Clinical protection of goats against CpHV-1 induced genital disease with a BoHV-4-based vector expressing CpHV-1 gD

Abstract

Caprine herpesvirus type 1 (CpHV-1) is an alphaherpesvirus causing genital disease leading to abortion in adult pregnant goats and a systemic disease with high morbility and mortality in kids. Further, Caprine herpesvirus 1 infection represents a valuable large animal model for human herpesvirus induced genital disease, exploitable for pathogenic studies, new vaccines and antiviral molecules testing. Here, the bovine herpesvirus 4 (BoHV-4) based vector derived from an apathogenic isolate of BoHV-4 and expressing the immunodominant CpHV-1 glycoprotein D (BoHV-4-A-gD(cp)gD(106)ΔTK) was constructed and its ability to protect goats against CpHV-1 induced genital disease evaluated. The subcutaneous route of recombinant BoHV-4 administration was first tested in vivo/ex vivo by in vivo image analysis and in vitro by goat skin primary cultures preparation and transduction. Next, an exploratory immunization and safety study in goats was performed with two recombinant BoHV4, BoHV-4-A-gD(cp)gD(106)ΔTK or BoHV-4-CMV-IgK-gE2gD-TM. In both cases no clinical signs were evident but a good titer of serum neutralizing antibodies was produced in all inoculated animals. When a challenge experiment was performed in a new group of animals using a highly pathogenic dose of CpHV-1, all the vaccinated goats with BoHV-4-A-gD(cp)gD(106)ΔTK were protected toward CpHV-1 induced genital disease respect to the unvaccinated control which showed typical vaginal lesions with a high grade of clinical score as well as a long lasting viral shedding. In summary, the data acquired in the present study validate BoHV-4-based vector as a safe and effective viral vector for goat vaccination against CpHV-1 induced genital disease and pave the way for further applications.

Figure 1

A) gD_cpgD₁₀₆ peptide sequence and predicted amino-acid product. In red CapHV-1 gD whereas in black the gD₁₀₆ tag. B) Diagram (not to scale) showing the pCMV-gD_cpgD₁₀₆ construct: In blue the cytomegalovirus enhancer promoter (CMV), in red the CapHV-1 gD, in black the gD₁₀₆ tag and in white the bovine growth hormone polyadenylation signal (PA). C) western immune-blotting of pCMV-gD_cpgD₁₀₆ transfected HEK cells lysates at different time post transfection (24, 48 and 72 hours). Untransfected HEK cells as negative control (−) and pIgKE2gD-TM transfected HEK cells lysate as a positive control (+).

Figure 2

A) Diagram showing (not to scale) the targeting 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-gD_cpgD₁₀₆ expression cassette. B) Representative colonies tested by HindIII restriction enzyme analysis, agar gel electrophoresis, and Southern blotting performed with a probe for the CMV promoter sequence, where the 2,612 bp band is present only for the targeted clones bat not for the untargeted control. C) Stability of the pBAC-BoHV-4-A-gD_cpgD₁₀₆ΔTK plasmid in E. coli SW102 cells. D) Representative fluorescent microscopic images of plaques formed by viable reconstituted recombinant BoHV-4-A-gD_cpgD₁₀₆ΔTK after DNA electroporation into BEK cells or in BEK cells expressing cre recombinase. Magnification, ×10. E) Replication kinetics of BoHV-4-A-gD_cpgD₁₀₆ΔTK growth on cre-expressing cells, compared with those of the parental 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).

Figure 3

A) Diagram not on scale of BoHV-4LucΔTK delivering the CMV-Luc expression cassette. CMV promoter is represented by the blue box, the luciferase ORF by the yellow box whereas the polyadenylation signal by the white box. B) Representative image of the site of BoHV-4LucΔTK injection, under the goat tail (red arrow), and the skin explant visualized by in vivo image analysis. C) Representative image of flasks containing the BoHV-4LucΔTK infected or uninfected goat skin explant organotypic cultures and visualized by in vivo image analysis.

Figure 4

A) Diagram not on scale of BoHV-4EGFPΔTK delivering the CMV-EGFP expression cassette and BoHV-4-A-gD_cpgD₁₀₆ΔTK delivering the CMV-gD_cpgD₁₀₆ expression cassette. CMV promoter is represented by the blue box, the enhanced green fluorescent protein ORF by the green box, the caprine gD ORF by the red box, the gD₁₀₆ tag by the grey box, whereas the polyadenylation signal by the white box. B) Representative phase contrast and fluorescence images of BoHV-4EGFPΔTK infected goat subcutaneous primary cell cultures at 24 hours post infection. C) Representative phase contrast and fluorescence images of BoHV-4EGFPΔTK infected goat subcutaneous primary cell cultures at 48 hours post infection and showing cytopathic effects. D) BoHV-4EGFPΔTK titer at 24 and 48 hours post infection as log₁₀ TCID50/ml. 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). E) Western immunoblotting of BoHV-4-A-gD_cpgD₁₀₆ΔTK infected goat subcutaneous epithelial and stromal primary cell cultures lysates. 5, 10 and 20 are the different amount as µl of the lysates.

Figure 5

A) Kinetics of the humoral immune responses of goats immunized with BoHV-4-A-gD_cpgD₁₀₆ΔTK and BoHV-4-A-CMV-IgK-gE2gD-TM (B). Anti-BoHV-1 [Bovine herpesvirus 1 (grey bars)], anti-BVDV [Bovine viral diarrhea virus (red bars)] and anti-CapHV-1 [Caprine herpesvirus 1 (black bars)] 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). T1, T2, T3, T4, T5, T6, T7, T8 and T26 is the time (T) expressed as week (1,2,3,4,5,6,7,8 and 26) post inoculation. S(T26) is the sentinel animal at 26 weeks. T0 is the pre immune sera.

Figure 6. Representative image of typical CpHV-1 induced vaginal lesions in unvaccinated goat (D) respect to vaccinated goats (A, B, C).

Figure 7. Clinical scores of vaccinated and control goats following CpHV-1 vaginal challenge.

Figure 8. Virus titers in vaginal swabs of vaccinated and control goats after CpHV-1 challenge.