Protective Immunity and Antibody-Secreting Cell Responses Elicited by Combined Oral Attenuated Wa Human Rotavirus and Intranasal Wa 2/6-VLPs with MutantEscherichia coliHeat-Labile Toxin in Gnotobiotic Pigs

Abstract

Two combined rotavirus vaccination regimens were evaluated in a gnotobiotic pig model of rotavirus infection and disease and were compared to previously tested rotavirus vaccination regimens. The first (AttHRV/VLP2؋) involved oral inoculation with one dose of attenuated (Att) Wa human rotavirus (HRV), followed by two intranasal (i.n.) doses of a rotavirus-like particle (2/6-VLPs) vaccine derived from Wa (VP6) and bovine RF (VP2) rotavirus strains. The 2/6-VLPs were coadministered with a mutant Escherichia coli heat-labile toxin, LT-R192G (mLT) adjuvant. For the second regimen (VLP2؋/AttHRV), two i.n. doses of 2/6-VLPs؉mLT were given, followed by one oral dose of attenuated Wa HRV. To compare the protective efficacy and immune responses induced by the combined vaccine regimens with individual rotavirus vaccine regimens, we included in the experiments the following vaccine groups: one oral dose of attenuated Wa HRV (AttHRV1؋ and Mock2؋/AttHRV, respectively), three oral doses of attenuated Wa HRV (AttHRV3؋), three i.n. doses of 2/6-VLPs plus mLT (VLP3؋), three i.n. doses of purified double-layered inactivated Wa HRV plus mLT (InactHRV3؋), mLT alone, and mock-inoculated pigs. The isotype, magnitude, and tissue distribution of antibody-secreting cells (ASCs) in the intestinal and systemic lymphoid tissues were evaluated using an enzyme-linked immunospot assay. The AttHRV/VLP2؋ regimen stimulated the highest mean numbers of intestinal immunoglobulin A (IgA) ASCs prechallenge among all vaccine groups. This regimen induced partial protection against virus shedding (58%) and diarrhea (44%) upon challenge of pigs with virulent Wa HRV. The reverse VLP2؋/AttHRV regimen was less efficacious than the AttHRV/VLP2؋ regimen in inducing IgA ASC responses and protection against diarrhea (25% protection rate) but was more efficacious than VLP3؋ or InactHRV3؋ (no protection). In conclusion, the AttHRV/VLP2؋ vaccination regimen stimulated the strongest B-cell responses in the intestinal mucosal immune system at challenge and conferred a moderately high protection rate against rotavirus disease, indicating that priming of the mucosal inductive site at the portal of natural infection with a replicating vaccine, followed by boosting with a nonreplicating vaccine at a second mucosal inductive site, may be a highly effective approach to stimulate the mucosal immune system and induce protective immunity against various mucosal pathogens.

Cytokine Responses in Gnotobiotic Pigs after Infection with Virulent or Attenuated Human Rotavirus

“…The VirHRV infection caused diarrhea and villous atrophy and 100% rectal and nasal virus shedding, with the presence of viral antigen within villous epithelial cells and infectious rotavirus in the serum (viremia) (4,45). Strong intestinal and systemic B-and T-cell responses were generated against VirHRV, and pigs that recovered from VirHRV infection were fully protected against homotypic rotavirus reinfection (46,49,51). In comparison, AttHRV does not cause diarrhea or villous atrophy (45).…”

“…The pair of virulent and attenuated Wa HRV strains provides a valuable tool to study the relationship between cytokine profiles, kinetics, and magnitude of responses and their relationships to the pathogenicity and immunity induced by these two rotavirus strains that differ in virulence (34,35). The pathogenicity of and immune responses to VirHRV and AttHRV have been well defined in gnotobiotic pigs (3,4,45,46,49,51). The VirHRV infection caused diarrhea and villous atrophy and 100% rectal and nasal virus shedding, with the presence of viral antigen within villous epithelial cells and infectious rotavirus in the serum (viremia) (4,45).…”

“…The immune responses to rotavirus infection or vaccination have been assessed mainly by measuring antibody responses in serum and fecal samples and cellular immunity (3,(48)(49)(50)(51). Only a few investigators have described selected cytokine responses to rotavirus infection and their potential roles in pathogenicity and immunity.…”

Virus-specific intestinal IFN-γ producing T cell responses induced by human rotavirus infection and vaccines are correlated with protection against rotavirus diarrhea in gnotobiotic pigs

“…To improve the efficacy of rotavirus vaccines, an improved understanding of both B and T cell arms of rotavirus protective immunity is essential. The distribution and magnitude of antibodysecreting cell (ASC) and memory B cell responses to virulent (Vir) human rotavirus (HRV) Wa strain (P1A [8]G1), the attenuated (Att) Wa HRV or the combined vaccine regimen of AttHRV oral priming followed by 2/6-virus-like particle (2/6VLP) intranasal (IN) boosting (AttHRV-2/6VLP) have been delineated in our previous studies of gnotobiotic (Gn) pigs [2][3][4][5][6]. The protective role of rotavirus-specific IgA effector and memory B cells in intestinal lymphoid tissues and IgA antibodies in the serum, intestinal contents or feces against rotavirus diarrhea have been indicated in the Gn pig model of HRV diarrhea [3,[5][6][7][8] and in studies of rotavirus natural infection of humans [9][10][11].…”

Viremia and Nasal and Rectal Shedding of Rotavirus in Gnotobiotic Pigs Inoculated with Wa Human Rotavirus

“…Recombinant baculoviruses expressing rotavirus proteins VP2 (bovine RF strain), provided by the late J. Cohen (Virologie Moleculaire et Structurale, UMR CNRS-INRA, Gif-sur-Yvette, France), and VP6 (human Wa strain) were used to produce 2/6 virus-like particles (VLPs) by coinfection of Sf9 (Spodoptera frugiperda) insect cells (6). The virus-like particles were purified as previously described and used at 250 g/dose as a nonreplicating control inoculum (1,39).…”