Department of Animal Health and Biomedical Sciences, University of Wisconsin-Madison, 1655 Linden Drive, Madison, WI 53706, USA1
Author for correspondence: Gary Splitter.Fax +1 608 262 7420. e-mail splitter{at}ahabs.wisc.edu
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Abstract |
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Main text |
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Immunization with gB evokes neutralizing antibody and a specific T cell response (Gao et al., 1994 ; Leary & Splitter, 1990
) and confers protection against virulent BHV-1 challenge (Gao et al., 1994
, 1995
). Passive immunization with a pool of monospecific antibodies to gB, gC or gD failed to protect cattle against BHV-1 infection (Marshall & Letchworth, 1988
). CD4+ T cells proliferate to gB (Hutchings et al., 1990
; Leary & Splitter, 1990
; Leary et al., 1992
) and, since antibodies alone to envelope glycoproteins of BHV-1 may not protect calves, T cells specific for BHV-1 glycoproteins may be important for protection. Defining the major antigenic region(s) recognized among a large number of outbred animals will determine the extent to which this domestic population can recognize a series of epitopes within BHV-1 gB, which would be important in developing alternative vaccines.
Previously, the extracellular domain of gB was shown to be sufficient to induce a protective immune response (Gao et al., 1994 , 1995
). In the present study, truncated gB segments expressed by the baculovirus system localized major antigenic gB regions that induced lymphocyte proliferation. Next, overlapping synthetic peptides spanning the antigenic region were produced to map T and B cell epitopes. These present findings define T cell epitopes of gB and position them in the context of antibody epitopes by using the immune response of the natural host.
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Recombinant truncated gB segments |
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To avoid false reactions on Western blot with recombinant gB protein, TN5 cells were passaged three times in EX-CELL-400 (JRH Biosciences) medium without foetal bovine serum (FBS) before infecting with virus from purified plaques. TN5 insect cell monolayers were infected with recombinant virus (either BacPAKgB-StuI, BacPAKgB-SmaI, BacPAKgB-SacI or BacPAKgB-PvuII) at an m.o.i. of 10 at room temperature for 2 h before addition of FBS-free EX-CELL-400 medium. Cell supernatant was collected after 3 days incubation, centrifuged to remove cellular debris and dialysed against PBS for 3 days. Supernatants were adjusted to pH 7·3 for T cell proliferation assays.
Proteins in the recombinant baculovirus-infected cell supernatants were separated under reducing conditions (Gao et al., 1994 ) and detected by Western blot analysis with a gBb-specific antibody (Fig. 1a
). For each sample, 50 µl cell supernatant was separated on a reducing SDSPAGE gel and blotted onto a PVDF membrane. The specific gB segments were detected by gBb-specific monoclonal antibody 510604, provided by G. Letchworth (University of Wisconsin, USA). The gB-PvuII gBb subunit appeared at 60 kDa, gB-SacI at 60 kDa, gB-SmaI at 30 kDa and gB-StuI as a broad band at 2225 kDa. The complete gB-PvuII segment was observed at 90 kDa in 35S-immunoprecipitation experiments under non-reducing conditions (data not shown). No reactivity was observed for supernatants of BacPAKGLU-infected TN5 cells (a control that expressed E. coli ß-glucuronidase; Fig. 1a
, lane 9) or mock-infected TN5 cells (not shown). Fig. 1(a)
also shows the affinity-purified gB as gBa (130 kDa) and reduced subunit gBb (74 kDa). The gBb of recombinants gB-SacI and gB-PvuII had lower molecular masses than native gBb (60 kDa vs. 74 kDa), resulting from different glycosylation in the baculovirus expression system, as documented previously (Reynolds et al., 1992
). The concentration of truncated gB fragments produced by the baculovirus expression system was determined by using twofold dilutions of known gB protein and the NIH Image 1.59 program to measure density.
