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Hepatotropic pathogens, such as hepatitis B (HBV) and hepatitis C (HCV), often escape cellular immune clearance resulting in chronic infection. As HBV and HCV infections are the most common causes of hepatocellular carcinoma (HCC), prevention of these infections is believed to be key to the prevention of HCC. It is believed that an effective immune therapy must induce strong cytotonic T lymphocytes (CTLs) that can migrate into the liver, where they can clear infected hepatocytes. Here, we compared the induction of CD8 T cells by two different DNA immunization methods for T-cell differentiation, function, memory programming and their distribution within relevant tissues in a highly controlled fashion. We used hydrodynamic tail vein injection of plasmid to establish liver-specific LCMV-gp antigen (Ag) transient expression, and studied CD8 T cells induced using the P14 transgenic mouse model. CD8 T cells from this group exhibited unique and limited expansion, memory differentiation, polyfunctionality and cytotoxicity compared with T cells generated in intramuscularly immunized mice. This difference in liver-generated expansion resulted in lower memory CD8 T-cell frequency, leading to reduced protection against lethal viral challenge. These data show an unusual induction of naive CD8 T cells contributed to the lower frequency of Ag-specific CTLs observed after immunization in the liver, suggesting that limited priming in liver compared with peripheral tissues is responsible for this outcome. [ABSTRACT FROM AUTHOR]

Melanoma is the most deadly type of skin cancer, constituting annually ∼75% of all cutaneous cancer-related deaths due to metastatic spread. Currently, because of metastatic spread, there are no effective treatment options for late-stage metastatic melanoma patients. Studies over the past two decades have provided insight into several complex molecular mechanisms as to how these malignancies evade immunological control, indicating the importance of immune escape or suppression for tumor survival. Thus, it is essential to develop innovative cancer strategies and address immune obstacles with the goal of generating more effective immunotherapies. One important area of study is to further elucidate the role and significance of myeloid-derived suppressor cells (MDSCs) in the maintenance of the tumor microenvironment. These cells possess a remarkable ability to suppress immune responses and, as such, facilitate tumor growth. Thus, MDSCs represent an important new target for preventing tumor progression and escape from immune control. In this study, we investigated the role of MDSCs in immune suppression of T cells in an antigen-specific B16 melanoma murine system utilizing a novel synthetic tyrosinase (Tyr) DNA vaccine therapy in both prophylactic and therapeutic models. This Tyr vaccine induced a robust and broad immune response, including directing CD8 T-cell infiltration into tumor sites. The vaccine also reduced the number of MDSCs in the tumor microenvironment through the downregulation of monocyte chemoattractant protein 1, interleukin-10, CXCL5 and arginase II, factors important for MDSC expansion. This novel synthetic DNA vaccine significantly reduced the melanoma tumor burden and increased survival in vivo, due likely, in part, to the facilitation of a change in the tumor microenvironment through MDSC suppression. [ABSTRACT FROM AUTHOR]

There are well over a quarter of a billion chronic hepatitis B virus (HBV) carriers across the globe. Most carriers are at high risk for development of liver cirrhosis and subsequent progression to hepatocellular carcinoma. It is therefore imperative to develop new approaches for immunotherapy against this infection. Antibodies and cytotoxic T cells to different HBV antigens are believed to be important for reducing viral load and clearing HBV-infected cells from the liver. Some of the major challenges facing current vaccine candidates have been their inability to induce both humoral and cellular immunity to multiple antigenic targets and the induction of potent immune responses against the major genotypes of HBV. In this study, highly optimized synthetic DNA plasmids against the HBV consensus core (HBc) and surface (HBs) antigens genotypes A and C were developed and evaluated for their immune potential. These plasmids, which encode the most prevalent genotypes of the virus, were observed to individually induce binding antibodies to HBs antigens and drove robust cell-mediated immunity in animal models. Similar responses to both HBc and HBs antigens were observed when mice and non-human primates were inoculated with the HBc-HBs cocktails. In addition to the cytotoxic T lymphocyte activities exhibited by the immunized mice, the vaccine-induced responses were broadly distributed across multiple antigenic epitopes. These elements are believed to be important to develop an effective therapeutic vaccine. These data support further evaluation of multivalent synthetic plasmids as therapeutic HBV vaccines. [ABSTRACT FROM AUTHOR]

