Engineering and evaluation of molecular adjuvants for DNA immunogens based on the HIV transactivator (Tat) proteins from Indian isolates

Abstract

HIV infections have reached alarming proportions worldwide. UNAIDS estimated that up to 42 million people were living with HIV/AIDS at the end of 2002. In India, NACO figures suggest that up to 4 million people are infected. Subtype-C strains of HFV-l have emerged as the dominant clade globally. With chemotherapy being expensive and the virus rapidly acquiring resistance to currently available drugs, a vaccine is being increasingly seen as a priority. HIV vaccine approaches haven't been successfiil partly owing to the overwhelming emphasis on the envelope as a candidate antigen. Mutant strains escaping the vaccine-induced immune response rapidly emerge, establish the infection and ultimately lead to the development of AIDS. Search for alternative or adjunct antigens to env has identified the structural proteins such as Gag and pol or the regulatory genes such as Tat, Rev and Nef as possible candidates. The regulatory genes have the advantage of expressing early during the viral life cycle. Additionally they are also conserved to a greater level and are less immunodominant. The viral transactivator protein. Tat, has been studied as a candidate vaccine in several formats. Although these studies have reported mixed results, the essence of these analyses appears to be that Tat could be an important component of a multivalent vaccine. We have evaluated the immunogenic potential of the Subtype-C Tat (C-Tat) protein of HIV-1, as a candidate DNA vaccine, in a mouse model. Considering that HIV-2 infections are also common in India, the Tat gene of HIV-2 has also been included in the study. Since Tat is not strongly immunogenic in a natural infection, and that the DNA vaccine as a medium of immunization is not efficient in eliciting strong immune responses, especially in larger animals, we have engineered a few molecular approaches to augment immune responses to Tat. This thesis investigated the potential of three different molecular approaches to enhance immune responses to C-Tat delivered as a DNA immunogen. a) Codon optimization of Tat genes for mammalian expression b) Use of Ubiquitin as an adjuvant to prime cellular immune responses c) Use of complement protein C3d to prime humoral immune responses Codon optimization of AT-rich genes of HIV, to match that of the host mammahan system enhanced translational efficiency and immune responses to several viral antigens. We assembled synthetic HFV-l and HIV-2 Tat genes comprising of the frequently used mammalian codons. The amino acid sequence encoded by the HIV-l Tat gene belonged to a consensus Subtype-C sequence while that of the HIV-2 Tat gene corresponded to a Subtype-A sequence obtained from India. The synthetic genes were compared with the respective wildtype counterparts at the molecular and immunological levels. Enhanced translational efficiency was noted from the synthetic Tat genes (Tatco)- Tatco complemented virus production from a Tatdeficient cell-line (HLM-1) earlier than the wild type genes. Tatco also generated sfronger immune responses than the wildtype genes. The assays used to test the immune responses were lymphoproliferation, CTL assay and ELISPOTs for the cytokines IL-4 and IFN-7. Importantly, immunization with wildtype Tat genes promoted immune responses tihed towards the Th-2 type with higher levels of IL-4 precursor T-cells than IFN-7 precursors. The cytokine profile was reversed in immunization with the codon-optimized Tat genes where a predominant Th-1 type response was observed. Pepscan analysis identified the core-domain of Tat as the immunodominant region. Cell depletion studies, selectively removing CD4 or CDS cells, revealed that the epitope is possibly of a T-helper nature. Intracellular stability of the proteins is dependent on several parameters. The nature of the amino acid located at the N-terminal end of the protein is one important factor that could critically influence protein stability. According to the 'N-end' rule, proteins with a destabilizing amino acid such as phenylalanine are rapidly targeted to the proteasomal-degradation pathway. Rapid processing of the proteins, in turn leads to enhanced levels of cellular immune responses, especially CTL responses against the antigen. To target Tat for rapid processing, we expressed the viral antigens as fusion proteins with ubiquitin. Control Tat proteins that are not expected to be subjected to rapid processing, following ubiquitin mediated cleavage of the chimera protein, were also included in the study. Biochemical analysis suggested that the half-life of the rapidly processed Tat (rp-Tat) is nearly three times smaller than that of the appropriate control protein in vivo. In a functional assay, the rp-Tat generated less reporter activity than the stable Tat (stb-Tat). Evaluation of the immune responses, however, demonstrated that in various assays, rp-Tat generated significantly stronger immune responses as compared to the stb-Tat. This observation confirmed that the molecular approach indeed targeted Tat for rapid protein processing thereby leading to elevated immune response. Pepscan analysis suggested that the immune response was primarily focussed on the core domain of the Tat protein. In a third approach, to elicit antigen specific humoral immime response. Tat was expressed as a fusion protein with multiple copies of C3d. Tat can exit cells via a leader-less pathway and a host of pathogenic properties has been ascribed to the extracellular Tat. A strong humoral immune response is believed to be protective by sequestering the extra-cellular Tat. C3d is a complement fragment that is covalently cross-linked to the antigens. C3d could cross link CR2 on the antigen presenting Bcells and enhance antigen-specific response by several fold. In our experiments. Tat expressed as a recombinant protein with 3 tandem copies of C3d and with the secretory signal sequence of insulin growth factor 1, generated the strongest humoral immune response in mice. The antisera neutralized extracellular Tat in the culture medium and abrogated Tat-mediated complementation of a defective pro virus. Future work is projected in the direction of evaluating some of these promising molecular approaches in a primate model with Tat alone or as a component of a multivalent vaccine.