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Alphavirus replicons were evaluated as potential vaccine candidates for Venezuelan equine encephalitis virus (VEEV), western equine encephalitis virus (WEEV), or eastern equine encephalitis virus (EEEV) when given individually or in combination (V/W/E) to mice or cynomolgus macaques. Individual replicon vaccines or the combination V/W/E replicon vaccine elicited strong neutralizing antibodies in mice to their respective alphavirus. Protection from either subcutaneous or aerosol challenge with VEEV, WEEV, or EEEV was demonstrated out to 12 months after vaccination in mice. Individual replicon vaccines or the combination V/W/E replicon vaccine elicited strong neutralizing antibodies in macaques and demonstrated good protection against aerosol challenge with an epizootic VEEV-IAB virus, Trinidad donkey. Similarly, the EEEV replicon and V/W/E combination vaccine elicited neutralizing antibodies against EEEV and protected against aerosol exposure to a North American variety of EEEV. Both the WEEV replicon and combination V/W/E vaccination, however, elicited poor neutralizing antibodies to WEEV in macaques, and the protection conferred was not as strong. These results demonstrate that a combination V/W/E vaccine is possible for protection against aerosol challenge and that cross-interference between the vaccines is minimal. Importance: Three related viruses belonging to the genus Alphavirus cause severe encephalitis in humans: Venezuelan equine encephalitis virus (VEEV), western equine encephalitis virus (WEEV), and eastern equine encephalitis virus (EEEV). Normally transmitted by mosquitoes, these viruses can cause disease when inhaled, so there is concern that these viruses could be used as biological weapons. Prior reports have suggested that vaccines for these three viruses might interfere with one another. We have developed a combined vaccine for Venezuelan equine encephalitis, western equine encephalitis, and eastern equine encephalitis expressing the surface proteins of all three viruses. In this report we demonstrate in both mice and macaques that this combined vaccine is safe, generates a strong immune response, and protects against aerosol challenge with the viruses that cause Venezuelan equine encephalitis, western equine encephalitis, and eastern equine encephalitis., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

We have previously shown that a hydrophobic alkylating compound, 1,5-iodonaphthyl-azide (INA) can efficiently inactivate the virulent strain of Venezuelan equine encephalitis virus (VEEV), V3000 in vitro. In this study, we have evaluated the safety of INA-inactivated V3000 and V3526 and the protective efficacy of INA-inactivated V3000. INA-inactivated V3000 and V3526 did not cause disease in suckling mice. RNA isolated from the INA-inactivated V3000 and V3526 was also not infectious. Immunization of adult mice with INA-inactivated V3000 induced an anti-VEEV antibody response and protected mice from virulent VEEV challenge. The protective efficacy of INA-inactivated V3000 increased with the use of adjuvants. Results suggest that inactivation of enveloped viruses by INA may occur by two independent mechanisms and the INA-inactivated VEEV elicit a protective antibody response in mice., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

V3526, a genetically modified strain of Venezuelan equine encephalitis virus (VEEV), was formalin inactivated for evaluation as a next generation vaccine candidate for VEEV. In this study, we tested formalin-inactivated V3526 (fV3526) with and without adjuvant for immunogenicity and efficacy in BALB/c mice and results were compared to the existing inactivated VEEV vaccine, C84. Mice were vaccinated intramuscularly (IM) or subcutaneously (SC) with fV3526 formulations and challenged with VEEV IAB Trinidad donkey (VEEV TrD) strain by SC or aerosol exposure. Efficacy following SC or aerosol challenge was not significantly different between the fV3526 formulations or compared to C84 despite C84 being administered in more doses and higher concentration of viral protein per dose. These data support further evaluation of fV3526 formulations as a next generation VEEV vaccine., (Copyright 2010 Elsevier Ltd. All rights reserved.)

A multisystem approach was used to assess the efficiency of several methods for inactivation of Venezuelan equine encephalitis virus (VEEV) vaccine candidates. A combination of diverse assays (plaque, in vitro cytopathology and mouse neurovirulence) was used to verify virus inactivation, along with the use of a specific ELISA to measure retention of VEEV envelope glycoprotein epitopes in the development of several inactivated VEEV candidate vaccines derived from an attenuated strain of VEEV (V3526). Incubation of V3526 aliquots at temperatures in excess of 64 degrees C for periods >30 min inactivated the virus, but substantially reduced VEEV specific monoclonal antibody binding of the inactivated material. In contrast, V3526 treated either with formalin at concentrations of 0.1% or 0.5% (v/v) for 4 or 24 h, or irradiated with 50 kGy gamma radiation rendered the virus non-infectious while retaining significant levels of monoclonal antibody binding. Loss of infectivity of both the formalin inactivated (fV3526) and gamma irradiated (gV3526) preparations was confirmed via five successive blind passages on BHK-21 cells. Similarly, loss of neurovirulence for fV3526 and gV3526 was demonstrated via intracerebral inoculation of suckling BALB/c mice. Excellent protection against subcutaneous challenge with VEEV IA/B Trinidad donkey strain was demonstrated using a two dose immunization regimen with either fV3526 or gV3526. The combination of in vitro and in vivo assays provides a practical approach to optimize manufacturing process parameters for development of other inactivated viral vaccines., (2009 Elsevier B.V. All rights reserved.)

