Transgenic plants have been tested as an alternative host for the production and delivery of experimental oral vaccines. Here, we developed transgenic potatoes that express the major antigenic sites A and D of the glycoprotein S from transmissible gastroenteritis coronavirus (TGEV-S0.7) under three expression vector systems. The DNA integration and mRNA expression level of the TGEV-S0.7 gene were confirmed in transgenic plants by PCR and northern blot analysis. Antigen protein expression in transgenic potato was determined by western blot analysis. Enzyme-linked immunosorbent assay results revealed that based on a dilution series of Escherichia coli-derived antigen, the transgenic line P-2 had TGEV-S0.7 protein at levels that were 0.015% of total soluble proteins. We then examined the immunogenicity of potato-derived TGEV-S0.7 antigen in mice. Compared with the wild-type potato treated group and synthetic antigen treated group, mice treated with the potato-derived antigen showed significantly higher levels of immunoglobulin (Ig) G and IgA responses.

Passive protection provided by sows inoculated with the virulent Miller strain of transmissible gastroenteritis virus (TGEV), or the ISU-1 strain of porcine respiratory coronavirus (PRCV), or both was evaluated in nursing pigs challenge exposed with virulent TGEV. Four sows (group B) were inoculated with PRCV oronasally twice at 4 and 2 weeks before parturition; 1 sow (group C) was inoculated similarly, but in 2 subsequent pregnancies; and 2 sows (group D) were oronasally primed with PRCV at 4 weeks before parturition, and 2 weeks later were administered a booster inoculation of virulent TGEV. Two additional sows (group E) remained uninoculated and served as seronegative controls, and 1 sow (group A) that had been naturally infected with TGEV served as a seropositive control. The degree of passive immunity transferred by these sows to their litters was assessed by challenge exposing the pigs of sows in groups BE (only the second litter of group C) with virulent TGEV at 3 to 5 days of age. After challenge exposure, clinical signs of infection and mortality were noted and fecal and nasal shedding of virus was assessed by ELISA. The IgA, IgG, and IgM antibody titers to TGEV were quantified in colostrum and milk of the sows by use of an isotype-specific monoclonal antibody-capture ELISA, using biotinylated monoclonal antibodies against each porcine isotype as detecting reagents. A plaque-reduction assay was used to quantify neutralizing antibody titers in serum, colostrum, milk, and fractionated whey (IgG and IgA/IgM). In the sow naturally infected with TGEV (group A), there was a pronounced decrease in IgG antibody titers to TGEV in the transition from colostrum to milk, and IgA TGEV antibodies became predominant, with high titers maintained throughout lactation. The 4 group-B sows partially protected their pigs after TGEV challenge exposure; mean mortality was 67%, compared with 100% in pigs suckling the 2 TGEV seronegative control sows (group-E litters). Although IgA TGEV antibodies were detected in colostrum and milk of group-B sows, IgG TGEV antibodies were the most abundant. The sow of group C had a marked increase in IgA TGEV antibody titers in colostrum and milk after reinoculation with PRCV during the second pregnancy, before TGEV challenge exposure of the litter. Its pigs were passively protected to a high degree after TGEV challenge exposure (27% litter mortality). The sows in group D, primed with PRCV and boosted with TGEV, provided the best passive protection after TGEV challenge exposure of their pigs. Not only litter mortality (27%) but also morbidity was reduced, compared with those factors for the other challenge exposed litters, and the sows did not become ill. In these swine, the high degree of passive protection observed could not be associated with the presence of only IgA TGEV antibodies in the milk, but high IgM TGEV antibody titers also were detected in colostrum and milk. Results of this study suggest that PRCV-inoculated sows are able to partially protect their pigs from TGEV challenge exposure and, on the basis of preliminary data, the degree of protection may increase after multiple PRCV exposures or after secondary exposure to TGEV during pregnancy. Also, an IgA respiratory tract-mammary gland link may exist as evident by the low titer of IgA TGEV antibodies in the milk of PRCV-inoculated sows, but may not be as efficient in inducing lactogenic IgA immunity as is the gastrointestinal tract-mammary gland link.

