Antibody-secreting cell responses and protective immunity assessed in gnotobiotic pigs
inoculated orally or intramuscularly with inactivated human rotavirus.
Yuan L, Kang SY, Ward LA, To TL, Saif LJ
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.
Antigenic characteristics of polypeptides of Coxiella burnetii isolates.
To H, Hotta A, Zhang GQ, Nguyen SV, Ogawa M, Yamaguchi T, Fukushi H, Amano K, Hirai K
Department of Veterinary Microbiology, Faculty of Agriculture, Gifu University, Japan.
Eighteen Coxiella burnetii strains from a variety of clinical and geographical sources were screened for antigenic variation of polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with Coomassie brilliant blue (CBB) staining or immunoblotting. These polypeptide profiles showed the greatest variability in the region from 33 to 8.1 kDa. Such differences in the antigenicity of the polypeptides were also recognized by immunoblotting with 15 various mouse anti-C. burnetii antisera. In addition, we detected a polypeptide at about 28 kDa which was immunodominant in strains from human cases of acute Q fever, milk and ticks but not immunogenic in strains from human cases of chronic Q fever. These findings suggest that this polypeptide is a marker to distinguish between acute and chronic strains.
Clinical evaluation of a new PCR assay for detection of Coxiella burnetii in human serum samples.
Zhang GQ, Nguyen SV, To H, Ogawa M, Hotta A, Yamaguchi T, Kim HJ, Fukushi H, Hirai K
Department of Veterinary Microbiology, Faculty of Agriculture, Gifu University, Japan.
A nested PCR method was developed for the detection of Coxiella burnetii in human serum samples. Two pairs of oligonucleotide primers were designed to amplify a 438-bp fragment of the com1 gene encoding a 27-kDa outer membrane protein of C. burnetii. The primers amplified the predicted fragments of 21 various strains of C. burnetii but did not react with DNA samples from other microorganisms. The 438-bp amplification products could be digested with restriction enzymes SspI and SalI. The utility of the nested PCR was evaluated by testing human serum samples. The com1 gene fragment was amplified from 135 (87%) of 155 indirect immunofluorescence test (IF)-positive serum samples and from 11 (11%) of 100 IF-negative serum samples. The nested PCR with primers targeted to the com1 gene appeared to be a sensitive, specific, and useful method for the detection of C. burnetii in serum samples.
The specific variable domain of camel heavy-chain antibodies is encoded in the germline.
Nguyen VK, Muyldermans S, Hamers R
Department Ultrastructure, Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Belgium.
The variable domains of the functional heavy-chain antibodies (VHHs) discovered in camels are related to the human VH subgroup III. They are nevertheless clearly distinguishable from the VHs of conventional four-chain immunoglobulins by the presence of important amino acid substitutions, located in the solvent-exposed surface normally covered by the variable domain of the light chain. The analysis of an unrearranged dromedary DNA library revealed that the specific VHH gene with its characteristic amino acid substitutions is encoded in the germline. Therefore, it is concluded that the VHHs do not arise through an ontogenic process of somatic hypermutation. The presence of putative DNA recombination signals that are more prevalent in the camel VHH, compared to the VH germline gene, might play a role in the formation and efficient expansion of the VHH repertoire.
Comparison of llama VH sequences from conventional and heavy chain antibodies.
Vu KB, Ghahroudi MA, Wyns L, Muyldermans S
Department Ultrastructure, Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Belgium.
Forty different PCR clones encoding a llama variable heavy chain domain were analysed. The majority of these clones are derived from heavy-chain antibody cDNA in which the entire CH1 exon is absent. It appears from the amino acid within the VHH framework 1 and 3 that all the llama clones belong to the VH III family. However, the individual llama VHH sequences differ more substantially from each other than expected for members of the same family. Several remarkable amino acid substitutions in the framework 2 hinder the proper association of the VL. However, they lay the foundation for the secretion from the endoplasmic reticulum and good solubility behaviour of llama H2 antibodies. The repertoire of the llama VHHs may be extensive due to the presence of a long CDR3-loop, often constrained by a disulfide bridge and the occurrence of H1 and H2 loop conformations not yet encountered in mice or human VHs. The variability plot of the amino acids in the VHH shows that the first hypervariable region coincides with the structural H1 loop in contrast to the situation found in mice and man where the CDR1 and H1 are slightly offset. We propose that the amino acids of the llama H1 loop participate actively in the antigen binding. All these observations are characteristic for the llama VHHs of the homodimeric heavy-chain H2 antibodies, but are not maintained in the llama clones from conventional heterotetrameric H2L2 immunoglobulins.
Evaluation of the high-density agglutination test for Coxiella burnetii antibodies in animals.
Nguyen SV, To H, Minamoto N, Ogawa M, Yamaguchi T, Fukushi H, Hirai K
Department of Veterinary Microbiology, Faculty of Agriculture, Gifu University, Japan.
