The B-cell arm of the immune response plays a major role in controlling RV infection. This inference comes from many reports of experimental and clinical observations. For example, in RV infections of children (8
), mice (11
), and piglets (44
), serum and intestinal antibodies correlate with protection. Furthermore, infection experiments using B-cell-deficient mice indicate that B cells are necessary for protection (14
). Interestingly, chronic RV infection in Rag-2-deficient mice is resolved by transfer of mucosally targeted α4β7high
B cells, indicating that local B cells have a potent role in the control of RV infection (43
). However, the mechanisms by which B cells act against RV are unclear. Among unsolved issues is whether and how the immunodominant antibody response against the inner capsid VP2 and VP6 proteins plays a determining role in the defense against RV. Several experimental studies suggest that anti-VP6 and/or -VP2 antibodies can be involved in protection. Indeed, double-layer EDIM virus, i.e., EDIM virus without the external capsid, induces better protection in WT mice than in B-cell-deficient μMT mice (28
). Furthermore, in the mouse backpack model, a hybridoma producing anti-VP6 IgA protected the mice against an RV ECw challenge (4
). In this report, we further show that adult mice with an inherent block in IgA and IgM transcytosis via the pIgR demonstrate a delay in clearing RV and are not protected against a murine RV challenge by immunization with heterologous bovine VLP2/6. The J chain−/−
mice's failure to control RV infection cannot be attributed to known altered B- or T-cell responses. In fact, we verified that the anti-RV-specific B-cell distributions in spleen and mesenteric lymph node of the two types of mice were similar. In addition, anti-RV IgA-secreting cells were detected by the enzyme-linked spot assay in both immune WT and J chain−/−
mouse intestine (data not shown). FACS analysis of splenocytes from the J chain−/−
mice using monoclonal antibodies to CD3, CD4, and CD8 revealed staining patterns indistinguishable from those of WT littermates (17
). In addition, T-cell-dependent IgG responses were comparable in J chain−/−
mice and WT controls (unpublished data).
The significant delay in clearance of RV infection in J chain−/−
mice in the context of preserved B-cell responses supports the role of mucosal immunoglobulins transported by the pIgR in the control of RV primary infection. This finding suggests that secretion of mucosal IgA and IgM directed to the whole viral particle participates in the clearance of RV infection, either in the lumen of the intestine or possibly inside the epithelial cells, as suggested for human immunodeficiency virus (1
), influenza virus (25
), and Sendai virus (26
) infections. Previous works using genetically engineered mice have shown that B cells are major players in the protection against RV reinfection but minor actors in the resolution of infection (14
). However, these findings were obtained with mice bred on a C57BL/6 × 129 (14
) or on a C57BL/6 (26
) background that probably emphasizes the impact of CD8 T cells on viral clearance, as C57BL/6 mice are genetically biased towards potent cytotoxic responses.
VLP2/6 has been shown to induce protection against RV infection in mouse (30
) and rabbit models (7
) using intranasal and parenteral vaccination routes, respectively. In this study, nasally delivered VLP2/6 induced protection in the adult mouse against infection with a 104
SD50 challenge dose of ECw virus, which is 103
higher than the dose previously used by O'Neal et al. (30
). By contrast, immunization with VLP2/6 did not protect J chain−/−
mice under these challenge conditions. This finding strongly suggests that transcytosis of IgA and IgM directed to VLP2/6 is required to fully protect mice against a high virus dose challenge. As for other viruses (1
), intracellular neutralization of RV has been proposed by Burns et al. (4
). Conceivably, transcytosis of anti-VP2 or -VP6 polymeric Ig associated to pIgR may allow intracellular interaction between the Ig and the corresponding structural protein VP6 or VP2, inhibiting a crucial step of the viral cycle such as transcription or assembly. It has been recently shown that some protective IgA in a backpack mouse model inhibits the assembly of the RV shell by preventing association of the outer capsid VP7 protein (15
). The subcellular compartment where the transcytosing IgA interacts with the RV particle has yet to be identified.
Whereas the intracellular neutralization is an attractive mechanistic hypothesis, it still remains possible that the mucosal secretory anti-VP6 antibodies act inside the intestinal lumen, after their intracellular transit. Although monoclonal and polyclonal anti-VP6 antibodies have never been found to be neutralizing in in vitro assays, some secretory antibodies to VP6 may be able to prevent viral infection in vivo. Actually, secretory IgA exhibits higher stability and avidity as well as an increased neutralization potential compared to monomeric IgA or IgG (33
). In addition, due to a high sugar moiety content, immune complexes made of secretory IgA are highly hydrophilic and interact efficiently with mucins, leading to an efficient elimination of undesired antigens through the mucus layer (40
). Thus, another possible explanation of our data is that secretory IgA (or IgM) directed to VP6-accessible epitopes may actually bind to infectious RV particles efficiently enough to lead to the viral immune exclusion within the mucus layer. This mechanism would be particularly efficient in rodents, as huge amounts of secretory IgA are found in the intestinal lumen due to the biliary excretion of IgA.
