Sin Nombre virus (SNV) and Andes virus (ANDV) cause most of the hantavirus pulmonary syndrome (HPS) cases in North and South America, respectively. The chances of a patient surviving HPS are only two in three. Previously, we demonstrated that SNV and ANDV DNA vaccines encoding the virus envelope glycoproteins elicit high-titer neutralizing antibodies in laboratory animals, and (for ANDV) in nonhuman primates (NHPs). In those studies, the vaccines were delivered by gene gun or muscle electroporation. Here, we tested whether a combined SNV/ANDV DNA vaccine (HPS DNA vaccine) could be delivered effectively using a disposable syringe jet injection (DSJI) system (PharmaJet, Inc). PharmaJet intramuscular (IM) and intradermal (ID) needle-free devices are FDA 510(k)-cleared, simple to use, and do not require electricity or pressurized gas. First, we tested the SNV DNA vaccine delivered by PharmaJet IM or ID devices in rabbits and NHPs. Both IM and ID devices produced high-titer anti-SNV neutralizing antibody responses in rabbits and NHPs. However, the ID device required at least two vaccinations in NHP to detect neutralizing antibodies in most animals, whereas all animals vaccinated once with the IM device seroconverted. Because the IM device was more effective in NHP, the Stratis® (PharmaJet IM device) was selected for follow-up studies. We evaluated the HPS DNA vaccine delivered using Stratis® and found that it produced high-titer anti-SNV and anti-ANDV neutralizing antibodies in rabbits (n=8/group) as measured by a classic plaque reduction neutralization test and a new pseudovirion neutralization assay. We were interested in determining if the differences between DSJI delivery (e.g., high-velocity liquid penetration through tissue) and other methods of vaccine injection, such as needle/syringe, might result in a more immunogenic DNA vaccine. To accomplish this, we compared the HPS DNA vaccine delivered by DSJI versus needle/syringe in NHPs (n=8/group). We found that both the anti-SNV and anti-ANDV neutralizing antibody titers were significantly higher (p-value 0.0115) in the DSJI-vaccinated groups than the needle/syringe group. For example, the anti-SNV and anti-ANDV PRNT50 geometric mean titers (GMTs) were 1,974 and 349 in the DSJI-vaccinated group versus 87 and 42 in the needle/syringe group. These data demonstrate, for the first time, that a spring-powered DSJI device is capable of effectively delivering a DNA vaccine to NHPs. Whether this HPS DNA vaccine, or any DNA vaccine, delivered by spring-powered DSJI will elicit a strong immune response in humans, requires clinical trials.

Polyclonal immunoglobulin-based medical products have been used successfully to treat diseases caused by viruses for more than a century. We demonstrate the use of DNA vaccine technology and transchromosomal bovines (TcBs) to produce fully human polyclonal immunoglobulins (IgG) with potent antiviral neutralizing activity. Specifically, two hantavirus DNA vaccines [Andes virus (ANDV) DNA vaccine and Sin Nombre virus (SNV) DNA vaccine] were used to produce a candidate immunoglobulin product for the prevention and treatment of hantavirus pulmonary syndrome (HPS). A needle-free jet injection device was used to vaccinate TcB, and high-titer neutralizing antibodies (titers >1000) against both viruses were produced within 1 month. Plasma collected at day 10 after the fourth vaccination was used to produce purified α-HPS TcB human IgG. Treatment with 20,000 neutralizing antibody units (NAU)/kg starting 5 days after challenge with ANDV protected seven of eight animals, whereas zero of eight animals treated with the same dose of normal TcB human IgG survived. Likewise, treatment with 20,000 NAU/kg starting 5 days after challenge with SNV protected immunocompromised hamsters from lethal HPS, protecting five of eight animals. Our findings that the α-HPS TcB human IgG is capable of protecting in animal models of lethal HPS when administered after exposure provides proof of concept that this approach can be used to develop candidate next-generation polyclonal immunoglobulin-based medical products without the need for human donors, despeciation protocols, or inactivated/attenuated vaccine antigen.

