Noroviruses and Epidemic Gastroenteritis

The Caliciviridae is a family of positive-strand RNA viruses and consists of five genera designated: (1) Norovirus (with species Norwalk virus); (2) Sapovirus (with species Sapporo virus); (3) Vesivirus (with species, feline calicivirus and vesicular exanthema of swine virus); (4) Lagovirus (with species rabbit hemorrhagic disease virus and European brown hare syndrome virus) and (5) Nebovirus (with species Newbury-2 virus). The Caliciviruses Section in LID is focused on one major goal: lessen the disease burden from caliciviruses that cause disease in humans. Noroviruses are the primary focus of our research because they are the major calicivirus pathogens in humans. They are antigenically-diverse and cause the majority of nonbacterial epidemic gastroenteritis outbreaks. Noroviruses are important also as agents of sporadic, acute gastroenteritis in infants and young children and they can cause life-threatening diarrhea in immunocompromised individuals. <P> Because a major technical challenge in the study of these viruses is the inability to grow them in cultured cells, our laboratory has explored the development of calicivirus replication systems and animal models to establish parameters of infection and immunity. Our chimpanzee animal model proved important in the evaluation of virus-like particles (VLPs) as vaccine candidates (Bok et al., 2011). We showed that homologous protection was achieved against Norwalk virus when animals were immunized with Norwalk VLPs; however, immunization with a heterotypic norovirus VLP (GII.4) did not induce protection. The sera from protected versus non-protected animals were used to explore the role and specificity of serum antibodies. Animals resistant to Norwalk virus infection developed antibodies that blocked binding of Norwalk VLPs to HBGA carbohydrates, confirming the use of carbohydrate blocking as an effective surrogate assay in the absence of an in vitro neutralization test. Small intestinal biopsies from the animals enabled us to identify monoclonal antibodies that detect expression of norovirus antigens in tissue, leading to the visualization of permissive cell types in the chimpanzee gut. We are now using the same methodology to identify permissive cell types in human intestinal biopsies. Because chimpanzees will no longer be used in most biomedical research supported by NIH, we are working to develop new models and systems in which to evaluate norovirus vaccines and therapeutic drugs. In addition, we have established collaboration with a leading company in norovirus vaccine development, LigoCyte Pharmaceuticals, Inc., to address the immunogenicity of new norovirus vaccine candidates. <P> Our collaborative work this year was published in the journal Vaccine (Parra et al., 2012), and is highlighted below as a major scientific advance.One approach under consideration for the treatment of norovirus disease is therapeutic antibodies. Our laboratory has generated panels of norovirus-specific monoclonal antibodies following immunization of mice. One such library was characterized in detail, and a GII.4 antigenic map was generated (Parra et al., 2012). Importantly, antibodies that blocked binding of GII.4 VLPs to HBGA carbohydrates bound to multiple distinct epitopes on the capsid surface, with some of the sites overlapping those known to evolve during the emergence of new variants. It is likely that some of the GII.4 monoclonal antibodies in our panel would neutralize virus infectivity in an in vitro neutralization assay, with a possible therapeutic potential. We are continuing to develop and map additional antibodies in the laboratory, generated by a variety of technologies. <P> One goal is to map cross-reactive antigenic sites that might serve as targets for the design of broadly-protective vaccines. In this regard, our long-standing collaboration with Dr. Al Kapikian has been important: we have identified some of the oldest known norovirus strains in his archived epidemiologic samples. Analysis of these viruses has been useful in establishing the evolutionary rates of predominant circulating noroviruses, such as the GII.3 noroviruses, published last year (Boon et al., 2011). We have investigated the etiology of diarrhea in a World Health Organization (WHO) study that was conducted in the 1970s to assess the need for vaccines. Noroviruses were grossly underestimated as etiologic agents of diarrhea back then, and our application of new diagnostics technology to the analysis of the WHO samples has yielded new insight into the antigenic types that were present then and now (Rackoff et al., in preparation). These samples have been invaluable in our antibody mapping studies that search for potent neutralizing epitopes that are conserved over time.