Susceptibility of Human Volunteers With Different Histo-Blood With Different Histo-Blood Group Antigens to Norovirus

Noroviruses are single-stranded, positive-sense RNA viruses that cause acute gastroenteritis in humans. The prototype Norwalk virus (NV) was identified in a large outbreak of acute gastroenteritis in an elementary school in Norwalk, Ohio, in 1968. Since then, many strains have been described and named by the locations of their first isolation. Genetically, Noroviruses belong to one of four genera within Caliciviridae (CV). Phylogenetic analysis has shown that the Norovirus genus is divided into five genogroups and each genogroup can be further divided into genetic clusters; at least 30 genetic clusters of Noroviruses have been identified.

Noroviruses have been recognized as the most important cause of non-bacterial epidemics of acute gastroenteritis, affecting individuals of all ages, in both developing and developed countries. Such outbreaks usually have high attack rates and have occurred in child care centers, schools, restaurants, summer camps, hospitals, nursing homes, ships (both civilian and military) as well as deployed military troops. The viruses are highly contagious and are able to survive in the environment for extended periods of time. They are spread by contact with environmental surfaces and person-to-person transmission. Noroviruses normally cause moderate-to-severe diarrhea and the disease is often incapacitating and can be fatal in the young and elderly. The most important public health concern is that Noroviruses can cause large water- and food-borne outbreaks, resulting in the virus being classified as a Category B agent.

Noroviruses have also been implicated in several military outbreaks. The "Desert Shield virus" (DSV) isolated in US troops in the 1991 Gulf War was subsequently identified as a Norovirus. Similarly the recently reported "mysterious" attacks of acute gastroenteritis affecting the British troops in Afghanistan were also caused by a Norovirus. Large outbreaks of Norovirus-associated gastroenteritis are common on US Navy ships, similar to those that occurred on board cruise ship lines. Surveillance of acute gastroenteritis on board five US Navy combat ships in 1998-1999 showed that all the ships experienced large outbreaks of Norovirus acute gastroenteritis during their deployment in the Pacific and Indian Oceans, following port visits. Because of the rapid spread of the disease, many departments and units on board the ships were unable to function during the peak of the outbreaks.

Noroviruses are difficult to study because: 1) they are genetically and antigenically highly diverse and multiple strains with distinct genetic identities co-circulate in the same communities, making diagnosis and disease control extremely difficult; and 2) the virus cannot be cultivated in cell culture and 3) no small animal model for studying Noroviruses exists. The molecular cloning of the prototype Norwalk Virus (NV) in 1990 by one of the co-investigators of this application has allowed rapid progress in studying Noroviruses and other genera; however, many issues on Norovirus infection, pathogenesis, immunology and host range remain unknown.

The discovery of Norovirus receptors provides new opportunities to evaluate the pathogenesis and epidemiology of Noroviruses. A host genetic factor involving Norovirus infection was originally suggested by many researchers before the cloning of Noroviruses in the 1990s because about 20-30% of individuals who did not have antibodies against Noroviruses were never infected following a challenge with NV, and in outbreaks, individuals who remained healthy were clustered in families that had the same exposure to Noroviruses as the ill families. Further, pre-existing antibodies against NV were not protective against NV infection, while individuals who had high levels of antibodies against NV were more susceptible to NV than individuals who did not have the antibodies. These observations indicate that, in addition to immune responses, there must be another factor(s) that controls the host-specificity of Norovirus infection.

We recently discovered that Noroviruses recognize human histo-blood group antigens as receptors. Using recombinant capsid proteins from different Noroviruses representing different genetic clusters, we have identified four binding patterns of Noroviruses to human histo-blood group antigens, which are defined by the host Lewis, secretor and ABO blood types. This finding provides a new approach to develop strategies to control the disease. Specifically, if Noroviruses rely on the histo-blood group antigens on the surfaces of the intestinal epithelial cells as receptors, a compound that inhibits the interaction between the virus and receptors could be developed as an antiviral drug for Noroviruses. This finding provides the basis for this protocol which seeks to confirm the relationship between specific Norovirus susceptibility and specific histo-blood group antigen presence as well as establish a model to evaluated future interventions (drugs and vaccines).

The specificity of the NV interaction with the sugar moieties of histo-blood group antigens was shown by specific blocking of the binding by human milk from a secretor, by monoclonal antibodies specific for secretor human blood-group antigens (HBGAs), by synthetic oligosaccharide conjugates containing secretor antigens, and by treatment of the tissues with a1,2 fucosidase. NV also binds to differentiated CaCo2 cells and differentiated CaCo2 cells express human histo-blood group antigens. Transfection of Chinese Hamster Ovary (CHO) cells with a a1,2-linked fucosyl-transferase cDNA allowed attachment of NV virus-like particles (VLPs). The bound rNV VLPs were internalized following incubation of the cells at 37oC.

To further investigate the association between secretor status and susceptibility to Norwalk virus (NV), we collaborated with Dr. Christine Moe at Emory University's School of Medicine on a volunteer challenge study. This study utilized the prototype NV, the first Norovirus strain to be used for human challenge studies. Saliva samples from 77 NV-challenged volunteers were tested for secretor status and for NV-binding. Fifty-five (71%) of the 77 volunteers were secretors. Among the 55 secretors, 34 (62%) were infected with NV following challenge and saliva from 41 (75%) bound NV recombinant VLPs. None of the saliva samples from the 22 non-secretor volunteers bound NV virus-like particles (VLPs) and none of them became infected following NV challenge; strongly suggesting that the susceptibility to NV is determined at least in part by the ability of the host to bind the norovirus strain.

More recently we performed a study to determine whether other strains of Noroviruses have a similar pattern of host-specificity and have extended the binding patterns to 7 based on examination of fourteen Noroviruses. The 7 patterns have been classified into two groups according to their interactions with three major epitopes (A/B, H and Lewis) of HBGAs: the A/B binding group and the Lewis-binding group. Strains in the A/B binding group recognize the A and/or B and H antigens, but not the Lewis antigens, while strains in the Lewis-binding group react only to the Lewis and/or H antigens. This classification also resulted in a model of the Norovirus/HBGA interaction, in which a maximum of two binding sites within a receptor binding interface are involved in NV/HBGA interaction. The A, B and H side chains are responsible for binding by the A/B binding strains while the Lewis epitopes are responsible for the Lewis binding strains. Phylogenetic analyses showed that strains with identical or closely related binding patterns tended to be clustered, but strains in both binding groups can be found in both genogroups I and II. Our results suggest that Noroviruses have a wide spectrum of host range and human HBGAs play an important role in Norovirus evolution.

Since human Noroviruses cannot be cultivated in vitro or infect animals, it is necessary to perform human volunteer challenge studies to study the pathobiology of the infection. The study will further the concept that Norovirus has a narrow host range which is fundamental in the pathogenesis and immunology of Noroviruses. Additionally, we recently have identified more than a dozen compounds that specifically block Norovirus attachment to their receptors. The human challenge study should also establish a model that can be used in the evaluation of these new compounds in regards to their capacity to block virus replication (anti-viral drug). Furthermore, our study will re-evaluate whether acquired immunity is an independent factor in Norovirus infections which may provide additional information in host immunity potentially useful in vaccine development. Finally, the study will allow for banking of the Norovirus challenge strain by collecting the diarrheal stools passed by the study subjects. Banking of additional specimen, as mentioned above, is critical for future clinical trials because the virus cannot be cultivated in vitro and must be isolated from humans infected with the virus.

A specific challenge study would only use one challenge strain and any challenge strain used in human clinical trials would have a separate IND and FDA approval prior to administration to subjects.