A highly conserved epitope-vaccine candidate against varicella-zoster virus induces neutralizing antibodies in mice

Abstract

Varicella-zoster virus (VZV) is a highly infectious agent of varicella and herpes zoster (HZ). Vaccination is by far the most effective way to prevent these diseases. More safe, stable and efficient vaccines, such as epitope-based vaccines, now have been increasingly investigated by many researchers. However, only a few VZV neutralizing epitopes have been identified to date. We have previously identified a linear epitope between amino acid residues 121 and 135 of gE. In this study, we validated that this epitope is highly conserved amongst different VZV strains that covered five existing phylogenetic clades with an identity of 100%. We evaluated the immunogenicity of the recombinant hepatitis B virus core (HBc) virus-like particles (VLPs) which included amino acids (121–135). VZV-gE-specific antibodies were detected in immunized mouse serum using ELISA. The anti-peptide antiserum positively detected VZV via Western blot and immunofluorescent staining assays. More importantly, these peptides could neutralize VZV, indicating that these peptides represented neutralizing epitopes. These findings have important implications for the development of epitope-based protective VZV vaccines.

Introduction

Varicella-zoster virus (VZV) is a member of the human α-herpesvirus subfamily with a double-stranded DNA genome of approximately 124,884 bp which encodes 71 open reading frames (ORFs) [1]. It is the causative agent of varicella (chickenpox) and herpes zoster (shingles, HZ). Humans are the only natural hosts for this virus. VZV causes varicella with systemic features, such as fever and a generalized pruritic vesicular rash in childhood and becomes latent in the sensory ganglia after the primary infection [2]. HZ is caused by VZV reactivation and manifests as a painful, blistering skin eruption in a unilateral and dermatomal distribution that usually lasts approximately 2 weeks, which may be followed by a common and debilitating complication, post-herpetic neuralgia (PHN), with a weakened immune system that occurs with aging [3].

Currently, two live-attenuated VZV Oka vaccines are licensed, one against varicella, another against HZ, which have remarkable and beneficial effects in preventing varicella and HZ [4], [5]. With the development of biochemistry, molecular vaccinology and genetic engineering technology and increased demand for vaccine safety, the need for more safe and stable vaccines in various forms continues to grow. Plasmids containing the gene encoding the full-length or truncated form of gE (prototype DNA vaccines), which could stimulate immunity in mice, were constructed by Hasan et al. [6], [7]. Mullane et al. [8] reported that the heat-treated zoster vaccine was generally safe and immunogenic in immunocompromised adults. Significantly, an adjuvanted HZ subunit vaccine based on VZV glycoprotein E (gE) was well tolerated and immunogenic, and more importantly, this vaccine induced a robust immune response and had a clinically acceptable safety [9], [10], [11]. In view of this, more safe, stable and efficient vaccines including epitope-based vaccines now more prevalent. Epitope peptides are considered to be promising candidates for new generation vaccines, especially those with conserved and neutralizing epitopes [12]. The potential advantages of epitope-based vaccines include increased safety; economical technology as they are easily produced and have simplistic compositions; the reduction of allergic reactions; and the ability of focus immune responses on selected epitopes [13], [14].

VZV is an enveloped virus that expresses at least nine membrane proteins, of which gE is the most essential and abundant. Only a few neutralizing VZV epitopes have been identified to date. Yasushi et al. [15] reported that the gH neutralizing epitopes were conformational and comprised a cluster of the seven independent protein portions of gH. Marius et al. [16] generated a recombinant monoclonal anti-VZV antibody that recognized by a conformational epitope within the gH/L protein complex, which could effectively neutralize VZV. Residues 1-134 and 101-161 of gE were identified as immunodominant regions by Western blotting and ELISA analyses, using serum from both varicella and zoster patients [17]. Additionally, immunizations with the recombinant hybrid Ty-virus-like particles presenting the gE (aa1-134) and gE (aa101-161) regions in small animals could induce neutralizing antibody responses [18]. Furthermore, monoclonal antibody (mAb) 3B3 recognized an 11-amino acid (residues 151-161; QRQYGDVFKGD) epitope in the ectodomain of VZV gE by using the recombination PCR technique [19]. Therefore, pinpoint neutralizing epitopes and epitope-based vaccines against VZV are still lacking in-depth research. Therefore, we are interested in developing a recombinant epitope-based vaccine with neutralizing abilities against VZV.

Our group previously generated a VZV-gE-specific complement-dependent mAb (named 4A2) by immunization with a recombinant gE antigen expressed by insect cells and the 4A2 epitope was identified as the amino acid sequence (SAQEDLGDDTGIHIV) [20]. In this study, we validated the gE-epitope that we identified and found that it was highly conserved. Then, we generated a fusion protein with 149 aa of the hepatitis B virus core (HBc) protein and the epitope, and confirmed that this protein was able to self-assemble into virus-like particles (VLPs) by electron microscopy. We further found that the VZV gE-specific IgG antibody in mice immunized with chimeric VLPs HBc-gE (aa121-135) were not lower than purified rgE protein and heated-inactivated vOka groups, in high-dose groups. More importantly, we proved that these chimeric particles were assessed for an ability to induce neutralizing antibodies to gE-specific epitope in mice. Our results suggest that chimeric HBc particles carrying the neutralizing epitope of gE can induce protective immunity against VZV. These findings have important implications for the development of epitope-based protective VZV vaccines