Intranasal administration of 2/6-rotavirus-like particles with mutant Escherichia coli heat-labile toxin (LT-R192G) induces antibody-secreting cell responses but not protective immunity in gnotobiotic pigs

We investigated the immunogenicity of recombinant double-layered rotavirus- like particle (2/6-VLPs) vaccines derived from simian SA11 or human (VP6) W a and bovine RF (VP2) rotavirus strains. The 2/6-VLPs were administered to gnotobiotic pigs intranasally (i.n.) with a mutant Escherichia coil heat-la bile toxin, LT-R192G (mLT), as mucosal adjuvant, Pigs were challenged with virulent Wa (P1A[8],G1) human rotavirus at postinoculation day (PID) 21 (tw o-dose VLP regimen) or 28 (three-dose VLP regimen). In vivo antigen-activat ed antibody-secreting cells (ASC) (effector B cells) and in vitro antigen-r eactivated ASC (derived from memory B cells) from intestinal and systemic l ymphoid tissues (duodenum, ileum, mesenteric lymph nodes [MLN], spleen, per ipheral blood lymphocytes [PBL], and bone marrow lymphocytes) collected at selected times were quantitated by enzyme-linked immunospot assays. Rotavir us-specific immunoglobulin M (IgM), IgA and IgG ASC and memory B-cell respo nses were detected by PID 21 or 28 in intestinal and systemic lymphoid tiss ues after i.n. inoculation,vith two or three doses of 2/6-VLPs with or with out mLT. Greater mean numbers of virus-specific ASC and memory B cells in a ll tissues prechallenge were induced in pigs inoculated,vith two doses of S A11 2/6-VLPs plus mLT compared to SA11 2/6-VLPs without mLT, After challeng e, anamnestic IgA and IgG ASC and memory B-cell responses were detected in intestinal lymphoid tissues of all VLP-inoculated groups, but serum virus-n eutralizing antibody titers were not significantly enhanced compared to the challenged controls. Pigs inoculated with Wa-RF 2/6-VLPs (with or without mLT) developed higher anamnestic IgA and IgG ASC responses in ileum after c hallenge compared to pigs inoculated with SA11 2/6-VLPs (with or without mL T). Three doses of SA11 2/6-VLP plus mLT induced the highest mean numbers o f IgG memory B cells in MLN, spleen, and PBL among all groups postchallenge . However, no significant protection against diarrhea or virus shedding was evident in any of the 2/6-VLP (with or without mLT)-inoculated pigs after challenge with virulent Wa human rotavirus. These results indicate that 2/6 -VLP vaccines are immunogenic in gnotobiotic pigs when inoculated i.n. and that the adjuvant mLT enhanced their immunogenicity. However, i.n. inoculat ion of gnotobiotic pigs with 2/6-VLPs did not confer protection against hum an rotavirus challenge.