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Research with 'Piglet' Has Life-Saving Potential for Human Infants
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14- T. L. To (1), L. A. Ward, L. Yuan and L. J. Saif*. Serum and Intestinal Isotype Antibody Responses and Correlates of Protective Immunity to Human Rotavirus in a Gnotobiotic Pig Model of Disease. J. Gen. Virol, (1998)in press.
Department of Veterinary Preventive Medicine, Ohio Agriculture Research and Development Center, The Ohio State University, Wooster 44691-4096, USA.
(1) Permanent address : Department of Virology, National Institute of Veterinary Research, 61 Truong-Chinh Street, Bach-mai, Hanoi, Vietnam.
We examined the antibody responses and homotypic protection to a heterologous rotavirus in gnotobiotic (Gn) pigs. Pigs were perorally inoculated twice with live attenuated (PO, 2x, group 1), or once with live virulent (PO, 1x, group 2), or twice with BEI-inactivated (PO, 2x, group 3), or intramuscularly inoculated twice with BEI-inactivated (IM, 2x, group 4) Wa strain (G1P1A[8]) human rotavirus (HRV). The pigs were challenged orally at post-inoculation day (PID) 21 with virulent Wa HRV. After challenge, 83% of group 1 pigs, 0% of group 2 pigs, but 100% of groups 3 and 4 pigs shed virus, whereas 29% of group 1 pigs, 86 % of group 2 pigs, 0% of group 3 pigs and only 3% of group 4 pigs were protected against HRV diarrhea. Intramuscular inoculation of pigs with adjuvanted, inactivated-HRV induced high serum anti-HRV antibody GMT in comparison with the other route of inoculation. However, there is no correlation between the degree of protection and the neutralizing antibody GMT of serum, LIC, SIC and RS samples for any groups of pigs. Antibodies to HRV (IgM, IgA, or IgG) were detected in serum and intestinal contents of pigs of all groups after virus inoculation or challenge, but GMT varied among the group for the different samples and at various times assayed. In general, the HRV antibody GMT from intestinal contents, although of lower magnitude, showed similar kinetics to the serum responses. There was a correlation between intestinal content and serum IgM GMT (r =0.77), intestinal content and serum IgA GMT (r =0.71), but no correlation between intestinal content and serum IgG GMT (r =0.37). There was a correlation between serum IgG and VN GMT (r =0.88), but no correlation either between serum IgG and intestinal VN GMT (r =0.12), or between serum and intestinal IgA or IgM and serum and intestinal VN GMTs. There was a correlation (p=0.00001) between protection and serum IgA GMT (r =0.9), intestinal content IgA (r=0.7), and IgG GMT (r = 0.8) for all groups of pigs. The correlation between protection and serum IgA GMT and protection and intestinal IgA GMT of group 2 pigs suggests that serum IgA antibodies to HRV could serve as an indicator for IgA antibody in the intestine after rotavirus infection in humans. The virulent Wa HRV elicited protective immunity and greater intestinal content antibody responses of IgA antibody class compared to live attenuated and inactivated Wa HRV. These findings suggest that more efficient mucosal delivery systems and/or adjuvants are needed to enhance the intestinal immune responses to live attenuated or inactivated HRV, if successful vaccinationtion is to be achieved in neonates.
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13- Yuan L, Kang SY, Ward LA, To TL, Saif LJ. Antibody-secreting cell responses and protective immunity assessed in gnotobiotic pigs inoculated orally or intramuscularly with inactivated human rotavirus. Virol 1998 Jan;72(1):330-338.
Department of Veterinary Preventive Medicine, Ohio Agriculture Research and Development Center, The Ohio State University, Wooster 44691-4096, USA.
