The holy grail for HIV vaccine development is an immunogen that elicits persisting antibodies with broad neutralizing activity against field strains of the virus. Unfortunately, very little progress has been made in finding or designing such immunogens. Using the SIV model, we have taken a markedly different approach: delivery of an adeno-associated virus (AAV) gene transfer vector to muscle for the expression of antibodies or antibody-like immunoadhesins having predetermined anti-SIV specificity. With this approach, anti-SIV molecules are endogenously synthesized in myofibers and passively distributed to the circulatory system. Using such an approach in monkeys, we have now generated long-lasting neutralizing activity in serum and observed complete protection against intravenous challenge with virulent SIV. In essence, this strategy bypasses the adaptive immune system and holds significant promise as a novel approach to an effective HIV vaccine.

Recombinant adeno-associated virus (rAAV) vectors are promising human gene transfer vectors, because they mediate long-term gene expression in vivo. The vector DNA form responsible for sustained gene expression has not been clearly defined, but it has been presumed that the vector integrates to some degree and persists in this manner. Using two independent methods, we were unable to identify rAAV integrants in mouse muscle. In the first approach, we were unable to recover host cell-vector DNA junctions from a lambda phage library generated using transduced mouse muscle DNA that contained a high vector copy number. Following this result, we devised a PCR assay based on the principle that integrated rAAV vector sequences could be amplified using primers specific for mouse interspersed repetitive sequences (B1 elements). Using this assay, we analyzed transduced mouse muscle DNA isolated from 6 to 57 weeks after injection and did not detect amplification above background levels. Based on the demonstrated sensitivity of the assay, these results suggested that >99.5% of vector DNA was not integrated. Additional analyses using a novel DNA exonuclease showed that the majority of the rAAV vector DNA in muscle persisted over time as transcriptionally active monomeric and concatameric episomes.

Infection with wild-type adeno-associated virus (AAV) is common in humans, but very little is known about the in vivo biology of AAV. On a molecular level, it has been shown in cultured cells that AAV integrates in a site-specific manner on human chromosome 19, but this has never been demonstrated directly in infected human tissues. To that end, we tested 175 tissue samples for the presence of AAV DNA, and when present, examined the specific form of the viral DNA. AAV was detected in 7 of 101 tonsil-adenoid samples and in 2 of 74 other tissue samples (spleen and lung). In these nine samples, we were unable to detect AAV integration in the AAVS1 locus using a sensitive PCR assay designed to amplify specific viral-cellular DNA junctions. Additionally, we used a second complementary assay, linear amplification-mediated-PCR (LAM-PCR) to widen our search for integration events. Analysis of individual LAM-PCR products revealed that the AAV genomes were arranged predominantly in a head-to-tail array, with deletions and extensive rearrangements in the inverted terminal repeat sequences. A single AAV-cellular junction was identified from a tonsil sample and it mapped to a highly repetitive satellite DNA element on chromosome 1. Given these data, we entertained the possibility that instead of integrated forms, AAV genomes were present as extrachromosomal forms. We used a novel amplification assay (linear rolling-circle amplification) to show that the majority of wild-type AAV DNA existed as circular double-stranded episomes in our tissues. Thus, following naturally acquired infection, AAV DNA can persist mainly as circular episomes in human tissues. These findings are consistent with the circular episomal forms of recombinant AAV vectors that have been isolated and characterized from in vivo transduced tissues.