Epitope-specific antibody responses in market-stressed calves to bovine herpesvirus type 1

Epitope-specific antibody responses in market-stressed calves to bovine herpesvirus type 1 Virginia K. Ayers*, James K. Collins and Charles W. Purdy* Reciprocal competition ELISA (rcELISA) was conducted to map monoclonal antibodies (mAbs) reactive with 9L 9111 and 9IV 9lycoproteins o f bovine herpesvirus type 1 (BHV-1) into epitope 9roups. mAbs to 91ycoproteins 9I and 9IV were divided into six epitope 9roups each, while 9111 mAbs had been previously divided into .four areas, mAbs were chosen from each epitope 9roup to compete in cELISA wih bovine sera collected during a typical regimen of vaccination and transportation from farm to auction to feedlot. The immunodominant epitopes were identified for each BHV-1 9lycoprotein. With 9lycoprotein 91, three epitopes defined by mAbs 1FIO, D9 and 4807 were the most dominant; with 9lycoprotein g i l l epitopes defined by mAbs G2 and 1507, and with 9lycoprotein 9IV epitopes defined by mAbs 1102, 1106, 3C1, 3402 and 3E7 showed the maximum responses. The overall cELISA responses to each 91ycoprotein amon9 two vaccination 9roups were also compared and it was shown that cELISA responses were significantly higher for each 9lycoprotein in calves receivin9 two vaccinations, one on the farm of origin and one at auction, than in calves receivin9 only one vaccination at auction. Keywords: Bovine herpesvirus type 1; epitopes; reciprocal competition ELISA; vaccination; bovine respiratory disease

Bovine herpesvirus type 1 (BHV-1) is a major viral disease agent in cattle. It is a primary cause of upper respiratory tract disease, capable of inducing classic infectious bovine rhinotracheitis (IBR) t, and it also plays a major role in undifferentiated respiratory tract disease commonly encountered in cattle feedlots 2. Bovine respiratory disease can be initiated by various infectious agents which are encountered in cattle feedlots and which rapidly colonize and infect the respiratory tract 2'3. Stress is an important factor in susceptibility to infection. Calves which have been transported over long distances, deprived of food and water, mixed and introduced into confinement operations encounter BHV-1 and other infectious agents at a time of maximal physiological stress. As a result, they rapidly succumb to infection and are frequently unable to mount successful physiological and immunological defences. Primary infections with BHV-1 typically predispose cattle to secondary bacterial infections 2'4. Vaccinations have traditionally been used in attempts to induce immunity to BHV-1, and thus prevent respiratory disease, but they are only partially effective5 7 Virus

infections and outbreaks of respiratory disease, including those of high morbidity and mortality, occur in appropriately vaccinated feedlot animals 8-11. Because of this, and because of the important economic loss due to bovine respiratory disease 12, new strategies should be developed for inducing appropriate and effective immunological responses. Before new strategies of immunization can be developed, the antigenic determinants which are recognized and may be important in protection and recovery must be determined for the infecting agents. It is known that the major glycoproteins of BHV-1 are the dominant targets of the bovine immune response 13'14. These glycoproteins include gI, partially homologous to the herpes simplex virus (HSV) gB glycoprotein 15.16; gIII, partially homologous to the HSV gC glycoprotein 15; and gIV, partially homologous to the HSV gD glycoprotein 17. We have previously reported on the epitope responses to gIII glycoprotein of BHV-1 in cattle that were naturally exposed or experimentally infected is. In the present study, we extend our analysis to include all three glycoproteins.

Department of Microbiology, Colorado State University, Fort Collins, CO 80523, USA. *United States Department of Agriculture, Agriculture Research Service, Conservation and Production Research Laboratory, Bushland, TX 79012, USA. tTo whom correspondence should be addressed. (Received 30 April 1993; revised 29 November 1993; accepted 29 November 1993)

MATERIALS AND METHODS

0264-410X/94/10/0940-07 © 1994 Butterworth-HeinemannLtd 940

Vaccine 1994 Volume 12 Number 10

Monoclonal antibodies

Monoclonal antibodies were derived previously: D9, F2, G219; 4407, 4807, 5106, 5606, 1507, 2905, 1102, 3402, 4906, 500620; 1Ell, 1F8, 1F102x; 10C2, 3C1, 3E7, 9D622; 3D9S 23.