Cell-mediated immunity was evaluated in intestinal, respiratory, and systemic lymphoid tissues of pigs exposed when 11 days old to virulent transmissible gastroenteritis virus (TGEV), attenuated TGEV, or porcine respiratory coronavirus (PRCV), 3 antigenically related porcine coronaviruses with distinct enteric and respiratory tissue tropisms. Mononuclear cells were prepared from mesenteric lymph nodes (MLN), bronchial lymph nodes (BLN), and spleens of pigs and tested for virus-specific responses by use of lymphocyte proliferation assays. Vigorous MLN and BLN proliferation responses to virulent TGEV and PRCV, respectively, at postinoculation days 8 to 24 were strongly associated with prior detection of TGEV in rectal swab samples and PRCV in nasal swab samples. Gastrointestinal disease and intestinal virus replication, assessed on the basis of rectal virus shedding, were almost exclusively found in the virulent TGEV-inoculated pigs, even though virulent TGEV and a high dose of attenuated TGEV elicited the highest proliferation responses in MLN. Pigs exposed to PRCV or attenuated TGEV did not have clinical signs of disease, and only 1 pig given a high dose of attenuated TGEV shed virus in feces. Porcine respiratory coronavirus replicated in the respiratory tract after either oronasal or aerosol inoculation of virus and induced strong BLN, but not MLN, proliferation responses. A high dose of attenuated TGEV (4 X 10(8) plaque-forming units) was more effective than a lower dose of attenuated TGEV (7 X 10(6) plaque-forming units) in eliciting significant lymphocyte proliferation in MLN and BLN. Cellular immune function, assessed on the basis of mitogen-induced proliferation of lymphocytes, was comparable for all 3 sources of lymphocytes and was not adversely affected by exposure to any of the pigs. The tissue tropism of TGEV and PRCV was associated with induction of virus-specific cell-mediated immune responses, as evidenced by substantial lymphocyte proliferation responses in MLN and BLN, mucosa-associated lymph nodes adjacent to the primary sites of virus replication. The failure of PRCV strain ISU-1 to replicate in the intestinal tract correlated with poor virus-specific cellular immune responses in MLN.

Hybridomas secreting monoclonal antibodies (MAB) to transmissible gastroenteritis virus (TGEV) were produced by fusion of SP2/0 myeloma cells and splenic lymphocytes of BALB/c mice immunized with the virulent cell-passaged Miller strain of TGEV. The MAB secreted by these hybridomas were partially characterized; 4 of them (MA4, MA5, MH11, MB2) had high-neutralization titer for TGEV. The remaining 7 (MC6, MD9, ME5, MG5, MF2, ME9, MG7) did not neutralize TGEV at 1:25 dilution. All 4 neutralizing and 2 of the nonneutralizing MAB reacted with the E2 protein of TGEV in a radioimmunoprecipitation assay. The remaining 5 MAB reacted with the E1 protein of TGEV. Reactivity of the MAB was tested in an indirect immunofluorescent assay with 3 cell culture-adapted strains of TGEV (Miller, Purdue, and Illinois) and 13 wild-type isolates of TGEV. Neutralizing MAB reacted with all 13 wild-type isolates and the 3 cell culture-adapted strains of TGEV. In contrast, nonneutralizing MAB that reacted with the Miller strain of TGEV varied in their reactivity with the wild-type TGEV isolates. Reactivity of neutralizing MAB was also tested, using plaque-reduction neutralization assays with Miller, Purdue, and Illinois strains and 5 wild-type isolates. All 4 neutralizing MAB neutralized the 8 virus isolates, but the neutralization titer was higher with the homologous virus than with the heterologous virus isolates. However, neutralization titers of the 4 neutralizing MAB were 4 to 16 times higher for the homologous Miller strain of TGEV than for the heterologous Illinois and Purdue strains, and were 4 to 1,000 times higher than for the wild-type isolates. Extensive antigenic heterogeneity was observed among TGEV isolates on epitopes recognized by the nonneutralizing MAB directed against either E1 or E2 protein.

