Mapping T and B lymphocyte epitopes of bovine herpesvirus-1 glycoprotein B

Four pBacPAK vectors (Clontech), termed pBacPAK9gB-StuI, pBacPAK8gB-SmaI, pBacPAK8gB-SacI and pBacPAK8gB-PvuII (Fig. 1a), containing sequences encoding differently C-terminally truncated gB were generated as follows. The plasmid PE5 (Gao et al., 1995 ) contained the HinfI segment of gB encoding amino acids 1296 (Leary & Splitter, 1990 ). This fragment was subcloned into the transfer vector pBacPAK9 and the new plasmid was digested with StuI to create a gB segment encoding amino acids 1172, termed pBacPAK9gB-StuI. Similarly, the gB MscIXbaI fragment was subcloned into pBacPAK8 and digested with SmaI to create a gB segment encoding amino acids 1253, termed pBacPAK8gB-SmaI. PE5gB-StuI was digested with RsrII and PvuII. The RsrIIPvuII fragment released was cloned into pBacPAK8gB-SmaI between the RsrII and SmaI sites to form pBacPAK8gB-PvuII, encoding amino acids 1742. Finally, pBacPAK8gB-PvuII was digested with SacI to create a gB segment encoding amino acids 532742, termed pBacPAK8gB-SacI. The plasmids pBacPAK9gB-StuI, pBacPAK8gB-SmaI, pBacPAK8gB-SacI and pBacPAK8gB-PvuII carry coding sequences of gB with the same N terminus but different C termini (Fig. 1a). The authenticity of the recombinant transfer vectors was confirmed by DNA sequence analysis.

(137K): Fig. 1. Truncated gB segments expressed by the baculovirus system. (a) Western blot analysis of truncated gB segments in recombinant baculovirus-infected cell supernatants. Lanes 14 represent twofold dilutions of affinity-purified gB as the positive control and density standard. Lanes 58 represent gB expressed from BacPAKgB-PvuII, BacPAKgB-SacI, BacPAKgB-SmaI and BacPAKgB-StuI. Lane 9 shows a BacPAKGLU control (C). (b) PBMC proliferation to truncated gB segments expressed by recombinant baculovirus. Animal numbers are in the upper right of each box. PBMC (2x105) from immune calves were stimulated with 50 µl 1:5 (open bars), 1:25 (hatched bars) or 1:125 (filled bars) diluted supernatant from TN5 cells infected with BacPAKgB-PvuII (pvuII), BacPAKgB-SacI (sacI), BacPAKgB-SmaI (smaI), BacPAKgB-StuI (stuI) or BacPAKGLU (GLU) or mock-infected (TN5) or medium alone (m). For some animals, only a 1:25 dilution of recombinant virus supernatant was tested. Statistical significance of differences relative to the BacPAKGLU control is shown: *, P<0·01; **, P<0·001.

TN5 insect cell monolayers (High Five, Invitrogen) were co-transfected with pBacPAK6 virus DNA and recombinant vectors pBacPAK9gB-StuI, pBacPAK8gB-SmaI, pBacPAK8gB-SacI or pBacPAK8gB-PvuII. Transfected TN5 cell monolayers were incubated for 3 days at 27 °C and the supernatant was collected. Recombinant virus-containing supernatant was plated on a fresh monolayer of TN5 cells to create individual plaques. Plaques were picked and amplified on TN5 cells and recombinant gB protein supernatant was analysed by Western blotting. Positive viruses, termed BacPAKgB-StuI, BacPAKgB-SmaI, BacPAKgB-SacI and BacPAKgB-PvuII, were amplified.

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.

Holstein bull calves (34 months old) of different parentage were immunized intranasally or subcutaneously at the base of the ear with recombinant truncated gB spanning the extracellular domain (Gao et al., 1994 , 1995 ). Cows 3886 (Holstein), 912 (Brown Swiss) and 602 (Jersey) were immunized intranasally eight times, twice a year, with attenuated live BHV-1 (TSV-2, SmithKline). Supernatants containing truncated gB segments expressed by baculovirus, diluted 1:5 (30 µg/ml), 1:25 (15 µg/ml) and 1:125 (7·5 µg/ml), were added to cultures of PBMC obtained 2 weeks after the second gB immunization. Results from 11 calves are shown in Fig. 1(b). All animals except animal 912 failed to recognize the gB-StuI-encoded fragment. Only three animals (2126, P0·001; 382, P0·01; and 602, P0·05) recognized gB-SmaI significantly more strongly than gB-StuI. Importantly, PBMC from all calves of different parentage and breeds (Holstein, Jersey and Brown Swiss) recognized gB-SacI- and gB-PvuII-encoded segments. Since the four recombinant gB segments shared a common N-terminal sequence but differed at their C termini, differences in proliferation to each protein were due to variations in the C-terminal sequence. Thus, an antigenic region recognized by PBMC is located at the C terminus of the gB-SacI sequence between amino acids 254532. Animals vaccinated with BHV-1 (animals 602, 912 and 3886) or immunized with gB protein (15 animals) responded similarly to the gB-PvuII-encoded protein segment. Supernatants from TN5 cells mock-infected or infected with recombinant BacPAKGLU induced proliferation similar to medium alone (Fig. 1b). Twenty-three peptides (21-mers) spanning amino acids 247532 of gB were synthesized (Chiron Mimotopes, Raleigh, NC, USA). The last nine amino acids of each peptide overlapped the first nine amino acids of the next peptide. Synthetic peptides included an acetyl group at the N terminus and diketopiperazine at the C terminus, as this format stimulates T cell responses during T cell epitope-mapping experiments (Allen et al., 1989 ; Gammon et al., 1991 ). Peptides were dissolved in 100 µl DMSO (Sigma) and 900 µl PBS as a stock solution, aliquoted and stored at -70 °C.

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), γδ (CC15) or B cells (antibody 33) had no effect (data not shown).

Table 1. gB peptide sequences and antibody response

(34K): Fig. 2. Partial T cell epitope mapping. Synthetic peptides (3 µg/ml final concentration) spanning amino acids 247532 of gB were screened by lymphocyte proliferation assay of PBMC from immune calves (animal numbers are in the upper right of each box). The horizontal lines represent an arbitrary value that was considered as a positive response.

Peptides were tested by ELISA (Gao et al., 1995 ) for reactivity to bovine anti-gB sera collected 2 weeks after the second gB immunization and the results are summarized in Table 1. Peptides 8, 20, 21 and 23 contained antibody epitopes recognized by antisera (Table 1). Animals vaccinated with BHV-1 responded as follows: animal 3886 responded to peptides 8, 20 and 23, while animals 912 and 602 responded to peptides 8, 20 and 21 (data not shown). Peptide 3 comprised a non-specific antibody epitope, as all sera tested, including normal sera, reacted positively (data not shown). Normal sera, however, did not react to any of the remaining peptides (data not shown).

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-2K^b-binding motifs and that peptide 15 contained HLA-DR2a-, 2b- and I-E^K-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-E^K-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.

We thank Nancy Soeurt, Patric Lundberg and Hsia-Hua Lisa Lin for their helpful discussions. This work was supported by the College of Agricultural and Life Sciences and USDA grants 93-37204-9205 and 96-35204-3670.