Glycoprotein antigens of BHV-1: V.K. Ayers

Competition ELISA The reciprocal competition ELISA (rcELISA) assay used to determine interrelationships among mAbs and the cELISA used to analyse the bovine serum samples for antibody to BHV-1 have been described previously 18. Briefly, dilutions of ascites or serum samples were added to microtitre plates that had been coated with purified BHV-1 antigen and were incubated at 37°C for 30 min. Horseradish peroxidase-conjugated monoclonal antibody at the appropriate dilution was added to this and further incubated for 30 min. The plates were then washed and o-phenylene diamine substrate was added and the plates were read in a Titertek multiscan plate reader. Percentage competition was calculated as the percentage reduction of the absorbance (A) reading for each serum sample compared with the reading for a negative control (fetal calf serum or non-reactive ascites).

Experimental design A group of 100 calves from five different farms in Tennessee underwent treatment regimens similar to those described previously 24-26. Several vaccines were given on the farm of origin, including Nasalgen IBR/PI-3 intranasal vaccine (SmithKline Beecham Animal Health, Lincoln, NE). This group of 100 is referred to as the 'Tops' group (for Texas Optimum Processing System). Another group of 50 from the same farms did not receive the intranasal vaccine. This group is referred to as the 'non-Tops' group. Four months after this initial treatment, the calves were transported to auction where all 150 received IBR/PI-3 MLV intramuscular vaccine (SmithKline Beecham). Nine days after arrival at the auction barn, the calves were transported to a feedlot in Bushland, TX. Serum samples were taken on the farm of origin, on arrival at auction (2 October), and four times at 1-2-week intervals at the feedlot (11, 18 and 25 October, and 8 November). From this group of 150, 30 calves were randomly selected for this study (half of them were from the Tops group and the other half were from the non-Tops group).

Clinical disease The system of scoring and treatment of calves has been described previously25. Clinical signs were recorded at the start of the experiment (on the farm of origin) and at

Table 1

e t al.

each of the serum collection times. Calves were judged to be sick or well on presence or absence of signs of pneumonia: eye and nasal drainage, lethargy, cough and fever >/104°C. Sick animals were treated for 4-9 days with antibiotics, as described previously26.

Statistical methods Data were statistically analysed using methods described by Armitage 27. RESULTS

Characterization and selection of epitopes rcELISA was performed with mAbs to each of the three major glycoproteins of BHVI.1, gI, gIII and gIV, in order to select mAbs reactive with different epitopes. Eleven gI mAbs were analysed by rcELISA and grouped by epitope, as shown in Table 1. mAbs D9, 3F3, 1Ell and 1F10 each represented unique epitopes, rcELISA with the remaining mAbs suggested a possible structural relationship of overlapping antigenic areas (Table 1). ,From these 11 mAbs, six were selected to analyse the bovine sera by cELISA (one from each epitope region: D9, 3F3, 4807, 5106, I E l l and 1F10). A panel of 11 gIV mAbs was analysed in the same way, and six antigenic regions were defined (Table 2). Six mAbs, representing differing epitope specificities, were chosen to analyse the bovine antibody response: 3D9S and 3402, which represented unique epitopes, and 1102, 3E7, 1106, and 3C1 from related or overlapping antigenic areas.

The results of rcELISA analysis with a panel of gIII mAbs have been published previously 18. Of the gIII mAbs used to compete with bovine sera, G1, G2 and 1507 are in separate epitope areas, while F2, 2905 and 6003 are included in an area of partial overlap or antigenic relatedness.

