Vesicular Stomatitis Virus as a Vector to Deliver Virus-Like Particles of Human Norovirus: A New Live Vectored Vaccine for Human Norovirus.

摘要

Human norovirus (NoV) is the leading cause of acute non-bacterial gastroenteritis worldwide. Despite the significant health, emotional, and economic burden caused by human NoV, there are no vaccines or therapeutic interventions for this virus. This is due in major part to the lack of a cell culture system and an animal model for human NoV infection.;Thus, a vector-based vaccine may be ideal for controlling this disease. The major capsid gene (VP1) of a human NoV was inserted into the VSV genome at the glycoprotein (G) and large (L) polymerase gene junction. Recombinant VSV expressing VP1 protein (rVSV-VP1) was recovered from an infectious cDNA clone of VSV. Expression of the capsid protein by VSV resulted in the formation of human NoV virus-like particles (VLPs) that are morphologically and antigenically identical to the native virions. Recombinant rVSV-VP1 was attenuated in cultured mammalian cells as well as in mice. Mice inoculated with a single dose of rVSV-VP1 stimulated a significantly stronger humoral and cellular immune response compared to baculovirus-expressed VLP vaccination. These results demonstrated that that the VSV-based human NoV vaccine induced strong humoral, cellular, and mucosal immunity in a mouse model.;To further improve the safety and efficacy of the VSV-based human NoV vaccine, the gene for the 72kDa heat shock protein (HSP70) was inserted into rVSV and rVSV-VP1 vectors as an adjuvant, which resulted in construction of recombinant VSV expressing HSP70 (rVSV-HSP70) and VSV co-expressing human NoV VP1 protein and HSP70 (rVSV-HPS70-VP1), respectively. At the same inoculation dose, both rVSV-HSP70-VP1 and rVSV-VP1 triggered similar levels of specific immunity, even though VP1 expression by rVSV-HSP70-VP1 was approximately five-fold less than that of rVSV-VP1. To compensate for the reduced VP1 expression levels, the inoculation dose of rVSV-HSP70-VP1 was increased five-fold or same dosage of rVSV-VP1 and rVSV-HSP70 was combined vaccinated. Mice immunized with five does of rVSV-HSP70-VP1 or those receiving combined vaccination generated significantly higher mucosal and/or T cell immunity than those immunized with rVSV-VP1 alone (P<0.05). Therefore, this data indicates that insertion of HSP70 into the VSV vector further attenuates the VSV-based vaccine and HSP70 enhances the human NoV-specific immunities.;To determine whether the VSV-based human NoV vaccine confers protection from human NoV challenge, a gnotobiotic pig model was developed. Newborn gnotobiotic piglets vaccinated intranasally with rVSV-based vaccine (rVSV-VP1) produced high levels of specific serum IgG and fecal and vaginal IgA antibody. Three weeks after vaccination, piglets were orally challenged with human NoV. All three piglets in the unvaccinated challenged group developed histopathologic lesions typical of human NoV infection in the duodenum and proximal jejunum on day 5 post-challenge. However, only one of five vaccinated piglets exhibited focal epithelium loss and villous atrophy, and mild edema in the small intestines. Immunofluorescent assay showed that a large amount of human NoV antigens were detected in duodenum, jejunum, and ileum of the challenge control group but not vaccinated group. These results demonstrate that the rVSV-based human NoV vaccine triggered partially protective immunity in swine and protected gnotobiotic pigs from challenge by human NoV.