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PBMC recognition of truncated gB segments expressed by the baculovirus system |
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T cell-epitope mapping with synthetic peptides |
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In order to locate T cell epitopes, the 23 overlapping peptides spanning amino acids 247532 of the gB-SacI-encoded segment (Table 1) were tested in proliferation assays at a final concentration of 3 µg/ml with PBMC from gB- and BHV-1-immunized calves (Fig. 2
). Two major epitopes located in peptides 7 and 15 were recognized by five of nine animals. Animal 602 had no reactivity to any synthetic peptides and apparently recognized an epitope(s) N-terminal of the region spanned by the synthetic peptides, since its response to gB-SmaI was similar to that to gB-SacI (Fig. 1b
). Depletion of CD4 cells (antibody CC30 and complement) resulted in no proliferation of PBMC in response to the peptide containing the T cell epitope, while depletion of CD8 (CC58),
(CC15) or B cells (antibody 33) had no effect (data not shown).
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Antibody epitopes |
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Our earlier results demonstrated that the extracellular domain of gB protected cattle against BHV-1 challenge (Gao et al., 1994 , 1995
). Here, we have determined the major antigenic region and epitopes within the gB extracellular domain recognized by animals of different parentage. One major antigenic region, amino acids 319508, was identified that contained two T cell epitopes and three antibody epitopes.
Because the T lymphocyte response is MHC restricted, differences in cell response among individuals could be a major obstacle to the development of vaccines, since some individuals may not develop T cell responses to these molecules. Although gB can initiate a protective immune response, little is known about the relationship of T cell and antibody epitopes of gB. Initially, the antigenic region recognized by T cells was identified by using recombinant baculoviruses that expressed truncated gB segments, and PBMC from nine outbred calves recognized a region between amino acids 254 and 532 of gB. By using synthetic peptides spanning this major antigenic region, two T cell epitopes were identified in peptides 7 (amino acids 319340) and 15 (aa 415436) that induced lymphocyte proliferation in five of nine calves. Analysis by the TSITES program to predict T cell epitopes indicated that peptide 7 contained amphiphilic helices of seven or eight amino acids and that peptide 15 contained the Rothbard/Taylor motif (Rothbard & Taylor, 1988 ) of five residues. Alternatively, potential T cell epitopes can be determined by MHC-binding motifs and anchor residues (Rotzschke et al., 1991
). The program MHCPEP (version 1.2) indicated that peptide 7 contained HLA-DR1- and H-2Kb-binding motifs and that peptide 15 contained HLA-DR2a-, 2b- and I-EK-binding motifs. All motifs from these programs overlapped peptides 7 and 15. Interestingly, peptide 15 contained the HLA-A2.1-binding motif for CTL recognition included within the DR2a-, 2b- and I-EK-binding motifs.
By using synthetic peptides to map linear antibody epitopes, three epitopes were identified in peptide 8 (aa 331352), 20 (aa 475496) and 21 (aa 487508) (Table 1). By using monoclonal antibodies, five linear antibody epitope regions on gB were identified previously (Fitzpatrick et al., 1990
). Consistent with our results, epitope III (aa 469492) was included within peptide 20 (475496) (Fitzpatrick et al., 1990
). Epitope V, which included our T cell epitope (peptide 15), is closely linked to another T cell epitope (peptide 7) and a B cell epitope (peptide 8). On the basis of the results of the present study, using cells and sera from the natural host for BHV-1, the region spanning peptides 7 to 21 (aa 319508) is critical for both T cell and B cell recognition. Peptide 21 includes the protein-cleavage site responsible for the production of the 74 and 55 kDa gB subunits that remain linked by disulphide bonds. In summary, our study defines T cell recognition of BHV-1 gB by the natural host and positions this information in the context of antibody recognition to understand the immune-related functional domains of gB. Defining the major antigenic fragments of gB that elicit an immune response in different outbred individuals whose immune systems were primed to gB alone or intact virus supports a central role for a few select regions of gB in eliciting both T cell and antibody responses.
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Acknowledgments |
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References |
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Received 2 March 1999;
accepted 19 June 1999.