Competition of bovine sera with representative mAbs Sera collected at each of the processing dates from the two groups of calves were used to compete with the representative mAbs in cELISA (Figures 1-3). At the initial date, which was arrival at auction from individual farms, the Tops calves, which received the intranasal (i.n.) vaccination on the farm, had higher average levels of

Reciprocal cELISA analysis of a panel of mAbs to glycoprotein gl a Ascites

Conjugate

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+ -P P -------

ND ND ND ND ND -----

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-

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aMonoclonal ascites fluid were competed against each of the peroxidase--conjugated mAbs for binding to denote > 7 0 % , 40-70%, and < 4 0 % competition, respectively ND, not done

purified

BHV-1. The symbols + , P, and --

V a c c i n e 1994 V o l u m e 12 N u m b e r

10

941

Glycoprotein antigens of BHV-I: V.K. Ayers et al. Table 2

Reciprocal cELISA analysis of a panel of mAbs to glycoprotein glVa Ascites

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942

V a c c i n e 1994 V o l u m e

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Glycoprotein antigens of BHV-I: V.K. Ayers et al. 1 1 0 6

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competing antibody than the non-Tops calves for all mAbs. Both groups of calves received the intramuscular (i.m.) vaccine at this time, resulting in elevation of antibody levels at later dates. The maximum level of competition reached for each mAb varied, and the difference between Tops and non-Tops calves gradually diminished at the later sampling dates. With respect to glycoprotein gI (Figure 1), the average competition from all calves with mAb 1F10 on 8 November (the last sampling date), was significantly higher than the other five mAbs (Student's t test, p < 0.05). Competition with mAb D9 and 4807 also reached

consistently higher levels, 90-100%, while competition with mAbs 5106 and 1Ell reached only 60-70%. Glycoprotein gIV competition levels reached 80-90% for all mAbs except 3D9S (Figure 2). Competition with this mAb reached only about 60-70%, and on the final sampling date (8 November) the response was statistically lower than with other gIV mAbs (Student's t test, p < 0.05). For glycoprotein gIII (Figure 3), levels of 70-80% were reached with mAbs F2, 2905, 6003 and G1, while competition with mAbs G2 and 1507 approached 90-100%. Competition with mAb 1507 was significantly

Vaccine 1994 V o l u m e 12 N u m b e r 10

943

Glycoprotein antigens of BHV-I: V.K. Ayers et al. Gl

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higher than the other gIII mAbs on 8 November (Student's t test, p < 0.05). By considering the selected glycoprotein-specific mAbs as representative of the overall immune responses to each individual glycoprotein, the responses to each glycoprotein among the groups of calves could be compared. The competition levels for the six epitopes for each glycoprotein on each date were averaged. It could be shown by computing the standard error of the difference between the means that the level of competition by the Tops calves was significantly higher than that of the

944

at.

Date

Vaccine 1994 V o l u m e 12 N u m b e r 10

non-Tops calves at each of the first four sampling dates for each glycoprotein (p<0.003). The difference was greatest on 11 October (Table3). Since the i.m. vaccination received on 2 October represented a secondary immunization for the Tops calves (following the i.n. vaccine received on the farm earlier) and a primary immunization for the non-Tops calves, the results of increased competition in the Tops calves on 11 October could be interpreted as reflecting a secondary or anamnestic immune response. On this date, the responses by the Tops calves were significantly higher than those

Glycoprotein antigens of BHV-I: V.K. Ayers et al. 3 Average percentage competition for each glycoprotein obtained from serum at entry into feedlot with mAbs representative of each glycoprotein Table

Primary response n Secondary response a Glycoprotein (non-Tops calves) (Tops calves)

p value b

gl gill glV

< 0.001 <0.001 <0.001

56.9 _+27.4 63.24-17.1 45.1 _+24.1

81.4 4-16.3 80.34-17.8 78.34-14.9

aPercentage competition was averaged by glycoprotein_+standard deviation for serum samples taken on 11 October bThestandard error of the difference between the means was computed for each non-Tops vesus Tops pair, and the p values derived from this are given. Comparison of the responses between glycoproteins (non-Tops calves): p =0.046 for gl versus gill; p =0.001 for gl versus glV; and p <0.001 for gill versus glV; secondary: p>0.1 for each glycoprotein pair

