DNA vaccine cocktail expressing genotype A and C HBV surface and consensus core antigens generates robust cytotoxic and antibody responses in mice and Rhesus macaques.

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.

Inducing humoral and cellular responses to multiple sporozoite and liver-stage malaria antigens using exogenous plasmid DNA.

A vaccine candidate that elicits humoral and cellular responses to multiple sporozoite and liver-stage antigens may be able to confer protection against Plasmodium falciparum malaria; however, a technology for formulating and delivering such a vaccine has remained elusive. Here, we report the preclinical assessment of an optimized DNA vaccine approach that targets four P. falciparum antigens: circumsporozoite protein (CSP), liver stage antigen 1 (LSA1), thrombospondin-related anonymous protein (TRAP), and cell-traversal protein for ookinetes and sporozoites (CelTOS). Synthetic DNA sequences were designed for each antigen with modifications to improve expression and were delivered using in vivo electroporation (EP). Immunogenicity was evaluated in mice and nonhuman primates (NHPs) and assessed by enzyme-linked immunosorbent assay (ELISA), gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assay, and flow cytometry. In mice, DNA with EP delivery induced antigen-specific IFN-γ production, as measured by ELISpot assay and IgG seroconversion against all antigens. Sustained production of IFN-γ, interleukin-2, and tumor necrosis factor alpha was elicited in both the CD4(+) and CD8(+) T cell compartments. Furthermore, hepatic CD8(+) lymphocytes produced LSA1-specific IFN-γ. The immune responses conferred to mice by this approach translated to the NHP model, which showed cellular responses by ELISpot assay and intracellular cytokine staining. Notably, antigen-specific CD8(+) granzyme B(+) T cells were observed in NHPs. Collectively, the data demonstrate that delivery of gene sequences by DNA/EP encoding malaria parasite antigens is immunogenic in animal models and can harness both the humoral and cellular arms of the immune system.

Molecular adjuvant HMGB1 enhances anti-influenza immunity during DNA vaccination.

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.

Prototype development and preclinical immunogenicity analysis of a novel minimally invasive electroporation device.

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.

Effect of sialidase on blood group specificity of hog submaxillary glycoproteins.

Hogs can be divided into three phenotypes A, H and In, depending on the ability of extracts of their submaxillary glands to inhibit the agglutination of human A cells with human anti-A, and human O cells with eel-anti-H serum. Evidence is here provided that the inactive glycoprotein, In, represents H active glycoprotein containing additional N-glycolyl neuraminic acid, which stereospecifically masks the H determinant, alpha (1 --> 2)-L-fucosyl residue.