Identification of Conserved Linear Epitopes on Viral Protein 2 of Foot-and-Mouth Disease Virus Serotype O by Monoclonal Antibodies 6F4.D11.B6 and 8D6.B9.C3.

Foot-and-mouth disease (FMD) is a highly infectious disease of cloven-hoofed animals with a significant economic impact. Early diagnosis and effective prevention and control could reduce the spread of the disease which could possibly minimize economic losses. Epitope characterization based on monoclonal antibodies provide essential information for developing diagnostic assays and vaccine designs. In this study, monoclonal antibodies raised against FMD virus (FMDV) were produced. Sixty-six monoclonal antibodies demonstrated strong reactivity and specificity to FMDV. The purified monoclonal antibodies were further used for bio-panning to select phage expressing specific epitopes from phage-displayed 12 mer-peptide library. The phage peptide sequences were analyzed using multiple sequence alignment and evaluated by peptide ELISA. Two hybridoma clones secreted monoclonal antibodies recognizing linear epitopes on VP2 of FMDV serotype O. The non-neutralizing monoclonal antibody 6F4.D11.B6 recognized the residues 67-78 on antigenic site 2 resinding in VP2, while the neutralizing monoclonal antibody 8D6.B9.C3 recognized a novel linear epitope encompassing residues 115-126 on VP2. This information and the FMDV-specific monoclonal antibodies provide valuable sources for further study and application in diagnosis, therapeutics and vaccine designs to strengthen the disease prevention and control measures.

A Single-Cycle Influenza A Virus-Based SARS-CoV-2 Vaccine Elicits Potent Immune Responses in a Mouse Model.

The use of virus-vectored platforms has increasingly gained attention in vaccine development as a means for delivering antigenic genes of interest into target hosts. Here, we describe a single-cycle influenza virus-based SARS-CoV-2 vaccine designated as scPR8-RBD-M2. The vaccine utilizes the chimeric gene encoding 2A peptide-based bicistronic protein cassette of the SARS-CoV-2 receptor-binding domain (RBD) and influenza matrix 2 (M2) protein. The C-terminus of the RBD was designed to link with the cytoplasmic domain of the influenza virus hemagglutinin (HA) to anchor the RBD on the surface of producing cells and virus envelope. The chimeric RBD-M2 gene was incorporated in place of the HA open-reading frame (ORF) between the 3' and 5' UTR of HA gene for the virus rescue in MDCK cells stably expressing HA. The virus was also constructed with the disrupted M2 ORF in segment seven to ensure that M2 from the RBD-M2 was utilized. The chimeric gene was intact and strongly expressed in infected cells upon several passages, suggesting that the antigen was stably maintained in the vaccine candidate. Mice inoculated with scPR8-RBD-M2 via two alternative prime-boost regimens (intranasal-intranasal or intranasal-intramuscular routes) elicited robust mucosal and systemic humoral immune responses and cell-mediated immunity. Notably, we demonstrated that immunized mouse sera exhibited neutralizing activity against pseudotyped viruses bearing SARS-CoV-2 spikes from various variants, albeit with varying potency. Our study warrants further development of a replication-deficient influenza virus as a promising SARS-CoV-2 vaccine candidate.

The immunogenicity of the secretory GΔTM protein of bovine ephemeral fever virus stably expressed by mammalian cells.

Bovine ephemeral fever virus (BEFV) causes an acute febrile disease in cattle and water buffalo. The disease has an impact on dairy and beef production in tropical and subtropical countries. Vaccination is used for disease prevention and control. In this study, we developed a recombinant lentivirus to produce mammalian stable cells expressing histidine-tagged BEFV G protein with a deleted transmembrane domain (GΔTM) as a secretory protein. In addition, guinea pigs were immunised with the purified GΔTM protein and booster immunised at a 3-week interval. The mammalian stable cells were able to continuously produce GΔTM protein for a minimum of 25 passages. All of the mammalian stable cells expressing GΔTM protein could react specifically with a BEFV convalescent bovine serum. Serum samples from the immunised guinea pigs could react strongly and specifically with the purified GΔTM protein. Moreover, post-immunised guinea pig sera contained antibodies that could neutralise BEFV. These results indicate that the G protein without a transmembrane domain can be used as a subunit vaccine for the prevention and control of BEFV. The availability of the mammalian stable cells, which constitutively express GΔTM protein, could facilitate the potential use of the secretory protein for BEFV diagnosis and vaccine development.

Genesis and genetic constellations of swine influenza viruses in Thailand.

Swine influenza virus (SIV) is one of the most important zoonotic agents and the origin of the most recent pandemic virus. Asia is considered to be the epicenter for genetic exchanging of influenza A viruses and Southeast Asia including Thailand serves as a reservoir to maintain the persistence of the viruses for seeding other regions. Therefore, searching for new reassortants in this area has been routinely required. Although SIVs in Thailand have been characterized, collective information regarding their genetic evolution and gene constellations is limited. In this study, whole genomes of 30 SIVs isolated during clinical target surveillance plus all available sequences of past and currently circulating Thai SIVs were genetically characterized based on their evolutionary relationships. All genetic pools of Thai SIVs are comprised of four lineages including classical swine (CS), Eurasian swine (EAs), Triple reassortants (TRIG) and Seasonal human (Shs). Out of 84 isolates, nine H1N1, six H3N2 and one H1N2 strains were identified. Gene constellations of SIVs in Thailand are highly complex resulting from multiple reassortments among concurrently circulating SIVs and temporally introduced foreign genes. Most strains contain gene segments from both EAs and CS lineages and appeared transiently. TRIG lineage has been recently introduced into Thai SIV gene pools. The existence of EAs and TRIG lineages in this region may increase rates of genetic exchange and diversity while Southeast Asia is a persistent reservoir for influenza A viruses. Continual monitoring of SIV evolution in this region is crucial in searching for the next potential pandemic viruses.