Vol. 66, No. 11

JOURNAL OF VIROLOGY, Nov. 1992, p. 6502-6508 0022-538X/92/116502-07$02.00/0 Copyright © 1992, American Society for Microbiology

Epitope Specificity of Protective Lactogenic Immunity against Swine Transmissible Gastroenteritis Virus MARIBEL DE DIEGO,' MARIA D. LAVIADA,1 LUIS ENJUANES,2 AND JOSE' M. ESCRIBANO1* Departamento de Sanidad Animal-Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria, Embajadores 68, 28012 Madrid, 1 and Centro de Biologia Molecular, CSIC-UAM, Universidad Aut6noma de Madnid, Canto Blanco, 28049 Madrid, 2 Spain Received 15 May 1992/Accepted 5 August 1992

The epitope specificity of the protective immune response against swine transmissible gastroenteritis (TGE) has been investigated by using circulating and secretory antibodies. This study was carried out with sows vaccinated with TGEV or the antigenically related porcine respiratory coronavirus (PRCV). TGEV vaccination of sows resulted in greater lactogenic protection of suckling piglets against TGEV challenge and a higher secretory immune response than PRCV vaccination did. These differences in the immune response were conditioned by the route of antigen presentation as a result of the different tropism of each virus. Epitopes on S protein, and in particular those contained in its antigenic site A, were more immunogenic than epitopes on N and M proteins in both groups of vaccinated sows, as determined by a competitive radioimmunoassay. Minor differences in antibody response against the previously defined antigenic subsites Aa, Ab, and Ac were also detected, with subsite Ab being the most antigenic in both TGEV- and PRCV-immune sows. These findings suggest that antigenic site A on S protein, involved in virus neutralization, is the immunodominant site in pregnant sows that confer lactogenic protection. They also validate, in experiments with secretory antibodies, the antigenic maps made with murine monoclonal antibodies. Therefore, this antigenic site should be considered for vaccine or diagnostic development.

factors, the class of immunoglobulin induced by relevant epitopes may be important. Immunoglobulin A seems to be more valuable in passive protection (24, 25). The quantitative factors refer to the relative amounts of antibodies directed against epitopes inducing neutralizing antibodies (18). In the work described in this paper, the quantitative factors were studied by characterizing the immunogenicity of different epitopes on the three viral structural proteins S, N, and M. A panel of MAbs that recognize different epitopes on the three structural proteins has been used in a competition assay with antibodies present in serum, colostrum, and milk from TGEV- or PRCV-vaccinated sows. By using this method, we have quantified the antibody representation of the TGEV or PRCV epitopes in sows which confer protection to the suckling piglets, and hence we should be able to define the most antigenic viral epitopes that should be included in a hypothetical subunit vaccine.

Transmissible gastroenteritis virus (TGEV) is a member of the Coronaviridae family that infects pigs of all age groups but causes high mortality only in pigs under 2 weeks of age (3, 27). Since the highest mortality from TGE results when newborn pigs are infected, there has been much interest in providing passive immunity to suckling pigs. Pregnant sows exposed orally to virulent TGEV develop immunity to the virus and confer passive protection to the suckling piglets via colostrum and milk containing high neutralizing antibody titers (4). A respiratory tropism variant of TGEV has been described (21). This variant is porcine respiratory coronavirus (PRCV), which has a high degree of antigenic similarity to TGEV (26). PRCV virus induces a degree of passive protection against TGEV when it is used to immunize pregnant sows (2), although there is controversy about the levels of this protection. TGEV is composed of three structural proteins, S, N, and M, which contain 1,447, 382, and 282 amino acids, respectively (14, 15, 22). Only the S glycoprotein has been involved in virus neutralization in the absence of complement (10, 13). In this protein, at least four antigenic sites (A, B, C, and D) have been defined (6, 8, 11). Site A is the major inducer of neutralizing antibodies and has been divided into three antigenic subsites: Aa, Ab, and Ac (6). Other sites (B and D) are also involved in virus neutralization (5). In addition, antibody-mediated complement-dependent virus neutralization has been reported with M-specific monoclonal antibodies (MAbs) (28). Antigenic sites A, B, and D, as well as the epitopes defined by the M-specific MAbs 9DB4 and 3BB3, are exposed to the antibodies on the surface of infected cells and mediate cell lysis by antibody and complement (17). Qualitative and quantitative factors may play a role in passive protection against TGEV. Among the qualitative *

