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DNA vaccination of transchromosomal bovines (TcBs) with DNA vaccines expressing the codon-optimized (co) glycoprotein (GP) genes of Ebola virus (EBOV) and Sudan virus (SUDV) produce fully human polyclonal antibodies (pAbs) that recognize both viruses and demonstrate robust neutralizing activity. Each TcB was vaccinated by intramuscular electroporation (IM-EP) a total of four times and at each administration received 10 mg of the EBOV-GPco DNA vaccine and 10 mg of the SUDV-GPco DNA vaccine at two sites on the left and right sides, respectively. After two vaccinations, robust antibody responses (titers > 1000) were detected by ELISA against whole irradiated EBOV or SUDV and recombinant EBOV-GP or SUDV-GP (rGP) antigens, with higher titers observed for the rGP antigens. Strong, virus neutralizing antibody responses (titers >1000) were detected after three vaccinations when measured by vesicular stomatitis virus-based pseudovirion neutralization assay (PsVNA). Maximal neutralizing antibody responses were identified by traditional plaque reduction neutralization tests (PRNT) after four vaccinations. Neutralizing activity of human immunoglobulins (IgG) purified from TcB plasma collected after three vaccinations and injected intraperitoneally (IP) into mice at a 100 mg/kg dose was detected in the serum by PsVNA up to 14 days after administration. Passive transfer by IP injection of the purified IgG (100 mg/kg) to groups of BALB/c mice one day after IP challenge with mouse adapted (ma) EBOV resulted in 80% protection while all mice treated with non-specific pAbs succumbed. Similarly, interferon receptor 1 knockout (IFNAR -/-) mice receiving the purified IgG (100 mg/kg) by IP injection one day after IP challenge with wild type SUDV resulted in 89% survival. These results are the first to demonstrate that filovirus GP DNA vaccines administered to TcBs by IM-EP can elicit neutralizing antibodies that provide post-exposure protection. Additionally, these data describe productio

Type I interferons (IFNs) are potent mediators of the innate immune response to viral infection. IFNs released from infected cells bind to a receptor (IFNAR) on neighboring cells, triggering signaling cascades that limit further infection. Subtle variations in amino acids can alter IFNAR binding and signaling outcomes. We used a new gene crossbreeding method to generate hybrid, type I human IFNs with enhanced antiviral activity against four dissimilar, highly pathogenic viruses. Approximately 1400 novel IFN genes were expressed in plants, and the resultant IFN proteins were screened for antiviral activity. Comparing the gene sequences of a final set of 12 potent IFNs to those of parent genes revealed strong selection pressures at numerous amino acids. Using three-dimensional models based on a recently solved experimental structure of IFN bound to IFNAR, we show that many but not all of the amino acids that were highly selected for are predicted to improve receptor binding., Published in Antiviral Research, v92 p461-469, 2011.

We employed a directed molecular evolution approach consisting of in vitro multigene DNA recombination and screening technologies in an attempt to improve the cross-reactivity and immunogenicity of Venezuelan equine encephalitis virus (VEEV) envelope glycoproteins E1 and E2. DNA encoding E1 and E2 from three VEEV subtypes (IA/B, IIIA, IE) and one strain each of eastern (PE-6) and western (CBA87) equine encephalitis viruses (EEEV and WEEV) were used to create libraries of recombined variants. Five different libraries were generated by recombining one or both parent glycoprotein genes. Several hundred of the recombined variants were used as naked plasmid DNA to vaccinate mice, and their serum samples were screened for the presence of antibodies in a virus-specific ELISA. Selected sera were further assayed for the presence of neutralizing antibodies by plaque reduction neutralization tests (PRNT). We found that approximately 10% of the recombined variants elicited greater levels of cross-reactive antibodies against all five viral antigens as compared to DNA vaccines expressing the parental genes. In addition, representative variants from a library in which only E2 genes were recombined elicited significantly increased neutralizing VEEV antibody titers as compared to the parent DNA vaccine and provided improved protection against aerosol VEEV challenge. These data demonstrate that in vitro, multigene DNA recombination and associated screening can lead to the selection of antigens that exhibit improved cross-reactivity, immunogenicity, and protective efficacy as compared to the wild-type proteins and suggest a broad utility for these technologies in optimizing vaccine antigens., Pub. in Vaccine, v27, p4152-4160, 2009. Sponsored in part by Defense Advanced Research Project Agency.