Velpandi Ayyavoo

Targeted antigen delivery to antigen-presenting cells including dendritic cells by engineered Fas-mediated apoptosis

Immunity to tumors as well as to viral and bacterial pathogens is often mediated by cytotoxic T lymphocytes (CTLs). Thus, the ability to induce a strong cell-mediated immune response is an important requirement of novel immunotherapies. Antigen-presenting cells (APCs), including dendritic cells (DCs), are specialized in initiating T-cell immunity. Harnessing this innate ability of these cells to acquire and present antigens, we sought to improve antigen presentation by targeting antigens directly to DCs in vivo through apoptosis. We engineered Fas-mediated apoptotic death of antigen-bearing cells in vivo by co-expressing the immunogen and Fas in the same cell. We then observed that the death of antigen-bearing cells results in increased antigen acquisition by APCs including DCs. This in vivo strategy led to enhanced antigen-specific CTLs, and the elaboration of T helper-1 (Th1) type cytokines and chemokines. This adjuvant approach has important implications for viral and nonviral delivery strategies for vaccines or gene therapies.

Development of a multicomponent candidate vaccine for HIV-1.

Nucleic acid or DNA immunization represents a novel approach to both vaccine and immune therapeutic development. DNA vaccination induces antigen-specific cellular and humoral immune responses through the delivery of non-replicating transcription units which drive the synthesis of specific foreign proteins within the inoculated host. We have previously reported on the potential use of DNA immunization as a novel vaccine strategy for HIV-1. We found that both antigen-specific cellular and humoral immune responses could be induced in vivo with various DNA vaccine constructs against different antigenic targets within HIV-1. In order to enhance the DNA vaccines ability to elicit cell-mediated immune responses, we co-delivered plasmids encoding costimulatory molecule B7 and interleukin-12 genes with DNA vaccine for HIV-1. We observed a dramatic increase in both antigen-specific T helper cell proliferation and CTL response. Eventual development of successful vaccines for HIV-1 would likely involve targeting multiple antigenic components of the virus to direct and empower the immune system to protect the host from viral infection. We present here the utility of multicomponent DNA immunization to elicit specific humoral and cell-mediated immune responses against different antigenic targets of HIV-1 as well as the ability of this immunization strategy to achieve significant enhancements of antigen-specific cellular immune responses.

In vivo engineering of a cellular immune response by co-administration of IL-12 expression vector with a DNA immunogen

Recent studies support the importance of investigating a DNA vaccination approach for the immunologic control of HIV-1. In this regard, it may be important to specifically engineer immune responses in order to improve on first generation vaccine attempts. Especially for HIV, induction of cell-mediated immunity may be an important feature for any candidate vaccine. In an attempt to engineer in vivo the enhancement of cellular immune response and to direct Ag-dependent immune response from Th2 to Th1 type, we investigated the role of codelivery of genes for IL-12 and granulocyte-macrophage-CSF along with DNA vaccine formulations for HIV-1 Ag. We found that codelivery of IL-12 expression cassettes with DNA vaccines for HIV-1 in mice resulted in splenomegaly as well as a shift in the specific immune responses induced. The codelivery of IL-12 genes resulted in the reduction of specific Ab response, while the coinjection of granulocyte-macrophage-CSF genes resulted in the enhancement of specific Ab response. In addition, we observed a significant Ag-specific stimulation of T cells with codelivery of both cytokines. Most importantly, we observed a dramatic increase in specific CTL response from the group coimmunized with the HIV-1 DNA vaccine and IL-12 genes. This work demonstrates the power of DNA delivery in vivo for both the production of a new generation of more effective and targeted vaccines or immunotherapies as well as an analytic tool for the molecular dissection of the mechanisms of immune function.

Costimulatory Molecule Immune Enhancement in a Plasmid Vaccine Model Is Regulated in Part Through the Ig Constant-Like Domain of CD80/86

There is great interest in understanding the role of costimulatory molecules in immune activation. In both the influenza and HIV DNA immunization models, several groups have reported that coimmunization of mice with plasmids encoding immunogen and CD86, but not CD80, effectively boosts Ag-specific T cell activation. This difference in immune priming provided an opportunity to examine the functional importance of different regions of the B.7 molecules in immune activation. To examine this issue, we developed a series of chimeric CD80 and CD86 constructs as well as deletion mutants, and examined their immune activating potential in the DNA vaccine model. We demonstrate that the lack of an Ig constant-like region in the CD80 molecule is critically important to the enhanced immune activation observed. CD80 C-domain deletion mutants induce a highly inflammatory Ag-specific cellular response when administered as part of a plasmid vaccine. The data suggest that the constant-like domains, likely through intermolecular interactions, are critically important for immune regulation during costimulation and that engineered CD80/86 molecules represent more potent costimulatory molecules and may improve vaccine adjuvant efficacy.