The usefulness of the high-density particle agglutination (HDPA) test as a potential tool for the detection of anti-Coxiella burnetii antibodies in animal sera was studied by using 619 cow, 589 dog, and 150 cat serum samples and antisera from rabbits, guinea pigs, and mice. The sensitivity and specificity of the test versus those of the reference microimmunofluorescence test were determined at two different threshold titer values. At the cutoff value of 1:16, the sensitivities of the HDPA test for cow, dog, and cat sera were 94.3, 95, and 91.3%, respectively, and the specificities were 95.5, 95.3, and 91.3%, respectively. At the cutoff value of 1:32, the sensitivities were 86.7, 88.3, and 82.6%, respectively, and the specificities were 99, 99.2, and 98.4%, respectively. For the group of immune laboratory animals all samples from rabbits, guinea pigs, and mice were positive by the HDPA test. The erythrocyte-sensitizing substance from phase II C. burnetii was found to contain protein and carbohydrate, and both fractions are immunoreactive. The study results show that the HDPA test is a useful tool in the epizootiological survey of Coxiella infection in animals.
Comparative studies of the pathogenesis, antibody immune responses, and homologous
protection to porcine and human rotaviruses in gnotobiotic piglets.
Saif L, Yuan L, Ward L, To T
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.
Rapid method for detection of Coxiella burnetii antibodies using high-density particle agglutination.
Nguyen SV, Otsuka H, Zhang GQ, To H, Yamaguchi T, Fukushi H, Noma A, Hirai K
Department of Veterinary Microbiology, Faculty of Agriculture, Gifu University, Japan.
A high-density particle agglutination test, using erythrocyte-sensitizing substance from phase II Coxiella burnetii adsorbed to high-density composite particles, was developed for rapid serodiagnosis of Q fever. The test was compared with the microimmunofluorescence test for sensitivity and specificity by using 3,036 human serum samples collected in Gifu Prefecture, Japan. An excellent agreement was found between the two tests for the acute-phase group and paired serum samples, but some discordant results were observed in the single-sample group. The sensitivity and specificity of the high-density particle agglutination test were both 100% in the former group and 81.6 and 99.9%, respectively, in the latter group. The test is a very promising tool for routine serodiagnosis of Q fever because of its simplicity, sensitivity, and specificity.
Development of mucosal and systemic lymphoproliferative responses and protective
immunity to human group A rotaviruses in a gnotobiotic pig model.
Ward LA, Yuan L, Rosen BI, To TL, Saif LJ
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.
Systematic and intestinal antibody-secreting cell responses and correlates of protective
immunity to human rotavirus in a gnotobiotic pig model of disease.
Yuan L, Ward LA, Rosen BI, To TL, Saif LJ
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.
Q fever pneumonia in children in Japan.
To H, Kako N, Zhang GQ, Otsuka H, Ogawa M, Ochiai O, Nguyen SV, Yamaguchi T, Fukushi H, Nagaoka N, Akiyama M, Amano K, Hirai K
Department of Veterinary Microbiology, Faculty of Agriculture, Gifu University, Japan.
The prevalence of Q fever pneumonia among children with atypical pneumonia from whom only an acute-phase serum sample was available was traced by using an indirect immunofluorescence (IF) test, nested PCR, and isolation. Twenty (34.5%) of 58 sera were found to have both polyvalent and immunoglobulin M antibodies to the phase II antigen of Coxiella burnetii by the IF test. Q fever pneumonia was present in 23 (39.7%) of 58 patients as determined by both the nested PCR and isolation and in 20 patients as determined by the IF test. The sensitivities for nested PCR and isolation were 100%, and that for the IF test was 87%. Our results indicate that nested PCR was faster and more sensitive than isolation and the IF test in the diagnosis of acute Q fever when a single acute-phase serum was available. These findings suggest that C. burnetii is an important cause of atypical pneumonia in children in Japan.
The gnotobiotic piglet as a model for studies of disease pathogenesis and immunity to human
rotaviruses.
Saif LJ, Ward LA, Yuan L, Rosen BI, To TL
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.
Transmissible gastroenteritis coronavirus: the development and applications of the fixed-cell
immunoperoxidase technique.
To LT, Bernard S, Aynaud JM
National Institute of Veterinary Research, Department of Virology, Hanoi, Vietnam.
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.
Effect of fixation on the detection of transmissible gastroenteritis coronavirus antigens by the
fixed-cell immunoperoxidase technique.
To LT, Bernard S.
National Institute of Veterinary Research, Department of Virology, Hanoi, Vietnam.
The 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.
Transmissible gastroenteritis coronavirus: surface antigens induced by virulent and
attenuated strains.
To LT, Bernard S, Bottreau E
National Institute of Veterinary Research, Department of Virology, Hanoi, Vietnam.
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.
Fixed-cell immunoperoxidase technique for the study of surface antigens induced by the
coronavirus of transmissible gastroenteritis (TGEV).
To LT, Bernard S, Lantier I
National Institute of Veterinary Research, Department of Virology, Hanoi, Vietnam.
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.
