A Simple and Versatile Method for Ex Vivo Monitoring of Goat Vaginal Mucosa Transduction by Viral Vector Vaccines

Abstract

Goat may represent a valid large animal model for human pathogens and new vaccines testing. Appropriate vaccine administration is a critical component of a successful immunization program. The wrong route of administration may reduce the efficacy of the vaccine, whereas the proper administration strategy can enhance it. Viral vectors have been employed successfully for goat and sheep immunization; however, no data concerning the vaginal route are available. A viral vector's ability to transduce the site of inoculation is of primary interest. In this study, a fast and reliable ex vivo assay for testing the transduction capability of an Ad5-based vector when intravaginally administered was developed. An Ad5 vector delivering an expression cassette with a bicistronic reporter gene, Ad5-CMV-turboGFP-IRES-Luc2, was constructed. We demonstrated Ad5-CMV-turboGFP-IRES-Luc2's ability to transduce caprine vaginal mucosa by ex vivo bioluminescent imaging (BLI) employing a simple CCD camera apparatus for chemiluminescence western immunoblotting. These data, though simple, provide valuable insights into developing a vaginal immunization strategy using a viral vector-based vaccine to protect against pathogens causing genital diseases.

Keywords: animal model; bioluminescent imaging; genital diseases; goat; vaginal mucosa vaccines; viral vectors.

Figure 1

Generation and transduction efficiency of Ad5-CMV-turboGFP-IRES-Luc2. (A) Schematic diagram (not to scale) of Ad5-CMV-turboGFP-IRES-Luc2 genomic composition: 5′ and 3′ inverted terminal repeat (ITR; grey), adenoviral packaging signal (Ψ; blue), human cytomegalovirus immediate early enhancer/promoter (CMV; azure), open reading frame coding for turbo GFP (green), internal ribosomal entry site (IRES; turquoise), human codon usage adapted luciferase ORF (Luc2; orange), bovine growth hormone polyadenylation signal (pA; blue), and E1A/B and E3 deleted human adenovirus type 5 genome backbone (ΔE1-E3Ad5 backbone; yellow). (B) Phase contrast and fluorescence images of Ad5-CMV-turboGFP-IRES-Luc2 infected HEK293T (scale bar corresponds to 100 µm) at 24 and 48 h post infection (pi). (C) Respective viral titters expressed as log10 per mL of transducing units (TUs) of viral particles released at 24 and 48 h pi (*** p < 0.01). Values are the means ± standard errors of three independent experiments. (D) Phase contrast and fluorescence images of Ad5-CMV-turboGFP-IRES-Luc2 transduced fetal caprine kidney primary cell cultures at different MOIs (1 and 10) and the untransduced control. (E) Representative images of the efficiency of transduction, as quantified by cytofluorimetric analysis, when cells were transduced with 1 MOI of Ad5-CMV-turboGFP-IRES-Luc2; 89% transduction efficiency was obtained with respect to the untransduced control. The experiment was repeated three times with similar results.

Figure 2

Bioluminescence imaging (BLI) and quantification of Ad5-CMV-turboGFP-IRES-Luc2 transduction in CFK cells and vaginal organotypic cultures. (A) Representative images of Ad5-CMV-turboGFP-IRES-Luc2 transduced (1 and 10 MOIs) and untransduced (0 MOI) CFK cells’ monolayers expressing luciferase, as observed by BLI in grey or pseudocolor mode and (B) their respective quantification expressed as pixel squared (Supplementary File S1). Data presented are the means ± standard errors of triplicate measurements (measured by the Student’s t-test). (C) Representative BLI images of Ad5-CMV-turboGFP-IRES-Luc2 transduced and untransduced vaginal organotypic cultures, as observed by BLI in grey or pseudocolor mode, as well as their unexposed and merge images. (D) Photon emissions, quantified as in (B). Data presented are means ± standard errors of triplicate measurements (measured by the Student’s t-test).