Evolution Mechanism Of Major Epidemic Norovirus Gâ…¡.4Strain And Passive Immunization Based On P Particle

Noroviruses (NoVs) are the leading cause of nonbacterial acute gastroenteritis in both developed and developing countries, with high infection rate people of all ages worldwide. Data from CDC of America showed almost10%of the population of United States experienced gastroenteritis caused by NoV each year (23million cases); and NoV accounts for a substantial extent of disease and potential economic loss (35-75million dollars).NoVs are difficult to study due to no cell culture or small animal model. Up to date, there is still no vaccines or effective antivirals against NoV. An effective vaccine is highly demanded for populations exposed to NoV infection, especially for the young and elderly. Despite the difficulty in studying NoV, significant advances in understanding the genomic structure, capsid proteins, and virus-host interaction of the virus have been made. The discovery of P particle is an important advancement as P particle has the similar structure, immunogenicity and receptor binding with that of VLPs. Through the crystal structure study of P protein and HBGA antigen oligosaccharide A and B, the receptor binding site is located at P2sub-domain, the outer layer of P particle. All these suggest P particle is a good model to explore the interaction between NoV/HBGA receptor, and development of vaccine against NoV. Based on our previous study, to elucidate host factors’(HBGA receptor) role in NV evolution, we performed the sequences and phylogenic analysis of NoV GⅡ.4variants from1974to2012. By expressing P particles of different clusters, the HBGA binding patterns of P particles and antigenic relatedness determined by human challenge sera were studied. At the same time, passive immunization effect of IgY against NoV or/and RV were evaluated by immunizing P particle and P-VP8*.1. Molecular evolution mechanism of epidemic NoV GⅡ.4variantsThe GⅡ.4noroviruses (NVs) are a single genotype that is responsible for over60%of NV gastroenteritis epidemics in the world population. However, GⅡ.4NVs have been found genetically variable and new variants of GⅡ.4NVs emerged every1-3years, suggesting a selection by the host herd immunity in NV evolution. Human NVs are also found to recognize the human histo-blood group antigens (HBGAs) that also play an important role in NV evolution. To elucidate these host factors in NV evolution, we performed a genetic analysis of NVs based on available crystal structures of NVs. The HBGA binding interfaces of NVs are formed by a highly conserved receptor binding pocket that interacts with a key residue of HBGAs and a relatively variable region surrounding the binding pocket that interact with additional saccharides of HBGAs. Using serum samples from patients involved in a challenge study of a GⅡ.4virus, we observed a significant level of cross-reactive antigenic types among14GⅡ.4variants from1998to2012by both ELISA and HBGA receptor blocking assays. The abilities of GⅡ.4NV binding to the ABH HBGAs were maintained although the binding affinity to variable A, B and H antigens varied. We hypothesize that the highly conserved HBGA binding pocket region is strongly selected by the HBGAs which may be important for vaccine development against different GⅡ.4variants. The variable regions could be selected by both HBGAs and host immunity. Since the binding pocket region is also conserved among other GⅡ NVs, further study to determine the antigenic relatedness among all GⅡ NVs is necessary for a broadly reactive vaccine against NVs.2. Evaluation of anti-norovirus IgY from egg yolk of chickens immunized with norovirus P particlesNoroviruses (NoVs) are a leading cause of epidemic acute gastroenteritis affecting millions of people worldwide. Understanding of NoV remains limited due to the lack of a cell culture system and small animal models. Currently, there are no available vaccines or antivirals against NoVs. In this study, an approach for large-scale production of anti-NoV antibodies for use as a potential treatment for NoV disease using passive immunization was evaluated. NoV-specific immunoglobulins (IgY) were produced by immunizing chickens with NoV P particles. The birds continuously produced high titers of antibodies in their eggs for at least3months, in which NoV-specific antibody levels reached4.7-9.2mg/egg yolk. The egg yolk antibodies strongly reacted with NoV P particles by both ELISA and Western blot and blocked NoV virus-like particle (VLP) and P particle binding to the histo-blood group antigen (HBGA) receptors with a BT50of about1:800. The blocking activity of the chicken IgY remained after an incubation at70oC for30min or treatment at pH4to9for3h. These data suggested that chicken IgY could be a practical strategy for large-scale production of anti-NoV antibodies for potential use as passive immunization against NoV infection, as well as for diagnostic purposes.3. A Dual Chicken IgY Against Rotavirus and NorovirusRotavirus (RV) and norovirus (NoV) are the two most important causes of viral gastroenteritis. While vaccine remains an effective prophylactic strategy, development of other approaches, such as passive immunization to control and treat clinical infection and illness of the two pathogens, is necessary. Previously we demonstrated that high titers of NoV-specific IgY were readily developed by immunization of chickens with the NoV P particles. In this study, we developed a dual IgY against both RV and NoV through immunization of chickens with a divalent vaccine comprising neutralizing antigens of both RV and NoV. This divalent vaccine, named P-VP8*particle, is made of the NoV P particle as a carrier with the RV spike protein VP8*as a surface insertion. Approximately45mg of IgY were readily obtained from each yolk with high titers of anti-P particle and anti-VP8*antibodies detected by ELISA, Western blot, HBGA blocking (NoV and RV) and neutralization (RV) assays. Reductions of RV replication were observed with viruses treated with the IgY before and after inoculation into cells, suggesting an application of the IgY as both prophylactic and a therapeutic treatment. Collectively, our data suggested that the P-VP8*based IgY could serve as a practical approach against both NoV and RV.