1103. Augmentation of Immunity Against AAV- Encoded Transgenes by Use of the Genetic Adjuvants C3d3 and CTLA4

Abstract

Top of pageAbstract AAV transduction enables long-term transgene expression in immuno-competent hosts because of minimal activation of the innate immune system. However, it is possible to elicit transgene-specific immunity but this is dependent upon the antigen and route of immunization. With this view, we are exploiting the unique biology of AAV as a genetic vaccine vector. The cDNA encoding MSP4 (from Plasmodium falciparum), MSP1-19 or MSP4/5 (both from Plasmodium yoelli) were fused with a TPA leader sequence cDNA (for antigen secretion) and cloned downstream of the CMV immediate/early promoter within an AAV provirus (pTRUF-2). AAV genomes were packaged into AAV1 capsids using a helper virus-free system and injected (1012 vg) into the hindlimb muscle (im) of 8|[ndash]|10 week old female Balb/C mice. Antibody responses specific for the transgene were detected against each vector although those generated against MSP1-19 were poor. No protective efficacy was afforded by vaccination with rAAV- MSP4/5 as tested by challenge with P.yoelli in a model where high titre antibodies to MSP4/5 correlates with protection. We concluded that augmentation of transgene-specific immunity is required to confer protection in this model and we have therefore investigated two genetic adjuvant strategies. The first strategy was the genetic fusion of antigen to three tandem copies of C3d, a complement protein, which has been effective at augmenting immunity in other vaccine vector systems. Unexpectedly, rather than augmenting immunity, im injection of rAAV-MSP4/5- C3d3 did not stimulate detectable antibody responses against MSP4/5. This contrasts with the positive control experiment where genetic fusion of C3d3 with hen egg lysozyme (HEL) was shown to augment anti-HEL antibody responses in the context of an rAAV vector. The second strategy tested was genetic fusion of MSP4/5 with Cytotoxic T-Lymphocyte Activation antigen-4 (rAAV-CTLA4- MSP4/5), which is thought to promote immunity by targeting antigen to antigen-presenting cells. Vectors were initially tested over the range 109|[ndash]|1012 vg/injection and responses to rAAV-CTLA4-MSP4/5 were detected in 3/5 animals given 109 vg. This is in contrast to 1010 vg as the minimal amount of rAAV-MSP4/5 vector required to stimulate detectable antibodies. A subsequent study over the range 106|[ndash]|109 vg showed detectable antibody against |[ge]|108 vg rAAV-CTLA4-MSP4/5 indicating that this strategy can augment immunity 100-fold with low dose vector. Antibody responses against rAAV- CTLA4-MSP4/5 peaked earlier than those against rAAV-MSP4/5 in all groups and immunity was not augmented with |[ldquo]|CTLA4- fused|[rdquo]| vector with |[ge]|1011vg. We conclude that it is possible to augment immunity against AAV encoded antigens using the genetic adjuvants C3d3 and CTLA4 in an antigen and adjuvant-dependent manner. These strategies may prove advantageous for the promotion of immunity against other antigens in the context of AAV, particularly where responses are initially poor, and studies are ongoing to determine whether they may provide protection in the murine model of P.yoelli infection.