MATERIALS AND METHODS Cells and viruses. The attenuated strain PUR46 and the virulent strain MAD88 of TGEV and the BEL85-83 strain of PRCV (16, 26) were grown in swine testicular (ST) cells. Infectious virus for in vivo immunization was grown on confluent cell monolayers infected with TGEV MAD88 or PRCV BEL85-83 at a multiplicity of infection of 0.5 in medium supplemented with 2% fetal bovine serum. After 20 h (20% cytopathic effect), medium containing extracellular virus was harvested. Cellular debris were removed from the fluid by low-speed centrifugation. Supernatants were supplemented with 20% fetal bovine serum and stored at -70°C. After titration by the 50% tissue culture infective dose (TCID50) method (23), these inocula were used for vaccination and challenge. Partially purified virus preparations were obtained by using confluent monolayers infected with TGEV PUR46 or

Corresponding author. 6502

VOL. 66, 1992

MAD88 at a multiplicity of infection of 5. When cells reached 80 to 90% cytopathic effect, extracellular virus was harvested as described above. Virions were then semipurifled by centrifugation through a continuous sucrose gradient (60 to 20%), and the viral band was sedimented. The pellet was resuspended in TNE buffer (0.001 M Tris-HCl, 0.1 M NaCl, 1 mM disodium EDTA [pH 7.5]), and its titer was determined antigenically in microtiter plates with a hyperimmune serum. These virus preparations were used for Western immunoblot assay, enzyme-linked immunosorbent assay (ELISA), and competitive radioimmunoassay (cRIA). Vaccination and challenge procedures. Six pregnant Large White sows, each weighing about 100 kg, were orally inoculated twice (at 84 and 104 days of pregnancy) with 2.5 x 109 TCID50 of TGEV MAD88 per inoculation. Two sows with the same characteristics were oronasally inoculated twice with an aerosol containing the same TCID50 of PRCV BEL85-83 at the same days of pregnancy as the TGEVinoculated sows. One pregnant sow was left unimmunized as a negative control. Piglets (7 days old) were isolated, orally exposed to 6.4 x 108 TCID50 of TGEV MAD88, and then breast fed 1 h later. Antibodies. All sows were bled before each immunization and on the day of farrowing. Colostrum was collected from each sow on the day of farrowing. Milk samples from each sow were collected on the second day after farrowing and on the day of piglet challenge. Casein and free fat were removed from colostrum and milk by centrifugation at 10,000 x g for 3 h. The whey was further clarified by centrifugation at 10,000 x g for 3 h. Lipoproteins were precipitated by adding 0.02 ml of 10% dextran sulfate 500 and 0.1 ml of 1 M CaCl2 per ml of serum or whey. The heavy precipitate was removed by centrifugation at 1,500 x g for 10 min. Piglets were bled before challenge and on days 7 and 15 after challenge. The previously characterized TGEV-specific MAbs (6, 11, 13, 26) were obtained from hybridoma culture supernatants and stored at -70°C until use. ELISA. Semipurified TGEV MAD88, at the optimal concentration determined by titer determination (about 0.44 ,ug per well), was used to coat microtiter plate wells. Wells were blocked with blocking buffer (0.05% Tween 20 and 30% fetal bovine serum in phosphate-buffered saline [PBS; pH 7.2]). A 100-,ul portion of the serial dilutions of sow serum, colostrum, and milk was added and incubated for 1 h at 37°C. After the wells had been washed, protein A conjugated with peroxidase (Sigma Chemical Co., St. Louis, Mo.) was added. Plates were incubated for 1 h at 37°C and, after being washed again, were incubated with 3-methylaminobenzoic acid (DMAB)-methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate (MBTH) substrate (Sigma). The reaction was stopped by addition of H2SO4. Finally, the reactions were read spectrophotometrically (optical density at 620 nm was measured). The antibody titer was considered the reciprocal of the highest dilution with a positive absorbance value (>0.3 optical density unit). Seroneutralization test. The neutralization titers of serum, colostrum, and milk samples were determined as described previously (29). Briefly, 100 TCID50 was added per well in a microtiter plate and incubated with serial dilutions of antibody samples. After 1 h, 1.5 x 104 cells per well were added and the plate was incubated for 3 days at 37°C. Antibody titers were calculated as the highest dilution that neutralized 100% of the cytopathic effect. The titer was expressed as the reciprocal of the dilution to the log2 value. Radioimmunoprecipitation (RIPA), SDS-PAGE, and Western blot analyses. The ST cells inoculated with TGEV

