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The discovery that genes can be functionally transferred from bacteria to mammalian cells has suggested the possible use of bacterial vectors as gene delivery vehicles for vaccines. Attenuated invasive human intestinal bacteria, such as Salmonella and Shigella , have been used as plasmid DNA vaccine carriers and their potency has been evaluated in several animal models. This delivery system allows the administration of DNA vaccines together with associated bacterial immunostimulators directly to professional antigen presenting cells via human mucosal surfaces. Various strategies have been taken to improve the use of this delivery system to achieve robust immune responses at both mucosal and systemic sites of the immunized animals. [ABSTRACT FROM AUTHOR]

An abstract of the conference paper "Reinventing the nucleic acid vaccine with self-amplifying RNA" by A.J. Geall and colleagues is presented.

The effectiveness of cationic microparticles with adsorbed DNA at inducing immune responses was investigated in mice, guinea pigs, and rhesus macaques. Plasmid DNA vaccines encoding human immunodeficiency virus (HIV) Gag and Env adsorbed onto the surface of cationic poly(lactide-coglycolide) (PLG) microparticles were shown to be substantially more potent than corresponding naked DNA vaccines. In mice immunized with HIV gag DNA, adsorption onto PLG increased CD8(+) T-cell and antibody responses by approximately 100- and approximately 1,000-fold, respectively. In guinea pigs immunized with HIV env DNA adsorbed onto PLG, antibody responses showed a more rapid onset and achieved markedly higher enzyme-linked immunosorbent assay and neutralizing titers than in animals immunized with naked DNA. Further enhancement of antibody responses was observed in animals vaccinated with PLG/DNA microparticles formulated with aluminum phosphate. The magnitude of anti-Env antibody responses induced by PLG/DNA particles was equivalent to that induced by recombinant gp120 protein formulated with a strong adjuvant, MF-59. In guinea pigs immunized with a combination vaccine containing HIV env and HIV gag DNA plasmids on PLG microparticles, substantially superior antibody responses were induced against both components, as measured by onset, duration, and titer. Furthermore, PLG formulation overcame an apparent hyporesponsiveness of the env DNA component in the combination vaccine. Finally, preliminary data in rhesus macaques demonstrated a substantial enhancement of immune responses afforded by PLG/DNA. Therefore, formulation of DNA vaccines by adsorption onto PLG microparticles is a powerful means of increasing vaccine potency.

DNA vaccines have been extensively studied in the past ten years and much is now known about their effectiveness and mode of action in animal models. Several DNA vaccines have been tested in phase I clinical trials, with mixed results. That is, DNA vaccines appear safe and well tolerated, but lack potency. This has led to the search for technologies that will enable sufficient potency for effectiveness in humans.

DNA vaccines have been the subject of intense investigation for the past 10 y, during which time several tuberculosis (TB) DNA vaccines have been shown to confer protective immunity in animal models. So far, proof of principle for priming of immune responses by a naked DNA vaccine (malaria) has been demonstrated in humans, but potency remains a significant limitation. However, new DNA vaccine formulations and delivery systems are being developed with markedly improved potency in animal models. Therefore, there is a clear path to the human clinical testing of TB DNA vaccines.

There is reasonable evidence that both cross-priming and direct transfection of antigen-presenting cells (APCs) play a role in induction of immune responses by DNA vaccines. It is not known which mode is more important for priming cytotoxic T cell responses, but both are sufficient and neither alone is necessary. Hence, a rational strategy for increasing DNA vaccine potency would be to facilitate both pathways. With regard to cross-priming, a better understanding of the nature of the antigen transferred and the molecules/cells involved may suggest ways to design DNA vaccines to enhance this pathway. With respect to transfection of APCs, certain DNA formulations or delivery systems may be able to target APCs for increased DNA uptake. Other considerations include recruitment of APCs to the site of DNA injection and manipulation of these cells to ensure the proper activation state for priming immune responses. The burgeoning scientific literature in these areas indicates that much effort is currently being directed toward these goals.

