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 human-pathogenic 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.

FIG. 1.

Schematic representation of the recombinant SIN-83S virus genome and the junction between SINV and VEEV sequences in the region of the subgenomic promoter. Bold letters represent SINV-specific sequence, and underlined letters represent VEEV-specific sequence. Lowercase letters indicate mutations introduced into the VEEV sequence to make the junction more Sindbis-like and to preserve the putative secondary structure of the 5′ UTR in the VEEV subgenomic RNA.

FIG. 2.

Analysis of plaque morphology and virus replication in BHK-21 cells. (A) The originally designed SIN-83S virus, stock prepared from plaque-purified virus, which formed large plaques, designated SIN-83 lp, and SIN-83 virus containing the S₇₉₅→T mutation in nsP2 found in SIN-83 lp were titrated on monolayers of BHK-21 cells. Plaques were allowed to develop for 36 h prior to fixation and staining. (B) BHK-21 cells were infected with SIN-83S, SIN-83 lp, and SIN-83 viruses at an MOI of 5 PFU/cell. At the indicated times, media were replaced, and virus titers were determined as described in the Materials and Methods. The experiment was performed twice with very similar results.

FIG. 3.

Analysis of virus replication in BHK-21 and Vero cells. BHK-21 (A) or Vero (B) cells were infected with VEE TC-83, SIN-83, and SIN Toto1101 viruses at an MOI of 1 PFU/cell. At the indicated times, media were replaced, and virus titers were determined as described in the Materials and Methods. The experiments were also performed at MOI of 0.1 and 10 PFU/cell with very similar differences in virus growth (data not shown).

FIG. 4.

Analysis of protein synthesis and replication of virus-specific RNAs in infected cells. BHK-21 cells were infected with VEE TC-83, SIN-83, and SIN Toto 1101 viruses at an MOI of 10 PFU/cell. (A) At 12 h postinfection, the cells were labeled with [³⁵S]methionine and analyzed on a sodium dodecyl sulfate-10% polyacrylamide gel as described in Materials and Methods. The additional ³⁵S-labeled protein bands in the lysate of SIN-83-infected cells are marked by stars. (B) At 2 h postinfection, medium in the wells was replaced by 1 ml of Alpha MEM supplemented with 10% FBS, dactinomycin (1 μg/ml), and [³H]uridine (20 μCi/ml). After 3 h of incubation at 37°C, RNAs were isolated from the cells and analyzed by agarose gel electrophoresis as described in Materials and Methods.

FIG. 5.

Survival of mice infected with VEE TC-83 and SIN-83 viruses. Six-day-old NIH Swiss mice were inoculated i.c. (A) or s.c. (B) with indicated doses of viruses. Animals were monitored for 2 months. No deaths occurred after day 7 postinfection in any of these experiments.

FIG. 6.

Mouse body weights after infection with VEE TC-83 or SIN-83 viruses. Six-day-old NIH Swiss mice were inoculated s.c. with 2 × 10⁶ PFU of VEE TC-83 or SIN-83 viruses. Ten animals were used per virus, and six noninfected animals of the same age were included as a control group. The body weight of each animal was determined weekly. Weights of the VEEV TC-83-infected mice that died were excluded from the study. Error bars indicate standard deviations.