Production of herpes B virus recombinant glycoproteins and evaluation of their diagnostic potential

Abstract

B virus (cercopithecine herpesvirus 1) is the only deadly alphaherpesvirus that is zoonotically transmissible from macaques to humans. The detection of humoral immune responses is the method of choice for the rapid identification of B virus-infected animals. We evaluated the diagnostic potential of recombinant B virus glycoproteins for the detection of immunoglobulin G (IgG) antibodies in monkey and human sera. Glycoproteins B, C, and E and secreted (sgG) and membrane-associated (mgG) segments of glycoprotein G (gG) were expressed in the baculovirus expression system, while gD was expressed in CHO cells. We developed recombinant protein-based IgG enzyme-linked immunosorbent assays (ELISAs) and compared their diagnostic efficacies by using B virus antibody-negative (n = 40) and -positive (n = 75) macaque sera identified by a whole antigen-based ELISA and Western blotting. The diagnostic sensitivities of the gB-, gC-, gD-, and mgG-ELISAs were 100, 97.3, 88.0, and 80.0%, respectively. The specificities of the gB-, gC-, and gD-ELISAs and of the mgG-ELISA were 100 and 97.5%, respectively. In contrast, the sensitivities and specificities of sgG- and gE-ELISAs were low, suggesting that sgG and gE are less effective diagnostic antigens. Sera from nonmacaque monkeys cross-reacted with gB, gC, and gD, and only baboon sera reacted weakly with mgG. Human herpes simplex virus type 1 (HSV-1)- and HSV-2-positive sera pools reacted with gB and gD, whereas sera from B virus-infected individuals reacted with all four antigens. These data indicate that gB, gC, gD, and mgG have a high diagnostic potential for B virus serodiagnosis in macaques, whereas mgG may be a valuable antigen for discrimination between antibodies induced by B virus and those induced by other, closely related alphaherpesviruses, including HSV-1 and -2.

FIG. 1.

Construction of baculovirus donor plasmids for expression of B virus glycoproteins. (A) Schematic diagram of the modified expression cassette in the donor plasmid pFBHV5. The HBM secretion signal was incorporated upstream and V5- and His tag-encoding sequences were added downstream of the multiple cloning site. The expression of the recombinant proteins was driven by the polyhedrin promoter (P_PH). (B) Schematic diagrams of B virus glycoproteins. Numbers above the diagrams refer to the predicted amino acid sequence boundaries of the signal peptides (hatched bars) and extracellular (open bars), transmembrane (black bars), and cytoplasmic (gray bars) domains of B virus glycoproteins. Lollipop symbols indicate the locations of the predicted N-glycosylation sites. The dashed line in the gG ectodomain shows the predicted protease cleavage site. The secreted gG fragment (sgG) and the viral membrane-associated gG fragment (mgG) were expressed separately. Amplification of the extracellular domains by the use of specific primers (arrows) resulted in the generation of DNA fragments containing BamHI sites at the 5′ ends and EcoRI sites at the 3′ ends for cloning into the corresponding pFBHV5 sites.

FIG. 2.

Characterization of recombinant glycoproteins. The purified recombinant proteins gB, gC, gE, sgG, and mgG were separated by SDS-10% PAGE, followed by Coomassie staining (A) or immunoblotting (B and C). For panel B, the blot was probed with an anti-V5 MAb (diluted 1:2,000). For panel C, the blot was incubated with pooled sera from B virus antibody-positive rhesus macaques (diluted 1:100). The positions of the molecular size markers are given on the left.

FIG. 3.

Western blot analysis of rabbit antisera produced by immunization with the recombinant proteins. Uninfected (lanes 1) and B virus-infected (lanes 2) Vero cell lysates were separated by SDS-10% PAGE, followed by immunoblotting with sera from rabbits immunized with the purified recombinant proteins gB (1:2,000), gC (1:2,000), gE (1:2,000), sgG (1:400), and mgG (1:2,000) and with pooled sera from B virus-positive macaques (1:200). Molecular masses of marker standards are indicated on the left.

FIG. 4.

Reactivities of macaque serum pools with B virus recombinant antigens. ELISA plates were coated with 50 ng (gB) or 100 ng (gC, gD, gE, sgG, and mgG) of antigen per well and incubated with B virus antibody-positive (solid bars) or -negative (open bars) macaque serum pools at a dilution of 1:50. ODs were read at 405 nm.

FIG. 5.

Distribution of Rec-ELISA reactivities for individual macaque sera. ELISA plates were coated with 50 ng (gB) or 100 ng (gC, gD, and mgG) of antigen per well and incubated with individual B virus antibody-positive (n = 75) (▴) or -negative (n = 40) (▪) macaque sera at a dilution of 1:50. ODs were read at 405 nm. The solid lines indicate the mean OD values and the dashed lines indicate the cutoff OD values for each group.