Intramuscular injection of DNA vaccines elicits potent humoral and cellular immune responses in mice. However, DNA vaccines are less efficient in larger animal models and humans. To gain a better understanding of the factors limiting the efficacy of DNA vaccines, we used fluorescence-labeled plasmid DNA in mice to 1) define the macroscopic and microscopic distribution of DNA after injection into the tibialis anterior muscle, 2) characterize cellular uptake and expression of DNA in muscle and draining lymph nodes, and 3) determine the effect of modifying DNA distribution and cellular uptake by volume changes or electroporation on the magnitude of the immune response. Injection of a standard 50-microl dose resulted in the rapid dispersion of labeled DNA throughout the muscle. DNA was internalized within 5 min by muscle cells near the injection site and over several hours by cells that were located along muscle fibers and in the draining lymph nodes. Histochemical staining and analysis of mRNA expression in isolated cells by RT-PCR showed that the transgene was detectably expressed only by muscle cells, despite substantial DNA uptake by non-muscle cells. Reduction of the injection volume to 5 microl resulted in substantially less uptake and expression of DNA by muscle cells, and correspondingly lower immune responses against the transgene product. However, expression and immunogenicity were restored when the 5-microl injection was followed by electroporation in vivo. These findings indicate that distribution and cellular uptake significantly affect the immunogenicity of DNA vaccines.

Along with the elucidation of the role of cytotoxic T lymphocytes in the immune responses against a number of pathogens and cancer, and with the increased understanding of the cellular processing mechanisms of antigens for generation of these cells, has come an increased focus on vaccines that can generate cellular immunity along with antibodies. Promising approaches based on the delivery of genes, either as plasmid DNA or by viral vectors, have been extensively evaluated pre-clinically and in early-phase clinical trials. Although the first generation of DNA plasmid vaccines were broadly effective in animal disease models, early clinical immunogenicity pointed towards the need for increased potency. This manuscript reviews recent developments for gene-based vaccines, specifically, new approaches for formulating and delivering plasmid DNA and alphaviral replicon vectors, all of which have resulted in increased potency of gene-based vaccines.

The immunology of hepatitis C virus (HCV) infection should be studied in the context of HCV antigen expression in the liver, because HCV primarily infects this organ. Indeed, the nature, function, and fate of T cells primed after antigen expression in the liver might differ from those primed when antigens are expressed systemically or in other organs, because the nature of the antigen-presenting cells (APCs) involved may be different. In addition, the normal liver contains a resident population of lymphocytes that differ from those present at other sites. Thus, we investigated whether HCV-specific CD8(+) cytotoxic T cells (CTLs) could be elicited following portal vein (PV) injection of plasmid DNA in mice whose hepatic veins were transiently occluded. We show that PV injection of mice with "naked" DNA expressing the HCV-NS5a protein, under the control of a liver-specific enhancer/promoter, resulted in NS5a expression in the liver and the priming of HCV-specific CTLs. These results suggested that such a model might be relevant to the study of HCV-specific immune responses primed during natural infection.

DNA vaccines have been demonstrated to be potent in small animals but are less effective in primates. One limiting factor may be inefficient uptake of DNA by cells in situ. In this study, we evaluated whether cellular uptake of DNA was a significant barrier to efficient transfection in vivo and subsequent induction of immune responses. For this purpose, we used the technique of electroporation to facilitate DNA delivery in vivo. This technology was shown to substantially increase delivery of DNA to cells, resulting in increased expression and elevated immune responses. The potency of a weakly immunogenic hepatitis B surface Ag DNA vaccine was increased in mice, as seen by a more rapid onset and higher magnitude of anti-hepatitis B Abs. In addition, the immunogenicity of a potent HIV gag DNA vaccine was increased in mice, as seen by higher Ab titers, a substantial reduction in the dose of DNA required to induce an Ab response, and an increase in CD8+ T cell responses. Finally, Ab responses were enhanced by electroporation against both components of a combination HIV gag and env DNA vaccine in guinea pigs and rabbits. Therefore, cellular uptake of DNA is a significant barrier to transfection in vivo, and electroporation appears able to overcome this barrier.

