Mark Selby

Increased DNA Vaccine Delivery and Immunogenicity by Electroporation In Vivo

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.

Distribution of DNA Vaccines Determines Their Immunogenicity After Intramuscular Injection in Mice

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-μl 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 μl 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-μl injection was followed by electroporation in vivo. These findings indicate that distribution and cellular uptake significantly affect the immunogenicity of DNA vaccines.

Synthesis of a novel hepatitis C virus protein by ribosomal frameshift

Hepatitis C virus (HCV) is an important human pathogen that affects ∼100 million people worldwide. Its RNA genome codes for a polyprotein, which is cleaved by viral and cellular proteases to produce at least 10 mature viral protein products. We report here the discovery of a novel HCV protein synthesized by ribosomal frameshift. This protein, which we named the F protein, is synthesized from the initiation codon of the polyprotein sequence followed by ribosomal frameshift into the −2/+1 reading frame. This ribosomal frameshift requires only codons 8–14 of the core protein‐coding sequence, and the shift junction is located at or near codon 11. An F protein analog synthesized in vitro reacted with the sera of HCV patients but not with the sera of hepatitis B patients, indicating the expression of the F protein during natural HCV infection. This unexpected finding may open new avenues for the development of anti‐HCV drugs.

Characterization of Hepatitis C Virus Core-Specific Immune Responses Primed in Rhesus Macaques by a Nonclassical ISCOM Vaccine

Current therapies for the treatment of hepatitis C virus (HCV) infection are only effective in a restricted number of patients. Cellular immune responses, particularly those mediated by CD8+ CTLs, are thought to play a role in the control of infection and the response to antiviral therapies. Because the Core protein is the most conserved HCV protein among genotypes, we evaluated the ability of a Core prototype vaccine to prime cellular immune responses in rhesus macaques. Since there are serious concerns about using a genetic vaccine encoding for Core, this vaccine was a nonclassical ISCOM formulation in which the Core protein was adsorbed onto (not entrapped within) the ISCOMATRIX, resulting in ∼1-μm particulates (as opposed to 40 nm for classical ISCOM formulations). We report that this Core-ISCOM prototype vaccine primed strong CD4+ and CD8+ T cell responses. Using intracellular staining for cytokines, we show that in immunized animals 0.30–0.71 and 0.32–2.21% of the circulating CD8+ and CD4+ T cells, respectively, were specific for naturally processed HCV Core peptides. Furthermore, this vaccine elicited a Th0-type response and induced a high titer of Abs against Core and long-lived cellular immune responses. Finally, we provide evidence that Core-ISCOM could serve as an adjuvant for the HCV envelope protein E1E2. Thus, these data provide evidence that Core-ISCOM is effective at inducing cellular and humoral immune responses in nonhuman primates.

Enhancement of DNA vaccine potency by electroporation in vivo

The potential of electric current-mediated delivery technology to enhance DNA delivery and DNA vaccine potency was evaluated. Higher levels of reporter gene expression were observed in muscle cells of mice inoculated with luciferase or beta-galactosidase DNA followed by the application of electrical current, compared with DNA injected with no current. Similarly, substantially higher levels of immune responses (up to 20-fold) were demonstrated in mice vaccinated with HIV gag DNA and electric current. These enhanced responses were observed after one or two inoculations, and were maintained for at least 12 weeks. Therefore, the present studies demonstrate the utility of electroporation for enhancement of DNA vaccine potency in animals.

5' end-dependent translation initiation of hepatitis C viral RNA and the presence of putative positive and negative translational control elements within the 5' untranslated region.

Hepatitis C virus (HCV) is a distant relative of pestiviruses and flaviviruses, but it has a 5 untranslated region (UTR) with some features structurally similar to that of picornaviruses. In order to test the role of the 5 UTR in controlling the expression of the HCV polyprotein, we fused full-length or deleted versions of the 5 UTR of HCV-1 RNA to chloramphenicol acetyl transferase (CAT) mRNA to monitor CAT activity in vivo. We found: (1) the full-length 5 UTR of HCV-1 RNA is translationally inactive while 5 deletions which mimic a 5 subgenomic RNA detected in vivo are active, (2) an efficient cis-acting element which represses translation is found at the 5 terminus, (3) a putative element which enhances translation is found near the 3 terminus of the 5 UTR, (4) additional cis-acting elements including small open reading frames (ORFs) upstream from the putative enhancer element downregulate translation. We did not find evidence supporting the existence of an internal ribosome entry site in the 5 UTR of HCV-1 RNA. These data suggest that HCV may employ a distinctive translation control strategy such as the generation of subgenomic viral mRNA in infected cells. Translational control of HCV might be responsible for some of the characteristic pathobiology seen in viral infection.

Relative Potency of Cellular and Humoral Immune Responses Induced by DNA Vaccination

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.

The Hepatitis C Virus: Genetic Organization, Persistence, and Vaccine Strategies

The proteins encoded by the hepatitis C virus (HCV) positive-stranded RNA genome have been identified in mammalian cells transfected with cloned HCV cDNA. The putative nucleocapsid protein C, (20kDa) and envelope glycoproteins (El, 33kDa and E2, 70kDa) have been identified along with putative nonstructural proteins 2 (23kDa), 3 (70kDa), 4a (10kDa), 4b (27kDa), 5a (56kDa), and 5b (70kDa). These proteins are processed from a poly protein precursor through the combined action of host and viral encoded proteases. The immune response to HCV has been investigated in patients with chronic hepatitis. Essentially, all patients have circulating antibodies to the envelope glycoproteins, and HCV-specific cytotoxic lymphocytes have been isolated from many individuals. These preliminary data indicate that persistence of HCV is not simply due to the absence of an immune response. Purified recombinant E1 and E2 glycoproteins have been used to immunize seven chimpanzees. Following experimental challenge with homologous HCV-1, five animals were protected from both HCV infection and disease. Although infected, the course of disease may have been inhibited in the remaining two vaccinees. These data encourage the use of an HCV vaccine to prevent infection and the carrier state.

Mechanisms of action of DNA vaccines

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.