52
. Notably, none of the three animals that displayed anti-transgene antibody responses have shown clinical signs or symptoms of disease.
A second caveat was that traditional in vitro
neutralization assays did not appear to faithfully represent in vivo
activity. While animals without measurable neutralizing activity on the day of challenge were all infected, the opposite was not true; the presence of in vitro
neutralizing activity, even at high titer, did not guarantee protection. Animal 05C053 exhibited an anti-immunoadhesin antibody response that was directed at the Fc domain of the chimeric protein that did not inhibit in vitro
neutralizing activity. In fact, this animal had the highest neutralizing titer of all the immunized animals on the day of challenge (). These data suggested that immunoglobulin effector functions not measured in standard in vitro
assays (like Fc activity) might be very important for in vivo
neutralizing activity 53
. Interestingly, our data appear to confirm the recent work of Hessell et al., where the Fc effector function of a neutralizing monoclonal antibody was inactivated by mutation and the in vivo
protective effect of the antibody was blunted54
. These observations suggest that optimizing antibody functions, over and above antigen binding, might be of benefit in future iterations of antiviral transgenes.
A final caveat was that we used the intravenous route for the SIV challenge. Since most new HIV infections occur across a mucosal surface, it will be important to show protection against a mucosal challenge. However, there is cause for optimism. Several studies have shown that traditional systemic passive immunization with antibody preparations can protect against an SIV or SHIV mucosal challenge 16-18
. Moreover, there is ample evidence to suggest that systemic immunization with a non-replicating immunogen can protect from natural infection at a mucosal surface. A recent example is the vaccine against human papillomavirus, which is a non-replicating virus-like particle that when given intramuscularly elicits antibodies that protect against infection of the female genital tract 55. Importantly, the animals immunized by gene transfer maintained high levels of circulating antibodies that likely permeated most body tissues and mucosal surfaces.
Although significant hurdles remain, the HIV immunization approach outlined here appears to be a viable alternative to more traditional strategies. To ultimately succeed, more and better neutralizing monoclonal antibodies against primary HIV isolates that can be tested in gene transfer experiments will be needed. Also, more non-antibody inhibitors like CD4 and its derivatives should be developed in anticipation of combinatorial approaches that target multiple steps along the HIV entry pathway. And finally, as we have shown here, the SIV-monkey model can be used to investigate a variety of variations on this theme.