Kyeong-Ok Chang

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 µg or 25 µg; (3, 4) three IN immunizations with 2/6 VLP–ISCOM at VLP concentrations of 250 µg or 25 µg (2/6 VLP–ISCOM); (5) three oral immunizations with AttHRV (3×AttHRV); (6) one oral immunization with AttHRV (1×AttHRV); (7) controls (ISCOM matrix and/or diluent). The pigs that received 3×AttHRV 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 3×AttHRV and Att + 2/6 VLP250–ISCOM groups than for the 1×AttHRV 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.

Systemic and intestinal antibody secreting cell responses and protection in gnotobiotic pigs immunized orally with attenuated Wa human rotavirus and Wa 2/6-rotavirus-like-particles associated with immunostimulating complexes

The undesirable side effects and variable efficacy of some oral live rotavirus vaccines in infants have necessitated alternative vaccine approaches. We evaluated a recombinant RFVP2/WaVP6 rotavirus-like-particle (2/6VLP) oral vaccine, using an immunostimulating complex (ISCOM) matrix as adjuvant, in a gnotobiotic (Gn) pig model of human rotavirus (HRV) disease. The 2/6VLPs adhered to the ISCOM-matrix (2/6VLP-ISCOM ) and were antigenic, but they failed to induce protection. However, when combined with attenuated (Att) HRV for oral priming, the 2/6VLP-ISCOM vaccine was effective as a booster and induced partial protection against virulent Wa HRV. The 250 microg 2/6VLP dose was more effective than 100 microg. The highest mean numbers of IgA antibody secreting cells evaluated by ELISPOT in intestinal lymphoid tissues were in pigs receiving AttHRV+2/6VLP-ISCOM or three doses of AttHRV and were associated with the highest protection rates.

Lactogenic antibody responses in cows vaccinated with recombinant bovine rotavirus-like particles (VLPs) of two serotypes or inactivated bovine rotavirus vaccines

Triple-layered virus-like particles (VLPs) were produced in a baculovirus expression system from the two prevalent bovine rotavirus (BRV) serotypes, IND (P[5]G6) and 2292B (P[11]G10). Five groups of pregnant cows were inoculated intramuscularly and intramammarily with IND VLPs [BRV RF VP2, and IND VP4, 6, and 7, 250 microg per dose], 2292B VLPs [RF VP2, Cr VP4 (P[11]), and 2292B VP6 and 7, 250 microg per dose], combined IND/2292B VLPs (125 microg each VLP per dose), inactivated IND BRV (5x10(7)PFU per dose, pre-inactivation), or cell supernatant (mock-controls) in incomplete Freunds adjuvant. Serum, colostrum and milk were collected and tested for isotype-specific antibodies, and homologous and heterologous neutralizing antibodies (VN) to BRV by ELISA and VN tests, respectively. After vaccination, the IgG1 and homologous VN geometric mean antibody titers (GMTs) to BRV in serum of vaccinated groups were significantly (P<0.05) higher than in the mock-controls through postpartum day (PPD) 30. In colostrum, the IgG1 and IgA, and the homologous and heterologous VN GMTs of the IND VLP, 2292B VLP, combined IND/2292B VLP and the inactivated IND groups were significantly enhanced compared to the mock-controls, except for the heterologous VN GMTs in the inactivated IND group. However, the VLP vaccine groups had significantly higher homologous and heterologous VN GMTs than the inactivated IND group. The VN GMTs of the IND/2292B VLP group were statistically similar to the homologous VN GMTs of the IND or 2292B VLP groups, although the IgG1 GMT was lower. In milk, the IgG1 and homologous VN GMTs of the VLP groups were significantly higher than the inactivated IND or the mock-control groups through PPD30. However, the heterologous and homologous VN GMTs of inactivated IND group were statistically similar to the mock-control group at PPD0 and 30, respectively. These results demonstrate that the BRV antibody titers in serum, colostrum and milk are significantly enhanced by the use of triple-layered VLPs and inactivated IND vaccines, but significantly higher antibody responses were observed in the VLP vaccinated cows. The combined IND/2292B VLP vaccine induced comparable VN responses to BRV in serum, colostrum and milk compared to those induced by the individual IND or 2292B VLP vaccines, suggesting that at least two different serotypes can be mixed to confer maximum antibody responses to the incorporated serotypes.