Priming of strong cellular immune responses to hepatitis C (HCV) is thought to be important for eradication of infection. Although productive infection of HCV occurs only reproducibly in humans and chimpanzees, definition of HCV-specific T cell epitopes in mice is necessary to screen efficiently HCV vaccine strategies for their ability to prime cellular immune responses. Out of seven strains of mice screened for immunodominant CTL epitopes against HCV-1a Core, E2, NS5a and NS5b, only one epitope (p214K9) in only one mouse strain was identified. Enumeration of p214K9-specific CD8+ cells by flow cytometry revealed that the number of epitope specific CTL primed by 'naked' DNA immunization was lower than that reported during viral infection. The p214K9 epitope described here, combined with analysis of CTL responses by flow cytometry, should be instrumental in ranking various HCV vaccine strategies for their ability to prime CTL responses.

DNA vaccines induce protective humoral and cell-mediated immune responses in several animal models. When compared with conventional vaccines, however, DNA vaccines often induce lower antibody titers. We have now found that formulation of a DNA vaccine encoding hepatitis B surface antigen with calcium- or aluminum phosphate adjuvants can increase antibody titers by 10-100-fold and decrease the immunogenic dose of DNA by 10-fold. Furthermore, boosting an HBs protein-primed response with the adjuvanted DNA vaccine resulted in a dramatic increase in the HBs-specific IgG2a response reflecting a shift towards a TH1 response. The mechanism by which aluminum phosphate exerts its adjuvant effect is not through increased expression of HBsAg in vivo; rather, the adjuvant appears to increase the number and affinity of HBs peptide antigen-specific IFN-gamma and IL-2 secreting T cells.

DNA vaccines can induce cytotoxic T lymphocyte (CTL) responses in various species including mice, non-human primates and humans. It is now well established that antigen presenting cells (APCs) are required for induction of these responses. However, it is not yet known whether this is a function of antigen expression within or acquisition of antigen by these cells, or a combination of both. Cross-priming has been demonstrated to occur from cells (including muscle cells) to APCs in vivo. In addition, there is evidence that APCs can be transfected after DNA vaccination. Hence, efforts to facilitate cross-priming and to increase transfection of APCs will be important for increasing the potency of DNA vaccines.

DNA vaccines can prime broad-based immune responses in small animal models. In the present study, we sought to evaluate the relative ability of DNA vaccines to induce humoral and cellular immune responses. Using a DNA vaccine encoding HIV gag in mice, we observed that CD8+ T cell responses were primed more readily than were antibody responses, particularly at low doses of DNA. These CD8+ T cell responses were detected in spleen cells, as well as at local sites such as the lung and draining lymph nodes. The potency of the HIV gag DNA vaccine used was sufficient to prime strong CTL responses in macaques, but only low to undetectable antibody responses. Therefore, DNA vaccines appear able to prime strong, broad CTL but only modest antibody responses. These results may have implications on the development of vaccines against infectious diseases where both CTL and antibody responses are desired, such as HIV., (Copyright 2001 S. Karger AG, Basel.)

The immunogenicity and protective efficacy of DNA vaccines have been amply demonstrated in numerous animal models of infectious disease. However, the feasibility of DNA vaccines for human use is not yet known. In order to investigate potential means of increasing the potency of DNA vaccines, conventional adjuvants such as aluminum salts were tested. Coadministration of these adjuvants with DNA vaccines substantially enhanced the ability of these vaccines to induce antibody responses up to 100-fold in mice and guinea pigs, and 5-10-fold in non-human primates. Effective formulations had no demonstrable effect on the levels of antigen expression in situ and consisted of adjuvants that did not form complexes with the plasmid DNA; rather they exerted their effects on antigen after expression in situ. Therefore, the potency of DNA vaccines both in laboratory rodents and in non-human primates can be substantially increased by simple formulation with conventional aluminum adjuvants.

