The porcine humoral immune response against pseudorabies virus specifically targets attachment sites on glycoprotein gC

Abstract

High titers of virus-neutralizing antibodies directed against glycoprotein gC of Pseudorabies virus (PRV) (Suid herpesvirus 1) are generally observed in the serum of immunized pigs. A known function of the glycoprotein gC is to mediate attachment of PRV to target cells through distinct viral heparin-binding domains (HBDs). Therefore, it was suggested that the virus-neutralizing activity of anti-PRV sera is directed against HBDs on gC. To address this issue, sera with high virus-neutralizing activity against gC were used to characterize the anti-gC response. Epitope mapping demonstrated that amino acids of HBDs are part of an antigenic antibody binding domain which is located in the N-terminal part of gC. Binding of antibodies to this antigenic domain of gC was further shown to interfere with the viral attachment. Therefore, these results show that the viral HBDs are accessible targets for the humoral anti-PRV response even after tolerance induction against self-proteins, which utilize similar HBDs to promote host protein-protein interactions. The findings indicate that the host's immune system can specifically block the attachment function of PRV gC. Since HBDs promote the attachment of a number of herpesviruses, the design of future antiherpesvirus vaccines should aim to induce a humoral immune response that prevents HBD-mediated viral attachment.

FIG. 1

Schematic representation of the gC protein. The location of the three fusion proteins gC-N-term, gC-middle, and gC-C-term is indicated by the respective amino acid positions. The positions delineating the HBDs HBD 1 (H1), HBD 2 (H2), and HBD 3 (H3) are shown as described previously (8), as is that of the identified antigenic ABD. Segments found to react with the porcine immune sera are shaded.

FIG. 2

Porcine immune sera exhibit reduced capacity to neutralize gC-negative PRV compared to WT PRV. Immune sera from inbred pigs 1 and 2 (numbered circles) and from outbred pigs 1 and 2 (numbered triangles) were diluted and used for plaque reduction assay (performed in triplicate) with WT PRV and an isogenic gC-negative mutant PRV (gC-minus) as described in Materials and Methods. The reciprocal serum dilutions resulting in 95 to 100% plaque reduction are depicted, as are the standard errors (error bars).

FIG. 3

Western blot analysis of gC fusion proteins. The fusion proteins gC-N-term (N), gC-middle (M), and gC-C-term (C) were obtained by urea extraction and analyzed by SDS–15% PAGE followed by Coomassie blue staining (A) or Western blot analysis (B to D). The shown results of the Western blot analysis are representative of the reactivities of all sera tested (Table 1), and the porcine preimmune serum (B) and immune sera from inbred pig 1 (C) and inbred pig 2 (D) were diluted 1:500. The specifically induced proteins are marked by arrowheads. Migration of the molecular weight (MW) standards (in thousands) is indicated to the left.

FIG. 4

Identification of linear B-cell epitopes in the N-terminal part of gC. Overlapping synthetic peptides spanning amino acids 55 to 114 of gC were coated onto 96-well plates and tested by ELISA with diluted (1:200) immune sera (open columns) of inbred pig 1 (A) and inbred pig 2 (B) and with the respective preimmune sera (gray columns). Peptide numbers correspond to the first amino acid position of each peptide in the gC sequence. Each experiment was performed in triplicate, with the standard deviations shown by error bars; no error bar is shown where the standard deviation was less than the extent of the bar.

FIG. 5

The identified ABD is exposed on the surface of PRV virions. Mouse antisera against peptide 65-79 (open symbols) and preimmune sera (closed symbols) were tested by ELISA for reactivity with peptide 65-79 (insert, squares) and an irrelevant control peptide (insert, circles), or with purified PRV (squares) and bovine serum albumin (circles). Each experiment was performed in triplicate, with the error bars indicating the standard deviations. The results are representative for five different mouse anti-peptide 65-79 serum samples.

FIG. 6

Amino acids corresponding to HBD 1 are essential for specific antibody recognition of ABD. Ninety-six-well plates were coated with synthetic peptides 65-79, 65-77, and 67-79 (designated as such because of the amino acids of gC that they comprise) and peptides with mutated amino acid 66 (T to G; 65-79/66G), amino acid 71 (P to G; 65-79/71G), or amino acid 77 (R to E; 65-79/77E) and tested by ELISA with diluted (1:200) immune serum from inbred pig 1 (open columns) and the respective preimmune serum (gray columns). Each experiment was performed in triplicate, with the error bars indicating the standard deviations; no error bar is evident where the standard deviation was less than the extent of the bar. Very similar results were obtained with the goat anti-PRV serum diluted at 1:2,000 (data not shown).

FIG. 7

Antibodies directed against ABD interfere with the attachment of PRV. Medium (A) and diluted goat anti-PRV serum (1:50) without peptide (B), with the indicated concentrations of peptide 65-79 (C), or with an irrelevant control peptide (D) were incubated overnight before the lacZ gene-expressing PRV strain PHY-B111 was added. After adsorption to MDBK cells in the cold, excess virus was removed and the cells were incubated at 37°C and analyzed 4 h later by flow cytometry as described in Materials and Methods. The percentage of infected cells is given in the upper right corner of each quadrant. The results are representative of three independent experiments. The y axis shows the relative number of cells; the x axis indicates fluorescence intensity.