Newborn gnotobiotic pigs were inoculated twice perorally (p.o.) (group 1) or intramuscularly (i.m.) (group 2) or three times i.m. (group 3) with inactivated Wa strain human rotavirus and challenged with virulent Wa human rotavirus 20 to 24 days later. To assess correlates of protection, antibody-secreting cells (ASC) were enumerated in intestinal and systemic lymphoid tissues from pigs in each group at selected postinoculation days (PID) or postchallenge days. Few virus-specific ASC were detected in any tissues of group 1 pigs prior to challenge. By comparison, groups 2 and 3 had significantly greater numbers of virus-specific immunoglobulin M (IgM) ASC in intestinal and splenic tissues at PID 8 and significantly greater numbers of virus-specific IgG ASC and IgG memory B cells in spleen and blood at challenge. However, as for group 1, few virus-specific IgA ASC or IgA memory B cells were detected in any tissues of group 2 and 3 pigs. Neither p.o. nor i.m. inoculation conferred significant protection against virulent Wa rotavirus challenge (0 to 6% protection rate), and all groups showed significant anamnestic virus-specific IgG and IgA ASC responses. Hence, high numbers of IgG ASC or memory IgG ASC in the systemic lymphoid tissues at the time of challenge did not correlate with protection. Further, our findings suggest that inactivated Wa human rotavirus administered either p.o. or parenterally is significantly less effective in inducing intestinal IgA ASC responses and conferring protective immunity than live Wa human rotavirus inoculated orally, as reported earlier (L. Yuan, L. A. Ward, B. I. Rosen, T. L. To, and L. J. Saif, J. Virol. 70:3075-3083, 1996). Thus, more efficient mucosal delivery systems and rotavirus vaccination strategies are needed to induce intestinal IgA ASC responses, identified previously as a correlate of protective immunity to rotavirus.
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12- Saif L, Yuan L, Ward L, To T. Comparative studies of the pathogenesis, antibody immune responses, and homologous protection to porcine and human rotaviruses in gnotobiotic piglets. Adv. Exp. Med. Biol, 1997,Department of Veterinary Preventive Medicine, Ohio Agricultural Research and Development Center, Ohio State University, Wooster 44691, USA.
Gnotobiotic piglets serve as a useful animal model for studies of rotavirus pathogenesis and immunity. An advantage over laboratory animal models is the prolonged susceptibility of piglets to rotavirus-induced disease, permitting an analysis of cross-protection and active immunity. Studies from our laboratory of the pathogenesis of human rotavirus infections in gnotobiotic piglets have confirmed that villous atrophy is induced in piglets given virulent but not attenuated human rotavirus (Wa strain) and have revealed that factors other than villous atrophy may contribute to the early diarrhea induced. To facilitate and improve rotavirus vaccination strategies, it is important to identify correlates of protective immunity. Comparison of antibody immune responses induced by infection with virulent porcine and human rotaviruses (mimic host response to natural infection) with those induced by live attenuated human rotavirus (mimic attenuated oral vaccines) in the context of homotypic protection has permitted an analysis of correlates of protective immunity. Our results indicate that the magnitude of the immune response is greatest in lymphoid tissues adjacent to the site of viral replication (small intestine). Secondly there was a direct association between the degree of protection induced and the level of the intestinal immune response, with primary exposure to virulent rotaviruses inducing significantly higher numbers of IgA ASC and complete protection against challenge. These studies thus have established basic parameters related to immune protection in the piglet model of rotavirus-induced disease, verifying the usefulness of this model to apply new strategies for the design and improvement of rotavirus vaccines.
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11- Ward LA, Yuan L, Rosen BI, To TL, Saif LJ. Development of mucosal and systemic lymphoproliferative responses and protective immunity to human group A rotaviruses in a gnotobiotic pig model. Clin Diagn Lab Immunol 1996 May;3(3):342-350.
Department of Veterinary Preventive Medicine, Ohio Agricultural Research and Development Center, Ohio State University, Wooster 44691-4096, USA.
