| The stable delivery of preselected, broadly neutralizing antibodies (NAbs) genes will provide protection against pathogenic simian human immunodeficiency virus/simian immunodeficiency virus (SHIV/SIV) challenge. Using this approach, effective humoral immunity can be supplied to the host to prevent or significantly blunt viral infection.;To improve the serum antibody expression levels following muscle mediated gene transfer, we first used the single-chain variable-fragment X5 (scFv X5) which is smaller than IgG1b12 and can be expressed by a single promoter. We also utilized AAV serotype 1 that has been demonstrated to efficiently mediate gene delivery to muscle. Additionally, we identified an optimized leader peptide (SL1) that improved antibody secretion efficiency. By utilizing a self-complementary (SC) rAAV1 vector, we improved the scFv X5 expression compared to standard single stranded (SS) rAAV1 vector by 3 to 14 fold. To address whether the dose is scalable from mice to primates, we performed a dosing study and showed that dosing on a per kg basis is scalable. However, the levels of circulating scFv X5 in rhesus macaques were unable to prevent a challenge virus infection.;Due to the short half-life of scFv X5, we looked another scFv antibody 3B3 and developed novel fusion constructs based on the constant region hinge-CH2-CH3 (hCC) of IgG1 to increase half-life. These genetic fusions increased serum levels by 100-1,000 fold. rAAV8 was demonstrated to mediate 2-fold higher transgene expression and the woodchuck hepatitis post-transcriptional regulatory element (WPRE) was shown to further increase levels 4-6 fold. Two strong ubiquitous promoters (cytomegalovirus [CMV] and CMV-chicken beta-actin hybrid [CAG]) showed equal strength in expressing the transgene in muscle tissue. These improvements resulted in circulating levels of transgene exceeding 600 mug/ml in mice and up to 43.0 mug/ml in the serum of rhesus macaques. However, challenge studies using a SHIV162P3 via the intra-rectal route failed to achieve protection in vaccinated macaques. Pre-existing neutralizing antibodies to AAV capsid may have had a role in the failure to protect.;Finally, we explored two rhesus-derived scFv SIV NAbs (4L6 and 5L7) and one CD4 based inhibitor (N4) consisting of part of the rhesus CD4 sequence. These three molecules were fused with a rhesus IgG2 hCC. In total, nine rhesus monkeys were injected with rAAV1 containing 4L6, 5L7, or N4 (three animals per group). Overall, we achieved significant improvement in transgene expression by using this approach. Long-lasting high level serum expression and neutralizing activities were achieved in 8 of the 9 monkeys. More importantly, six out of nine vaccinated monkeys were completely protected against a virulent SIV challenge through the intravenous (IV) route.;As a proof-of-concept experiment, we demonstrated the feasibility of using rAAV-mediated gene transfer into muscle tissue to achieve sufficient levels of circulating neutralizing antibodies to block virus infection and lower viral loads in the infected animals. This "reverse immunization" strategy bypasses the adaptive immune system and holds significant promise as a designer approach for an effective HIV vaccine. Considering the diversity of HIV-1, the success of our approach is predicted on the availability of potent and broadly NAbs and inhibitors. The impact of humoral immunity targeting the vector and transgene was demonstrated and has the potential to adversely affect our vaccination approach. In addition, this novel strategy can be applied not only as a prophylactic HIV vaccine, but also as a therapeutic vaccine against other infectious diseases and cancers. (Abstract shortened by UMI.)... |
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