In vivo electroporation enhances CTLA4-based DNA vaccine in both antigen presenting cell targeting and CD4 T cell-dependent ways

Abstract

This study focused on a strategy of DNA vaccine design that employed fusion of soluble cytotoxic T-lymphocyte-associated protein 4 (sCTLA4) with HIV-1 Gag p24. CTLA4 functions as a negative regulatory molecule for T cells through interaction with its native ligands CD80 and CD86 expressed on antigen presenting cells (APCs). The CTLA4-CD80/CD86 interaction causes T cells exhaustion and the blockade of this interaction may rescue the T cells functions during chronic viral infection. Vaccine strategy that targets APCs by utilizing the interaction of sCTLA4 with its ligands has been reported previously with significant enhancement in antibody responses that could protect mice from lethal viral challenge. The underlying mechanism, however, remains incompletely understood. Here, I hypothesize that in vivo electroporation (EP) could enhance the immunogenicity of sCTLA4-based DNA vaccines. I found that the in vivo EP not only enhanced antibody respond to HIV-1 antigen but also significantly enhanced cellular immune responses by eliciting higher frequencies of HIV-1 Gag-specific, broadly reactive cytotoxic CD8+ T cells. Such enhancement diminished when a mutant sCTLA4 that could not bind to CD80/CD86 was included for comparison. My findings demonstrated the importance of antigen uptake through the sCTLA4-CD80/CD86 interaction during immune activation. Similar results were also obtained when chicken Ovalbumin antigen was used as a test antigen. The immunogenicity of sCTLA4-based HIV-1 DNA vaccine was further compared with sPD1-based HIV-1 DNA vaccine as a mean to understand the underlying differences between these two strategies. PD1 is another negative regulatory signal for T cells through interaction with its native ligands PD-L1 and PD-L2 expressed on APCs. The upregulation of PD1 on T cells also indicates their functional exhaustion. By head-to-head comparison, we found that in vivo EP enhanced the immunogenicity of both sPD1- and sCTLA4-based HIV-1 DNA vaccines for inducing antigen-specific humoral and cellular immune responses. Confocal analysis of intracellular antigen trafficking indicated that only sPD1-based HIV-1 DNA vaccine engaged Rab14+ endosome that is essential for antigen cross-presentation. To confirm this finding, we conducted in vivo CD4 deletion during the entire course of vaccination. Interestingly, after CD4+ T cells were depleted, while sPD1-based HIV-1 DNA vaccine was still able to induce specific CD8+ T cells responses, sCTLA4-based DNA vaccine could not do so. Conversely, sCTLA4-based DNA vaccine still induced significantly higher levels of antigen-specific antibody responses whereas sPD1-based DNA vaccine could not do so. These results demonstrated that sPD1- and sCTLA4-based DNA vaccine strategies might engaged distinct pathways for activating adaptive immune response. In summary, my study demonstrated distinct in vivo immunogenicity profiles between sPD1- and sCTLA4-based DNA vaccine strategies through in vivo EP. While the enhanced cellular immune responses by sCTLA4-based DNA vaccine was CD4+ T cell-dependent, the humoral immune responses was elicited in a CD4+ T cell-independent way.