The nature of immunity to norovirus is a key determinant if considering the prospect of future prevention of disease by possible vaccines. To date, little is known about the protective immune response elicited by norovirus infection, that is largely due to the lack of cell culture systems for noroviruses pathogenic to humans.
In the absence of in vitro replicating viruses, VLPs represent a useful tool for investigating virus-ligand interactions and the anti-viral immune response, since they strictly resemble infectious viruses both antigenically and morphologically [4, 15, 28]. VLPs might also be suitable for vaccination against NoV infection , similar to vaccines already in use for other viruses such as papillomavirus .
In the present study, we used a baculovirus expression system to generate VLPs from a NoV GII.4 strain identified during a major gastroenteritis outbreak in Italy in 2000 . As previously reported by others [31–33], also the VLPs described in this study induced a strong immune response in Balb/c mice, eliciting elevated antibody levels (data not shown). The VLPs prepared in this study persisted during stressful CsCl centrifugation and proved antigenically stable at 4°C. They were thus suitable for investigating the cell-mediated immune response to GII.4 norovirus, which was done using peripheral blood cells from five asymptomatic adult volunteers. Although these subjects had not been affected by gastroenteritis in the 4 weeks before testing, they all resulted positive for NoV-antibodies in a NoV-VLP based ELISA test, indicating a previous infection with this or a similar norovirus genotype. This observation is not surprising since GII.4 is a pandemic genotype, and is in line with previous findings that between 90-100% healthy adult humans are seropositive for norovirus [3, 4, 34].
The innate and early adaptive immune response induced by GII.4 NoV-VLPs was evaluated on ex vivo stimulated PBMCs from the volunteers by means of activation/maturation phenotype analysis and cytokine production analysis. NoV-VLPs induced an increased expression of activation markers and co-stimulatory molecules in circulating APCs, particularly in CD14+ monocyte and CD11+ mDC cell populations. In particular, PBMC stimulation resulted in increased expression of surface activation/maturation markers (i.e. CD80, CD86, CD40 and HLA-DR), whose role in the initiation of the immune response is well defined. Both CD80 and CD86 are able to prime T cells, providing a co-stimulatory signal necessary for T cell activation and survival [35, 36]. In addition, CD40 expression on antigen-presenting cells is known to be enhanced by CD40L on activated T cells, in a loop of induction of the immune response . Finally, the HLA-DR molecule, which represents a ligand for the T-cell receptor (TCR), is up-regulated as well in response to antigenic stimuli . The overall results provide interesting clue on the responsiveness of ex vivo loaded circulating APCs.
Moreover, PBMC stimulation resulted in increased production of specific cytokines, such as TNF-α, IL-6, whose level persisted for the 6 days of stimulation, and IFN-γ, whose level increased only after prolonged stimulation.
Several studies have been performed to identify the cytokine profile induced by NoV. In particular, an investigation on the T-cell response, induced in humans after a challenge with a GII.2 norovirus strain, identified a predominant Th1 CD4-dependent cell response, characterized by significant IFN-γ secretion . Moreover, experimental infection of gnotobiotic piglets with a GII.4 human NoV strain was also reported to induce both antibodies and Th1/Th2 cytokine responses, locally and systemically . These authors detected persistently higher Th1-specific cytokines (low transient IFN-γ and high IL-12) in infected pig sera, but also Th2-specific IL-4 and IL-6 cytokines, although to a lower level. Notably, a delayed IFN-α response was also evident .
Several authors suggest that response to norovirus infection mainly involves IFN-γ secretion by CD4 Th1 T cells [17, 23, 41, 42].
Since the production of cytokines was not investigated in isolated cell subpopulations in the present study, no conclusion can be drawn about a specific pattern of Th1 versus Th2 response upon stimulation with GII.4 VLPs. However, our present observations suggest that despite an initial status characterized by IL-6 production, a prolonged stimulation may induce viable T cells to produce IFN-γ, implying that NoV-VLPs might activate autologous T cells.
The low reactivity shown by ELISA testing of some donors’ sera may be due to the antigenic differences between the strain(s) which had infected the five subjects in the past and the virus genotype corresponding to the VLPs used. The observation that IFN-γ became detectable only at later time points after in vitro stimulation might either indicate a switch in cytokine production or a low rate of secretion of this cytokine that would require a longer time to reach a detectable concentration in the culture supernatant.