Replication and Clearance of Venezuelan Equine Encephalitis Virus from the Brains of Animals Vaccinated with Chimeric SIN/VEE Viruses

Abstract

Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic pathogen. Recent outbreaks in Venezuela and Colombia in 1995, involving an estimated 100,000 human cases, indicate that VEEV still poses a serious public health threat. To develop a safe, efficient vaccine that protects against disease resulting from VEEV infection, we generated chimeric Sindbis (SIN) viruses expressing structural proteins of different strains of VEEV and analyzed their replication in vitro and in vivo, as well as the characteristics of the induced immune responses. None of the chimeric SIN/VEE viruses caused any detectable disease in adult mice after either intracerebral (i.c.) or subcutaneous (s.c.) inoculation, and all chimeras were more attenuated than the vaccine strain, VEEV TC83, in 6-day-old mice after i.c. infection. All vaccinated mice were protected against lethal encephalitis following i.c., s.c., or intranasal (i.n.) challenge with the virulent VEEV ZPC738 strain (ZPC738). In spite of the absence of clinical encephalitis in vaccinated mice challenged with ZPC738 via i.n. or i.c. route, we regularly detected high levels of infectious challenge virus in the central nervous system (CNS). However, infectious virus was undetectable in the brains of all immunized animals at 28 days after challenge. Hamsters vaccinated with chimeric SIN/VEE viruses were also protected against s.c. challenge with ZPC738. Taken together, our findings suggest that these chimeric SIN/VEE viruses are safe and efficacious in adult mice and hamsters and are potentially useful as VEEV vaccines. In addition, immunized animals provide a useful model for studying the mechanisms of the anti-VEEV neuroinflammatory response, leading to the reduction of viral titers in the CNS and survival of animals.Venezuelan equine encephalitis virus (VEEV) is an enveloped virus with a nonsegmented, positive-sense RNA genome of approximately 11.4 kb and belongs to the Alphavirus genus in the Togaviridae family. The 5Ј two-thirds of the genome contains four nonstructural proteins (nsP1 to nsP4) that form an enzyme complex required for viral replication (46-48). After release of the viral genome into the cytoplasm, a nonstructural polyprotein is translated directly from this RNA and utilized in the production of a full-length, negative-sense replicative RNA intermediate (45). The full-length RNA then serves as a template for the synthesis of positive-sense genomic RNA and for transcription of a subgenomic 26S RNA (46). The approximately 4-kb-long, subgenomic RNA corresponds to the 3Ј onethird of the viral genome and is translated into a structural polyprotein that is proteolytically cleaved into the capsid and the envelope glycoproteins E2 and E1 (34). Two hundred forty copies of the capsid protein enclose the genomic viral RNA to form an icosahedral nucleocapsid that buds from the plasma membrane, acquiring a lipid envelope with embedded protein spikes formed by E1/E2 heterodimers (41, 48).Venezuelan equine encephalitis virus is a zoonotic pathogen...

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 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.…”

“…To use a highly stringent model for neurovirulence, cohorts of twelve 6 day-old NIH Swiss mice were inoculated IC (5 log 10 PFU) with either SINV/EEEV chimeras, parental NA EEEV, SA EEEV, or SINV strains, the previously described chimeric SIN/VEEV [20], or the TC-83 [24,29] or V3526 [30] attenuated VEEV vaccine strain, and monitored for signs of neurologic disease (Fig. 6).…”

“…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.…”