Ubiquitin-linked E2 mRNA vaccine shows protection in a small piglet challenge study of classical swine fever
Background: Vaccine design addressing both protection and infection differentiation
A ubiquitin-linked E2 mRNA vaccine candidate showed protection in a small piglet study after challenge with highly virulent classical swine fever virus. All four animals in this vaccine group survived for 14 days after challenge and had no fever. The findings do not establish long-term prevention on farms, but suggest that modifying antigen design could improve vaccine-induced immune responses.
Classical swine fever (CSF) is a livestock disease caused by classical swine fever virus (CSFV). The paper describes the effectiveness of the existing live attenuated C-strain vaccine and its cross-protection against some variants, while noting that serological tests have difficulty distinguishing antibodies induced by vaccination from those caused by natural infection. E2 glycoprotein-based subunit vaccines offer advantages for differentiating infected from vaccinated animals (DIVA), but inducing sufficient cellular immunity remains a challenge. This study focused on improving these immune responses by changing how the antigen is delivered and processed.
Key findings: All animals receiving either fusion construct survived, but fever patterns differed
The researchers encapsulated messenger RNA (mRNA) encoding E2 without its transmembrane region in lipid nanoparticles (LNPs). They compared three candidates: E2 alone, a fusion with the chemokine XCL1 intended to target dendritic cells, and a ubiquitin (Ub) fusion designed to promote antigen processing. Ub carried a G76A mutation to prevent cleavage of its linkage to E2.
Nineteen piglets were divided into five groups, with four animals in each vaccine group and three in the phosphate-buffered saline (PBS) control group. The mRNA vaccines were administered at 100 μg per animal and the E2 subunit vaccine at 50 μg, followed by a booster at the same dose two weeks later. The comparison therefore involved different formulations and doses. Twenty-eight days after the first vaccination, the animals were challenged with the highly virulent Shimen strain and monitored for 14 days.
All four animals in each of the Ub-E2 and XCL1-E2 groups survived. One animal died in each of the E2-only mRNA and subunit vaccine groups, while all animals in the PBS group died. The two fusion constructs had the same survival rate, but differed in fever patterns. The XCL1-E2 group experienced transient fever, whereas no rise in body temperature was observed in the Ub-E2 group. Viral RNA was not detected in blood or nasal, oral, and rectal swab samples from the Ub-E2 group. However, low levels of RNA were reported in tissues, so the findings cannot be interpreted as viral clearance from all samples.
Serum neutralizing activity was detected in the Ub-E2 group on day 10 after vaccination, and all four animals had become antibody-positive by day 14. Peak neutralizing antibody titers were significantly higher than in the E2-only mRNA and subunit vaccine groups. An analysis using peripheral blood mononuclear cells (PBMCs) also found more virus-specific interferon gamma (IFN-γ)-secreting cells, supporting a cellular immune response. In a separate rabbit study, the XCL1-E2 and Ub-E2 groups showed no fever after challenge with the attenuated C-strain, and viral RNA was not detected in the spleen.
Significance and outlook: Potential of an antigen-processing strategy and remaining validation
The researchers proposed that Ub fusion may have enhanced immune responses by promoting proteasomal degradation of the antigen and antigen presentation by major histocompatibility complex class I (MHC-I). This interpretation is consistent with the design rationale and observations, but the full causal mechanism responsible for protection in vivo has not been established.
Serum from the Ub-E2 group also neutralized a subgenotype 2.1d virus. However, this must be distinguished from demonstrating protection by challenging vaccinated animals with that strain. Tests for antibodies against Erns, another viral protein, were negative after vaccination and became positive after challenge, suggesting a possible DIVA application.
These findings provide evidence from a small preclinical animal study. Replication at a larger scale, protection against diverse strains, and duration of protection remain to be assessed. Although neutralizing activity appeared on day 10 after vaccination, the actual challenge was conducted on day 28, so early protection requires separate evaluation. Manufacturing processes and cost competitiveness also remain to be addressed before farm use.
Sources: Full text of the Journal of Virology paper, PubMed citation and abstract.
Classical swine fever (CSF), caused by classical swine fever virus (CSFV), continues to threaten the global swine industry. Current E2-based subunit vaccines are limited by their slow adaptation to emerging viral strains and insufficient cell-mediated immunity. To address these challenges, we developed mRNA-lipid nanoparticle (LNP) vaccines encoding modified forms of the CSFV E2 glycoprotein. In this study, we constructed three novel mRNA-LNP vaccines encoding engineered E2 proteins: E2-mRNA-LNP, XCL1-E2-mRNA-LNP containing the X-C motif chemokine ligand 1 (XCL1) to target dendritic cells, and Ub-E2-mRNA-LNP containing ubiquitin (Ub) to enhance antigen presentation through Ub-mediated degradation. These vaccines were evaluated in rabbits and piglets. Following challenge with the live attenuated CSFV C-strain, rabbits immunized with XCL1-E2-mRNA-LNP or Ub-E2-mRNA-LNP exhibited neither fever nor detectable viral RNA in the spleens. Notably, immunization with Ub-E2-mRNA-LNP conferred complete protection against challenge with the highly virulent CSFV Shimen strain in piglets, outperforming the E2-based subunit vaccine, the unmodified E2-mRNA-LNP, and XCL1-E2-mRNA-LNP. Further analysis revealed that Ub-E2-mRNA-LNP elicited more potent antibody and cell-mediated immune responses. Our study demonstrates that Ub-E2-mRNA-LNP is a promising vaccine candidate for CSF prevention. IMPORTANCECSF remains a major threat to the global swine industry. Recently, a moderately virulent subgenotype CSFV 2.1c strain was isolated and reported in China. Given that the mRNA platform enables rapid vaccine development to address emerging pathogens, we constructed three nucleoside-modified mRNA-LNP vaccines expressing engineered E2 glycoproteins of CSFV, including E2-mRNA-LNP, XCL1-E2-mRNA-LNP (targeting dendritic cells), and Ub-E2-mRNA-LNP (enhancing proteasomal degradation). Evaluated in rabbits, XCL1-E2- and Ub-E2-mRNA-LNPs conferred strong protection against CSFV C-strain challenge, with no
This study suggests that, even with the same E2 antigen, a ubiquitin-linked design could improve immune responses and the pattern of protection observed in a challenge study. It also supports evaluating vaccine candidates by considering survival alongside fever, viral RNA in different sample types, antibodies, and cellular immunity. If introduced into farm disease-control programs, the vaccine would need to be accompanied by infection surveillance in vaccinated animals. The Erns antibody test evaluated here is a candidate tool for a DIVA strategy that distinguishes animals that have only been vaccinated from infected animals. However, reduced transmission and economic benefits have not been demonstrated. Decisions about disease-control use require evidence on duration of protection, differentiation performance under field conditions, production costs, and supply conditions.