of the non-Tops calves for each glycoprotein ( T a b l e 3 , p<0.001). As time elapsed, the difference bewteen the groups diminished. It was also found that the overall competition levels between the glycoproteins (comparing gI versus gIII versus gIV) in the non-Tops calves were significantly different from each other (Table 3), reflecting a varying response after primary immunization to each glycoprotein. The secondary response (by the Tops calves) to each glycoprotein were not significantly different. Comparison of cELISA responses of calves with and without respiratory disease In order to determine whether there was a correlation between clinical disease outcome and response to specific epitopes, each of the two treatment groups were again subdivided into two groups (healthy and sick) based on development of clinical respiratory disease while in the feedlot. Of the 15 Tops and 15 non-Tops calves, five in each group developed clinical respiratory disease. Among the Tops calves, the average level of competition on 2 October for each mAb was higher in the calves that remained healthy in those that later became sick (data not shown). The difference between Tops healthy versus sick calves was not significant on any other date. In the non-Tops group of calves, no correlation was found between health status and levels of antibody at any date. DISCUSSION It is widely acknowledged that bovine herpesvirus type ! causes significant problems in the cattle industry, being a major factor involved in shipping fever pneumonia, and that current vaccines are often not adequate to control the disease. There have been several reports of studies examining the epitope specificity of the immune response to BHV-128-3o. Van Drunen Littel-van den Hurk et al. 3° determined that immunization with individual purified glycoproteins protected calves in a BHV-1 challenge model, and they showed that most of the epitopes were represented in the antibody response. Duque et al. zs demonstrated responses to all epitopes on gI and gIV and one epitope on gIII in cattle that were immunized with a formalin-inactivated BHV-1 vaccine. Mashall et al. 29 showed that calves experimentally or naturally infected with BHV-1 responded to most (experimental infection) or all (natural infection) of the epitopes on the three glycoproteins. However, it is not known which, if

any, specific epitopes correlate to a protective immune response, or how the antibody response is affected by the stress of marketing calves. This present study reports the relative significance of epitope responses to the major glycoproteins. Competition ELISA between the bovine sera and a panel of 18 mAbs showed that calf-to-calf variability was quite high for some epitopes (i.e. G2), and very low for others (i.e. 1507). The variability from one epitope to another in the level of competition by bovine antibodies with the different mAbs was also considerable and significant. Some of these epitopes elicited a higher response (1FI0, D9, 4807 on glycoprotein gI; 1507 on glycoprotein gIII) or lower response (3D9S on glycoprotein gIV) than others. This observed variability could have been due to differences in immunogenicity of each epitope. In the study by van Drunen Littel-van den Hurk et al. 3°, it was found that after vaccination of cattle with purified glycoproteins, most, but not all, epitopes on gI and gIII were recognized by the cattle. It was concluded that this lack of recognition was due to an inability of the cattle to respond well to these epitopes rather than destruction of the epitopes during purification, since cattle experimentally infected with BHV-1 also failed to respond to these epitopes. Marshall et al. 29 reported on an experimentally infected calf which apparently did not react to several gIII and gIV epitopes, based on a lack of competition with the representative mAbs. We believe that in the present study the observed differences in levels of competition with different mAbs were a direct reflection of the level of antibody to each epitope, and therefore demonstrated that the calves responded differently to each epitope. These differences could also be explained by differences in avidities between the mAbs. However, it should be noted, from the range of competition levels observed, that with every epitope there were bovine sera that competed to a level of 90% or greater. This suggested that the cattle were capable of producing antibodies of at least the same avidity of each mAb. The response to all three glycoproteins was significantly higher in the Tops group than in non-Tops at each sampling date except on the last date (where the difference in responses to gI was not significant). The greatest difference occurred on 11 October, and decreased at each later date. On this date, it was apparent that for each glycoprotein the response to secondary immunization represented by the Tops calves was significantly higher than the response to primary immunization, represented by the non-Tops calves. This would be expected of a memory response. In addition, the glycoproteins apparently differed in their ability to induce a (primary) response in the non-Tops calves. Glycoprotein gIV appeared to be the most different from the other two, inducing the lowest response. The glycoproteins did not differ significantly in their ability to induce a secondary response. Van Drunen Littel-van den Hurk et al. 3° also found that the glycoproteins differed in the level of antibody induced in calves after immunization with purified glycoproteins, as measured by cELISA, although in that study gIV appeared to induce the highest response. There were no significant differences between the glycoprotein responses after subsequent challenge. The comparison of cELISA responses of calves that remained healthy throughout the study with those that developed respiratory disease showed significant