Gnotobiotic pigs were orally inoculated with virulent Wa strain (G1P1A[8]) human rotavirus (group 1), attenuated Wa rotavirus (group 2) or diluent (controls) and were challenged with virulent Wa rotavirus 21 days later. On various postinoculation or postchallenge days, virus-specific responses of systemic (blood and spleen) and intestinal (mesenteric lymph node and ileal lamina propria) mononuclear cells (MNC) were assessed by lymphoproliferative assays (LPA). After inoculation, 100% of group 1 pigs and 6% of group 2 pigs shed virus. Diarrhea occurred in 95, 12, and 13% of group 1, group 2, and control pigs, respectively. Only groups 1 and 2 developed virus-specific LPA responses prior to challenge. Group 1 developed significantly greater mean virus-specific LPA responses prior to challenge and showed no significant changes in tissue mean LPA responses postchallenge, and 100% were protected against virulent virus challenge. By comparison, both group 2 and controls had significantly lower LPA responses at challenge and both groups showed significant increases in mean LPA responses postchallenge. Eighty-one percent of group 2 and 100% of control pigs shed challenge virus, and both groups developed diarrhea that was similar in severity postchallenge. The virus-specific LPA responses of blood MNC mirrored those of intestinal MNC, albeit at a reduced level and only at early times postinoculation or postchallenge in all pigs. In a separate study evaluating antibody-secreting-cell responses of these pigs (L. Yuan, L.A. Ward, B.I. Rosen, T.L. To, and L.J. Saif, J. Virol. 70:3075-3083, 1996), we found that the magnitude of a tissue's LPA response positively correlated with the numbers of virus-specific antibody-secreting cells for that tissue, supporting the hypothesis that the LPA assesses T-helper-cell function. The magnitude of LPA responses in systemic and intestinal tissues also strongly correlated with the degree of protective immunity elicited by the inoculum (p = 0.81). We conclude that blood may provide a temporary "window" for monitoring intestinal T cells and that the LPA can be used to assess protective immunity to human rotaviruses.
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10- Yuan L, Ward LA, Rosen BI, To TL, Saif LJ. Systematic and intestinal antibody-secreting cell responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease. J Virol 1996 May;70(5):3075-3083.
Food Animal Health Research Program, Department of Veterinary Preventive Medicine, Ohio Agricultural Research and Development Center, Ohio State University, Wooster 44691-4096, USA.
Neonatal gnotobiotic pigs orally inoculated with virulent (intestinal-suspension) Wa strain human rotavirus (which mimics human natural infection) developed diarrhea, and most pigs which recovered (87% protection rate) were immune to disease upon homologous virulent virus challenge at postinoculation day (PID) 21. Pigs inoculated with cell culture-attenuated Wa rotavirus (which mimics live oral vaccines) developed subclinical infections and seroconverted but were only partially protected against challenge (33% protection rate). Isotype-specific antibody-secreting cells (ASC were enumerated at selected PID in intestinal (duodenal and ileal lamina propria and mesenteric lymph node [MLN]) and systemic (spleen and blood) lymphoid tissues by using enzyme-linked immunospot assays. At challenge (PID 21), the numbers of virus-specific immunoglobulin A (IgA) ASC, but not IgG ASC, in intestines and blood were significantly greater in virulent-Wa rotavirus-inoculated pigs than in attenuated-Wa rotavirus-inoculated pigs and were correlated (correlation coefficients: for duodenum and ileum, 0.9; for MLN, 0.8; for blood, 0.6) with the degree of protection induced. After challenge, the numbers of IgA and IgG virus-specific ASC and serum-neutralizing antibodies increased significantly in the attenuated-Wa rotavirus-inoculated pigs but not in the virulent-Wa rotavirus-inoculated pigs (except in the spleen and except for IgA ASC in the duodenum). The transient appearance of IgA ASC in the blood mirrored the IgA ASC responses in the gut, albeit at a lower level, suggesting that IgA ASC in the blood of humans could serve as an indicator for IgA ASC responses in the intestine after rotavirus infection. To our knowledge, this is the first report to study and identify intestinal IgA ASC as a correlate of protective active immunity in an animal model of human-rotavirus-induced disease.