EPITOPE SPECIFICITY OF LACTOGENIC IMMUNITY

6503

(multiplicity of infection, 10) were incubated for 4 h and pulse-labeled for 2 h with 500 ,uCi of [35S]methionine (800 Ci/mmol; Amersham International, Amersham, England) per ml in Eagle minimal essential medium lacking methionine and supplemented with 1% fetal calf serum. Aliquots of 106 cpm of 5S-labeled cell extracts were immunoprecipitated with antibodies from vaccinated sows and Staphylococcus aureus as described previously (1). The antigen-antibody complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) as previously described (9) with acrylamide-N,N'diallyltartardiamine (DATD; Bio-Rad Laboratories, Richmond, Calif.) gels. The Western blotting technique was used as described previously (20), with a 1:30 dilution of the samples and using protein A-peronidase and 4-chloro-1-naphthol (Sigma) as substrates to detect the immunocomplexes. cRIA. Partially purified TGEV PUR46 was plated as described above for ELISA. Coated wells were incubated for 1 h at 37°C with 150 pl of 5% bovine serum albumin (BSA) in PBS and then washed in PBS containing 0.05% Tween 20. Competition assays were performed in a one-step test. Pretitrated MAbs and competitor antibodies were diluted in PBS-0.1% BSA and incubated together for 2 h at 37°C. Then plates were washed with PBS-0.05% Tween 20, and then 1"I-labeled anti-mouse immunoglobulin (105 cpm per well; Amersham International) diluted in PBS-0.1% BSA-5% TGEV-negative pig serum was added to plates and incubated at 37°C for 1 h. After six washes with PBS-0.05% Tween 20, the MAbs bound to the wells were eluted with SR1 x (2% SDS and 1 M mercaptoethanol in 50 mM Tris-HCl [pH 7]) and the radioactivity was counted. Results were expressed as the percentage of competition according to the following formula: percent blocking = 100 - (cpm bound with competitor sample/cpm bound without competitor sample). Each assay was done at least in triplicate. RESULTS

Circulating and secretory antibody response to TGEV and PRCV in vaccinated sows. Two groups of TGE-seronegative sows were vaccinated. Group I consisted of six sows vaccinated twice with the TGEV virulent strain MAD88. Group II consisted of two sows vaccinated twice with PRCV BEL8583. Piglets nursing from three sows belonging to group I and the two sows from group II and those nursing from a nonvaccinated sow were challenged with 6.4 x 108 TCID50 of TGEV at 7 days old. Morbidity and mortality rates of the challenged piglets were recorded (Table 1). All piglets nursing from the control sow showed clinical signs of infection by 24 to 48 h after challenge with the virulent TGEV. Characteristic clinical signs of TGE, i.e., vomiting and diarrhea, were observed in these piglets. All control piglets nursing from the seronegative sow died between 3 and 5 days after challenge. Fifteen piglets nursing from the PRCV-vaccinated sows also developed clinical signs of TGE at similar times after challenge exposure. However, the mortality rates in suckling piglets from this group were 37.5 and 71.5%. Of 18 pigs nursing from TGEV-vaccinated sows, 13 developed mild clinical signs of disease. The onset of disease was delayed until 2 to 4 days postchallenge, and the infection was frequently characterized by a mild and transient diarrhea. The morbidity rate in the different nursing pigs was 100% in two cases and 37.5% in the other case. One piglet in this group died 6 days after challenge. Sows vaccinated with TGEV or PRCV developed circu-

6504

-

J. VIROL.

DE DIEGO ET AL.

TABLE 1. Morbidity and mortality of piglets after challenge with virulent TGEV MAD88 Sow no.

1 2 3 4 5 6 7 8 9

No. of piglets: With Survived Total clinical after

Virus used for vaccination

None TGEV MAD88 TGEV MAD88 TGEV MAD88 TGEV MAD88 TGEV MAD88 TGEV MAD88 PRCV BEL85-83 PRCV BEL85-83

4 8 10 8 9 5 5 8 7

%

Survivablity

signs

challenge

4 _a

0

0

3b

8

100

5b

5 4 5 2

100 80 62.5 28.5

5b 8 7

a _ challenge with the virulent virus was not performed. Delayed appearance of mild clinical signs, with respect to control piglets.

b

lating and secretory antibodies which were collected from serum, colostrum, and milk on the day of farrowing and 1 day later. ELISA and neutralization titers of serum, colostrum, and milk showed a good correlation (Fig. 1). TGEVvaccinated sows had higher neutralization and ELISA titers than PRCV-vaccinated sows did. The lower antibody induction by PRCV than TGEV was corroborated by analysis of antibody titers of sera from pigs naturally infected with PRCV, which were similar to those obtained in our experiment (data not shown). In TGEV-vaccinated sows, colostrum and milk showed higher antibody titers than serum, by both ELISA and neutralization tests. In contrast, PRCVvaccinated sows did not show differences in the titers in serum, colostrum, and milk. The specificity of antibodies induced by the two types of vaccination was tested by Western blotting and RIPA (Fig.