The role of anti-VP6 antibodies in protection could not be demonstrated with anti-VP6 IgA-producing hybridomas in a neonatal mouse infection model (34
). The discordant findings between studies regarding the protective capacities of anti-VP6 hybridomas may be due to different biological conditions between the adult and the neonatal mouse models of infection. The protective mechanisms involved, such as pIgR transcytosis, secretory IgA interaction with the intestinal milieu, or complementation with undefined immune factors, may be modified with age. In addition, the lack of protection observed in the neonatal mouse model of infection with the chosen anti-VP6 IgA-producing hybridomas may have resulted from anti-VP6 IgA unable to interfere with the viral life cycle, in either an extracellular or an intracellular compartment. Actually, the epitopes that are recognized by the anti-VP6 IgA are probably essential to achieving antiviral effects. As vaccination with VLP2/6 elicits a wide panel of anti-VP6 and -VP2 antigenic specificities, it is likely that anti-VP2 or -VP6 IgA (or IgM) of adequate reactivity, efficient at blocking the viral cycle, was produced in our immune mice.
The major role of mucosal Ig in protection against RV that can be deduced from the J chain−/−
model is not in accordance with the observation that lack of IgA synthesis in IgA−/−
mice does not impair protection against secondary RV infection (31
). This discrepancy may result from the fact that J chain−/−
mice have a defect in secretion of both IgM and IgA whereas IgA−/−
mice can produce secretory IgM. In fact, IgM from intestinal human plasma B cells shows accumulations of somatic mutations in the variable region to the same extent as IgA, implying that IgM could functionally replace IgA in mucosa (13
). A compensatory role of IgM in IgA−/−
mice immunized against reovirus (37
) and against CT (16
) has been suggested, as these mice showed increased specific IgM levels in their feces compared to WT mice. However, RV-specific IgM levels could not be detected in the IgA−/−
), nor were they found in our study (Table ). This lack of detection could result from the stability of secretory IgM being lower than that of secretory IgA, rendering IgM detection in feces unreliable (35
). Intestinal IgG secretion was suggested to compensate for IgA absence in the IgA−/−
). However, although immune J chain−/−
mice had higher levels of anti-inner core fecal IgG than WT mice, they were unable to efficiently control the viral challenge. Finally, infection of the IgA−/−
mice, which were bred onto the C57BL/6 genetic background, could elicit immune effectors that compensate for the lack of IgA in the IgA−/−
mice and that may not be elicited in our J chain−/−
BALB/c mice. Although CD4 and CD8 T cells did not seem to be involved in the protection of immune IgA−/−
), subpopulations of intraepithelial T cells or γ/δ T cells could have played a determining compensatory role.
mice have been used in several infectious models to assess whether secretory polymeric immunoglobulins play a dominant role in protection against pathogens. J chain is not required for the cross-protective immunity against influenza A virus (9
) or for protection against herpes simplex virus type 2 disease following immunization with an attenuated virus (18
). Thus, in some instances, lack of J chain does not interfere with the establishment of an efficient immune response to ensure protection of mucosa. By contrast, lack of J chain was associated with a marked decrease in the resistance to a CT challenge in small intestinal ligated loops of orally immunized mice (23
). This finding indicates that epithelial transport of specific IgA and/or IgM can prevent toxin-induced symptoms, i.e., loss of epithelial barrier integrity. In our study, we further show that the epithelial transport of IgM and/or IgA can actually control RV spreading through the intestine.
Other pathways of IgA secretion through the epithelial layer of the intestine have been suggested. J chain−/−
mice show large amounts of monomeric IgA in milk and in nasal and intestinal washes (19
), although the extent of IgA representation in intestinal secretions was not consistent between studies (23
). The accumulation of monomeric IgA in the intestinal lumen may result from passive leakage or from alternative intraepithelial routing of IgA, as several IgA receptors that are not specific for dimeric IgA have been described (29
). In any event, the possible alternative pathways of monomeric IgA secretion in the intestine were not efficient at controlling RV infection, nor were they efficient at preventing CT-induced damages.
Overall, our data support that pIgR-mediated transcytosis of IgA and/or IgM directed to the inner capsid proteins plays a major role in protection against RV infection in an adult mouse model. This finding encourages the development of mucosal vaccination strategies to ensure an optimal defense of the intestine through secretory immunoglobulins. Our study also presents additional data in favor of heterotypic virus-based strategies in the field of RV vaccine design.