The prevalence of hepatitis B virus (HBV) infection in Asia and sub-Sahara Africa is alarming. With quarter of a billion people chronically infected worldwide and at risk of developing liver cancer, the need for a prophylactic or therapeutic vaccination approach that can effectively induce protective responses against the different genotypes of HBV is more important than ever. Such a strategy will require both the induction of a strong antigen-specific immune response and the subsequent deployment of immune response towards the liver. Here, we assessed the ability of a synthetic DNA vaccine encoding a recombinant consensus plasmid from genotype A through E of the HBV core antigen (HBcAg), to drive immunity in the liver. Intramuscular vaccination induced both strong antigen-specific T cell and high titer antibody responses systematically and in the liver. Furthermore, immunized mice showed strong cytotoxic responses that eliminate adoptively transferred HBV-coated target cells. Importantly, vaccine-induced immune responses provided protection from HBcAg plasmid-based liver transfection in a hydrodynamic liver transfection model. These data provide important insight into the generation of peripheral immune responses that are recruited to the liver-an approach that can be beneficial in the search for vaccines or immune-therapies to liver disease. [ABSTRACT FROM AUTHOR]

DNA-based vaccines, while highly immunogenic in mice, generate significantly weaker responses in primates. Therefore, current efforts are aimed at increasing their immunogenicity, which include optimizing the plasmid/gene, the vaccine formulation and method of delivery. For example, co-immunization with molecular adjuvants encoding an immunomodulatory protein has been shown to improve the antigen (Ag)-specific immune response. Thus, the incorporation of enhancing elements, such as these, may be particularly important in the influenza model in which high titered antibody (Ab) responses are critical for protection. In this regard, we compared the ability of plasmid-encoded high-mobility group box 1 protein (HMGB1), a novel cytokine in which we have previously mutated in order to increase DNA vaccine immunogenicity, with boost Ag-specific immune responses during DNA vaccination with influenza A/PR/8/34 nucleoprotein or the hemagglutinin of A novel H1N1/09. We show that the HMGB1 adjuvant is capable of enhancing adaptive effector and memory immune responses. Although Ag-specific antibodies were detected in all vaccinated animals, a greater neutralizing Ab response was associated with the HMGB1 adjuvant. Furthermore, these responses improved CD8 T+-cell effector and memory responses and provided protection against a lethal mucosal influenza A/PR/8/34 challenge. Thus, co-immunization with HMGB1 has strong in vivo adjuvant activity during the development of immunity against plasmid-encoded Ag. [ABSTRACT FROM AUTHOR]

The magnitude of the immune response to a DNA vaccine depends on three criteria-the optimized vector design, the use of a suitable adjuvant and the successful delivery and subsequent expression of the plasmid in the target tissue. In vivo electroporation (EP) has proved to be particularly effective in efficiently delivering DNA immunogens to the muscle and the skin, and indeed several devices have entered into human clinical trials. Here, we report on a novel concept of DNA delivery to the dermal tissue using a minimally invasive EP device, which is powered using low-voltage parameters. We show that this prototype device containing a novel 4 × 4-electrode array results in robust and reproducible transfection of dermal tissue and subsequent antigen expression at the injection site. Using DNA encoding for NP and M2e influenza antigens, we further show induction of potent cellular responses in a mouse model as measured by antigen-specific T-cell ELISpot assays. Importantly, 100% of the immunized animals were protected when challenged with VN/1203/04 (H5N1) strain of influenza. We have also extended our findings to a guinea-pig model and demonstrated induction of HI titers greater than 1:40 against a pandemic novel H1N1 virus showing proof of concept efficacy for DNA delivery with the prototype device in a broad spectrum of species and using multiple antigens. Finally, we were able to generate protective HI titers in macaques against the same novel H1N1 strain. Our results suggest that the minimally invasive dermal device may offer a safe, tolerable and efficient method to administer DNA vaccinations in a prophylactic setting, and thus potentially represents an important new option for improved DNA vaccine delivery in vivo. [ABSTRACT FROM AUTHOR]