A

14

t12

4

*

4

s

6

.. CY)

..a

i

4

2

0

14

12

2

S

SOW NUMBER

FIG. 1. Antibody titers against TGEV in an unvaccinated sow (sow 1), TGEV-vaccinated sows (sows 2 to 7), and PRCV-vaccinated sows (sows 8 and 9), measured by ELISA (A) and seroneutralization (B) in sera (M), colostrum (n), and milk (l). Error bars show the standard deviation of the mean of three determinations.

2). By Western blotting analysis, N protein apparently was the most antigenic protein, whereas by RIPA analysis, S and M proteins were the best recognized by the antibodies. These differences could be explained by the conformational exposure of the epitopes on the different proteins. Epitopes present on S and M proteins seem to be more conformational than those present on N protein, since they did not react as strongly as those on N protein in Western blotting under partially denaturing conditions. In general, antibodies present in the sera from TGEV-vaccinated sows reacted less strongly with the three viral structural proteins than did antibodies present in colostrum and milk both in Western blotting and in RIPA. Differences in the reactivity of the antibodies from PRCV-vaccinated sows against viral proteins were detected only in the milk from sow 9, which had less reactivity in Western blotting and RIPA. Epitope specificity of the immune response to S protein in TGEV- and PRCV-immunized sows. Vaccination with virulent TGEV induced protection against disease and, in one case, against infection. Vaccination with PRCV also induced a degree of protective lactogenic immunity against TGE. We expected to find common denominators mainly in the epitope recognition in the S protein by antibodies obtained from the two groups of vaccinated sows. For this purpose, we performed cRIAs to determine whether serum, colostrum, or milk from vaccinated sows would block the binding of MAbs specific for S protein. These MAbs bind to sites B and D and subsites Aa, Ab, and Ac. Site B was not investigated in experiments with PRCV-vaccinated sows because it is absent in this virus. In TGEV-vaccinated sows the immune response against the epitopes studied was higher than in PRCV-vaccinated sows. At a 1:2 dilution all the MAbs reacting with the different subsites of antigenic site A were blocked more than 90% by sera, colostrum, and milk from all TGEV-vaccinated sows (Fig. 3). MAbs against sites B and D were less inhibited by antibodies from sows, with blocking percentages between 60 and 84% (Fig. 3). At higher dilutions of the competitor antibodies, differences between competition percentages obtained with circulating and secretory antibodies were detected in all TGEV-vaccinated sows. Antigenic subsite Ab, recognized by MAb 1DE7, was the most immunogenic since it showed the highest competition percentages with secreted antibodies contained in colostrum and milk from this group of sows (Fig. 3). In PRCV-vaccinated sows only the MAbs against some subsites of antigenic site A showed percentages of competition higher than 70% when competitor antibodies were analyzed at low dilutions (Fig. 3). Differences in the antibody specificity of circulating and secretory antibodies from both sows (sows 8 and 9) were found. In sow 8, which gave about 60% protection of nursing piglets, the antibody-binding activity for most of the S epitopes was similar in circulating and secretory (only colostrum) antibodies. However, in sow 9, which gave about 30% passive protection of nursing piglets, the binding of antibodies to the S epitopes was higher in circulating than in secretory antibodies (Fig. 3). Milk obtained from sows 6 and 7 on the day of challenge, 7 days after farrowing, showed a decrease in the level of S protein-specific antibodies (Fig. 4). At this time, sites B and D were low represented in antibody. Absorption of TGEVspecific antibodies by suckling piglets was very efficient as determined by the cRIAs of their sera when different antigenic site-specific MAbs were present (Fig. 4). A scheme of the specificity of secreted antibodies against S protein in milk from TGEV- and PRCV-vaccinated sows is

VOL. 66, 1992

;t\)A

EPITOPE SPECIFICITY OF LACIOGENIC IMMUNITY

TGEV

SCMSCM SCM SCM "EI

..

PRCV

RIPA

WB

WB

SCMSCMSCMSCM a unikiiii haA

SCMSCM

RIPA

P

SCMSCM

S

INWIRMIRIN I

_0bd&

t

6505

i

Ab 4w

-P~ -

MOM

aS

E

q-m.-qw4mmm

2 3 4 5

2 3 4 5 8 9 SOW NUMBER

A- m

8 9

FIG. 2. Antibody protein specificity of sera (lanes S), colostrum (lanes C), and milk (lanes M) obtained from TGEV-vaccinated (sows 2 to 5) or PRCV-vaccinated (sows 8 and 9) sows as determined by Western blotting (WB) and RIPA. The reactive proteins by the two techniques were compared with a [35S]methionine-labeled protein pattern of TGEV-infected cells at 8 h postinfection (lane P).