We recently developed a gamma-irradiation method to inactivate V3526, a live-attenuated Venezuelan equine encephalitis virus (VEEV) vaccine candidate. Dosage and schedule studies were conducted to evaluate the immunogenicity and efficacy of gamma-irradiated V3526 (gV3526). Subcutaneous (SC) and low dosage intramuscular (IM) administration of gV3526 were highly effective in protecting mice against a SC challenge with VEEV IA/B Trinidad Donkey strain, but not against an equivalent aerosol challenge. More robust immune responses and increased protective efficacy were noted when the IM dosage of gV3526 was increased. IM administration of gV3526 formulated with either CpG or CpG plus Alhydrogel further augmented the immune response in mice and resulted in 100% protection against aerosol challenge.

Non-human primates (NHP) are considered to be the most appropriate model for predicting how humans will respond to many infectious diseases. Due to ethical and monetary concerns associated with the use of NHP, rodent models that are as predictive of responses likely to be seen in human vaccine recipients are warranted. Using implanted telemetry devices, body temperature and activity were monitored in inbred and outbred mouse strains following administration of the live-attenuated vaccine for Venezuelan equine encephalitis virus (VEEV), V3526. Following analysis of individual mouse data, only outbred mouse strains showed changes in diurnal temperature and activity profiles following vaccination. Similar changes were observed following VEEV challenge of vaccinated outbred mice. From these studies, we conclude, outbred mouse strains implanted with telemeters are a sensitive model for predicting responses in humans following vaccination.

In an attempt to improve the current live, attenuated vaccine (TC-83) for Venezuelan equine encephalitis virus (VEEV), specific mutations associated with attenuation of VEEV in rodent models were inserted into a full-length cDNA clone of the Trinidad donkey strain of VEEV by site-directed mutagenesis. Because some viruses have been reported to be more pathogenic when introduced by mosquito bite than the same virus introduced by needle inoculation, there were concerns that the presence of mosquito saliva, or changes in the virus caused by replication in a mosquito, might allow the virus to overcome the protective effects of prior vaccination with V3526. Therefore, we determined if hamsters vaccinated with V3526 were protected from challenge with the virulent Trinidad donkey strain of VEEV. All non-vaccinated hamsters died after intraperitoneal challenge or after being fed on by VEEV-inoculated Aedes taeniorhynchus. In contrast, hamsters vaccinated with V3526 were resistant to intraperitoneal challenge and infection by VEEV-infected Ae. taeniorhynchus. Therefore, the V3526 candidate vaccine elicits protection against VEEV infection by mosquito bite.

The heightened concerns about bioterrorism and the use of biowarfare agents have prompted substantial increased efforts towards the development of vaccines against a wide range of organisms, toxins, and viruses. An increasing variety of platforms and strategies have been analyzed for their potential as vaccines against these agents. DNA vectors, live-attenuated viruses and bacteria, recombinant proteins combined with adjuvant, and viral- or bacterial-vectored vaccines have been developed as countermeasures against many potential agents of bioterrorism or biowarfare. The use of viruses, for example adenovirus, vaccinia virus, and Venezuelan equine encephalitis virus, as vaccine vectors has enabled researchers to develop effective means for countering the threat of bioterrorism and biowarfare. An overview of the different viral vectors and the threats they counter will be discussed.

Two live, attenuated strains of Venezuelan equine encephalitis virus (VEE), IE1150K and V3526, were administered to macaques to determine if they could elicit protection against an aerosol challenge with virulent VEE virus of the IE variety (VEEV-IE). These viruses were rescued from full-length cDNA clones of 68U201 (VEEV-IE variety) and Trinidad donkey (VEEV-IA/B variety), respectively, and both have a furin cleavage site deletion mutation and a second-site resuscitating mutation. Both vaccines elicited neutralizing antibodies to viruses of the homologous variety but not to viruses of the heterologous variety. Eight weeks after vaccination, the macaques were challenged by aerosol exposure to virulent 68U201. Macaques vaccinated with V3526 were protected as well as macaques inoculated with IE1009, the wild-type infectious clone of 68U201. However, IE1150K failed to significantly protect macaques relative to controls. V3526 has now been shown to protect macaques against both IA/B [Pratt WD, Davis NL, Johnston RE, Smith JF. Genetically engineered, live attenuated vaccines for Venezuelan equine encephalitis: testing in animal models. Vaccine 2003;21(25-26):3854-62] and IE strains of VEE viruses.