Two federally licensed attenuated live transmissible gastroenteritis (TGE) virus vaccines (an IM vaccine and an oral-IM vaccine) and 1 nonlicensed nonattenuated live TGE virus vaccine were evaluated and compared in sows free of TGE virus-neutralizing antibodies. Litters from the sows were challenge exposed at 3 and 5 days of age, and results were combined according to the vaccine administered to the sows. The survivability of pigs suckling sows vaccinated with the nonattentuated vaccine was significantly (P less than 0.01) greater than that of pigs suckling sows vaccinated with the IM attenuated vaccine, significantly (P less than 0.05) greater than that of pigs suckling sows vaccinated with the oral-IM attenuated vaccine, and significantly (P less than 0.05) greater than that of pigs suckling sows that had not been vaccinated. The differences, however, between survivability of litters from sows vaccinated with the IM attentuated vaccine or the oral-IM attenuated vaccine and that of litters from the sows not vaccinated were not significant (P greater than 0.10). The nonattenuated TGE vaccine, although giving a higher level of protection than the attenuated vaccine was eventually overwhelmed. Dexamethasone did not increase the incidence of diarrhea, and levamisole did not potentiate the lactogenic immunity in sows after given their first dose of the nonattenuated vaccine. Survivability in litters suckling sows that developed diarrhea after given their first dose of the nonattenuated vaccine was not greater than that in litters suckling sows that did not develop diarrhea. The best results were obtained when 3-day-old suckling pigs were challenge exposed with virulent TGE virus.

Monoclonal antibodies (MAB) to subsite A (25C9) and subsite D (44C11) of the S protein of transmissible gastroenteritis virus (TGEV) were used in a blocking ELISA on fixed TGEV-infected swine testis cells to differentiate sera from pigs experimentally inoculated with either TGEV or porcine respiratory coronavirus (PRCV). Serum samples were obtained from pigs at various intervals from postinoculation day (PID) 0 through at least PID 22 to 40. Eleven-day-old pigs, seronegative for TGEV-neutralizing antibodies at the time of inoculation, were inoculated orally and nasally with either the virulent Miller (M5C) strain or the attenuated Purdue (P115) strain of TGEV, or with the ISU-1 strain of PRCV. Gastroenteritis was observed in 100% of the M5C-TGEV-inoculated pigs; but clinical signs of disease were not observed in either the P115-TGEV- or PRCV-inoculated pigs. Virus-neutralization (VN) antibody titer in sera was determined by use of a plaque-reduction assay. Blocking ELISA antibody titer for subsites A and D was determined from the serum dilution that produced 50% reduction in the absorbance values when it competed with biotinylated MAB 25C9 and 44C11, respectively. In sera from the inoculated pigs, the VN antibody titer began to increase by PID 7 and reached maximum by PID 15 to 16. For pigs inoculated with TGEV M5C, subsite A and subsite D blocking antibody titers in the serum paralleled the VN antibody titer, began to increase after PID 7, and reached maximum by PID 15 to 16. The blocking antibody titer to subsites A and D began to increase in the P115-TGEV-inoculated pigs after PID 15 to 16 and reached maximum by PID 22 to 26. Blocking antibody titer to subsite A in PRCV-inoculated pigs behaved similarly to blocking antibody titer to subsite A in the M5C-TGEV-inoculated pigs, reaching maximum by PID 15 to 16; however, blocking antibody titer was not detected for subsite D up to PID 24 (the latest time point examined) in sera from the PRCV-inoculated pigs. Serum antibody responses and clinical signs of disease were monitored in pigs initially inoculated with either M5C-TGEV or -PRCV and challenge-exposed with M5C-TGEV on PID 24. Clinical signs of gastroenteritis were not observed in the M5C-TGEV-inoculated pigs after challenge-exposure with M5C-TGEV. Low increases in VN antibody titer and in subsite A or D blocking antibody titer were detected in the M5C-TGEV-inoculated and challenge-exposed pigs. Of the 12 pigs initially inoculated with PRCV then challenge-exposed with M5C-TGEV, 5 pigs developed diarrhea; the VN and subsite A antibody blocking titers began to increase by postchallenge-exposure day (PCD) 2 and reached maximal titer by PCD 9, increasing approximately 100-fold above the prechallenge-exposure titer. Subsite D antibody-blocking titer began to appear after PCD 9 and, by PCD 12, had reached nearly the same level as that for the primary response to the M5C-TGEV inoculation.