shown in Fig. 5. Site A, and especially subsite Ab, was the most immunogenic (Fig. 5A). A comparison of the antibody

representation of antigenic A subsites in milk from TGEVand PRCV-vaccinated sows is also shown (Fig. 5B). Epitope specificity of the immune response to N and M proteins in TGEV- and PRCV-immunized sows. cRIA studies with N- and M-specific MAbs and circulating and secretory antibodies from both groups of immunized sows were performed. The antigenicity of the epitopes tested on N and M proteins was lower than that of the epitopes tested on S protein. Most of the secreted antibodies specific for M protein in TGEV-vaccinated sows were specific for the epitope defined by MAb 9DB4 (Fig. 6), which is absent in

&A

S

8B ~ICC12(Aod lDSI2

F

0

ISh

'C

IE3 -i

8 | ~~ID912 IDE7(Ab)

Lli

PRCV

S$

/O

O

>

1 2

SOW NUMBER TGEV 5 3

!!11; &A ICC12 A 1SICUI I I 1 ~~AC3(AC) /IIC2 _ _E_3-A _I1-

O :

MAb

PRCV. In general, with the exception of sow 6, in which the specific N and M MAb blocking percentages were very low, N protein seems to be more immunogenic than M protein in the TGEV-vaccinated sows. For N protein, the antibody response against different epitopes was very variable among different TGEV-vaccinated sows, including sow 7, in which, interestingly, a preferential selection of a circulating antibody response against this protein was detected (Fig. 6). However, in PRCV-vaccinated sows, M protein seems to be more immunogenic than N protein for the epitopes tested. Only in serum and colostrum was an antibody response against these proteins detected in the two sows (Fig. 6). The epitope on M protein defined by MAb 3DE3 was the only

U)CN es.cN

_

_* I

U_

.~~~~~~~ 89

3

CD

_I_ _

Di GID AC3(Ad l GAC3CAd)f

A

9

I

Di

IDE?3

1/ W oij~~ E 8 ,. . ^,

FIG. 3. Competition binding assays to TGEV of different dilutions of sera (S), colostrum (C), and milk (M) from an unvaccinated sow (sow 1), TGEV-vaccinated sows (sows 2 to 7), and PRCV-vaccinated sows (sows 8 and 9) with MAbs directed toward antigenic sites A, B, and D (site B only in TGEV-vaccinated sows). MAbs 1CC12, 1DE7, and 6AC3 represented antigenic subsites Aa, Ab, and Ac respectively. Symbols: O, El, and M, competition percentages of 0 to 35%, 36 to 70%, and 71 to 100%, respectively.

6506

~

DE DIEGO ET AL.

MAb

6

___ 24

ICC12 .0

/2/

2/

/1/ /4 2/

0

S6AC3 @00 IDB12 ei IDG3

Di

NHjD "*S'|1lt2o-_ ulPRCV

SOW NUMBER 6

7

@0

2/

7

4

ANTIBODY

//OLUITION

S@

6 4 5 2 3 PIGLET NUMBER FIG. 4. Competition binding assays to TGEV of milk from two TGEV-vaccinated sows (sows 6 and 7) and the sera from six piglets nursing from these sows (piglets 1 through 6) at 1/2 and 1/4 dilutions, obtained on the day of challenge with the virulent TGEV strain (7 days after farrowing), with MAbs directed at antigenic sites A, B, and D on TGEV S protein. Symbols represent the same competition percentages as in Fig. 3.

antibody represented in the colostrum from both sows in this group, and the same was true of the epitope defined by MAb 3BB3 in sow 9 (Fig. 6). DISCUSSION

The systemic and secretory immune response to TGEV structural proteins in sows vaccinated with TGEV or PRCV has been analyzed. Both TGEV- and PRCV-immune sows confer lactogenic immunity to their piglets. The level of protection in the vaccinated sows in the two groups ranged from 28.5 to 100%, and there was a correlation between the level of neutralizing antibody in the colostrum and milk and the passive protection in the suckling piglets. These passiveprotection experiments were performed by using severe challenge conditions with a virulent field strain, since no piglet from the unvaccinated sow survived these conditions. In the studies with TGEV-vaccinated sows, we selected the litters from a sow presenting high antibody titers and those from two sows showing the lowest antibody titers to be challenged with the virulent TGEV. From litters of sow 4, with high antibody titers, about 60% of the offspring were protected against the infection and only 1 of the 18 piglets nursing in this group of sows died (2 days after challenge). This confirms the previous description (4) of good lactogenic protection that is conferred by inoculation of sows with a virulent TGEV strain. We observed cross-protection between PRCV and TGEV, with mortality rates for litters from PRCV-vaccinated sows of 37.5 and 71.5%. The levels of 536

