Induction of humoral responses to BHV-1 glycoprotein D expressed by HSV-1 amplicon vectors

Abstract

Herpes simplex virus type-1 (HSV-1) amplicon vectors are versatile and useful tools for transferring genes into cells that are capable of stimulating a specific immune response to their expressed antigens. In this work, two HSV-1-derived amplicon vectors were generated. One of these expressed the full-length glycoprotein D (gD) of bovine herpesvirus 1 while the second expressed the truncated form of gD (gDtr) which lacked the trans-membrane region. After evaluating gD expression in the infected cells, the ability of both vectors to induce a specific gD immune response was tested in BALB/c mice that were intramuscularly immunized. Specific serum antibody responses were detected in mice inoculated with both vectors, and the response against truncated gD was higher than the response against full-length gD. These results reinforce previous findings that HSV-1 amplicon vectors can potentially deliver antigens to animals and highlight the prospective use of these vectors for treating infectious bovine rhinotracheitis disease.

Fig. 1

Amplicon plasmids constructs. (A) Amplicon plasmid pAEUA2 contained sequences required for amplicon replication (Ori-S) and packaging (a). The multiple cloning site (MCS) located between the human cytomegalovirus promoter (H_CMV) and a polyadenylation signal of simian virus 40 (pA_SV40) contained unique NotI, XbaI, and NheI restriction sites. The enhanced green fluorescent protein (EGFP) reporter gene was placed between the herpes simplex virus type 1 (HSV-1) immediate-early promoter (P_IE4/5) and bovine growth hormone polyadenylation signal (pA_BGH). Amp R: ampicillin resistance, colE1: plasmid origin of replication. (B) Schematic diagram of glycoprotein D (gD) of bovine herpesvirus 1 showing the signal peptide, ectodomain, and transmembrane region. (C) Schematic diagram of truncated form of gD.

Fig. 2

Glycoprotein D (gD) and truncated form of gD (gDtr) expression in Gli36 cells. The upper panels show Gli36 cells transfected with pAgD BHV-1 (A), pAgDtr BHV-1 (B), and pAEUA2 (C). The lower panels show Gli36 cells infected with vAgD BHV-1 (D), vAgDtr BHV-1 (E), and vAEUA2 (F). gD glycoprotein was detected by immunofluorescence using the monoclonal antibody 1106 and secondary antibody anti-mouse IgG H+L Alexa Fluor 555 (red). EGFP expression was observed and appeared green. Scale bar = 50 µm.

Fig. 3

Antibody response against BHV-1 gD in immunized BALB/c mice. Six 8-week old mice were vaccinated twice at 21-day intervals with vAEUA2, vAgD BHV-1, and vAgDtr BHV-1. Groups of three mice were injected intramuscularly with 5 × 10⁵ TU/mL of amplicon vector. In panel A, the bars represent the antibody titers for mice treated with (a) the control amplicon vector vAEUA2 expressing no gD, (b) amplicon vector vAgD BHV-1 expressing full-length gD, and (c) amplicon vector vAgDtr BHV-1 expressing gDtr. Data represent the mean ± SD for two independent experiments. ^*p < 0.05 and ^**p < 0.01 (determined by Student's t-test relative to the control). In panel B, the reactivity of mice sera was tested by Western blotting. Serum pool (day 36 p.i.) of mice immunized with vAgDtr BHV-1 (Lane a), vAgD BHV-1 (Lane b), or vAEUA2 (Lane c) were evaluated. A pool of pre-immune mouse serum (Lane d) was used as a negative control. Positions of molecular weight markers are indicated on the left of the image.