Background

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) causes significant losses in the swine industry by inducing reproductive failure in sows and respiratory diseases in piglets. The primary difficulty in prevention is the high genetic diversity of the virus. As variant strains continuously emerge in different regions and farms, they can evade the immunity induced by existing vaccines.

Currently used live attenuated vaccines induce relatively strong immune responses, but their ability to block heterologous viral strains is inconsistent, and there are rare concerns regarding reversion to virulence and recombination. Inactivated vaccines offer high safety but may not provide sufficient cellular immunity and neutralizing antibody responses. Researchers focused on the messenger RNA (mRNA) platform, which allows for rapid antigen redesign without administering the virus itself. The target selected was GP5, a major glycoprotein of the PRRSV envelope that includes the neutralizing antibody binding site.

Key Findings

Researchers amplified the full ORF5 gene of the highly pathogenic PRRSV HeN-HC strain, inserted it into the pIVT5 vector, and created the recombinant plasmid pIVT5-GP5. Using this as a template, GP5-mRNA was synthesized via in vitro transcription and encapsulated in lipid nanoparticles (LNP). The resulting particles had a primary size of approximately 135 nanometers and a zeta potential of approximately 25 millivolts.

Whether the mRNA is actually translated into an antigen was verified in monkey kidney-derived Marc-145 cells. Indirect immunofluorescence assays showed specific fluorescence in GP5-mRNA-transfected cells, and Western blotting detected a GP5 protein band at approximately 25 kilodaltons. This provides evidence that the designed mRNA produces the target protein within cells.

Immunogenicity assessment was conducted using 60 6-week-old specific pathogen-free (SPF) BALB/c mice, divided into GP5 mRNA-LNP, commercial live attenuated vaccine, and medium control groups. Intramuscular injections were administered on days 0, 14, and 28, with serum collected on days 14, 28, 35, and 42. GP5-specific immunoglobulin G (IgG) levels were higher than those in the control group, and neutralizing activity against the homologous HeN-HC strain was detected from day 28 after the initial vaccination, persisting until day 42.

In the day 42 analysis of plasmacytoid dendritic cells, CD3-positive/CD4-positive T cells were 9.59% in the mRNA vaccine group and 0.96% in the medium control group. CD3-positive/CD8-positive T cells were 14.3% and 0.28%, respectively. The corresponding percentages in the commercial vaccine group were 5.73% and 6.39%. Spleen cells stimulated with the antigen secreted interleukin-2, interferon-gamma, and interleukin-4, with the interferon-gamma response being higher than that of the commercial vaccine group. This supports the interpretation that both humoral and cellular immunity were induced.

Implications and Outlook

These results demonstrate the potential of mRNA-LNP encoding a single GP5 antigen to simultaneously induce binding antibodies, neutralizing activity, and helper/cytotoxic T cell responses. A major strength is the ability to redesign candidates for circulating variants simply by changing the sequence without a virus cultivation process. There is also potential to expand this approach to multivalent vaccines that combine various GP5 mutations or include other structural proteins.

However, this study is a preliminary immunogenicity evaluation. Since mice are not the natural hosts for PRRSV and no viral challenge was performed, it remains unknown whether the vaccine actually inhibits disease or reduces viremia. Neutralization tests were also limited to the same HeN-HC strain as the vaccine antigen. Future development must verify appropriate dosages, vaccination intervals, duration of immunity, protection against heterologous variants, and LNP safety in pigs. In particular, because GP5 is highly variable and can induce immune-dominant non-neutralizing antibodies, the addition of conserved antigens and optimization of antigen combinations will be key to subsequent development.