Nanoplasmid Vectors Co-expressing Innate Immune Agonists Enhance DNA Vaccines for Venezuelan Equine Encephalitis Virus and Ebola Virus.

DNA vaccines expressing codon-optimized Venezuelan equine encephalitis virus (VEEV) and Ebola virus (EBOV) glycoprotein genes provide protective immunity to mice and nonhuman primates when delivered by intramuscular (IM) electroporation (EP). To achieve equivalent protective efficacy in the absence of EP, we evaluated VEEV and EBOV DNA vaccines constructed using minimalized Nanoplasmid expression vectors that are smaller than conventional plasmids used for DNA vaccination. These vectors may also be designed to co-express type I interferon inducing innate immune agonist genes that have an adjuvant effect. Nanoplasmid vaccinated mice had increased antibody responses as compared to those receiving our conventional pWRG7077-based vaccines when delivered by IM injection, and these responses were further enhanced by the inclusion of the innate immune agonist genes. The Nanoplasmid VEEV DNA vaccines also significantly increased protection against aerosol VEEV challenge as compared to the pWRG7077 VEEV DNA vaccine. Although all mice receiving the pWRG7077 and Nanoplasmid EBOV DNA vaccines at the dose tested survived EBOV challenge, only mice receiving the Nanoplasmid EBOV DNA vaccine that co-expresses the innate immune agonist genes failed to lose weight after challenge. Our results suggest that Nanoplasmid vectors can improve the immunogenicity and protective efficacy of alphavirus and filovirus DNA vaccines.

The genetic adjuvant IL-12 enhances the protective efficacy of a DNA vaccine for Venezuelan equine encephalitis virus delivered by intramuscular injection in mice.

We have previously shown that DNA vaccines expressing codon-optimized alphavirus envelope glycoprotein genes protect both mice and non-human primates from viral challenge when delivered by intramuscular electroporation (IM-EP). To determine if we could achieve equivalent immunogenicity and protective efficacy in the absence of electroporation, we co-delivered our Venezuelan equine encephalitis virus (VEEV) DNA vaccine with DNA plasmids expressing genetic adjuvants designed to augment immune responses. We tested the Th1-inducing cytokine IL-12 as well as the granulocyte growth factor GM-CSF, both of which have demonstrated significant adjuvant effect when included in clinical DNA vaccine formulations. Additionally, as multiple reports have described the necessity of IFN-αß in DNA vaccine immunogenicity, we tested vaccine plasmids encoding a potent stimulator of the IFN-αß pathway. Our data suggest that IM vaccination of mice with plasmid DNA encoding genetic adjuvants enhances VEEV vaccine immunogenicity, resulting in improved T cell responses, as well as skewing of the anti-VEEV IgG antibody isotype. Additionally, IM vaccination of VEEV DNA vaccine and IL-12 provided complete protection against aerosol VEEV challenge. Overall, our data suggest that co-delivery of genetic adjuvants with alphavirus DNA vaccines using IM delivery can influence the type of immune response obtained and provide comparable protective immunity to that achieved by IM-EP delivery of the vaccine without adjuvants.

Glycoprotein-Specific Antibodies Produced by DNA Vaccination Protect Guinea Pigs from Lethal Argentine and Venezuelan Hemorrhagic Fever.

UNLABELLED: Several members of the Arenaviridae can cause acute febrile diseases in humans, often resulting in lethality. The use of convalescent-phase human plasma is an effective treatment in humans infected with arenaviruses, particularly species found in South America. Despite this, little work has focused on developing potent and defined immunotherapeutics against arenaviruses. In the present study, we produced arenavirus neutralizing antibodies by DNA vaccination of rabbits with plasmids encoding the full-length glycoprotein precursors of Junín virus (JUNV), Machupo virus (MACV), and Guanarito virus (GTOV). Geometric mean neutralizing antibody titers, as measured by the 50% plaque reduction neutralization test (PRNT(50)), exceeded 5,000 against homologous viruses. Antisera against each targeted virus exhibited limited cross-species binding and, to a lesser extent, cross-neutralization. Anti-JUNV glycoprotein rabbit antiserum protected Hartley guinea pigs from lethal intraperitoneal infection with JUNV strain Romero when the antiserum was administered 2 days after challenge and provided some protection (∼30%) when administered 4 days after challenge. Treatment starting on day 6 did not protect animals. We further formulated an IgG antibody cocktail by combining anti-JUNV, -MACV, and -GTOV antibodies produced in DNA-vaccinated rabbits. This cocktail protected 100% of guinea pigs against JUNV and GTOV lethal disease. We then expanded on this cocktail approach by simultaneously vaccinating rabbits with a combination of plasmids encoding glycoproteins from JUNV, MACV, GTOV, and Sabia virus (SABV). Sera collected from rabbits vaccinated with the combination vaccine neutralized all four targets. These findings support the concept of using a DNA vaccine approach to generate a potent pan-arenavirus immunotherapeutic. IMPORTANCE: Arenaviruses are an important family of emerging viruses. In infected humans, convalescent-phase plasma containing neutralizing antibodies can mitigate the severity of disease caused by arenaviruses, particularly species found in South America. Because of variations in potency of the human-derived product, limited availability, and safety concerns, this treatment option has essentially been abandoned. Accordingly, despite this approach being an effective postinfection treatment option, research on novel approaches to produce potent polyclonal antibody-based therapies have been deficient. Here we show that DNA-based vaccine technology can be used to make potently neutralizing antibodies in rabbits that exclusively target the glycoproteins of several human-pathogenic arenaviruses found in South America, including JUNV, MACV, GTOV, and SABV. These antibodies protected guinea pigs from lethal disease when given post-virus challenge. We also generated a purified antibody cocktail with antibodies targeting three arenaviruses and demonstrated protective efficacy against all three targets. Our findings demonstrate that use of the DNA vaccine technology could be used to produce candidate antiarenavirus neutralizing antibody-based products.