Cellular targeting of engineered heterologous antigens is a determinant factor for bovine herpesvirus 4-based vaccine vector development

Abstract

In a previous study, an apathogenic strain of bovine herpesvirus 4 (BoHV-4) cloned as a bacterial artificial chromosome and expressing a chimeric peptide (gE2/gD) as a secreted form was described. Recombinant virus-inoculated animals produced antibodies against bovine viral diarrhea virus (BVDV) gE2 and BoHV-1 gD. However, neutralizing antibodies were produced only against BVDV, not against BoHV-1. In the present work a recombinant BoHV-4 expressing a membrane-linked form of gE2/gD chimeric peptide was constructed, and inoculated rabbits produced serum-neutralizing antibodies against both BVDV and BoHV-1. Protein cell sorting and targeting are a very important issue when immunodominant antigens are engineered for recombinant virus vaccine development.

FIG. 1.

(A) pCMV-IgK-gE2gD-TM vector diagram (not to scale) containing the cytomegalovirus enhancer promoter (CMV prom), the IgK signal peptide (IgK) in frame with the gE2 ectodomain (gE2), the full-length gD (gD) provided with its transmembrane domain (TM) together with the cytoplasmic tail (ct), and the growth hormone polyadenylation signal (pA). (B) Chimeric peptide sequence (IgK-gE2/gD-TM) and predicted amino acid product. Italic ocher letters indicate the signal peptide, and the underlined black letters indicate the linker sequence connecting the signal peptide and the gE2 ectodomain (in red). gE2 is followed by the gD ectodomain in black and its transmembrane domain in blue, containing the highly hydrophobic peptide (underlined) and the cytoplasmic tail (in black).

FIG. 2.

(A and B) Western immunoblotting of cell extract and supernatant from HEK 293T cells transfected with pCMV-IgK-gE2gD-TM or with pEGFP-C1 (plasmid expressing EGFP) and probed with both anti-gD (A) and anti-gE2 (B) monoclonal antibodies. (C to F) Fluorescence-activated cell sorting analysis of gE2/gD-TM chimeric peptide expression on the cell surface of pCMV-IgK-gE2gD-TM-transfected cells. The green line corresponds to mock-transfected cells, and the violet line corresponds to pCMV-IgK-gE2gD-TM-transfected cells. In panel C, pCMV-IgK-gE2gD-TM-transfected and mock-transfected cells were both assayed with the anti-isotype antibody (FITC), and no nonspecific signals for the two cell populations were observed, as shown by the complete overlapping of the curves. When mock- and pCMV-IgK-gE2gD-TM-transfected cells were assayed with anti-gD (D) or anti-gE2 (E) plus the secondary antibody, a significant shift was observed for the pCMV-IgK-gE2gD-TM-transfected cells, and such a shift was further increased when anti-gD and anti-gE2 were assayed together (F). The experiment was repeated three times, giving identical results.

FIG. 3.

(A) Diagram showing (not to scale) the retargeting by heat-inducible homologous recombination in SW102 containing pBAC-BoHV-4-A-TK-KanaGalK-TK, where the Kana/GalK cassette was removed and replaced with the CMV-IgK-gE2gD-TM expression cassette. (B) Representative colonies tested by HindIII restriction enzyme analysis, agar gel electrophoresis, and Southern blotting showing the right retargeting, where the 2,650-bp band (nonretargeted; indicated by an arrow) disappeared and a 2,260-kb band (retargeted; indicated by arrows), detected by Southern blotting with a gE2 probe, appeared. (C) Stability of the pBAC-BoHV-4-A-CMV-IgK-gE2gD-TM plasmid in E. coli SW102 cells. SW102 cells containing the pBAC-BoHV-4-A-CMV-IgK-gE2gD-TM were passaged for 21 consecutive days, and BAC DNA from the culture was prepared on the indicated days, analyzed by HindIII digestion and agarose gel electrophoresis, and compared to the parental unretargeted pBAC-BoHV-4-A-TK-KanaGalK-TK (control) growth in DH10B cells lacking recombinase.

FIG. 4.

