Assembly of feline calicivirus-like particle and its immunogenicity

Abstract

Virus-like particles (VLPs) were produced in insect cells infected with a recombinant baculovirus containing the capsid gene of feline calicivirus strain F9 (FCV-F9). The FCV VLPs were morphologically and antigenically similar to the native virus and contained a single capsid protein with a molecular weight of approximately 60 kDa that reacted with FCV antiserum. Moreover, following immunization of rabbits, VLPs were able to elicit neutralizing antibodies against several FCV strains isolated from clinical samples. Our preliminary results showed that FCV-VLP could be considered a candidate vaccine against FCV infections.

Introduction

Feline calicivirus (FCV), belonging to the Vesivirus genus in the Caliciviridae family, is a non-enveloped virus with a diameter of approximately 35 nm that contains a plus-sense single-stranded polyadenylated RNA genome of 7.7 kb. The genome is organized into three open reading frames (ORFs). The first ORF (ORF1) encodes a 200 kDa polyprotein that is processed by the 3C-like cysteine proteinase into six mature non-structural proteins, p5.6, p32, p39 (nucleoside triphosphatase), p30, VPg and Pro-pol. The second ORF (ORF2) of the FCV genome encodes a 73 kDa capsid precursor (preVP1) that is cleaved by the same virus-encoded proteinase to yield the mature 60 kDa capsid protein (VP1) by removal of the first 125 amino acids (aa) from the N-terminus (Carter et al., 1992). Tohya et al. (1997) showed that at least seven neutralizing epitopes are present on the FCV capsid protein. The third ORF (ORF3), located at the 3′-end of the genomic RNA encodes a small (12 kDa) minor structural protein of 106 amino acids, VP2, which is essential for the production of infectious virions (Sosnovtsev et al., 2005).

FCV infection causes an upper respiratory tract disease (URTD) in cats, especially those living in colonies (Marsilio et al., 2005). Following recovery from clinical disease, cats can develop persistent inapparent infection. These carriers represent a reservoir of infection for susceptible animals (Pedersen and Hawkins, 1995). Despite widespread vaccination with an F9 strain attenuated virus, FCV-related illnesses still occurs among vaccinated cats, and the proportion of chronic oral shedders is unchanged (Harbour et al., 1991).

Two different explanations exist for the persistence of FCV carriers and disease. Firstly, the conventional FCV vaccines induce only a partial or marginal cross-protection against field strains, because the virus changes its antigenic profile during infection selecting strains that are less efficiently neutralized by antibodies against the vaccine strain (Geissler et al., 1997, Lauritzen et al., 1997). Alternatively, the vaccine strain may persist in cat colonies, evolving over time and these changes may be associated with reversion to partial virulence (Radford et al., 2001). Results from studies by Pedersen and Hawkins (1995) regarding the residual virulence of live FCV-F9, showed that the vaccine strain was effective when introduced by the subcutaneous route but retained certain level of virulence when given orally. In vaccinated animals, mild disease symptoms are frequent and oral shedding is common and sometimes prolonged (Dawson et al., 1993).

Consequently, a new immunization approach that avoids the drawbacks like reversion and recombination associated with live vaccines is needed. In many cases, recombinant vaccines have proven to be an effective alternative to live virus vaccines. The capsid proteins from several strains of human calicivirus including Norwalk virus (NV), Mexico virus (MxV) and Snow Mountain agent have been expressed in insect cells infected with recombinant baculoviruses (Green et al., 1997, Hardy et al., 1997, Jiang et al., 1992, Jiang et al., 1995). The capsid proteins from these viruses spontaneously assemble into empty virus-like particles (VLPs) that are released into the medium of insect cell cultures, from which they can usually be purified in high yields (White et al., 1997). VLPs represent a specific class of subunit vaccine that mimic the structure of authentic virus particles. They are recognized readily by the immune system and present viral antigens in a more authentic conformation than other subunit vaccines (see review by Noad and Roy, 2003). In human, preliminary phase I to test the safety and immunogenicity of insect-cell-expressed Norwalk virus VLPs have confirmed that they are both safe and effective to stimulate IgG and IgA responses (Ball et al., 1999, Noad and Roy, 2003). However, to date VLPs using the baculovirus expression system for members of the Vesivirus genus including FCV have not been reported.

In this study, in order to develop a candidate vaccine for FCV, we expressed the capsid protein VP1 of FCV in insect cells and generated VLPs, which were morphologically identical to the virus capsid and could induce the immunogenicity in a model animal.