Specific mutations associated with attenuation of Venezuelan equine encephalitis (VEE) virus in rodent models were identified during efforts to develop an improved VEE vaccine. Analogous mutations were produced in full-length cDNA clones of the Cba 87 strain of western equine encephalitis (WEE) virus by site-directed mutagenesis in an attempt to develop an improved WEE vaccine. Isogenic viral strains with these mutations were recovered after transfection of baby hamster kidney cells with infectious RNA. We evaluated two of these strains (WE2102 and WE2130) for their ability to replicate in and be transmitted by Culex tarsalis, the principal natural vector of WEE virus in the United States. Each of the vaccine candidates contained a deletion of the PE2 furin cleavage site and a secondary mutation in the E1 or E2 glycoprotein. Both of these potential candidates replicated in mosquitoes significantly less efficiently than did either wild-type WEE (Cba 87) virus or the parental clone (WE2000). Likewise, after intrathoracic inoculation, mosquitoes transmitted the vaccine candidate strains significantly less efficiently than they transmitted either the wild-type or the parental clone. One-day-old chickens vaccinated with either of the two vaccine candidates did not become viremic when challenged with virulent WEE virus two weeks later. Mutations that result in less efficient replication in or transmission by mosquitoes should enhance vaccine safety and reduce the possibility of accidental introduction of the vaccine strain to unintentional hosts.

Chimeric cDNA clones, pMWE1000 and pMWE2000, differing by five nucleotides at their 5' termini, were constructed of the 5' two-thirds of the western equine encephalitis (WEE) virus genome (encoding nonstructural proteins) and the 3' one-third of the eastern equine encephalitis (EEE) virus genome (encoding structural proteins). The WEE virus sequences were derived from full-length cDNA clones, pWE1000 and pWE2000, which were isogenic except for five nucleotide differences at their 5' termini and were responsible for significant differences in mouse virulence. Each cDNA clone was placed downstream from a T7 promoter to allow in vitro transcription of full-length RNA. Transfection of BHK-21 cells with the chimeric RNA by electroporation gave rise to high-titer infectious virus. The in vitro characteristics of each chimera virus were determined by electrophoretic analysis of its structural proteins, plaque morphology, neutralization characteristics, replication kinetics, and rate of viral RNA synthesis. With the exception of plaque morphology, the in vitro characteristics of MWE1000 and MWE2000 were indistinguishable from the parental EEE virus. Subcutaneous inoculation of 5-week-old C57BL/6 mice with varying doses of MWE1000 or MWE2000 virus demonstrated that both chimeric viruses were significantly attenuated compared to the parental WEE virus (Cba 87) and EEE virus (PE-6). Animals infected with 10(5) PFU or more of either MWE1000 or MWE2000 were completely protected from lethal challenge with the virulent EEE virus, FL91-4679, but were not protected from virulent WEE virus Cba 87 challenge. Construction of viable virus chimeras often results in attenuated viruses that may hold promise as genetically engineered alphavirus vaccine candidates (R. J. Kuhn, D. E. Griffin, K. E. Owen, H. G. M. Niesters, and J. H. Strauss, 1996, J. Virol. 70, 7900-7909).

Rotavirus structural proteins VP4, VP6 and VP7 from Bovine Rotavirus Strain C486 were cloned and expressed in a baculovirus expression system. Combinations of the proteins were assembled into a series of virus-like particles, and a murine model was used to determine the capacity of the recombinant proteins and particles to induce protective immunity. All of the proteins induced humoral immunity as measured by an ELISA against whole virus. However, only the antisera from animals immunized with VP4 neutralized virus and inhibited haemagglutination. Challenge of neonates born to animals immunized with VP4 protein on assembled particles or in cell lysates showed protection against challenge with both homologous (bovine C486) and heterologous (SA-11) strains of rotavirus. In contrast, the offspring of mice immunized with VP6 were only partially protected. Neonates of animals immunized with virus-like particles composed of VP7 assembled on VP6 spherical particles were protected against challenge with the homotypic virus and significantly protected from a heterotypic challenge whereas unassembled VP7 protein provided only partial protection against challenge.