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IRES-driven expression of the capsid protein of the Venezuelan equine encephalitis virus TC-83 vaccine strain increases its attenuation and safety.

Guerbois M, Volkova E, Forrester NL, Rossi SL, Frolov I, Weaver SC - PLoS Negl Trop Dis (2013)

Bottom Line: Here we describe a second generation of the recombinant TC-83 in which the subgenomic promoter is maintained and only the capsid protein gene is translated from the IRES.This VEEV/IRES/C vaccine candidate did not infect mosquitoes, was stable in its attenuation phenotype after serial murine passages, and was more attenuated in newborn mice but still as protective as TC-83 against VEEV challenge.Thus, by using the IRES to modulate TC-83 capsid protein expression, we generated a vaccine candidate that combines efficient immunogenicity and efficacy with lower virulence and a reduced potential for spread in nature.

View Article: PubMed Central - PubMed

Affiliation: Institute for Human Infections and Immunity, Sealy Center for Vaccine Development, and Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America.

ABSTRACT
The live-attenuated TC-83 strain is the only licensed veterinary vaccine available to protect equids against Venezuelan equine encephalitis virus (VEEV) and to protect humans indirectly by preventing equine amplification. However, TC-83 is reactogenic due to its reliance on only two attenuating point mutations and has infected mosquitoes following equine vaccination. To increase its stability and safety, a recombinant TC-83 was previously engineered by placing the expression of the viral structural proteins under the control of the Internal Ribosome Entry Site (IRES) of encephalomyocarditis virus (EMCV), which drives translation inefficiently in insect cells. However, this vaccine candidate was poorly immunogenic. Here we describe a second generation of the recombinant TC-83 in which the subgenomic promoter is maintained and only the capsid protein gene is translated from the IRES. This VEEV/IRES/C vaccine candidate did not infect mosquitoes, was stable in its attenuation phenotype after serial murine passages, and was more attenuated in newborn mice but still as protective as TC-83 against VEEV challenge. Thus, by using the IRES to modulate TC-83 capsid protein expression, we generated a vaccine candidate that combines efficient immunogenicity and efficacy with lower virulence and a reduced potential for spread in nature.

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Related in: MedlinePlus

Survival in mice after infection with VEEV/IRES/C or VEEV TC-83 passaged in-vivo.Six-day-old CD1 pups received a 5×104 PFU dose SC of viruses passaged 10 times in CD1 mice (mp10). Parent unpassaged VEEV TC-83 and VEEV/IRES/C were injected at the same dose as controls. Animals were monitored daily for survival for 14 days. No deaths occurred after day 11 post-infection. *** = P<0.0001.
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pntd-0002197-g004: Survival in mice after infection with VEEV/IRES/C or VEEV TC-83 passaged in-vivo.Six-day-old CD1 pups received a 5×104 PFU dose SC of viruses passaged 10 times in CD1 mice (mp10). Parent unpassaged VEEV TC-83 and VEEV/IRES/C were injected at the same dose as controls. Animals were monitored daily for survival for 14 days. No deaths occurred after day 11 post-infection. *** = P<0.0001.

Mentions: VEEV/IRES/C was subjected to 5 serial passages in Vero cells or 10 serial passages in mouse brains. No discernible change was observed in plaque morphology after 5 serial passages in vitro or 10 passages in vivo (data not shown). Genetic stability was confirmed by full-genome sequencing of passaged viruses; no mutations were found in consensus sequences of Vero- or mouse-passaged viruses, aside from the deletion of one adenosine in a poly-A tract within the IRES itself, which appeared between passage 3 and 4 on Vero cells, and before passage 5 in mouse brains. No changes were detected in virulence for the mp10 VEEV/IRES/C compared to the parental strain (P = 0.95 for series A and P = 0.75 for series B) after SC injection of 6-day-old mice (Fig. 4), whereas a significant increase in virulence was observed for the mp10 TC-83 viruses compared to parental TC-83, as previously described [29].