144

N21

J. VIROL.

protection observed in this experience are similar to those obtained by other authors with naturally PRCV-infected sows (2). Apparently, an immunological link between the lungs and mammary glands exists in the sow since PRCVvaccinated sows secreted virus-specific antibodies in colostrum and milk, even though PRCV replicates only in the respiratory tract (7). This result is in concordance with a reduction in the number of TGE outbreaks in Europe concomitant with the development of PRCV seroconversion in the swine population (2). A major objective of this study was to determine which of the known antigenic sites on the TGEV or PRCV structural proteins were immunogenic in vaccinated sows, especially in terms of secreted antibodies. Since S glycoprotein was identified as the major inducer of neutralizing antibodies (8, 10, 13), it appears to be the primary candidate for an effective subunit vaccine for TGEV. In this work we have shown differences in the induction of secretory antibodies by different antigenic sites involved in virus neutralization, with site A being the most immunogenic. In site A, subsite Ab seems more antigenic than subsites Aa and Ac in the two groups of vaccinated sows. On the other hand, S protein also seems more antigenic than N and M proteins. The possibility that this result is conditioned by the epitopes on N and M proteins selected to perform this study cannot be ruled out, but this seems unlikely, since the epitopes analyzed were highly immunogenic in other studies (6). However, other authors (12) have described discrepancies in the levels of antigenicity of any particular epitope between different species. With respect to S protein, this study validates the antigenic maps made with murine MAbs in which antigenic site A is considered the immunodominant site (6, 8, 13). Adequate interpretation of the relative antibody response against the different viral epitopes by cRIA requires that the MAbs used, which bind the different antigenic sites, have similar isotype and relative affinities. All the MAbs used are -Yl with the exception of MAb 1CC12, which is y2. The relative affinities of the MAbs were shown by an RIA study of the binding of identical amounts of the purified immunoglobulins to TGEV. In the same assay, both the plateau (maximum binding) and the titers of all the MAbs were very similar (unpublished results). A similar conclusion has been reached by performing the same procedure in other viral systems (19). In addition, MAbs that bind to the same site inhibited the binding of all others, which were "2I labeled, in a cRIA (6). By performing Western blotting and RIPA analyses of the antibodies from vaccinated sows, we showed that the three

EiDE

RESIDUES

385

B

1ICOOHA I D Ac Ab~~~~~~~~.....SUBSITE AzAuA~~~~~~~~~ Ab .............1 SITE/SU9SITE A_AoAc A,b

E

TGEV

,

I~~~~~~~~~~~~~~~~~I L500 I

FIG. 5. (A) Scheme of the secretory antibody representation of the different antigenic sites and subsites on s protein localized in the sequence of this protein and deduced by the mean of the competition values obtained at different dilutions of milk from TGEV-vaccinated sows. (B) Antibody representation of the subsites Aa, Ab, and Ac on S protein comparatively in milk from TGEV- and PRCV-vaccinated sows, deduced by the mean of competition percentages of the MAbs shown in Fig. 3. Symbols represent the same competition percentages as in Fig. 3.

EPITOPE SPECIFICITY OF LACTOGENIC IMMUNITY

VOL. 66, 1992

MAb 9D84

M N

38D3

M

N M

N|

SlCS

3DE3

___

3CDS 3CE4

__ _ ___

9DO4

SOW NUMBER PRCV TGEV 2 s34 5 6 7 9 ,

I ___ _ __ _ _ _

* 000

0

SI

FIWI

0

@0 qq0

000

0

1/22

___

*

0. -1 *I-l0

0

00 0*

1i 11ii'11 0

@00 00

I

l

-- *I1II 1 1 1-II11 w1 Tl]I1 *U 1

I

[1l 1l 1l

3DE3 3DCIO

I IT1 l l

r-1

R ra r r rl Il Tl Il ]l

Eon.*

3CE4

3O D3

3DE3

3DCIO1

1

l l l

*00 @0- 0*0 e111 I I @01Il@ 000 1 *h17 0001 0| r I I I 00 l l l

00

0001

-

I

-

-s E

I I

l l

I01I I l I I lI I

l l l

T

l4

*

Il

1

1 1 II

lIl

I I

U)

I0-

E0. I1A,_,.8

7

0111*!!

