Elsevier

Vaccine

Volume 20, Issues 31–32, 1 November 2002, Pages 3744-3751
Vaccine

Induction of cytotoxic T-lymphocytes specific for bovine herpesvirus-1 by DNA immunization

https://doi.org/10.1016/S0264-410X(02)00375-4Get rights and content

Abstract

Cytotoxic T-lymphocytes (CTLs) are critical for the defense against herpesvirus infections, in which cell-to-cell spread occurs earlier than the hematogenous spread. The ability of bovine herpesvirus-1 (BHV-1) to undergo latency, to induce apoptosis of CD4+ T-lymphocytes, and to down-regulate the expression of major histocompatibility complex (MHC) class I molecules, necessitates the development of immunization strategies that do not involve the live virus. The objective of this study was to evaluate the feasibility of DNA immunization as a means of induction of CTLs against BHV-1. Mice were injected either by intramuscular (IM) or intradermal (ID) route with a Sindbis virus-based plasmid carrying the gene encoding the glycoprotein D (gD) of BHV-1. Splenocytes from the immunized mice were re-stimulated in vitro with gD-transduced syngeneic fibroblasts. The CTLs generated specifically lysed syngeneic targets, either transduced with gD or infected with BHV-1. IM route of inoculation induced a better CTL response when compared to ID route with respect to onset, magnitude and duration of immunity. These results indicate the feasibility of using a plasmid carrying the gene encoding BHV-1 gD as an immunogen to induce CTLs against BHV-1.

Introduction

Bovine herpesvirus-1 (BHV-1), an alphaherpesvirus, is an important etiological agent of bovine respiratory disease complex which costs over US$ 500 million annually to the US cattle industry alone [1]. The virus causes a wide variety of clinical manifestations that include infectious rhinotracheitis, pustular vulvo-vaginitis/balanoposthitis and neurological disease [2]. Infection with BHV-1 leads to secondary bacterial infection, particularly with Mannheimia (Pasteurella) haemolytica, resulting in fatal calf pneumonia [2]. The ability of BHV-1 to undergo latency and induce immunosuppression presents major difficulties in controlling this infection. The mechanisms of immunosuppression induced by BHV-1 include: (1) down-regulation of cell surface expression of major histocompatibility complex (MHC) class I molecules [3], [4]; and (2) induction of apoptosis of several subtypes of bovine leukocytes such as CD4+ T-lymphocytes, B cells and monocytes [5], [6], [7].

Both inactivated and modified live virus (MLV) vaccines against BHV-1 are available. Although the currently available inactivated vaccines induce a good neutralizing antibody response, they are poor inducers of cell-mediated immune (CMI) response. Moreover, the antibody response induced by the inactivated vaccines is short lived. Because the virus spreads via cell-to-cell transfer, and the infection proceeds in the presence of serum neutralizing antibodies, cytotoxic T-lymphocytes (CTLs) are critical for elimination of the virus [8]. Although the MLV vaccines help to control the clinical disease, they do not help in eliminating the virus infection since the vaccine strains can also undergo latency with subsequent reactivation and shedding [9]. Most importantly, the MLV vaccine strains, like the wild-type virus, down-regulate the cell surface expression of MHC class I molecules [3], [4] which is likely to compromise the development of CTLs against not only BHV-1, but also other viruses and intracellular pathogens. Therefore, immunization strategies that do not involve the use of live BHV-1 are better alternatives to the current immunization methods. Such alternatives include subunit vaccines, peptide vaccines, epitope-based vaccines and DNA vaccines [10].

Immunization with a DNA expression cassette encoding a particular immunogen for prophylactic and therapeutic purposes is a novel and revolutionary means in vaccine technology. Injection of a plasmid encoding a specific immunogen results in in vivo transfection of the host cells leading to the expression of the immunogen and development of antigen-specific immune response [11], [12]. DNA immunization of a mammalian host leads to the induction of long lasting antibody and CTL responses [13], [14]. Furthermore, the immune response can be directed towards Th1 or Th2 type of response based on the route of the plasmid delivery and, by co-administration of the plasmids encoding appropriate immunomodulatory cytokines [15], [16].

Earlier studies demonstrated the induction of antibody response against BHV-1 in mice and cattle by immunization with plasmids encoding glycoprotein D (gD) of BHV-1 [17], [18]. However, these studies did not demonstrate the induction of CTL response against BHV-1 by genetic immunization. In this paper, we report the induction of CTLs specific for BHV-1 by immunization with a Sindbis virus-based eukaryotic expression vector encoding BHV-1 gD (pSIN-gD).

Section snippets

Construction of vector encoding glycoprotein D (gD) of BHV-1

The gene encoding BHV-1 gD in the vector polink26 (pIBRxh) was a kind gift from Dr. S.I. Chowdhury (Kansas State University, Manhattan, KS). The Sindbis virus vector pSIN-gD was constructed by releasing the gene for BHV-1 gD from pIBRxh using XhoI and XbaI and inserting it into the same site of pSIN (Fig. 1A). The Sindbis virus vector pSIN-β-galactosidase (β-gal) encoding β-gal cloned into XhoI–NotI site of pSIN served as the negative control. The correct orientation and sequence of the

BHV-1 gD-transduced cells (BC-gD) express gD

BC-gD cell lysate was subjected to SDS-PAGE, followed by Western-blot analysis with the MAb 9D6 that is specific for BHV-1 gD. BC-β-gal cell lysate and BHV-1-infected cell lysate were used as the negative and positive controls, respectively. As expected, BHV-1 gD was present in BHV-1-infected cell lysate and BC-gD cell lysate, but not in BC-β-gal cell lysate (Fig. 2, panel A). A similar Western-blot experiment with the lysate of cells transfected with pSIN-gD and the gD-specific MAb 9D6

Discussion

Antibodies at the portal of entry prevent the entry of the virus into the host, and hence are an important component of anti-viral defense. However, once the virus enters and replicates within the host cells, its clearance requires either antibody-dependent cell cytotoxicity (ADCC) or virus-specific CTLs. ADCC is a relatively inefficient process since the effector function of virus-specific CTLs requires far fewer relevant functional molecules than ADCC to trigger an immunological response [29]

Acknowledgements

This article is published as ARD Journal Series no. 13345, with the approval of the University of Nebraska Agricultural Research Division. We are thankful to Mr. Anil K. Jayaprakash, Department of Biometry, University of Nebraska at Lincoln, for performing the statistical analyses.

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    Present address: Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland, OR 97201, USA.

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