Intramuscular injection of BALB/c mice with a DNA plasmid encoding nucleoprotein (NP) from influenza virus A/PR/8/34 (H1N1) provides cross-strain protection against lethal challenge with influenza virus A/HK/68 (H3N2). CTL specific for the H-2Kd-restricted epitope NP147-155 are present in these mice and are thought to play a role in the protection. To assess the effectiveness of NP DNA immunization in comparison with influenza virus infection in the induction of CTL responses, we monitored the frequency of CTL precursors (CTLp) in mice following i.m. injection with NP DNA or intranasal infection with influenza virus and showed that the CTLp frequency in NP DNA-immunized mice can reach levels found in mice that had been infected with influenza virus. We also measured the CTLp frequency, anti-NP Ab titers, and T cell proliferative responses in mice that were injected with titrated dosages of NP DNA and documented a correlation of the CTLp frequency and the Ab titers, but not proliferative responses, with the injection dose. Furthermore, we observed a positive correlation between the frequency of NP147-155 epitope-specific CTLp and the extent of protective immunity against cross-strain influenza challenge induced by NP DNA injection. Collectively, these results and our early observations from adoptive transfer experiments of in vitro activated lymphocytes from NP DNA-immunized mice suggest a protective function of NP-specific CTLp in mice against cross-strain influenza virus challenge.

DNA plasmids encoding foreign proteins may be used as immunogens by direct intramuscular injection alone, or with various adjuvants and excipients, or by delivery of DNA-coated gold particles to the epidermis through biolistic immunization. Antibody, helper T lymphocyte, and cytotoxic T lymphocyte (CTL) responses have been induced in laboratory and domesticated animals by these methods. In a number of animal models, immune responses induced by DNA vaccination have been shown to be protective against challenge with various infectious agents. Immunization by injection of plasmids encoding foreign proteins has been used successfully as a research tool. This review summarizes the types of DNA vaccine vectors in common use, the immune responses and protective responses that have been obtained in animal models, the safety considerations pertinent to the evaluation of DNA vaccines in humans and the very limited information that is available from early clinical studies.

Objective: To determine the efficacy of Ag85A-DNA against challenge with a highly virulent human clinical isolate of Mycobacterium tuberculosis (CSU37) and to compare the potencies of two types of Ag85A-DNA vaccines; those expressing secreted and non-secreted forms of the protein., Design: Ag85A-DNA vaccinated mice were challenged with a highly virulent clinical isolate of M. tuberculosis (CSU37) in order to compare the efficacy of these vaccines. In vitro studies were also performed., Results: Enhanced humoral and cellular responses were induced in mice vaccinated with the secreted Ag85A-DNA compared to the non-secreted Ag85A-DNA. In addition, secreted Ag85A-DNA conferred protective immunity against infection with M. tuberculosis (CSU37)., Conclusions: DNA vaccines encoding M. tuberculosis Ag85A have been shown to induce potent humoral and cellular immune responses leading to protection from M. tuberculosis (Erdman) challenge in mouse models. In this study we demonstrate that Ag85A can confer protection in a rigorous challenge model using a highly virulent human clinical isolate of M. tuberculosis (CSU37). This challenge model appears able to discriminate between DNA vaccines of differing potencies, as the more immunogenic DNA construct encoding a secreted form of Ag85A was protective, whereas the less immunogenic DNA construct encoding a non-secreted form of Ag85A was not.

The field of DNA vaccines can trace its inception to two papers which demonstrated that administration of plasmid DNA vectors expressing proteins resulted in expression in situ. Thereafter, the possible application of this technique to vaccine development was demonstrated through the induction of antibody responses in mice against a foreign protein, cellular immune responses against a viral antigen and protective efficacy in an infectious disease challenge model. Subsequently, the general utility of DNA vaccines in animal models of infectious and non-infectious disease has been established (for review, see [5]). Initially, most efforts were directed toward demonstration of effectiveness in particular disease models. Recently, however, more attention has been paid to gaining a better understanding of some of the underlying mechanisms of DNA vaccines. This review will focus on this new information and discuss it in the context of how it could benefit the development of more effective DNA vaccines.