111I

I D

I

3883 3CDS

6507

I

_.Q

i °

I

lll Ll I I I 111 FIG. 6. Competition binding assays to TGEV of different dilutions of sera (S), colostrum (C), and miLk (M) from an unvaccinated sow (sow 1), TGEV-vaccinated sows (sows 2 to 7), and PRCV-vaccinated sows (sows 8 and 9) with MAbs directed at M and N proteins. MAb 9DB4 was not used in PRCV-vaccinated sows because this epitope is absent in this virus. Symbols represent the same competition percentage as in Fig. 3. 3CD8 3CE4

structural proteins are antigenic during the experimental infection. Quantification of the antigenicity of these proteins was not possible because of differences in the reactivity in the methods used, probably as a result of the conformation dependence of the epitopes in the proteins. N protein epitopes seem to be exposed after denaturation by SDS and 2-mercaptoethanol, in contrast to epitopes in S and M proteins, since M protein was better recognized by antibodies in Western blotting than in RIPA. Despite the level of antibody induction by the two viruses used in this experiment, we detected differences in the recognition of viral epitopes by the circulating and secretory antibodies depending on the virus used in the immunization. In both cases, the secretory antibody induction against S protein epitopes seemed to be important in the surviving percentage rates of the litters. Antigenic site A on S protein is highly immunogenic in TGEV- and PRCV-vaccinated sows, and a secretory immune response to this site correlates well with the induction of passive protection. We can hypothesize that adequate presentation to the mucosal immune system of the pregnant sows of one or several epitopes belonging to this antigenic site would provide a protective passive immune response to the suckling piglets and that these epitopes should be included in a subunit vaccine. On the other hand, diagnostic procedures based on virus-induced antibody detection by peptides should also consider the use of antigenic site A. In addition, this antigenic site is highly conserved among TGEV and PRCV isolates, as well as between canine and feline coronaviruses (26). This suggests that antigenic sites that are not well conserved between viruses of this family are immunologically less important, and we can hypothesize about the existence in this virus of a relationship between structural conservation and immunologic response, as described for other viruses (12). As mentioned above, qualitative and quantitative factors can be important in the transference of immunity (18, 24, 25).

Qualitative studies on the isotype of immunoglobulin induced by the different viral epitopes during infection by TGEV or PRCV are in progress. ACKNOWLEDGMENTS We are grateful to S. Martin Rilo for providing the pregnant sows and to M. Sevilla for her excellent technical assistance. This work was supported by a grant from Instituto Nacional de Investigaci6n y Tecnologia Agraria y Alimentaria (INIA), Madrid,

Spain. REFERENCES 1. Alcaraz, C., M. D. Diego, M. J. Pastor, and J. M. Escribano. 1990. Comparison of a radioimmunoprecipitation assay to inmunoblotting and ELISA for detection of antibody to African swine fever virus. J. Vet. Diagn. Invest. 2:191-196. 2. Bernad, S., E. Bottreau, J. M. Aynaud, P. Have, and J. Szymansky. 1989. Natural infection with the porcine respiratory coronavirus induces protective lactogenic immunity against transmissible gastroenteritis. Vet. Microbiol. 21:1-8. 3. Bohl, E. H. 1975. Transmissible gastroenteritis, p. 168-208. In H. W. Dunne and A. D. Leman (ed.), Diseases of swine. Iowa State University Press, Ames. 4. Bohl, E. H. 1981. Transmissible gastroenteritis, p. 195-208. In A. D. Leman, R. D. Glock, W. L. Mengeling, R. H. C. Penny, E. School, and B. Straw (ed.), Diseases of swine. Iowa State University Press, Ames. 5. Correa, I., F. Gebauer, M. J. Bullido, C. Sun6, M. F. D. Baay, K A. Zawaagstra, W. P. A. Posthumus, J. A. Lenstra, and L. Enjuanes. 1990. Localization of antigenic sites of the E2 glycoprotein of transmissible gastroenteritis coronavirus. J. Gen. Virol. 71:271-279. 6. Correa, I., G. Jimenez, C. Sune, M. J. Bullido, and L. Enjuanes. 1988. Antigenic structure of the E2 glycoprotein from transmissible gastroenteritis coronavirus. Virus Res. 10:77-94. 7. Cox, E., M. B. Pensaert, P. Callebaut, and K Deun. 1990. Intestinal replication of a respiratory coronavirus closely related antigenically to the enteric transmissible gastroenteritis virus. Vet. Microbiol. 23:237-243. 8. Delmas, B., J. Gelfi, and H. Laude. 1986. Antigenic structure of

6508

9. 10.

11.

12.

13. 14. 15.

16. 17.

18.