Pigs were inoculated with various strains of transmissible gastroenteritis virus (TGEV) or with porcine respiratory coronavirus (PRCV), and antigenic site-specific antibody responses were compared. A blocking-ELSIA was used to study to what extent antibodies in convalescent sera interfered with the binding of monoclonal antibodies (MAB) 57.16 or 57.110 to the attenuated TGEV/Purdue virus. Monoclonal antibody 57.16 is directed against the A site on the peplomer, neutralizes virus, and recognizes TGEV and PRCV. Monoclonal antibody 57.110 is directed against the X site on the peplomer, but does not neutralize virus, and recognizes only TGEV. Antibodies directed against TGEV and PRCV could be detected in a blocking ELISA, using MAB 57.16 as a conjugate. Antibodies directed against both viruses were detectable as early as 1 week after inoculation. Antibody titers correlated well with those in a virus-neutralization test. Antibodies against TGEV could be detected in a blocking ELISA, using MAB 57.110 as a conjugate. Such antibodies were not induced by a PRCV infection. In the blocking ELISA, using MAB 57.110 as a conjugate, antibodies were detectable as early as 2 weeks after inoculation. There was a significant difference between antibody titers reached after infection with various TGEV strains, however. This difference is ascribed to a variation of the antigenic site defined by MAB 57.110 in TGEV strains. Conditions for a differential test for TGE serodiagnosis, and for serologic discrimination between TGEV- and PRCV-infected pigs, are discussed. It is concluded that by using a blocking ELISA with MAB 57.110, no definite distinction can be made between antibodies directed against TGEV and PRCV, because low antibody titers develop after infection with some TGEV strains, and because antibodies directed against antigenic sites that PRCV has in common with TGEV may interfere with the binding of MAB 57.110 to TGEV.

Five nonneutralizing monoclonal antibodies (MAb) generated to the virulent Miller strain of transmissible gastroenteritis virus (TGEV) and specific for the S protein were characterized. Competition assays between purified and biotinylated MAb indicated that MAb 75B10 and 8G11 mapped near a new subsite, designated V and 2 MAb, 44C11 and 45A8, mapped to a previously designated subsite D. A fifth MAb mapped between subsites V and E. These MAb were tested with 3 previously characterized MAb to subsites A, E, and F in fixed-cell ELISA and cell culture immunofluorescent assays against 5 reference and 9 field strains of TGEV and 2 US strains (ISU-1 and ISU-3 3) porcine respiratory coronavirus (PRCV). Subsites A, E, and F were conserved on all TGEV and PRCV strains examined. The 2 MAb to subsite V, 8G11 and 75B10, reacted only with the Miller TGEV strains (M5C, M6, and M60), except that 75B10 also recognized field strain U328. The MAb 11H8 did not react with 4 field strains or the Purdue strains of TGEV. The 2 MAb to subsite D reacted with all TGEV strains examined, but not with 2 US PRCV strains, 2 European PRCV strains, 1 feline infectious peritonitis virus strain, and 1 canine coronavirus strain. Because of this specificity for TGEV, but not PRCV, these latter 2 subsite D MAb may be useful for the development of competition ELISA to differentiate serologically between TGEV and PRCV infections in swine, similar to the currently used European subsite D MAb.

Pigs from 3 litters kept under gnotobiotic conditions were inoculated orally with virulent transmissible gastroenteritis (TGE) virus, a TGE vaccine, or Hank's balanced salt solution at 2 days of age and then euthanatized at intervals ranging from 1 to 7 days after inoculation. Pigs exposed to the vaccine had clinical evidence of diarrhea and weakness. Lesions resembling those of TGE were revealed grossly, microscopically, and by scanning electron microscopy. Viral antigen was seen in intestinal epithelial cells by the direct fluorescent antibody technique. The disease induced by the vaccine virus had a longer incubation period and lesions were less severe than that induced by the virulent virus.