Vaccines play a critical role in the control of viral diseases. Vaccines against both PRV and PPV are extensively used all over the world. However, the use of such vaccines in clinical practice is limited by considerations of time, cost, safety and poor PPV replication in vitro. Therefore, research is now focused on developing a bivalent vaccine.
The parent PRV strain SA215 used in this trial is an approved vaccine strain which is strongly immunogenic and has been widely used to prevent and control pseudorabies diseases in China. This PRV vaccine strain has been attenuated by deletion of the genes encoding three important virulence factors (TK, gE, and gI) of the Fa strain. It has been approved as a new veterinary medicine and is the first animal virus-based genetically engineered vaccine in China.
Many systems have been successfully used for the expression of the PPV VP2 gene resulting in self-assembly of VLPs. Such systems include adenovirus, yeast, and particularly, the insect cell-baculovirus system [7, 21, 22] which has been used for mass production. Their expression products assembled into VLPs were similar in size and morphology to the original virions. The highly immunogenic VLPs have been shown to protect breeding sows against reproductive failure following virulent virus challenge , which is in accordance with the data obtained in this study. However, the potential for contamination of PPV VLPs with the vectors used in these approaches has raised serious safety concerns about the use of these recombinant vaccines [24, 25]. In this study, the pseudorabies virus system, which was extensively applied to express other heterologous antigens, was used to express the PPV VP2 gene successfully, without any of the described safety issues.
Recently, PPV VLPs have been shown to express foreign polypeptides in certain positions, resulting in the successful production of many highly immunogenic peptides, and the induction of strong antibody, helper-T-cell, and cytotoxic T-lymphocyte responses . Sedlik et al. prepared the hybrid virus-like particles by self-assembly of the modified porcine parvovirus VP2 capsid protein, carrying a CD8+ T cell epitope from the lymphocytic choriomeningitis virus nucleoprotein. Immunization of mice with these hybrid pseudoparticles, without any additional adjuvant, induced strong cytotoxic T lymphocyte (CTL) responses against both peptide-coated and virus-infected target cells . Pan et al. used PPV VLPs as a new generation of non-replicative vectors for delivery of a PCV2 epitope, which was expressed in adenovirus. The expressed product significantly enhanced both antibody-specific and cell-mediated immune response to PPV and PCV2 . It can be speculated that the PRV SA215/VP2 constructed in this study could be used for the insertion of foreign polypeptides in specific positions of the VP2 gene to enhance immunogenicity of PRV and PPV, and furthermore, may be developed as a multivalent vaccine.
The VP2 and EGFP fusion protein facilitated screening and purification of the recombinant virus from infected Vero cells although the issue of correct folding of the VP2 protein was subsequently addressed. An independent VP2 expression cassette was constructed which induced a higher level of antibody production. Positive identification of VP2 protein by Western blot analysis and IFA assay using a polyclonal anti-VP2 rabbit antibody indicated correct folding of VP2 protein, thus, providing the essential component for vaccine development. Safety of the recombinant virus was indicated by the absence of clinical signs related to Aujeszky's disease in piglets and gilts immunized with recombinant virus. Furthermore, no difference in virulence was detected between the recombinant virus and the vector virus. Efficacy experiments demonstrated that piglets immunized with the recombinant virus elicited PRV-specific and PPV-specific humoral immune responses and provided complete protection against a lethal PRV challenge, which was in agreement with other reports [8, 10, 29]. Furthermore, gilts immunized with the recombinant virus induced PPV-specific antibodies and provided complete protection against challenge with the PPV-SC1 strain. These results demonstrated the immunogenicity of the recombinant virus.
Moreover, this genetically engineered vaccine also allowed differentiation between vaccinated and the naturally infected pigs, thus contributing to the Aujeszky's disease eradication program.