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. 2012 Feb 8;30(7):1276-82.
doi: 10.1016/j.vaccine.2011.12.121. Epub 2012 Jan 2.

A vaccine candidate for eastern equine encephalitis virus based on IRES-mediated attenuation

Jyotsna Pandya  1 , Rodion Gorchakov, Eryu Wang, Grace Leal, Scott C Weaver
Affiliations

A vaccine candidate for eastern equine encephalitis virus based on IRES-mediated attenuation

Jyotsna Pandya et al. Vaccine. .

Abstract

To develop an effective vaccine against eastern equine encephalitis (EEE), we engineered a recombinant EEE virus (EEEV) that was attenuated and capable of replicating only in vertebrate cells, an important safety feature for live vaccines against mosquito-borne viruses. The subgenomic promoter was inactivated with 13 synonymous mutations and expression of the EEEV structural proteins was placed under the control of an internal ribosomal entry site (IRES) derived from encephalomyocarditis virus (EMCV). We tested this vaccine candidate for virulence, viremia and efficacy in the murine model. A single subcutaneous immunization with 10(4) infectious units protected 100% of mice against intraperitoneal challenge with a highly virulent North American EEEV strain. None of the mice developed any signs of disease or viremia after immunization or following challenge. Our findings suggest that the IRES-based attenuation approach can be used to develop a safe and effective vaccine against EEE and other alphaviral diseases.

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Figures

Figure 1
Figure 1
Diagramatic representation of the mutated subgenomic promoter of EEEV/IRES. The position of the promoter is indicated by the open box and the mutations introduced to inactivate the subgenomic promoter in the vaccine candidate are shown in the lower case letters. The shaded codon in the wild type FL93-939 represents the stop codon.
Figure 2
Figure 2
Replication of FL93-939 and EEEV/IRES in Vero cells. Bars indicate standard deviations for triplicate infections.
Figure 3
Figure 3
Detection of the EEEV/IRES by RT-PCR in mosquito cells (C6/36) after infection with EEEV/IRES and serial passages. Lane 1: FL93-939; Lanes 2-6: passage 1-5; Lane 7: molecular weight marker. Arrow indicates the expected amplicon size of ~750 bps
Figure 4
Figure 4
Body temperature and body weight of NIH Swiss mice following vaccination with EEEV/IRES or PBS. (a) Body temperature post-vaccination (b) Body weight post-vaccination. Error bars indicate the standard deviations for triplicate experiments.
Figure 4
Figure 4
Body temperature and body weight of NIH Swiss mice following vaccination with EEEV/IRES or PBS. (a) Body temperature post-vaccination (b) Body weight post-vaccination. Error bars indicate the standard deviations for triplicate experiments.
Figure 5
Figure 5
Survival of 4-5-week-old NIH Swiss mice following vaccination with EEEV/IRES or sham vaccination with PBS, followed by challenge with EEEV strain FL93-939
Figure 6
Figure 6
Body temperature and weight of vaccinated (EEEV/IRES) or sham-vaccinated (PBS) NIH Swiss mice following intraperitoneal challenge with EEEV strain FL93-939. (a) Body temperature (b) Body weight. Error bars indicate the standard deviations for triplicate experiments.
Figure 6
Figure 6
Body temperature and weight of vaccinated (EEEV/IRES) or sham-vaccinated (PBS) NIH Swiss mice following intraperitoneal challenge with EEEV strain FL93-939. (a) Body temperature (b) Body weight. Error bars indicate the standard deviations for triplicate experiments.
Figure 7
Figure 7
Reverse transcription polymerase detection of viral replication in representative mosquitoes inoculated intrathoracically with EEEV/IRES, FL93-939 and PBS. Lanes 1-10: IRES/EEEV; Lanes 11-15: wt EEEV strain FL93-939; lane 16: PBS and Lanes 17-18: molecular weight markers.
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