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1994 V o l u m e 12 N u m b e r 10

945

G l y c o p r o t e i n a n t i g e n s o f B H V - I : V.K. A y e r s et al.

differences only on one date (2 October) in one group. These results suggested a correlation between antibody level and protection from disease in this group; however, since equal numbers of calves from the two vaccination groups became sick, vaccine differences, as well as cELISA responses, were not relevant to the development of respiratory disease. It was not known whether BHV-1 was involved in the respiratory illnesses of these calves. Possibly, factors other than BHV-l-specific antibody level, such as differences in the cell-mediated immune responses, exposure to other agents, or in on-farm handling/treatment regimens, played a role in determining health status for these calves. ACKNOWLEDGEMENTS This material is based upon work supported by the Cooperative State Research Service, US Department of Agriculture, under agreement no. 89-34116-4629, and by Colorado State University Agricultural Experiment Station.

REFERENCES 1 2

3 4

5

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Kahrs, R.F. Viral Disease of Cattle, Iowa State University Press, Ames, IO, 1981, pp. 135-156 Yates, W.P.G. A review of infectious bovine rhinotracheitis, shipping fever pneumonia, and viral-bacterial synergism in respiratory disease of cattle. Can. J. Comp. Med. 1982, 46, 226-263 Lillie, L.E. The bovine respiratory disease complex. Can. Vet. J. 1974, 15, 233-242 Bielfieldt-Ohmann, H. and Babiuk, L.A. Viral-bacterial pneumonia in calves: effect of bovine herpesvirus-1 on immunologic functions. J. Infect. Dis. 1985, 151,937-947 Babiuk, L.A., L'ltalien, J., van Drunen Littel-van den Hurk, S., Zamb, T.J., Lawman, M.J.P., Hughes, G. and Gifford, G.A. Protection of cattle from bovine herpesvirus type-1 (BHV-1) infection by immunization with individual viral glycoproteins. Virology 1987, 159, 57-66 Jericho, K.W.F. and Babiuk, L.A. The effect of dose, route, and virulence of bovine herpesvirus-1 vaccine on experimental respiratory disease incattle. Can. J. Comp. Med. 1983,47, 133-139 Gerber, J.D., Marron, A.E. and Kucera, C.J. Local and systemic cellular and antibody immune responses of cattle to infectious bovine rhinotracheitis virus vaccine administered intranasally or intramuscularly. Am. J. Vet. Res. 1978, 39, 753-760 Curtis, R.A. and Angulo, A. A field trial to evaluate an intranasal infectious bovine rhinotracheitis vaccine. Can. Vet. J. 1974, 15, 327~330 Jensen, R., Pierson, R.E., Braddy, P.M., Saari, D.S., Lauerman, L.H., England, J.J. et al. Shipping fever pneumonia in yearling feedtot cattle. J. Am. Vet. Med. Assoc. 1976, 169, 500-506 Martin, W., Willson, P., Curtis, R., Allen, B. and Acres, S. A field trial, of preshipment vaccination, with intranasal infectious bovine rhinotracheitis-parainfluenza-3 vaccines. Can. J. Comp. Med. 1983, 47, 245-249 Collins, J.K., Ayers, V.K. and Carman, J. Evaluation of an antigen capture ELISA for the detection of bovine herpesvirus type 1 shedding from feedlot cattle. Vet. Microbiol. 1988, 16, 101-107