A hurdle facing DNA vaccine development is the ability to generate strong immune responses systemically and at local immune sites. We report a novel systemically administered DNA vaccination strategy using intramuscular codelivery of CCL27 or CCL28, which elicited elevated peripheral IFN-γ and antigen-specific IgG while driving antigen-specific T-cell secretion of cytokine and antibody production in the gut-associated lymphoid tissue and lung. This strategy resulted in induction of long-lived antibody responses that neutralized influenza A/PR8/34 and protected mice from morbidity and mortality associated with a lethal intranasal viral challenge. This is the first example of the use of CCL27 and CCL28 chemokines as adjuvants to influence a DNA vaccine strategy, suggesting further examination of this approach for manipulation of vaccine-induced immunity impacting both quality and phenotype of responses. [ABSTRACT FROM AUTHOR]

In vivo electroporation has been used to efficiently deliver drugs and ‘therapeutic’ genes to tumors, including melanoma lesions. This study reports on the effect of intratumoral delivery of an optimized DNA plasmid expressing interleukin-15 (pIL-15) on established murine melanoma tumors. IL-15 has been demonstrated to have a pivotal role in the function of memory CD8+ T cells and natural killer cells, which are critical for tumor immunosurveillance. In this study, C57BL/6 mice were injected with B16.F10 melanoma cells and randomized into different experimental groups: untreated (P−V−E−), treated with pIL-15 (P+) or backbone plasmid (V+), with or without electroporation (E+ or E−). Treatment was performed intratumorally with 50 μg of plasmid on days 0, 4 and 7 and tumor volume/size, tumor regression and long-term survival were measured. At day 100 after initiation of treatment, the percentage of mice surviving with complete tumor regression in the P−V+E+, P+V−E−, P+V−E+ and P−V−E− treatment groups were 0, 12.5, 37.5 and 0%, respectively. These results demonstrate the ability of pIL-15 to mediate B16 melanoma regression, with the effect being significantly enhanced by electroporative delivery. This is the first description of the ability of a naked DNA plasmid expressing IL-15 to alone mediate complete regression of B16 melanoma tumors and underscores the potential clinical use of these plasmids for the treatment of malignant tumors when delivered with in vivo electroporation.Cancer Gene Therapy (2006) 13, 969–974. doi:10.1038/sj.cgt.7700973; published online 9 June 2006 [ABSTRACT FROM AUTHOR]

The destruction of CD4+ T cells and eventual induction of immunodeficiency is a hallmark of the human immunodeficiency virus type 1 infection (HIV-1). However, the mechanism of this destruction remains unresolved. Several auxiliary proteins have been proposed to play a role in this aspect of HIV pathogenesis including a 14 kDa protein named viral protein R (Vpr). Vpr has been implicated in the regulation of various cellular functions including apoptosis, cell cycle arrest, differentiation, and immune suppression. However, the mechanism(s) involved in Vpr-mediated apoptosis remains unresolved, and several proposed mechanisms for these effects are under investigation. In this review, we discuss the possibility that some of these proposed pathways might converge to modulate Vpr's behavior. Further, we also discuss caveats and future directions for investigation of the interesting biology of this HIV accessory gene.Cell Death and Differentiation (2005) 12, 962–970. doi:10.1038/sj.cdd.4401583 Published online 15 April 2005 [ABSTRACT FROM AUTHOR]