Establishment of a bovine herpesvirus 4 based vector expressing a secreted form of the bovine viral diarrhoea virus structural glycoprotein E2 for immunization purposes

Abstract

Background: The biological characteristics of BoHV-4 make it a good candidate as a gene delivery vector for vaccination purposes. These characteristics include little or no pathogenicity, unlikely oncogenicity, the capability to accommodate large amounts of foreign genetic material, the ability to infect several cell types from different animal species, and the ability to maintain transgene expression in both undifferentiated and differentiated cells.

Results: A recombinant bovine herpesvirus 4 (BoHV-4CMV-IgKE2-14 Delta TK) expressing an enhanced secreted form of the bovine viral diarrhea virus (BVDV) structural glycoprotein E2 (gE2-14), obtained by the removal of the putative transmembrane domain and addition of a 14 amino acids peptide at its carboxyl terminal and an immunoglobulin K signal peptide to the amino terminal, was successfully constructed using a Recombineering (recombination -mediated genetic engineering) approach on BoHV-4 cloned as bacterial artificial chromosome. The galactokinase - based recombineering system was modified by the introduction of a kanamycin expression cassette and a kanamycin selection step that allowed a significant reduction of the untargeted background clones. BoHV-4CMV-IgKE2-14 Delta TK infected cell lines highly expressed gE2-14, which maintained native antigenic properties in a serum neutralization inhibition test. When rabbits and sheep were immunized with BoHV-4CMV-IgKE2-14 Delta TK, high levels of serum neutralized antibodies against BVDV were generated.

Conclusion: This work highlights the engineerization of BoHV-4 genome as a vector for vaccine purposes and may provide the basis for BVDV vaccination exploiting the BoHV-4- based vector that delivers an improved secreted version of the BVDV structural glycoprotein E2.

Figure 1

Structure and evaluation of plasmid vectors expressing the secreted forms of gE2. a) Diagram (not to scale) showing the pCMV-IgKE2-14 vector along with the sequence, and predicted amino-acid product, containing: the CMV enhancer promoter (CMV, in black box), the IgK signal peptide (red box) in frame with the E2 ORF and 14 hydrophilic extra amino acids peptide (14aa, green box) and the bovine growth hormone polyadenylation signal (PA, white box). b) Diagram (not to scale) showing the pCMV-IgKE2-23 vector along with the sequence and the predicted amino-acid product, containing: the CMV enhancer promoter (black box), the IgK signal peptide (red box) in frame with the E2 ORF and 23 hydrophobic extra amino acids peptide (23aa, green box) and the bovine growth hormone polyadenylation signal (PA, white box). c) Hydrophilicity plot of the 14 aa and 23 aa (in d) peptides with the calculated net charge, iso-electric point and the average hydrophilicity. e) Western immunoblotting of cell extracts and supernatant of cells transfected with pIgKE2-14 and pIgKE2-23.

Figure 2

Overall strategy of BoHV-4CMV-IgKE2-14ΔTK generation. a) First targeting, where kanamycin resistant cassette (Kana) adjacent to the galactokinase cassette (GalK) and flanked by BoHV-4 thymidine Kinase gene and adjacent sequences (TK), are introduced into the TK gene of BoHV-4 genome cloned as bacterial artificial chromosome (BAC-BoHV-4) via heat inducible homologous recombination in SW 102 E. coli. Following a positive selection on (solid) minimal plates containing galactose as the only source of carbon and a positive selection on (liquid) medium containing Kanamycin. Selected clones, fermenting lactose (red colonies) on McConkey plates (representative plate). b) HindIII restriction enzyme analysis of 6 clones out of hundreds, all displaying the right targeting, where a new 2.6 kb band (indicated by arrow, 1 to 6) corresponding to the insertion of the Kana/GalK cassettes is present but missing in the untargeted control. c) Second targeting and counter selection, where the Kana/GalK cassettes were removed via heat inducible homologous recombination and replaced with CMV-IgKE2-14 expression cassette. All colonies tested through HindIII restriction enzyme analysis and agar gel electrophoresis (d) shows the right re-targeting. The disappearing of the 2.6 kb band and the appearing of a new band with a lower molecular size are indicated by arrows.

Figure 3

Expression analysis of BoHV-4CMV-IgKE2-14ΔTK infected cell lines. a) Western immunoblotting of supernatant coming from several bovine cell lines infected with BoHV-4CMV-IgKE2-14ΔTK. b) Time course of gE2 expression of cells sustaining different types of infection (persistent, permissive and unpermissive infection).

Figure 4

Inhibition of serum neutralization (SN) test, to assay the gE2-14 antigenic properties expressed and secreted by BoHV-4CMV-IgKE2-14ΔTK infected cells. (a) Diagram showing the principle of the assay, where BVDV neutralizing antibodies pre-incubated with medium containing gE2-14 are blocked, allowing the virus to infect and destroy the cell monolayer. (b) The quantitative assay performed in a 96-multiwell plate where 4 sera (1, 2, 3, and 4) containing neutralizing antibody against BVDV were tested at different dilutions in the presence of gE2-14 (+Sera +E2) and in the absence of gE2-14 (+Sera -E2). Control virus was established in the absence of sera and presence of gE2-14 and in the absence of sera and gE2-14. Crystal violet staining allows macroscopic evaluation of the integrity or the destruction of the cell monolayer. (c) Bar graph showing the quantification of serum neutralization made on the basis of three different experiments (** = P < 0.001). Results are expressed as the log2 of the highest dilution of the serum that inhibited the development of virus-induced cytopathic effect in cell culture.

Figure 5

Kinetics of the humoral immune responses of rabbits and sheep immunized with BoHV-4CMV-IgKE2-14ΔTK. Sera collected from rabbits (a) and sheep (b) before immunization (time 0) and after immunization (time 1, 2, 3, 4 and 5) were evaluated for anti-BVD neutralizing antibodies by serum neutralization (SN) test. Serum neutralizing antibodies against BVDV are expressed as the reciprocal of the highest dilution of the serum that inhibited the development of virus-induced CPE in MDBK cells. Virus neutralization (VN) titers of 2 (log2) were considered to be positive. In both panels, each value represents the mean response of 3 rabbits (a) (** = P < 0.001, * = P < 0,05) or 4 sheep (b) (** = P < 0.001, * = P < 0,05). Each test was repeated 3 times and statistical significance was evaluated by Student's t test.