High Prevalence of Infectious Adeno-associated Virus (AAV) in Human Peripheral Blood Mononuclear Cells Indicative of T Lymphocytes as Sites of AAV Persistence

Abstract

Seroepidemiology shows that infections with adeno-associated virus (AAV) are widespread, but diverse AAV serotypes isolated from humans or nonhuman primates have so far not been proven to be causes of human disease. In view of the increasing success of AAV-derived vectors in human gene therapy, definition of the in vivo sites of wild-type AAV persistence and the clinical consequences of its reactivation is becoming increasingly urgent. Here, we identify the presumed cell type for AAV persistence in the human host by highly sensitive AAV PCRs developed for the full spectrum of human AAV serotypes. In genomic-DNA samples from leukocytes of 243 healthy blood donors, 34% were found to be AAV positive, predominantly AAV type 2 (AAV2) (77%), AAV5 (19%), and additional serotypes. Roughly 11% of the blood donors had mixed AAV infections. AAV prevalence was dramatically increased in immunosuppressed patients, 76% of whom were AAV positive. Of these, at least 45% displayed mixed infections. Follow-up of single blood donors over 2 years allowed repeated detection of the initial and/or additional AAV serotypes, suggestive of fluctuating, persistent infection. Leukocyte separation revealed that AAV resided in CD3 ϩ T lymphocytes, perceived as the putative in vivo site of AAV persistence. Moreover, infectious AAVs of various serotypes could be rescued and propagated from numerous samples. The high prevalence and broad spectrum of human AAVs in leukocytes closely follow AAV seroepidemiology. Immunosuppression obviously enhances AAV replication in parallel with activation of human cytomegalovirus (HCMV) and human herpesvirus 6 (HHV-6), reminiscent of herpesvirus-induced AAV activation.IMPORTANCE Adeno-associated virus is viewed as apathogenic and replication defective, requiring coinfection with adenovirus or herpesvirus for productive infection. In vivo persistence of a defective virus requires latency in specialized cell types to escape the host immune response until viral spread becomes possible. Reactivation from latency can be induced by diverse stimuli, including infections, typically induced upon host immunosuppression. We show for the first time that infectious AAV is highly prevalent in human leukocytes, specifically T lymphocytes, and that AAV is strongly amplified upon immunosuppression, along with reactivation of latent human herpesviruses. In the absence of an animal model to study the AAV life cycle, our findings in the human host will advance the understanding of AAV latency, reactivation, and in vivo pathogenesis.

Detection of intra‐articular gene therapy in horses using quantitative real time PCR in synovial fluid and plasma

“…This could be due to normal individual variation or could reflect the presence of neutralizing antibodies (NAbs) against AAV which may have affected rAAV transduction efficiency in vivo. AAV viruses are common in human populations, where up to 70% of healthy people have been infected with one or more AAV serotypes 32–34 . The presence of neutralizing antibodies (NAbs) against AAV viruses have also been described in horses 35,36 .…”

Tetramer-Based Enrichment of Preexisting Anti-AAV8 CD8+ T Cells in Human Donors Allows the Detection of a TEMRA Subpopulation

“…The detection of whole infectious AAV particles in the bloodstream or in tissues would constitute the most accurate proof of ongoing AAV infections. However, this approach is hardly feasible considering the sensitivity of the detection techniques, and instead it is the presence of infectious AAV DNA in cells that is assessed (45)(46)(47)(48)(49)(50)(51).…”

Intrathymic adeno-associated virus gene transfer rapidly restores thymic function and long-term persistence of gene-corrected T cells

“…54,55,57 Moreover, it has recently been shown that T lymphocytes represent the in vivo site of AAV persistence after natural infection. 112 This is likely linked to the AAV vector-mediated long-term transgene maintenance (>10 months) that we detected in gene-corrected T lymphocytes. Indeed, our findings that AAV vector copy number in reconstituted T cells was approximately 1 and that long-term reconstituted T cells harbored integrated vector is likely to broaden the scope of immunologic deficiencies for which this type of approach can be envisioned.…”