Novel recombinant parapoxvirus vectors induce protective humoral and cellular immunity against lethal herpesvirus challenge infection in mice

Abstract

Orf virus (ORFV; Parapoxvirus ovis) was used to develop a novel vector system for the generation of effective and safe live vaccines. Based on the attenuated ORFV strain D1701-V, recombinants were produced that express the glycoproteins gC (D1701-VrVgC) or gD (D1701-VrVgD) of the alphaherpesvirus of swine, pseudorabies virus (PRV). Expression of gC and gD was also demonstrated on the surface of recombinant virus-infected murine cells that do not produce infectious ORFV. Single or combined immunization with the ORFV recombinants protected different mouse strains of a host species nonpermissive for ORFV against a fulminant, lethal PRV challenge infection equal to immunization with PRV live vaccine. Most notably, even a single immunization with D1701-VrVgC was protective, whereas two applications of D1701-VrVgD were required for immune protection. The higher protective capacity of D1701-VrVgC correlated with the induction of a strong specific humoral immune response. This suggestion was supported by transfer experiments using sera from recombinant-immunized mice, which resulted in partial gC but not gD antibody-mediated protection of the naïve recipients. Remarkably, immunization of different immune-deficient mice demonstrated that the application of the PRV gC-expressing recombinant controlled the challenge infection in the absence of either CD4(+) or CD8(+) T cells, B cells, or an intact perforin pathway. In contrast, D1701-VrVgD-immunized mice lacking CD4(+) T cells exhibited reduced protection, whereas animals lacking CD8(+) T cells, B cells, or perforin resisted the challenge infection. The present study demonstrates the potential of these new vector vaccines to efficiently prime both protective humoral and cell-mediated immune mechanisms in a host species nonpermissive for the vector virus.

FIG. 1.

Construction of the ORFV recombinants (A) The map locations of HindIII fragments and of the inverted terminal repeats (ITR) of the genome of ORFV strain D1701-V are depicted. (B) The PstI-HindIII fragment containing the VEGF-E and adjacent genes was cloned as plasmid pORF-PA. (C) The singular StyI restriction site in pORF-PA was used to delete the VEGF-E gene by a bidirectional Bal31 digest that resulted in plasmid pdV-550. (D) A synthetic linker covering the indicated restriction sites was inserted into the EcoRV site. The obtained plasmid, pdV-Rec1, contains the early promoter of VEGF-E (P_vegf-e) and the original early transcription stop motif T5NT. (E) The NcoI-HinfI fragment of plasmid pALM-20 containing the complete PRV gC gene was blunt-end ligated into the EcoRV site of pDV-Rec1, resulting in plasmid pdV-gC. (F) The use of the HindIII-BamHI fragment of plasmid pgDBSII allowed cloning of the complete gD gene of PRV in pdV-Rec1 to obtain plasmid pdVgD.

FIG. 2.

Expression of PRV gC and gD in ORFV recombinant-infected cells. Vero cells were infected (MOI of 10) with D1701-VrVgC (lanes 1), D1701-VrVgD (lanes 2), D1701-VrV (lanes 3), and PRV Begonia (lanes 5) or mock infected (lanes 4). For Northern blot hybridization using radioactively labeled probes specific for PRV gC (A), PRV gD (B) and the ORFV early gene ANK3 (C), total RNA was isolated from CH-treated Vero cells. The sizes of the respective specific transcripts are indicated in kilobase pairs to the left. Cell lysates were obtained 24 h p.i., and Western blot analysis was performed with the gC-specific MAb A18b (D), the gD-specific rabbit serum 016/00 (E), or the ORFV 39K-specific MAb 4D9 (F). The apparent molecular mass of the detected proteins is indicated in kilodaltons.

FIG. 3.

Surface expression of PRV gC and gD on ORFV recombinant-infected cells. Flow cytometry of the indicated cells 24 h after infection with D1701-VrVgC (A to C) or D1701-VrVgD (D to F). Nonfixed infected cells (dark lines) or noninfected cells (bright lines) were stained 24 h after infection with the antigen-specific antibodies. The diagrams show the number of counted cells exhibiting specific fluorescence intensities.

FIG. 4.

Single-step growth curve of D1701-VrVgC. Vero cells (solid triangles), 3T3 cells (open squares), or L929 cells (solid squares) were infected with a MOI of 10 (A and B) or 1.0 (C and D) and harvested at the indicated times after infection. Cell lysates (A and C) and supernatants (B and D) were separately titrated on Vero cells in triplicate to determine infectious virus progeny. Bars indicate standard deviations.

FIG. 5.

PRV-specific serum antibody response in immunized mice. Sera from immunized BALB/c (A), 129/Sv/Ev (B), and C57BL/6 (C) mice were taken 2 weeks after the final vaccination. PRV-specific IgG1 (shaded columns) and IgG2a (white columns) antibodies were determined by subclass-specific ELISA. The mice were immunized as indicated with D1701-VrVgC (D-gC) and D1701-VrVgD (D-gD) alone or in combination as well as with the PRV live vaccine Begonia. The ratios of IgG2a to IgG1 subclasses are given above the columns. Bars indicate standard deviations. (D) PRV-neutralizing antibody titers of the different sera. Note the different titer scale from those of panels A to C.

FIG. 5.

PRV-specific serum antibody response in immunized mice. Sera from immunized BALB/c (A), 129/Sv/Ev (B), and C57BL/6 (C) mice were taken 2 weeks after the final vaccination. PRV-specific IgG1 (shaded columns) and IgG2a (white columns) antibodies were determined by subclass-specific ELISA. The mice were immunized as indicated with D1701-VrVgC (D-gC) and D1701-VrVgD (D-gD) alone or in combination as well as with the PRV live vaccine Begonia. The ratios of IgG2a to IgG1 subclasses are given above the columns. Bars indicate standard deviations. (D) PRV-neutralizing antibody titers of the different sera. Note the different titer scale from those of panels A to C.

FIG. 6.

PRV challenge infection of mice after intravenous transfer of recombinant ORFV-immune sera. Sera were obtained from BALB/c mice after twofold immunization with each ORFV recombinant alone and passively transferred to naïve recipients. The survival times of individual animals are depicted after transfer of the indicated volumes of D1701-VrVgC immune sera (A) or D1701-VrVgD immune sera (B). Mice receiving serum from twofold D1701-VrV-immunized animals served as a control. Challenge infection with 300 LD₅₀s of PRV was performed 24 h after serum transfer.