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Venezuelan equine encephalitis virus (VEEV), a mosquito-borne alphavirus, causes periodic epizootics in equines and is a recognized biological defense threat for humans. There are currently no FDA-licensed vaccines against VEEV. We developed a candidate DNA vaccine expressing the E3-E2-6K-E1 genes of VEEV (pWRG/VEE) and performed a Phase 1 clinical study to assess the vaccine's safety, reactogenicity, tolerability, and immunogenicity when administered by intramuscular (IM) or intradermal (ID) electroporation (EP) using the Ichor Medical Systems TriGrid™ Delivery System. Subjects in IM-EP groups received 0.5mg (N=8) or 2.0mg (N=9) of pWRG/VEE or a saline placebo (N=4) in a 1.0ml injection. Subjects in ID-EP groups received 0.08mg (N=8) or 0.3mg (N=8) of DNA or a saline placebo (N=4) in a 0.15ml injection. Subjects were monitored for a total period of 360 days. No vaccine- or device-related serious adverse events were reported. Based on the results of a subject questionnaire, the IM- and ID-EP procedures were both considered to be generally acceptable for prophylactic vaccine administration, with the acute tolerability of ID EP delivery judged to be greater than that of IM-EP delivery. All subjects (100%) in the high and low dose IM-EP groups developed detectable VEEV-neutralizing antibodies after two or three administrations of pWRG/VEE, respectively. VEEV-neutralizing antibody responses were detected in seven of eight subjects (87.5%) in the high dose and five of eight subjects (62.5%) in the low dose ID-EP groups after three vaccine administrations. There was a correlation between the DNA dose and the magnitude of the resulting VEEV-neutralizing antibody responses for both IM and ID EP delivery. These results indicate that pWRG/VEE delivered by either IM- or ID-EP is safe, tolerable, and immunogenic in humans at the evaluated dose levels. Clinicaltrials.gov registry number NCT01984983., (Published by Elsevier Ltd.)

We have developed a rapid, reliable, and sensitive quantitative flow cytometric assay to measure the in vitro potency and stability of DNA vaccines to be delivered either by particle-mediated epidermal delivery (PMED) or by electroporation. The method involves transfecting cells with test DNA and comparing the measured antigen expression to that generated with expression from known quantities of reference material DNA. The assay was adapted for performance under Good Laboratory Practice (GLP) guidelines and was successfully utilized to perform potency testing in support of a Phase I study for two hantavirus DNA vaccines delivered by gene gun. The results from the potency assays conducted over a 24-month period using this method proved to be highly reproducible with high signal-to-noise ratios. The assay was also adapted to assess the in vitro potency and stability of a DNA vaccine for Venezuelan equine encephalitis virus that will be delivered by electroporation. Our results indicate that this assay can be readily applied to support potency and stability testing of numerous DNA vaccines delivered by various methods, including multiagent vaccines., (Published by Elsevier Ltd.)

A study to evaluate the immunogenicity and protective efficacy of a Venezuelan equine encephalitis virus (VEEV) DNA vaccine in an aerosol model of nonhuman primate infection was performed. Cynomolgus macaques vaccinated with a plasmid expressing the 26S structural genes of VEEV subtype IAB by particle-mediated epidermal delivery (PMED) developed virus-neutralizing antibodies. No serum viremia was detected in two out of three macaques vaccinated with the VEEV DNA after aerosol challenge with homologous virus, while one displayed a low viremia on a single day postchallenge. In contrast, all three macaques vaccinated with empty vector DNA developed a high viremia that persisted for at least 3 days after challenge. In addition, macaques vaccinated with the VEEV DNA had reduced febrile reactions, lymphopenia, and clinical signs of disease postchallenge as compared to negative control macaques. Therefore, although the sample size was small in this pilot study, these results indicate that a VEEV DNA vaccine administered by PMED can at least partially protect nonhuman primates against an aerosol VEEV challenge., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

We employed directed molecular evolution to improve the cross-reactivity and immunogenicity of the Venezuelan equine encephalitis virus (VEEV) envelope glycoproteins. The DNA encoding the E1 and E2 proteins from VEEV subtypes IA/B and IE, Mucambo virus (MUCV), and eastern and western equine encephalitis viruses (EEEV and WEEV) were recombined in vitro to create libraries of chimeric genes expressing variant envelope proteins. ELISAs specific for all five parent viruses were used in high-throughput screening to identify those recombinant DNAs that demonstrated cross-reactivity to VEEV, MUCV, EEEV, and WEEV after administration as plasmid vaccines in mice. Selected variants were then used to vaccinate larger cohorts of mice and their sera were assayed by both ELISA and by plaque reduction neutralization test (PRNT). Representative variants from a library in which the E1 gene from VEEV IA/B was held constant and only the E2 genes of the five parent viruses were recombined elicited significantly increased neutralizing antibody titers to VEEV IA/B compared to the parent DNA vaccine and provided improved protection against aerosol VEEV IA/B challenge. Our results indicate that it is possible to improve the immunogenicity and protective efficacy of alphavirus DNA vaccines using directed molecular evolution.