Adenoassociated Viral Vector for Vaccine Development

Adeno-associated viruses (AAVs) are non-pathogenic, transduce muscle cells well, and provide long lasting expression from primarily episomal molecular forms (Schnepp et al. 2003). As a non-enveloped virus, AAV exhibits high physical stability. Vectors derived from AAV have emerged as highly promising ones for use in gene therapy (Carter and Samulski 2000; Monahan and Samulski 2000). All virus-encoded genes are replaced with the gene of interest by flanking between the inverted terminal repeats (ITRs). The production of AAV vector presents some difficulties, as both AAV rep and cap functions and "helper" viral functions, typically adenovirus E1, E2, and E4, and VA RNAs, must be provided in trans. Integration of AAV vector is also a potential safety concern. However, a recent report suggests that AAV vector integrates no more frequently than naked DNA (Johnson et al. 2005).

The removal of all virus-encoded genes in AAV vectors results in comparatively low intrinsic immunogenicity against the viral antigens. In contrast to the adenoviral vector, AAV vector does not induce the expression of multiple inflammatory chemokines, including RANTES, interferon-inducible protein 10 (IP-10), interleu-kin-8 (IL-8), MIP-1beta, and MIP-2, above baseline levels despite 40-fold-greater titers than the adenoviral vector and greater amounts of intracellular AAV vector genomes according to Southern and slot blot analysis (Zaiss et al. 2002). Nevertheless, recent studies have shown that AAV vectors can elicit both cellular and humoral immune responses against the transgene product, depending on a number of variables, including the nature of transgene, the promoter used to drive the transgene expression, the route and site of administration, vector dose, and host factors (Sun et al. 2002; Zaiss and Muruve 2005).

AAV vectors have been evaluated as vaccine carriers for multiple antigens. Intramuscular injection of mice with an AAV serotype 2 (AAV2) vector expressing herpes simplex virus type 2 glycoprotein B (gB) led to the generation of both gB-specific major histocompatibility complex class I-restricted cytotoxic T lymphocytes and anti-gB antibody. AAV-mediated immunization was more potent than plasmid DNA or protein in generating antibody responses (Manning et al. 1997). A single intramuscular injection of AAV vaccine vectors expressing HIV-1 env, tat, and rev genes induced strong HIV-1-specific serum IgG and fecal secretory IgA antibodies as well as MHC class I-restricted CTL activity in BALB/c mice. The titer of HIV-1-specific serum IgG remained stable for 10 months. When AAV HIV vaccine vectors were co-administered with AAV vector expressing interleukin 2 (IL2), the HIV-specific cell-mediated immunity was significantly enhanced (Xin et al. 2001). A promising study in rhesus macaques showed that a single high dose of intramuscular injection of AAV serotype 2 vaccine vector encoding SIV antigens could elicit both SIV-specific T-cells and neutralizing antibodies. Furthermore, the immunized animals were able to significantly restrict replication of a live and virulent SIV challenge (Johnson et al. 2005). These data suggest that AAV vaccine vectors induced biologically relevant immune responses and thus warrant continued development as a viable HIV-1 vaccine candidate. A phase I human study of an AAV-based vaccine vectors encoding HIV Gag, protease, and deleted reverse transcriptase genes has been completed. The AAV vaccine vectors proved to be safe but only minimally immunogenic, with a 20% positive T-cell response rate in the group receiving the highest immunization dose. Although AAV vaccine vectors were able to elicit potent B-cell response, no HIV-specific antibodies were observed (Van, L.J., Mehendale, S. Clumeck, N. Bets, E., Rockstroh, J., Johnson, P. Schmidt, C., Excler, J., Kochhar, S., and Heald, A. A phase I study to evaluate the safety and immunogenicity of a recombinant adeno-associated virus vaccine. Poster 474; presented at the 14th Conference on Retroviruses and Opportunistic Infections, Los Angeles, CA, February 25-28, 2007).

In humans, the presence of serum antibodies directed against AAV2 is very common. The pre-existing immunity to wild type AAV2 in human is predominantly humoral, with a minority of subjects demonstrating marginal lymphocyte proliferation and interleukin 10 (IL-10) secretions in response to AAV2 proteins (Chirmule et al. 1999). Therefore, efforts to modify AAV2 capsid and to develop AAV vaccine vectors based on alternative serotypes are currently ongoing in order to address the poor immunogenicity of AAV2 vaccine vectors in humans exhibiting prior immunity. Several mutational analyses of AAV2 capsid proteins have been performed to both map areas of cell receptor binding and to identify points for insertion of peptides to modify vector tropisms (Girod et al. 1999; Shi et al. 2001). These studies laid the foundation of possible modification of AAV2 capsid, which elicits neutralizing responses. In addition, six peptides that were able to block human serum neutralizing activities against AAV2 were identified by screening human sera against a peptide library (Moskalenko et al. 2000). Such information may allow the design of reverse genetic approaches to circumvent the pre-existing immunity against AAV2. AAV serotype 5 (AAV5) is different from AAV serotypes 2, 3, 4, and 6 at the nucleotide level and at the amino acid level (Bentel-Schaal et al. 1999). It has been shown that AAV5 vaccine vector encoding HIV-1 Env gp160 exhibited higher tro-pism for both mouse and human dendritic cells and elicited more potent antigen-specific cellular and humoral responses in mice after intramuscular immunization than AAV2 vaccine vector encoding the same antigen (Xin et al. 2006). Furthermore, an expanded family of AAVs from human and nonhuman primates was discovered on the basis of recovery of latent forms of the genome using PCR techniques (Gao et al. 2003; Gao et al. 2004; Gao et al. 2002). Subsequently, AAV2 inverted terminal repeat was used to generate the pseudotyped AAV2-based novel AAV vectors. Mice were passively transferred with pooled human immunoglobulin at various doses to simulate the pre-existing antivector humoral immunity in humans. After intramuscular immunization, inhibition of antigen-specific immune responses induced by AAV2 vaccine vector encoding HIV-1 Gag occurred at doses of human immunoglobulin 10- to 20-fold less than that required to inhibit antigen-specific immunoge-nicity elicited by AAV2/7 and AAV2/8 pseudotyped vaccine vectors encoding the same antigen (Lin et al. 2008). These data suggested that the vaccine vectors based on these novel AAVs might be able to overcome the pre-existing immunity in human population. Furthermore, after intramuscular immunization in mice, AAV2/8 pseudotyped vaccine vector encoding HIV-1 Gag induced robust antigen-specific cellular and humoral responses. However, no CD8+ T-cell response generated by AAV2/8 vaccine vector was effectively boosted with a simian adenoviral vaccine vector expressing the same antigen. These results were consistent with the finding that most of CD8+ effector cells were quickly contracted and yielded few central memory cells. In contrast, the B-cell response to HIV-1 Gag encoded by AAV2/8 vaccine vector was quite vibrant and easily boosted with the simian adenoviral vaccine vector expressing the same antigen (Lin et al. 2007). Compared to Ad vaccine vectors, AAV vaccine vectors can induce largely comparable antigen-specific antibody responses after intramuscular immunization. However, the antigen-specific T-cell responses induced by AAV vaccine vectors might be different from that induced by Ad vaccine vectors after intramuscular injection.

Possible mechanisms that might explain the difference in antigen-specific T-cell responses induced by AAV or Ad vaccine vectors include little activation of innate immunity, insufficient CD4+ T-cell help, or T-cell exhaustion by possible long-term expression of the antigen following immunization with AAV vaccine vector compared to Ad vaccine vector. These results need to be better understood in order to take full advantage of the platform of AAV vaccine vectors.

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