Comparisons of nucleotide and deduced amino acid sequences of NSP4 genes of virulent and attenuated pairs of group A and C rotaviruses.

The NSP4 protein of rotavirus is a nonstructural glycoprotein and has a crucial function in virus morphogenesis during infection of host cells. It was recently reported that NSP4 may also function as a viral enterotoxin in the induction of rotavirus diarrhea by causing Ca++ influx in the cytoplasm of the infected cells. We sequenced and analyzed two (Wa and M strains) pairs of NSP4 genes of virulent (v) and attenuated (a) (after 30 to 40 passages in cell culture) human group A rotaviruses and a pair of NSP4 genes of virulent and attenuated porcine group C rotavirus (Cowden strain). These strains were previously identified as virulent (induce diarrhea) or attenuated (no diarrhea) in a gnotobiotic pig model of rotavirus infection [Bohl et al. (4), Saif et al. (13), Ward et al. (17)]. The NSP4 genes of the Wa, M and Cowden strains were amplified with RT-PCR using a proof reading polymerase (Tli) and the RT-PCR product was sequenced directly. Analysis of the NSP4 deduced amino acid sequences showed that only 3 (Wa) and 2 (M and Cowden) amino acids differed between the virulent and attenuated strains. For the Wa strain, the changes from the virulent to attenuated strain were in amino acids 13 (V to A), 16 (L to S) and 34 (P to L); in the M strain, the difference was in amino acids 53 (T to I) and 104 (K to E), and in the Cowden strains, amino acids 50 (L to F) and 97 (D to N) differed between virulent and attenuated strains. To our knowledge, this is the first sequence comparison between NSP4 of a virulent and attenuated pair of group C rotaviruses. The potential impact of these few amino acid changes on the pathogenesis of the NSP4 protein for piglets is unclear, relative to previous findings in mice (1), but requires further study using purified recombinant NSP4 proteins or peptides.

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.

Antibody-Secreting Cell Responses to Rotavirus Proteins in Gnotobiotic Pigs Inoculated with Attenuated or Virulent Human Rotavirus

ABSTRACT Because of their similarities to infants in mucosal immune responses and their susceptibility to human rotavirus (HRV) diarrhea, gnotobiotic pigs provide a useful model for rotaviral disease. In this study, we performed quantitative enzyme-linked immunospot (ELISPOT) assays to measure local and systemic isotype-specific antibody-secreting cell (ASC) responses to individual structural (VP4, VP6, and VP7) and nonstructural (NSP3 and NSP4) proteins of Wa HRV. TheSpodoptera frugiperda cells expressing each recombinant baculovirus HRV protein were formalin fixed and used as antigen for ELISPOT assays. Neonatal gnotobiotic pigs were orally inoculated once with virulent Wa (WaV) or three times with attenuated Wa (WaA) HRV or mock inoculated (Mock) and then were challenged with virulent Wa (WaV/PC) 28 days after the first inoculation. The ASCs from intestinal and systemic lymphoid tissues of pigs from each group were quantitated by ELISPOT assay at the day of challenge, at postinoculation day 28 (WaV, WaA, and Mock) or at postchallenge day (PCD) 7 (WaV+WaV/PC, WaA+WaV/PC, and Mock+WaV/PC). In all virus-inoculated pigs, regardless of the inoculum, lymphoid tissue, or isotype, VP6 induced the highest numbers of ASCs, followed by VP4; ASCs specific for VP7, NSP3, and NSP4 were less numerous. At challenge, total HRV- and HRV protein-specific immunoglobulin A (IgA) and IgG ASCs in intestinal lymphoid tissues were significantly greater in WaV- than in WaA-inoculated pigs, and WaV pigs were fully protected against diarrhea postchallenge, whereas the WaA pigs were partially protected. At PCD 7, there were no significant differences in ASC numbers for any HRV proteins between the WaV+WaV/PC and WaA+WaV/PC groups.