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Loan, R.W. Bovine Respiratory Disease, a Symposium (Ed. Loan, R.W.) Texas A&M University Press, College Station, TX, 1984 Van Drunen Littel-van den Hurk, S., van den Hurk, J.V., Gilchrist, J.E., Misra, V. and Babiuk, L.A. Interactions of monoclonal antibodies and bovine herpesvirus type 1 (BHV-1) glycoproteins: characterization of their biochemical and immunological properties. Virology 1984, 135, 466-479 Collins, J.K., Butcher, A.C. and Riegel, C.A. Immune response to bovine herpesvirus type-1 infections: virus-specific antibodies in sera from infected animals. J. Clin. Microbiol. 1985, 21,546-552 Fitzpatrick, D.R, Redmond, M.J., Attah-poku, S.K., van Drunen Littelwan den Hurk, S., Babiuk, L.A. and Zamb, T.J. Mapping of 10 epitopes on bovine herpesvirus type 1 glycoproteins gl and gill. Virology 1990, 176, 145-147 Misra, V., Nelson, R. and Smith, M. Sequence of a bovine herpesvirus type-1 glycoprotein gene that is homologous to the herpes simplex gene for the glycoprotein B. Virology 1988, 166, 542-549 Tikoo, S.K., Fitzpatrick, D.R., Babiuk, L.A. and Zamb, T.J. Molecular cloning, sequencing, and expression of functional bovine herpesvirus 1 glycoprotein glV in transfected bovine cells. J. Virol. 1990, 64, 5132-5142 Ayers, V.K., Riegel, C.A., Carman, J. and Collins, J.K. Epitope specificity of the bovine antibody response to the gill glycoprotein of bovine herpesvirus type 1. Viral Immunol. 1989, 2, 79-88 Collins, J.K., Butcher, A.C., Riegel, C.A., McGrane, V., Blair, C.D., Teramoto, Y.A. and Winston, S. Neutralizing determinants defined by monoclonal antibodies on polypeptides specified by bovine herpesvirus type 1. J. Virol. 1984, 52, 403-408 Marshall, R.L., Rodriguez, L.L. and Letchworth, G.J. Characterization of envelope proteins of infectious bovine rhinotracheitis virus (bovine herpesvirus 1) by biochemical and immunological methods. J. Virol. 1986, 57, 745-753 van Drunen Littel-van den Hurk, S., van den Hurk, J.V. and Babiuk, L.A. Topographical analysis of bovine herpesvirus-1 glycoproteins: use of monoclonal antibodies to identify and characterize functional epitopes. Virology 1985, 144, 216-227 Hughes, G., Babiuk, L.A. and van Drunen Littel-van den Hurk, S. Functional and topographical analyses of epitopes on bovine herpesvirus type 1 glycoprotein IV. Arch. Virol. 1988, 103, 47-60 van Drunen Littel-van den Hurk, S. and Babiuk, L.A. Synthesis and processing of bovine herpesvirus 1 glycoproteins. J. Virol. 1986, 59, 401-410 Loan, R.W. and Purdy, C.W. Vaccination for the prevention of bovine respiratory disease. In: Proc. 14th World Congress on Diseases of Cattle, Vol. 1 (Eds Hartigan, P.J. and Monaghan, M.L.) Dublin, 1986, pp. 65,3-658 Purdy, C.W., Livingston, C.W., Frank, G.H., Cummins, JM., Cole, N.A. and Loan, R.W. A live Pasteurella haemolytica vaccine efficacy trial. J. Am. Vet. Med. Assoc. 1986, 188, 589-591 McVey, D.S., Loan, R.W., Purdy, C.W. and Shuman, W.J. Specificity of bovine serum antibody to capsular carbohydrate antigens from Pasteurella haemolytica. J. Clin. Microbiol. 1990, 28, 1151-1158 Armitage, P. Statistical Methods in Medical Research Blackwell Scientific Publications, Oxford, 1987 Duque, H., Marshall, R.L., Israel, B.A. and Letchworth, G.L. Effects of formalin inactivation on bovine herpesvirus-1 glycoproteins and antibody response elicited by formalin-inactivated vaccines in rabbits. Vaccine 1989, 7, 513-520 Marshall, R.L., Israel, B.A. and Letchworth, G.J. Monoclonal antibody analysis of bovine herpesvirus-1 glycoprotein antigenic areas relevant to natural infection. Virology 1988, 165, 338-347 van Drunen Littel-van den Hurk, S., Gifford, G.A. and Babiuk, L.A. Epitope specificity of the protective immune response induced by individual bovine herpesvirus-1 glycoproteins. Vaccine 1990, 8, 358-358