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9- Saif LJ, Ward LA, Yuan L, Rosen BI, To TL. The gnotobiotic piglet as a model for studies of disease pathogenesis and immunity to human rotaviruses. Arch Virol Suppl 1996;12:153-161.
Ohio Agricultural Research and Development Center, Ohio State University, Wooster, Ohio, USA.
Gnotobiotic piglets serve as a useful animal model for studies of human rotavirus infections, including disease pathogenesis and immunity. An advantage of piglets over laboratory animal models is their prolonged susceptibility to human rotavirus-induced disease, permitting cross-protection studies and an analysis of active immunity. Major advances in rotavirus research resulting from gnotobiotic piglet studies include: 1) the adaptation of the first human rotavirus to cell culture after passage and amplification in piglets; 2) delineation of the independent roles of the two rotavirus outer capsid proteins (VP4 and VP7) in induction of neutralizing antibodies and cross-protection; and 3) recognition of a potential role for a nonstructural protein (NSP4) in addition to VP4 and VP7, in rotavirus virulence. Current studies of the pathogenesis of group A human rotavirus infections in gnotobiotic piglets in our laboratory have confirmed that villous atrophy is induced in piglets given virulent but not cell culture attenuated human rotavirus (G1, P1A, Wa strain) and have revealed that factors other than villous atrophy may contribute to the early diarrhea induced. A comprehensive examination of these factors, including a proposed role for NSP4 in viral-induced cytopathology, may reveal new mechanisms for induction of viral diarrhea. Finally, to facilitate and improve rotavirus vaccination strategies, our current emphasis is on the identification of correlates of protective active immunity in the piglet model of human rotavirus-induced diarrhea. Comparison of cell-mediated and antibody immune responses induced by infection with a virulent human rotavirus (to mimic host response to natural infection) with those induced by a live attenuated human rotavirus (to mimic attenuated oral vaccines) in the context of homotypic protection has permitted an analysis of correlates of protective immunity. Results of these studies have indicated that the magnitude of the immune response is greatest in lymphoid tissues adjacent to the local site of viral replication (small intestine). Secondly, there was a direct correlation between the degree of protection induced and the level of the intestinal immune response, with significantly higher local immune responses and complete protection induced only after primary exposure to virulent human rotavirus. These studies thus have established basic parameters related to immune protection in the piglet model of human rotavirus-induced disease, verifying the usefulness of this model to examine new strategies for the design and improvement of human rotavirus vaccines.
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8- To LT, Bernard S, Aynaud JM Transmissible gastroenteritis coronavirus: the development and applications of the fixed-cell immunoperoxidase technique. Rev Sci Tech 1993 Jun;12(2):537-558.Since the first demonstration in 1971 that solid-phase enzyme-linked immunosorbent assays (ELISAs) could be used for the quantitative determination of antigens and antibodies, this method has been widely applied in serodiagnosis of parasitic and infectious diseases. In addition to the classic ELISA variants using antigen or antibody to coat the plastic plates, there has recently been growing interest in the application of fixed-cell ELISA to research and diagnostic work on viral diseases. The authors discuss the development and applications of this technique to basic research and diagnosis of transmissible gastroenteritis, a highly contagious disease of swine. The success of this technique, as the name suggests, is largely due to the use of a suitable fixative, which preserves the antigenicity of the neo-synthesised viral proteins, and the presence of optimal conditions for viral antigen synthesis. In addition, various parameters are optimised, and this is discussed with reference to transmissible gastroenteritis virus. These parameters would help veterinarians and research workers to develop this technique in their own laboratories.