Nucleic Acid-Based Vaccines as an Approach to Immunization Against Human Immunodeficiency Virus Type-1

The spread of HIV-1 has resulted in significant morbidity and mortality worldwide. Traditional therapeutic approaches have included chemotherapy directed at several different viral components either alone or in combination. Studies evaluating combinations of two or three different anti-retroviral agents have recently generated optimism regarding the ability to control viral replication in vivo. It is too early to tell whether this approach is capable of controlling established infection without additional measures including immunotherapy. In any event, the majority of the worldwide at-risk population will not have easy access to these expensive therapeutic regimens. If achievable, protective vaccination represents the best solution to the worldwide problem of infection with HIV-1 and any strategy designed to control the epidemic is likely to rely heavily on mass immunization campaigns (Musgrove 1993). Unfortunately, the effort to design effective vaccines for HIV-1 has encountered a number of significant obstacles. In a 1993 poll by the journal Science (Cohen 1993) 150 top AIDS researchers agreed that the most significant obstacle has been the lack of consistent correlates of protection from natural or experimental infection. It is therefore not surprising that a wide variety of therapeutic modalities have been proposed in pursuit of effective therapy for HIV-1 (Table 1).

Construction of attenuated HIV-1 accessory gene immunization cassettes

Delivery of genetic expression cassettes into animals can effectively induce both humoral and cellular immunity to the expressed gene product. Previously, we used this strategy to immunize against HIV-1 structural and enzymatic proteins in mice, non-human primates and in humans. In contrast, the use of the accessory genes including vif, vpr, vpu and nef as immunotherapeutic vaccine targets has not been well characterized. Our goal is to design an effective genetic HIV vaccine, which includes the accessory genes as part of a multi-component immunogen. In order to develop accessory genes as genetic vaccines, we have molecularly cloned and analysed the sequence variation and immunogenic potential present in these genes derived from viral isolates obtained from HIV-1 infected patients and laboratory isolates. Prototype genetic variants were selected and their ability to induce humoral and cellular immune responses was studied in animal models. We observed that attenuated accessory genes can effectively induce both humoral and cellular responses in mice and the resulting immune response is directly correlated with DNA concentrations delivered and the number of boosts. This strategy can be used generally to develop an effective, safe DNA vaccine for any pathogen.

HIV-1 viral protein R (Vpr) as a regulator of the target cell

Among the putative accessory genes of HIV-1, the 96 amino acid virion-associated Vpr gene product has been described to have several novel biological activities. These include cytoplasmic-to-nuclear translocation thus empowering HIV to infect and replicate in nondividing cells and to function to increase viral replication, particularly in monocytes. Along with these viral effects, we describe the dramatic biological changes induced by HIV-1 Vpr in the target cells of HIV infection including induction of changes in transcriptional patterns and complete inhibition of proliferation which collectively is termed differentiation. These changes occur in the absence of other viral gene products and suggest that Vpr mediates its proviral effects partially or perhaps solely through modulation of the state of the target cell rather than directly on the virus. The inhibition of proliferation in T-cell lines has been proposed by several groups to demonstrate that the inhibition of proliferation specifically arrests the cell cycle further supporting the notion that Vpr activity is directed at cellular targets. We have recently described a role for Vpr in modulating the glucocorticoid pathway, a pathway involved in the regulation of the state of the cell in cytoplasmic-to-nuclear translocation and in the modulation of host cell transcription. Importantly, certain antiglucocorticoids have been shown to modulate Vpr activity in vitro. These results demonstrate that the cell contains specific receptor(s) molecule(s) through which Vpr mediates its activity and that these molecules have implications for cell biology in general. These results collectively demonstrate that Vpr represents a unique target for anti-HIV drug development and has significance for HIV-1 disease progression.