A West Nile virus (WNV) recombinant canarypox virus vaccine elicits WNV-specific neutralizing antibodies and cell-mediated immune responses in the horse

https://doi.org/10.1016/j.vetimm.2008.02.002Get rights and content

Abstract

Successful vaccination against West Nile virus (WNV) requires induction of both neutralizing antibodies and cell-mediated immune responses. In this study, we have assessed the ability of a recombinant ALVAC®-WNV vaccine (RECOMBITEK® WNV) to elicit neutralizing antibodies and virus-specific cell-mediated immune responses in horses. In addition, we examined whether prior exposure to ALVAC®-WNV vaccine would inhibit B and cell-mediated immune responses against the transgene product upon subsequent booster immunizations with the same vaccine. The results demonstrated that the recombinant ALVAC®-WNV vaccine induced neutralizing antibodies and prM/E insert-specific IFN-γ+ producing cells against WNV in vaccinated horses. Prior exposure to ALVAC®-WNV vaccine did not impair the ability of horses to respond to two subsequent booster injections with the same vaccine, although anti-vector-specific antibody and cell-mediated immune responses were induced in vaccinated horses. This report describes, for the first time, the induction of antigen-specific cell-mediated responses following vaccination with an ALVAC® virus recombinant vaccine encoding WNV antigens. Moreover, we showed that both WNV-specific IFN-γ producing cells and anti-WNV neutralizing antibody responses, are not inhibited by subsequent vaccinations with the same vector vaccine.

Introduction

An improved understanding of the contribution of the different immune responses to the prevention and control of infectious diseases is an important step towards rational design of new vaccines. Immune responses to viruses have been extensively studied in humans and mice. In particular, cell-mediated and innate immunity are generally recognized as important for the control and elimination of viral infections (Boehme and Compton, 2004, Doherty, 1996). Several functional assays have been developed to measure cell-mediated immune responses (O’Neill et al., 1999, Paillot et al., 2006). The results of these studies have shown important differences in the immune responses induced by conventional killed vaccines and those resulting from natural infection (Daly et al., 2004). In view of these qualitative differences, significant effort has been directed in the past decade towards the development of equine vaccines that induce strong cellular immune responses. These approaches include the use of plasmid DNA vaccines (Minke et al., 2006) and live vectored vaccines (Breathnach et al., 2004).

One of the most significant changes in the field of veterinary vaccines over the past few years has been the development of several recombinant vaccines based on the canarypox vector (ALVAC®) (Edlund Toulemonde et al., 2005, Karaca et al., 2005, Minke et al., 2007, Pardo et al., 1997, Poulet et al., 2003). Canarypox virus vectors undergo an abortive replication cycle in mammalian cells during which the inserted gene product (transgene) is expressed which results in the stimulation of both humoral and cell-mediated immune responses in a manner similar to a natural infection (Berencsi et al., 2001, Nacsa et al., 2004, Santra et al., 2002). Although not fully explained in its mechanism, canarypox virus vector allows for a strong immunogenicity of the recombinant antigen. Ignatius et al. reported that canarypox vectors were able to induce dendritic cells (DCs) maturation in vitro (Ignatius et al., 2000) but other mechanisms by which these vectors may influence immunogenicity of the transgene could exist. Very interestingly, canarypox vectors appear free of the inhibitory effects of vector immunity even after multiple injections (Franchini et al., 2004). In this regard, canarypox viruses successfully combine the safety of an inactivated vaccine with the efficacy of a modified live vaccine.

Recently, a recombinant West Nile virus (WNV) canarypox vector vaccine has been developed (Minke et al., 2004, Siger et al., 2004) for use in horses against WNV infection, a mosquito-transmitted flavivirus responsible for a potentially fatal or debilitating disease in horses and humans (Hayes and Gubler, 2006). Because horses infected with WNV would benefit from pre-existing virus-specific memory humoral and cell-mediated immune responses (Shrestha and Diamond, 2004), we investigated whether vaccination with the recombinant ALVAC®-WNV vaccine would induce these immune responses in the horse. In addition, we examined whether pre-existing immunity against the canarypox virus vector would inhibit subsequent immune responses against the transgene following repeated injections with this vaccine.

Section snippets

Vaccines

The WNV vaccine consisted of a commercial preparation of a freeze-dried vaccine containing recombinant canarypox virus (ALVAC®) expressing the prM/E genes derived from a 1999 New York isolate of WNV (Gould and Fikrig, 2004). Prior to immunization, the vaccine was reconstituted with 1 ml of diluent containing CARBOPOL® 974P adjuvant (BF Goodrich Chemicals Europe NV, Belgium) at a concentration of 4 mg/ml.

Animals and vaccination schedule

Ten horses (Welsh Mountain ponies and mixed breed) of both sexes were used in this study. The

WNV-specific IFN-γ+ cell responses

The WNV-specific IFN-γ+ responses in vaccinated and control horses are shown in Fig. 2. Only moderate non-specific IFN-γ signals were found at some time points in the non-vaccinated horses (Fig. 2B). In contrast, upon ex vivo stimulation with WNV antigens, prM/E-specific IFN-γ+ responses were found in all ALVAC®-WNV vaccinated horses following the primary vaccination course of two doses. These responses declined over time but were still detectable in two out of five horses on day 91, the last

Discussion

Several studies have examined immune correlates of protection against WNV in mouse models. Although neutralizing antibodies are thought to play a major role in eliminating viremia (Diamond et al., 2003a), the antibody response itself does not completely eliminate virus reservoirs in immunocompromised mice (Engle and Diamond, 2003). It has been established in flavivirus murine infection models that cell-mediated immune responses may contribute to viral protection (Diamond et al., 2003b), and

Acknowledgements

P.M. Guigal and the animal caretakers are gratefully acknowledged for their outstanding management of the horse trials.

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