(A) Representative fluorescent microscopic images of plaques formed by viable reconstituted recombinant BoHV-4-A-CMV-IgK-gE2gD-TM after DNA electroporation into BEK cells or in BEK cells expressing cre recombinase. Magnification, ×10. (B) Replication kinetics of BoHV-4-A-CMV-IgK-gE2gD-TM growth on cre-expressing cells, compared with those of the parental BoHV-4-A-CMV-IgK-gE2gD-TM still containing the BAC cassette and the BoHV-4-A isolate. The data presented are the means ± standard errors of triplicate measurements (P > 0.05 for all time points as measured by Student's t test). (C) Western immunoblotting of BoHV-4-A-CMV-IgK-gE2gD-TM-infected cell extracts. Uninfected BEK cell extract was used as a negative control.

FIG. 5.

(A to C) Representative pictures of BoHV-4-EFGPΔTK-infected RK-13 cells (A), RBMSC (B), and RAEC (C) at 24 and 96 h postinfection, visualized by fluorescence microscopy with a FITC filter for EGFP expression or with a DAPI (4′,6-diamidino-2-phenylindole) filter for nuclear counterstaining. (D to F) Representative phase-contrast pictures of BoHV-4-A-CMV-IgK-gE2gD-TM-infected RK-13 cells (D), RBMSC (E), and RAEC (F) at 24 and 96 h postinfection with respective titers (expressed as log₁₀ TCID₅₀ per ml) of viral particles released at 24 and 96 h postinfection. Values are the means ± standard errors of three independent experiments. Magnification, ×10 (all panels).

FIG. 6.

Kinetics of the humoral immune responses of rabbits immunized with BoHV-4-A-CMV-IgK-gE2gD-TM (transmembrane) and compared with those of rabbits immunized with BoHV-4-A-CMV-IgK-gE2gD-14 (secreted). (A) Diagram showing the rabbit immunization scheme and blood sample collection. (B and C) Sera collected from rabbits before immunization and after immunization were evaluated for anti-gD (B) and anti-gE2 (C) antibodies by ELISA. Antibodies detected were expressed as the optical density at 504 nm; each value represents the mean response of five rabbits ± the standard error of the mean (*, P < 0.005 as measured by Student's t test or one-way analysis of variance). (D and E) Anti-BoHV-1 (D) and anti-BVDV (E) serum-neutralizing antibodies (SN) as measured by serum neutralization test. SN antibodies were expressed as the reciprocal of the highest dilution of the serum that inhibited the development of virus-induced CPE in MDBK cells. Virus neutralization titers of >2 (log₂) were considered positive. Each value represents the mean response of five rabbits ± the standard error of the mean (*, P < 0.005 as measured by Student's t test or one-way analysis of variance).

FIG. 7.

(A) Incorporation of gE2/gD into recombinant BoHV-4 particles. Extracts of virus-infected cells and virus purified through CsCl₂ gradient centrifugation were analyzed by Western immunoblotting with an anti-gD monoclonal antibody. Positive and negative controls were performed with BoHV-4-A-CMV-IgK-gE2gD-TM-infected cell extract and BoHV-4-A purified virus, respectively. (B) Rabbit (1 to 5) or vaccinated bovine (1 to 3) sera containing neutralizing antibodies against both BVDV and BoHV-1 were tested against BVDV, BoHV-1, or BoHV-4-A-CMV-IgK-gE2gD-TM at a single dilution of 1:2 to increase the chances of detecting serum-neutralizing antibody against BoHV-4-A-CMV-IgK-gE2gD-TM. Control virus was established in the absence of sera or with sera from cows not infected with BVDV or BoHV-1. Crystal violet staining allows macroscopic evaluation of the integrity (dark wells) or the destruction (clear transparent wells) of the cell monolayer. The test was repeated three times, and the same results were obtained. (C) Representative microscopic IFAT images of sera from BoHV-4-A-CMV-IgK-gE2gD-TM-immunized rabbits. The presence of anti-BoHV-4 antibodies in the rabbit serum samples (5 weeks postimmunization) is detectable by green cells when observed with an FITC filter. Negative controls were established with preimmune sera as well as control wells containing BoHV-4-infected or uninfected cells treated only with the secondary antibody. Counterstaining with Evans blue dye observed with a tetramethyl rhodamine isothiocyanate (TRITC; red pictures) filter was used to monitor the integrity of the cell substrate. Magnification, ×10.