Formulations of a polymeric delivery system containing a 75/25 poly(DL-lactide-co-caprolactone dissolved in either N-methyl-2-pyrrolidone or dimethyl sulfoxide were injected both subcutaneously (SC) and intramuscularly (IM) into rhesus monkeys. Each monkey received an SC and IM injection of each of the two formulations, for a total injection volume of 4 mL. The monkeys were observed daily for overt signs of toxicity, and after 4 weeks biopsies of each implant site were fixed, stained, and evaluated histologically for tissue reaction to the polymer system. Tissue response was graded upon the presence and level of fibrous connective tissue and inflammatory cell infiltrate. The polymer formulations appeared to be safe, as the animals remained healthy and active throughout the study with no changes in food or water consumption, weight loss, or abnormal behavior observed. Tissue response to both formulations was considered mild and similar to that for other biodegradable polymers, in that the reaction was limited to tissue immediately adjacent to the residual polymer fragments and consisted of a mild fibroplasia with the presence of a few lymphocytes and macrophages. There were no differences between the two formulations in tissue response, and both formulations were considered acceptable for use as injectable implant systems.

Swine influenza is a highly contagious viral infection in pigs that significantly impacts the pork industry due to weight loss and secondary infections. There is also the potential of a significant threat to public health, as was seen in 2009 when the pandemic H1N1 influenza virus strain emerged from reassortment events among avian, swine, and human influenza viruses within pigs. As classic and pandemic H1N1 strains now circulate in swine, an effective vaccine may be the best strategy to protect the pork industry and public health. Current inactivated-virus vaccines available for swine influenza protect only against viral strains closely related to the vaccine strain, and egg-based production of these vaccines is insufficient to respond to large outbreaks. DNA vaccines are a promising alternative since they can potentially induce broad-based protection with more efficient production methods. In this study we evaluated the potentials of monovalent and trivalent DNA vaccine constructs to (i) elicit both humoral and gamma interferon (IFN-␥) responses and (ii) protect pigs against viral shedding and lung disease after challenge with pandemic H1N1 or classic swine H1N1 influenza virus. We also compared the efficiency of a needle-free vaccine delivery method to that of a conventional needle/syringe injection. We report that DNA vaccination elicits robust serum antibody and cellular responses after three immunizations and confers significant protection against influenza virus challenge. Needle-free delivery elicited improved antibody responses with the same efficiency as conventional injection and should be considered for development as a practical alternative for vaccine administration.

Andes virus (ANDV) and ANDV-like viruses are responsible for most hantavirus pulmonary syndrome (HPS) cases in South America. Recent studies in Chile indicate that passive transfer of convalescent human plasma shows promise as a possible treatment for HPS. Unfortunately, availability of convalescent plasma from survivors of this lethal disease is very limited. We are interested in exploring the concept of using DNA vaccine technology to produce antiviral biologics, including polyclonal neutralizing antibodies for use in humans. Geese produce IgY and an alternatively spliced form, IgYΔFc, that can be purified at high concentrations from egg yolks. IgY lacks the properties of mammalian Fc that make antibodies produced in horses, sheep, and rabbits reactogenic in humans. Geese were vaccinated with an ANDV DNA vaccine encoding the virus envelope glycoproteins. All geese developed high-titer neutralizing antibodies after the second vaccination, and maintained high-levels of neutralizing antibodies as measured by a pseudovirion neutralization assay (PsVNA) for over 1 year. A booster vaccination resulted in extraordinarily high levels of neutralizing antibodies (i.e., PsVNA80 titers >100,000). Analysis of IgY and IgYΔFc by epitope mapping show these antibodies to be highly reactive to specific amino acid sequences of ANDV envelope glycoproteins. We examined the protective efficacy of the goose-derived antibody in the hamster model of lethal HPS. α-ANDV immune sera, or IgY/IgYΔFc purified from eggs, were passively transferred to hamsters subcutaneously starting 5 days after an IM challenge with ANDV (25 LD50). Both immune sera, and egg-derived purified IgY/IgYΔFc, protected 8 of 8 and 7 of 8 hamsters, respectively. In contrast, all hamsters receiving IgY/IgYΔFc purified from normal geese (n=8), or no-treatment (n=8), developed lethal HPS. These findings demonstrate that the DNA vaccine/goose platform can be used to produce a candidate antiviral biological product capable of preventing a lethal disease when administered post-exposure.