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7- To LT, Bernard S Effect of fixation on the detection of transmissible gastroenteritis coronavirus antigens by the fixed-cell immunoperoxidase technique. J Immunol Methods 1992 Oct 2;154(2):195-204The effect of various fixatives and detergents on the in vitro detection of the viral determinants which are expressed in swine testis cells infected with the transmissible gastroenteritis coronavirus (TGEV) was studied using a microwell immunoperoxidase technique. When compared with glutaraldehyde and formaldehyde, 0.1% paraformaldehyde was found to be the fixative of choice for the detection of these determinants on the membranes of infected cells. Among dehydrating fixatives, 80% acetone or a mixture of acetone and ethanol or of acetone, methanol and ethanol were found to be the best fixatives for the detection of these viral determinants which are expressed in infected cells. In the case of acetone, the temperature of fixation and its concentration in the fixative preparation were found to be important. The treatment of 0.05% glutaraldehyde-fixed, infected cells with 0.1% saponin or 0.1% paraformaldehyde-fixed, infected cells with 1%NP-40 led to satisfactory detection of viral determinants. Using Triton X-100 to render cells permeable, the quantities of N and M antigen detected in TGEV-infected cells prefixed with either 0.05% glutaraldehyde or 0.1% paraformaldehyde were equal to those of 80% acetone-fixed, TGEV-infected cells while the quantity of S antigen detected was diminished. The effect of other detergents such as zwittergent, empigen BB, Chaps and N-lauroylsarcosine on the detection of viral determinants was also studied.
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6- To LT, Bernard S, Bottreau E Transmissible gastroenteritis coronavirus: surface antigens induced by virulent and attenuated strains. Res Virol 1992 Jul;143(4):241-248.Three strains of the transmissible gastroenteritis virus (TGEV) possessing different degrees of pathogenicity for piglets were examined for their capacity to express M and S glycoproteins on the infected cell surface using a microwell immunoperoxidase test. These two viral glycoproteins were easily detected on the plasma membrane of 0.1% paraformaldehyde-fixed swine testis (ST) or pig kidney (RP.D) cells which were infected with high-passaged Purdue-115 and low-passaged D-52 strains and a high-passaged attenuated (188-SG) mutant of TGEV. No significant differences were found between attenuated and virulent strains with regard to the viral antigen expression on the membrane of infected cells over a 14-h period.
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5- To LT, Bernard S, Lantier I Fixed-cell immunoperoxidase technique for the study of surface antigens induced by the coronavirus of transmissible gastroenteritis (TGEV). Vet Microbiol 1991 Nov;29(3-4):361-368.
An immunoperoxidase technique performed on the TGEV-infected cells was developed for detection of virus-induced antigens. The presence of M antigen of TGEV on the surface of infected cells was demonstrated by this technique. This finding is in contrast to the M protein of murine hepatitis coronavirus which migrates to the Golgi apparatus but is not transported to the plasma membrane. The time course of appearance M and S antigens on the surface of TGEV-infected cell can be studied by this technique.
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Sorry, the abstracts will be added later 4-Tran, M.C.,To, L.T., Le, T.T. Recherches des proprieres du virus vaccine (souche Janson) cultive sur oeufs embryonnes de poule et son efficacite vis a vis de la souche virulente vietnamienne de la peste de canard . Revue de Medecine Veterinaire INRV, 1983, n° 3, 12-19.
3-Tran, M.C.,To, L.T., Le T.N. Recherches des proprieres biologiques et immunologiques du virus vaccine (souche TN) et sa possibilite d'utilisation dans la protection des canetons contre l'hepatite du caneton. Revue de Medecine Veterinalre INRV, 1984, n° 5, 14-20.
2-Vu, N.C., Kieu, T.D., Dao, T.D.,To, L.T. Diagnostic de la maladie d'Aujeszky chez les porcs au Vietnam par la seroneutralisation en culture cellulaire. Revue de Medecine Veterinaire INRV, 1985, n 3,13-16.
1-To, L.T., Vu, N.C., Dao, T.D. Symptome clinique chez les porcs positifs vis a vis du virus Aujeszky. Revue de Medecine Veterinaire INRV, 1985, n° 4, 1-7.
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