Latent Adeno-Associated Virus Infection Elicits Humoral but Not Cell-Mediated Immune Responses in a Nonhuman Primate Model

Adeno-associated virus type 2 (AAV) is a nonpathogenic parvovirus that generally requires coinfection with a helper virus, such as an adenovirus (Ad) or herpesvirus, to undergo productive infection (3, 4, 7). In cultured cells infected with wild-type (wt) AAV in the absence of helper virus, AAV establishes a latent infection (5, 11, 22) and often integrates site specifically into a sequence located on the q arm of human chromosome 19, termed the AAVS1 site (19, 20, 29–31, 35). AAV proviral DNA remains in this latent state until rescued by a helper virus. Studies on the mode of viral integration suggest that tandem copies of the viral DNA are inserted into the host cell chromosome in a head-to-tail orientation via the inverted terminal repeats of the virus (28). Due to its lack of pathogenicity and its ability to establish persistent infections in human cells, AAV has gained acceptance as a potential vector for human gene therapy (18). Recombinant AAV (rAAV) vectors mediate stable in vivo expression in a wide range of different tissues including the lungs (16), muscles (12, 13, 26, 40), brain (2, 24, 27, 39), spinal cord (34), retinas (14, 32, 43), and liver (36). The use of AAV vectors has not been associated with significant toxicity in animal models. In addition, two phase I trials of recombinant AAV vectors have been undertaken in patients with cystic fibrosis (15, 38). Despite the growing body of data regarding the biology of AAV latency in vitro, very few studies have examined this phenomenon in vivo. Epidemiologic studies have shown a high prevalence of AAV2 seropositivity (6, 8, 9). However, it is not known whether seropositivity is indicative of previous productive infection or of latent infection. Although infectious wt AAV has been cultured from samples from the respiratory and gastrointestinal tract in association with productive helper virus infection (9), latent AAV DNA has been found only in peripheral blood mononuclear cells (PBMCs) (21). Since humans and monkeys are the only species known to possess the AAVS1 integration sequence (35), they are the only two in vivo models which would be expected to faithfully reflect the wt AAV latency pathway. One previous study examined the interactions between rAAV vectors, wt-AAV and Ad in a rhesus macaque model and demonstrated that the productive phase of the wt AAV life cycle could be reproduced in that model (1). In this study, we have examined latent wt AAV infection in that same rhesus model. Additional studies investigated the cell specificity of persistence, the integration state of latent viral genomes, and the immune responses to viral infection. In this setting, persistence was observed, although site-specific integration was infrequent. wt AAV genomes were detectable both at the site of administration and in circulating PBMCs. Neutralizing antibodies directed against wt AAV were observed in serum 21 days postinfection in animals infected intramuscularly or intravenously with wt AAV alone, but cell-mediated immune responses were elicited only in the presence of helper Ad.