Antibody responses to human rotavirus (HRV) in gnotobiotic pigs following a new prime/boost vaccine strategy using oral attenuated HRV priming and intranasal VP2/6 rotavirus-like particle (VLP) boosting with ISCOM.

Safer and more effective human rotavirus (HRV) vaccines are needed. We evaluated oral priming with attenuated WaHRV (AttHRV) followed by boosting with two intranasal (IN) doses of VP2/6 virus-like particles (2/6 VLP) with immunostimulating complexes (ISCOM) to determine if this regimen induces protection against diarrhoea and viral shedding in the gnotobiotic pig model. IgM, IgA and IgG antibody titres in serum and intestinal contents were quantified by enzyme-linked immunosorbent assay (ELISA) and serum neutralizing antibody titres were measured by a virus neutralization (VN) test. Seven groups of neonatal gnotobiotic pigs were vaccinated at post-inoculation days (PID) 0, 10 and 21 and challenged with virulent WaHRV at PID 28. The vaccine groups included: (1, 2) oral priming with AttHRV and boosting with two IN immunizations with 2/6 VLP-ISCOM (Att + 2/6 VLP-ISCOM) at VLP concentrations of 250 micro g or 25 micro g; (3, 4) three IN immunizations with 2/6 VLP-ISCOM at VLP concentrations of 250 micro g or 25 micro g (2/6 VLP-ISCOM); (5) three oral immunizations with AttHRV (3xAttHRV); (6) one oral immunization with AttHRV (1xAttHRV); (7) controls (ISCOM matrix and/or diluent). The pigs that received 3xAttHRV or Att + 2/6 VLP250-ISCOM had the highest protection rates against diarrhoea upon challenge at PID 28 with virulent WaHRV. The IgA antibody titres to HRV in intestinal contents were significantly higher in the Att + 2/6 VLP250-ISCOM group than in all other groups prechallenge (PID 28). Serum VN antibody titres were statistically similar after the first inoculation among the groups given AttHRV, but at PID 28 VN antibody titres were significantly higher for the 3xAttHRV and Att + 2/6 VLP250-ISCOM groups than for the 1xAttHRV group suggesting that boosting with 2/6 VLP also boosted VN antibody responses. In humans, intestinal IgA antibodies have been correlated with protection against symptomatic reinfection. Thus the vaccine regimen of one oral dose of AttHRV and two IN immunizations with 2/6 VLP250-ISCOM may be an alternative to multiple-dose live oral vaccines in humans.

Protection and antibody responses to oral priming by attenuated human rotavirus followed by oral boosting with 2/6-rotavirus-like particles with immunostimulating complexes in gnotobiotic pigs.

We evaluated antibody responses and protection induced by attenuated Wa human rotavirus (AttHRV) and VP2/6-rotavirus-like particles (VLP), 100 or 250 microg/dose, with immunostimulating complexes (ISCOM) (VLP/ISCOM) each given orally, alone or sequentially to gnotobiotic pigs. The AttHRV-VLP 250 microg/ISCOM and three-dose-AttHRV (AttHRV3x) groups had significantly higher serum IgA, IgG and intestinal IgA antibody titers to HRV pre-challenge than the three-dose-VLP 100 microg/ISCOM group (VLP/ISCOM3x) and controls (diluent/ISCOMmatrix). Protection rates against viral shedding and diarrhea were highest in the AttHRV-VLP250 microg/ISCOM and AttHRV3x groups, lower in the AttHRV-VLP 100 microg/ISCOM group, with no protection in the VLP/ISCOM3x group and controls. Thus, VLP/ISCOM boosted antibody titers and protection after priming with AttHRV.

Protective immunity and antibody-secreting cell responses elicited by combined oral attenuated Wa human rotavirus and intranasal Wa 2/6-VLPs with mutant Escherichia coli heat-labile toxin in gnotobiotic pigs.

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/VLP2x) 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 (VLP2x/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 (AttHRV1x and Mock2x/AttHRV, respectively), three oral doses of attenuated Wa HRV (AttHRV3x), three i.n. doses of 2/6-VLPs plus mLT (VLP3x), three i.n. doses of purified double-layered inactivated Wa HRV plus mLT (InactHRV3x), 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/VLP2x 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 VLP2x/AttHRV regimen was less efficacious than the AttHRV/VLP2x regimen in inducing IgA ASC responses and protection against diarrhea (25% protection rate) but was more efficacious than VLP3x or InactHRV3x (no protection). In conclusion, the AttHRV/VLP2x 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.

Inhibition of human immunodeficiency virus type 1 production in infected peripheral blood mononuclear cells by human leukocyte antigen class I-specific antibodies: evidence for a novel antiviral mechanism.

A well-characterized mechanism by which anti-HLA class I monoclonal antibodies (MAb) inhibit human immunodeficiency virus type 1 (HIV-1) propagation in in vitro cell cultures is the neutralization of the virus through interactions with HLA molecules associated with the virion envelope. Yet, the possibility that another mechanism of inhibition might affect a postbinding stage of the virus life cycle has been strongly suggested by our previous investigations. To demonstrate that the interaction of MAb B1-1G6 with the light chain of cell surface-expressed HLA class I molecules inhibits a postbinding step of the HIV-1 life cycle, peripheral blood mononuclear cells (PBMCs) were exposed to viruses grown in HLA class I-negative, CD4-positive cells (these viruses, which did not carry HLA class I molecules, cannot be neutralized by anti-HLA MAb during the first round of infection), and PCR was used at various times postexposure to search for the different forms of HIV-1 DNA and RNA in virus-exposed PBMCs cultured in either the presence or [correction of] absence of MAb B1-1G6. Although viral DNA was found in MAb B1-1G6-treated cells, spliced HIV-1 mRNA could not be detected in those cells. In contrast, HIV-1 gene expression was found in HIV-1-infected PBMCs treated with B9-12-1, another HLA class I-specific MAb which prevents infection of cells by cell-free viruses but which fails to inhibit cell-to-cell transmission of HIV-1. These results highlight a second antiviral mechanism by which anti-HLA MAb inhibit in vitro HIV-1 propagation.