DE DIEGO ET AL.

transmissible gastroenteritis virus. II. Domains in the peplomer glycoprotein. J. Gen. Virol. 67:1405-1418. Escribano, J. M., and E. Tabares. 1987. Proteins specified by African swine fever virus. V. Identification of immediate early, early and late proteins. Arch. Virol. 92:221-232. Garwes, D. J., M. H. Lucas, D. A. Higgins, B. V. Pike, and S. F. Cartwright. 1978. Antigenicity of structural components from porcine transmissible gastroenteritis virus. Vet. Microbiol. 3:179-190. Gebauer, F., W. P. A. Posthumus, A. Correa, C. Sune, C. Smerdou, C. M. Sanchez, J. A. Lenstra, R. H. Meloen, and L. Enjuanes. 1991. Residues involved in the antigenic sites of transmissible gastroenteritis coronavirus S glycoprotein. Virology 183:225-238. Henrickson, K. J., and A. Portner. 1990. Antibody response in children to antigen sites on human PIV-3 HN: correlation with known epitopes mapped by monoclonal antibodies. Vaccine 8:75-80. Jimenez, G., I. Correa, M. P. Melgosa, M. J. Bullido, and L. Enjuanes. 1986. Critical epitopes in transmissible gastroenteritis virus neutralization. J. Virol. 60:131-139. Kapke, P. A., and D. A. Brian. 1986. Sequence analysis of the porcine transmissible gastroenteritis coronavirus nucleocapsid protein gene. Virology 151:41-49. Laude, H., D. Rasschaert, and J. C. Huet. 1987. Sequence and N-terminal processing of the transmembrane protein El of the coronavirus transmissible gastroenteritis virus. J. Gen. Virol. 68:1687-1693. Laviada, M. D., M. A. Marcotegui, and J. M. Escribano. 1988. Diagn6stico e identificaci6n de un brote de gastroenteritis porcina transmissible en Espania. Med. Vet. 5:563-575. Laviada, M. D., S. P. Videgain, L. Moreno, F. Alonso, L. Enjuanes, and J. M. Escribano. 1990. Expression of swine transmissible gastroenteritis virus envelope antigens on the surface of infected cells: epitopes externally exposed. Virus Res. 16:247-254. Moxley, R. A., L. D. Olson, and R. F. Solorzano. 1989. Relationship among transmissible gastroenteritis virus antibody titers in serum, colostrum and milk from vaccinated sows, and protection in their suckling pigs. Am. J. Vet. Res. 50:119-125.

J. VIROL.

19. Nowinski, R. C., M. E. Lostrom, M. R. Tam, M. R. Stone, and W. N. Burnette. 1979. The isolation of hybrid cell lines producing monoclonal antibodies against the p15 (E) protein of murine leukemia viruses. Virology 93:111-126. 20. Pastor, M. J., M. Arias, and J. M. Escribano. 1990. Comparison of two antigens for use in an enzyme-linked immunosorbent assay to detect African swine fever antibody. Am. J. Vet. Res. 51:1540-1543. 21. Pensaert, M. B., P. Callebaut, and J. Vergote. 1986. Isolation of a new porcine respiratory, non enteric coronavirus related to transmissible gastroenteritis. Vet. Q. 8:257-261. 22. Rasschaert, D., and H. Laude. 1987. The predicted primary structure of the peplomer protein E2 of the porcine coronavirus transmissible gastroenteritis virus. J. Gen. Virol. 68:1883-1890. 23. Reed, I. J., and R. H. Muench. 1938. A simple method to estimating fifty percent end points. Am. J. Hyg. 27:493-497. 24. Saif, L. J., and E. H. Bohl. 1979. Role of secretory IgA in passive immunity of swine to enteric viral infection, p. 237-248. In P. Ogra and D. Dayton (ed.), Immunology of breast milk. Raven Press, New York. 25. Saif, L. J., and E. H. Bohl. 1979. Passive immunity in transmissible gastroenteritis of swine: immunoglobulin classes of milk antibodies after oral-intranasal inoculation of sows with a live low cell culture-passaged virus. Am. J. Vet. Res. 40:115-117. 26. Sanchez, C. M., G. Jimenez, M. D. Laviada, I. Correa, C. Sufin, M. J. Bullido, F. Gebauer, C. Smerdou, P. Callebaut, J. M. Escribano, and L. Enjuanes. 1990. Antigenic homology among coronaviruses related to transmissible gastroenteritis virus. Virology 174:410-417. 27. Siddell, S., H. Wege, and V. T. Meulen. 1983. The biology of coronaviruses. J. Gen. Virol. 64:761-776. 28. Woods, R. D., R. D. Wesley, and P. A. Kapke. 1987. Complement-dependent neutralization of transmissible gastroenteritis virus by monoclonal antibodies. Adv. Exp. Med. Biol. 218:493500. 29. Yus, E., M. D. Laviada, L. Moreno, J. M. Castro, J. M. Escribano, and I. Simarro. 1989. The prevalence of antibodies to influenza and respiratory coronaviruses among fattening pigs in Spain. J. Vet. Med. 36:551-556.