Recombinant Sindbis/Venezuelan Equine Encephalitis Virus Is Highly Attenuated and Immunogenic

Abstract

Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic virus. VEEV was a significant human and equine pathogen for much of the past century, and recent outbreaks in Venezuela and Colombia (1995), with about 100,000 human cases, indicate that this virus still poses a serious public health threat. The live attenuated TC-83 vaccine strain of VEEV was developed in the 1960s using a traditional approach of serial passaging in tissue culture of the virulent Trinidad donkey (TrD) strain. This vaccine presents several problems, including adverse, sometimes severe reactions in many human vaccinees. The TC-83 strain also retains residual murine virulence and is lethal for suckling mice after intracerebral (i.c.) or subcutaneous (s.c.) inoculation. To overcome these negative effects, we developed a recombinant, chimeric Sindbis/VEE virus (SIN-83) that is more highly attenuated. The genome of this virus encoded the replicative enzymes and the cis-acting RNA elements derived from Sindbis virus (SINV), one of the least humanpathogenic alphaviruses. The structural proteins were derived from VEEV TC-83. The SIN-83 virus, which contained an additional adaptive mutation in the nsP2 gene, replicated efficiently in common cell lines and did not cause detectable disease in adult or suckling mice after either i.c. or s.c. inoculation. However, SIN-83-vaccinated mice were efficiently protected against challenge with pathogenic strains of VEEV. Our findings suggest that the use of the SINV genome as a vector for expression of structural proteins derived from more pathogenic, encephalitic alphaviruses is a promising strategy for alphavirus vaccine development.Venezuelan equine encephalitis virus (VEEV) is a member of the Alphavirus genus in the Togaviridae family. VEEV is an enveloped virus with a nonsegmented, positive-sense RNA genome of approximately 11.5 kb. The 5Ј two-thirds of the genome encodes four nonstructural proteins (nsP1 to nsP4) that form an enzyme complex required for viral replication (45). After viral RNA entry into the cytoplasm, a nonstructural polyprotein is translated directly from the viral genome and utilized in the production of a full-length, negative-sense replicative RNA intermediate. This RNA is then used as a template for synthesis of positive-sense genomic RNA and for transcription of a subgenomic 26S RNA. The ca. 4-kb subgenomic RNA corresponds to the 3Ј one-third of the viral genome and is translated into a structural polyprotein that is proteolytically cleaved into the capsid and envelope glycoproteins E2 and E1 (39). Two hundred forty copies of the capsid protein combine with the genomic viral RNA to form an icosahedral nucleocapsid. Finally, the nucleocapsid buds from the plasma membrane to acquire a lipid envelope with embedded protein spikes containing E1-E2 heterodimers (42,45).VEEV was a significant human and equine pathogen for much of the past century, and recent epidemics (40, 50) indicate that VEEV still represents a serious public health threat. Furthermore, ...

Chimeric Sindbis/eastern equine encephalitis vaccine candidates are highly attenuated and immunogenic in mice

“…High yields of both chimeric viruses were produced in both cell types, similar to those of the parental SINV (Fig. 1); unlike the SIN/VEEV chimeras described previously [19,20], SIN/EEEV derived from electroporated cells produced large plaques on BHK and Vero cells and did not require further passages to replicate efficiently. The SIN/ NAEEEV chimeric virus replicated to slightly higher titers than SIN/SAEEEV in Vero cells, with peak titers about 10-fold higher (Fig.…”

“…The chimera including the NA EEEV structural protein genes was designated SIN/ NAEEEV, while that containing the SA EEEV structural protein genes was designated SIN/ SAEEEV. Specific activities of the transcribed RNAs determined by infectious center assays were similar to those of other alphavirus chimeric clones (data not shown) [19]. Parental and chimeric viruses recovered from BHK cells after RNA electroporation were tested for replication in Vero (an approved mammalian vaccine production substrate) and C7/10 mosquito cells at a MOI of 1.…”

“…The rationale for the chimeric approach was based on the previously described SIN/VEEV strains that are highly attenuated and immunogenic for mice [19,20] as well as a chimeric Western equine encephalitis/EEEV vaccine candidate [22]. Several chimeric flavivirus vaccines have a similar combination of structural proteins of the vaccine target and the nonstructural protein genes of the 17D attenuated Yellow fever vaccine strain [31].…”

“…The overall design of chimeric SIN/EEEV cDNA infectious clones was essentially the same as that described previously for SIN/Venezuelan equine encephalitis virus (VEEV) chimeras [19,20]. The chimera including the NA EEEV structural protein genes was designated SIN/ NAEEEV, while that containing the SA EEEV structural protein genes was designated SIN/ SAEEEV.…”

“…The rationale for the development of chimeric alphavirus vaccines included: 1) the consistently attenuated phenotype of all chimeric alphaviruses described to date, including recombinants between 2 highly virulent viruses [19][20][21][22], suggesting that their recombinant nature confers an attenuation phenotype, rather than point mutations that are subject to high rates of reversion. The latter may be explain the residual reactogenicity of alphavirus vaccines that rely on point mutations that accumulate during cell passages, including attenuated strains of Chikungunya virus [23] and VEEV [24]; 2) the success of the chimeric flavivirus vaccines also argues for this approach [25]; 3) live-attenuated vaccines have advantages over other approaches (e.g.…”