E2-based mRNA vaccine encapsulated in lipid nanoparticles protects pigs against classical swine fever virus

ABSTRACT

Classical swine fever (CSF), caused by the classical swine fever virus (CSFV), remains a significant threat to the global pig industry. Recent advances in mRNA vaccines offered a promising platform for combating CSFV. In this study, we designed and evaluated three lipid nanoparticle (LNP)-encapsulated mRNA vaccine candidates encoding the ectodomain of E2 glycoprotein (E2_EX), E2_EX fused with the transmembrane (TM) region of the PEDV S protein (E2tm), and E2_EX fused with the TM region of the influenza virus HA protein (E2tm-HA). Among these, the E2tm mRNA vaccine induced the most robust antibody responses in pigs. Immunization of piglets with the E2tm mRNA vaccine showed that its immunogenicity was not impaired by maternal antibodies. Comparative analysis of pseudouridine (Ψ)-modified (

Ψ E2tm) and unmodified (E2tm) mRNA vaccines revealed that E2tm induced significantly higher antibodies titers than

Ψ E2tm. All vaccinated pigs survived the CSFV challenge, with the 150 µg E2tm dose providing optimal protection, effectively suppressing viremia and preventing viral dissemination to tissues while also resulting in undetectable viral RNA in swab samples. Our findings provide a promising novel mRNA vaccine that could be used as an alternative vaccination strategy against CSFV infection.

IMPORTANCE Classical swine fever virus (CSFV) remains a significant threat to the global pig industry. While live attenuated and subunit vaccines are currently in use, there is an urgent need for more effective and safer vaccination strategies. Here, we present a novel mRNA vaccine encoding the CSFV E2 glycoprotein, which provides protective immunity against the CSFV challenge in pigs. Our findings underscore the promising efficacy of this mRNA-based vaccine platform and offer an alternative strategy for CSFV prevention and control.

Background:

Classical swine fever (CSF), caused by classical swine fever virus (CSFV), poses a significant threat to the global pig industry. It was first reported in America and then became endemic across Europe, South America, and Asia, causing significant economic losses. CSFV, belonging to the genus *Pestivirus* within the *Flaviviridae* family, is an enveloped RNA virus containing a positive-sense, single-stranded RNA genome of approximately 12.3 kb. The viral genome encodes a polyprotein which is cleaved into four structural proteins: a core protein (C) and three envelope glycoproteins (Eᵣⁿˢ, E1, and E2), and eight non-structural proteins. Among these, the E2 glycoprotein is a multifunctional glycoprotein. It plays an important role in viral entry and replication and is also associated with viral pathogenicity in pigs. In addition, the E2 glycoprotein is immunogenic and can induce high levels of neutralizing antibodies, making it an ideal target for vaccine development.

While the modified live vaccine (MLV) C‑strain has been widely used with good efficacy, its effectiveness can be affected by factors such as maternal antibodies. A recent study demonstrated that sporadic outbreaks have occurred in C‑strain vaccinated pigs. In addition, the MLV C‑strain lacks a serological marker to differentiate infected from vaccinated animals (DIVA). Although subunit vaccines have been developed, they require higher doses to achieve protective immunity comparable to that of live‑attenuated vaccines. Therefore, the development of novel vaccines would be highly beneficial for the prevention and control of this disease.

mRNA vaccines have shown great promise as a vaccine strategy and have been extensively explored since their success against severe acute respiratory syndrome coronavirus 2 (SARS‑COV‑2). The use of mRNA offers several advantages. mRNA vaccines can encode various antigens, and protein production can be enhanced by engineering the untranslated regions (UTRs) of the mRNA sequences. mRNA vaccines offer potential safety advantages, as their transient expression minimizes risks of insertional mutagenesis and genomic integration. mRNA vaccines possess self‑adjuvant properties and can induce both humoral and cellular responses. The manufacturing process for mRNA vaccines is relatively straightforward and scalable, allowing rapid development and adaptation for various infectious diseases.

Methods:

We designed and evaluated three lipid nanoparticle (LNP)‑encapsulated mRNA vaccine candidates encoding the ectodomain of E2 glycoprotein (E2_EX), E2_EX fused with the transmembrane (TM) region of the PEDV S protein (E2tm), and E2_EX fused with the TM region of the influenza virus HA protein (E2tm‑HA). Immunization of piglets with the E2tm mRNA vaccine showed that its immunogenicity was not impaired by maternal antibodies. Comparative analysis of pseudouridine (Ψ)‑modified (ΨE2tm) and unmodified (E2tm) mRNA vaccines revealed that E2tm induced significantly higher antibody titers than ΨE2tm.

Results:

Among these, the E2tm mRNA vaccine induced the most robust antibody responses in pigs. All vaccinated pigs survived the CSFV challenge, with the 150 µg E2tm dose providing optimal protection, effectively suppressing viremia and preventing viral dissemination to tissues while also resulting in undetectable viral RNA in swab samples.

Data Summary:

E2tm induced significantly higher antibody titers than ΨE2tm. The 150 µg E2tm dose provided optimal protection, as all vaccinated pigs survived the challenge, viremia was suppressed, viral dissemination to tissues was prevented, and viral RNA was undetectable in swab samples.

Conclusions:

Our findings provide a promising novel mRNA vaccine that could be used as an alternative vaccination strategy against CSFV infection.

Practical Significance:

Classical swine fever virus (CSFV) remains a significant threat to the global pig industry. Our findings underscore the promising efficacy of this mRNA‑based vaccine platform and offer an alternative strategy for CSFV prevention and control.