IRES-driven expression of the capsid protein of the Venezuelan equine encephalitis virus TC-83 vaccine strain increases its attenuation and safety.

Guerbois M, Volkova E, Forrester NL, Rossi SL, Frolov I, Weaver SC - PLoS Negl Trop Dis (2013)

Survival in mice after infection with VEEV/IRES/C or VEEV TC-83 passaged in-vivo.Six-day-old CD1 pups received a 5×104 PFU dose SC of viruses passaged 10 times in CD1 mice (mp10). Parent unpassaged VEEV TC-83 and VEEV/IRES/C were injected at the same dose as controls. Animals were monitored daily for survival for 14 days. No deaths occurred after day 11 post-infection. *** = P<0.0001.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3649961&req=5

pntd-0002197-g004: Survival in mice after infection with VEEV/IRES/C or VEEV TC-83 passaged in-vivo.Six-day-old CD1 pups received a 5×104 PFU dose SC of viruses passaged 10 times in CD1 mice (mp10). Parent unpassaged VEEV TC-83 and VEEV/IRES/C were injected at the same dose as controls. Animals were monitored daily for survival for 14 days. No deaths occurred after day 11 post-infection. *** = P<0.0001.
Mentions: VEEV/IRES/C was subjected to 5 serial passages in Vero cells or 10 serial passages in mouse brains. No discernible change was observed in plaque morphology after 5 serial passages in vitro or 10 passages in vivo (data not shown). Genetic stability was confirmed by full-genome sequencing of passaged viruses; no mutations were found in consensus sequences of Vero- or mouse-passaged viruses, aside from the deletion of one adenosine in a poly-A tract within the IRES itself, which appeared between passage 3 and 4 on Vero cells, and before passage 5 in mouse brains. No changes were detected in virulence for the mp10 VEEV/IRES/C compared to the parental strain (P = 0.95 for series A and P = 0.75 for series B) after SC injection of 6-day-old mice (Fig. 4), whereas a significant increase in virulence was observed for the mp10 TC-83 viruses compared to parental TC-83, as previously described [29].

Bottom Line: Here we describe a second generation of the recombinant TC-83 in which the subgenomic promoter is maintained and only the capsid protein gene is translated from the IRES.This VEEV/IRES/C vaccine candidate did not infect mosquitoes, was stable in its attenuation phenotype after serial murine passages, and was more attenuated in newborn mice but still as protective as TC-83 against VEEV challenge.Thus, by using the IRES to modulate TC-83 capsid protein expression, we generated a vaccine candidate that combines efficient immunogenicity and efficacy with lower virulence and a reduced potential for spread in nature.

View Article: PubMed Central - PubMed

Affiliation: Institute for Human Infections and Immunity, Sealy Center for Vaccine Development, and Department of Pathology, University of Texas Medical Branch, Galveston, Texas, United States of America.

ABSTRACT
The live-attenuated TC-83 strain is the only licensed veterinary vaccine available to protect equids against Venezuelan equine encephalitis virus (VEEV) and to protect humans indirectly by preventing equine amplification. However, TC-83 is reactogenic due to its reliance on only two attenuating point mutations and has infected mosquitoes following equine vaccination. To increase its stability and safety, a recombinant TC-83 was previously engineered by placing the expression of the viral structural proteins under the control of the Internal Ribosome Entry Site (IRES) of encephalomyocarditis virus (EMCV), which drives translation inefficiently in insect cells. However, this vaccine candidate was poorly immunogenic. Here we describe a second generation of the recombinant TC-83 in which the subgenomic promoter is maintained and only the capsid protein gene is translated from the IRES. This VEEV/IRES/C vaccine candidate did not infect mosquitoes, was stable in its attenuation phenotype after serial murine passages, and was more attenuated in newborn mice but still as protective as TC-83 against VEEV challenge. Thus, by using the IRES to modulate TC-83 capsid protein expression, we generated a vaccine candidate that combines efficient immunogenicity and efficacy with lower virulence and a reduced potential for spread in nature.

Show MeSH
Related in: MedlinePlus