Norovirus infection is common among individuals throughout the world, regardless of economic status, resulting in significant morbidity and loss of productivity. Recent reports of consecutive norovirus outbreaks on cruise ships, involving hundreds of passengers and crew, garnered substantial media attention and dramatically illustrate the potentially explosive community spread of these viruses (8
). Although the severity of the disease is usually moderate and short term, an effective vaccine would be advantageous to specific segments of the population, such as food handlers, care providers, and military personnel. The major obstacles to norovirus vaccine development are the great degree of antigenic heterogeneity within the family, the possibility that immunity to noroviruses may be short-lived, and the lack of understanding of the correlates of protective immunity. An effective vaccine should also provide protective immunity against an ever-growing list of norovirus strains. Evidence from outbreak investigations and human challenge studies suggests that these viruses are highly infectious, yet the biology and immunology of norovirus infection remain poorly understood (32
Noroviruses are members of the Caliciviridae
family of single-stranded, positive-sense RNA viruses. The majority of the human noroviruses are classified into genogroup 1 (G1) and G2 (47
) Each of these genogroups is further divided into genetic clusters. Norwalk virus (NV), Snow Mountain virus (SMV), and Hawaii virus (HV) are the prototype strains of genetic clusters G1.1, G2.2, and G2.1 and are the causative agents of an estimated 5, 8, and 7% of norovirus outbreaks, respectively (13
). Strains within a genetic cluster typically show ≥80% amino acid identity in the major capsid protein sequence. Strains within the same genogroup show ≥60% identity, and strains in different genogroups show ≤50% identity. This high degree of genetic variability translates into a high degree of antigenic variability within the noroviruses.
Early human challenge studies demonstrated that some volunteers remained uninfected even when challenged with high doses of NV (30
), suggesting that some individuals may be genetically resistant to NV infection. Recently, we demonstrated that the FUT2
gene that encodes an α1,2-fucosyltransferase responsible for the histo-blood group antigen (BGA) H type 1 is a susceptibility allele for NV infection (32
). Volunteers who were homozygous for the inactive FUT2
allele were resistant to infection at all dose levels studied. Other BGAs displayed weak modulating effects on NV interaction and pathogenesis. We and other groups have shown that NV virus-like particles (VLPs) bind to H type 1-related BGAs secreted in saliva (14
) and membrane bound on mucosal cells (34
) and also to synthetic H type 1-related BGA (20
). This H type 1-dependent in vitro binding pattern is so far restricted to NV, as other strains of noroviruses demonstrate different BGA binding affinities. Salivary binding assays have suggested that SMV VLP binding is associated with expression of the B BGA, while HV VLPs did not bind to any saliva sample (20
). These data suggest that noroviruses use multiple methods for attachment to cells and hence for infection. It is unclear whether the FUT2
allele determines susceptibility to all norovirus infections.
Short-term immunity to NV has been observed in previous human challenge studies (48
). However, this immunity did not necessarily extend to heterologous virus challenge, as volunteers who became ill upon challenge with NV also became ill upon subsequent challenge with HV at the same rate as volunteers not previously infected with NV (48
). Further rechallenge studies have shown that long-term immunity is not conferred by a single NV challenge, as some volunteers who were susceptible to an initial NV infection were susceptible to infection in subsequent challenges 27 to 42 months later (41
Previously, in both volunteer studies and outbreak investigations, immunity to noroviruses has almost exclusively been studied by monitoring serum immunoglobulin M (IgM) and IgG responses to virus (35
). IgM was shown to be a marker of recent NV infection (5
). In many studies, a fourfold increase in anti-norovirus serum IgG titer has been considered indicative of norovirus infection (17
). Attempts to study fecal IgA in NV infection have been unsuccessful in associating secretory IgA (sIgA) with infection outcome (40
). However, our recent study utilizing saliva samples as a source of sIgA showed a correlation between protection from NV infection and the presence of a memory sIgA response in uninfected susceptible volunteers (32
). Importantly, some susceptible volunteers who were not infected did not have strong salivary IgA responses, indicating that non-sIgA mechanisms are also involved in immunity to noroviruses.
In addition to antibody-producing B cells, T cells play an important role in the control of viral pathogens. The cytokines secreted by antigen-specific CD4+
helper T cells guide the adaptive immune response. T helper 1 (Th1) and Th2 responses can be activated by both the same and different epitopes within the same pathogen, but one response type will often dominate over the other. A Th2-favored immune response is primarily characterized by interleukin 4 (IL-4) and IL-10 secretion by CD4+
cells, resulting in production of IgG2 and other anti-inflammatory factors. A Th1-favored response is primarily characterized by secretion of gamma interferon (IFN-γ) by CD4+
cells, resulting in macrophage activation, B-cell differentiation to IgG1 synthesis, and support for cytotoxic T lymphocytes (CTLs). CTLs are important in the control of human immunodeficiency virus (HIV) (4
), Epstein-Barr virus (42
), influenza virus (26
), lymphocytic choriomeningitis virus (52
), and numerous other viruses. To date, cellular immunity has not been studied following norovirus infection, but we expect effector T cells to play a fundamental role in viral clearance and immunity.
In this study, we demonstrated that SMV infection was independent of secretor status and BGA expression and that the serum IgG, salivary IgA, and effector T cells elicited following infection were cross-reactive between strains within a genogroup but not between strains in different genogroups. This is the first study to examine the association between mucosal expression of BGAs and infection with a G2 norovirus in humans and to study T-cell activation after challenge with any live norovirus.