CA-CAS-01-A: A Permissive Cell Line for Isolation and Live Attenuated Vaccine Development Against African Swine Fever Virus.

African swine fever virus (ASFV) is the causative agent of the highly lethal African swine fever disease that affects domestic pigs and wild boars. In spite of the rapid spread of the virus worldwide, there is no licensed vaccine available. The lack of a suitable cell line for ASFV propagation hinders the development of a safe and effective vaccine. For ASFV propagation, primary swine macrophages and monocytes have been widely studied. However, obtaining these cells can be time-consuming and expensive, making them unsuitable for mass vaccine production. The goal of this study was to validate the suitability of novel CA-CAS-01-A (CAS-01) cells, which was identified as a highly permissive cell clone for ASFV replication in the MA-104 parental cell line for live attenuated vaccine development. Through a screening experiment, maximum ASFV replication was observed in the CAS-01 cell compared to other sub-clones of MA-104 with 14.89 and log10 7.5 ± 0.15 Ct value and TCID50/ml value respectively. When CAS-01 cells are inoculated with ASFV, replication of ASFV was confirmed by Ct value for ASFV DNA, HAD50/ml assay, TCID50/ml assay, and cytopathic effects and hemadsoption were observed similar to those in primary porcine alveolar macrophages after 5th passage. Additionally, we demonstrated stable replication and adaptation of ASFV over the serial passage. These results suggest that CAS-01 cells will be a valuable and promising cell line for ASFV isolation, replication, and development of live attenuated vaccines.

High-Level Expression of Recombinant VHSV Glycoprotein Using Transformed C. vulgaris and Verification of Vaccine Efficacy.

The demand for aquaculture is increasing, but production is declining due to high feed costs and disease outbreaks. Viral hemorrhagic septicemia (VHS) is a viral disease that seriously affects seawater and freshwater fish in aquaculture, including the olive flounder (Paralichthys olivaceus), a major aquaculture fish in Korea. However, very few vaccines are currently available for viral hemorrhagic septicemia virus (VHSV). The nutrient-rich microalga Chlorella vulgaris has been used as a feed additive in aquaculture and as a host for the industrial production of recombinant VHSV glycoprotein as an oral vaccine. In this study, VHSV glycoprotein was cloned with a salt-inducible promoter, and high levels of expression up to 41.1 mg/g wet C. vulgaris, representing 27.4% of total extracted soluble protein, were achieved by growing the transformed C. vulgaris for 5 days in the presence of 250 mM NaCl. The production of a neutralizing antibody was detected in the serum of fish given feed containing 9% VHSV glycoprotein-expressing C. vulgaris. Furthermore, relative survival rates of 100% and 81.9% were achieved following challenges of these fish with VHSV at 106 and 107 pfu/fish, respectively, indicating that C. vulgaris could be used as a platform for the production of recombinant proteins for use as oral vaccines in the control of viral diseases in aquaculture.

Efficacy of a Novel Multiepitope Vaccine Candidate against Foot-and-Mouth Disease Virus Serotype O and A.

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease in cloven-hoofed animals. To prevent the spread of FMD virus (FMDV), traditional inactivated vaccines are used to immunize susceptible animals in disease-endemic countries. However, the inactivated FMD vaccine has several limitations, including safety concerns. To overcome these limitations, subunit proteins have been studied as alternative vaccine candidates. In this study, we designed two multiepitope recombinant proteins (OVM and AVM) containing antigenic sites (residue of VP1 132-162 and residue of VP1 192-212) of three topotypes of FMDV serotype O or three topotypes of FMDV serotype A. Each recombinant protein was efficiently expressed in Escherichia coli with high solubility, and the immunogenicity and protective efficacy of the proteins as FMD vaccine candidates were evaluated. The results showed that OVM and AVM emulsified with ISA201 adjuvant induced effective antigen-specific humoral and cell-mediated immune responses and successfully protected mice from O/Jincheon/SKR/2014, O/VET/2013, and A/Malaysia/97 viruses. In addition, intramuscular immunization of pigs with the OVM and AVM emulsified with ISA201 elicited effective levels of neutralizing antibodies to the viruses with homologous epitopes. Importantly, OVM-AVM emulsified with CAvant®SOE-X adjuvant conferred 100% protection against the O/Jincheon/SKR/2014 virus with homologous residues and 75% protection against A/SKR/GP/2018 with heterologous residues. The results presented in this study suggest that the combination of OVM and AVM protein with an effective adjuvant could yield an effective and safe vaccine candidate for the prevention and control of foot-and-mouth disease. In addition, our results provide a vaccine platform that can safely, cost-efficiently, and rapidly generate protective vaccine candidates against diverse FMDVs.

Botulinum Toxin A Ameliorates Neuroinflammation in the MPTP and 6-OHDA-Induced Parkinson's Disease Models.

Recently, increasing evidence suggests that neuroinflammation may be a critical factor in the development of Parkinson's disease (PD) in addition to the ratio of acetylcholine/dopamine because dopaminergic neurons are particularly vulnerable to inflammatory attack. In this study, we investigated whether botulinum neurotoxin A (BoNT-A) was effective for the treatment of PD through its anti-neuroinflammatory effects and the modulation of acetylcholine and dopamine release. We found that BoNT-A ameliorated MPTP and 6-OHDA-induced PD progression, reduced acetylcholine release, levels of IL-1ß, IL-6 and TNF-α as well as GFAP expression, but enhanced dopamine release and tyrosine hydroxylase expression. These results indicated that BoNT-A had beneficial effects on MPTP or 6-OHDA-induced PD-like behavior impairments via its anti-neuroinflammation properties, recovering dopamine, and reducing acetylcholine release.

CAvant® WO-60 as an Effective Immunological Adjuvant for Avian Influenza and Newcastle Disease Vaccine.

Despite the immunogenicity of vaccines currently used in poultry, several pathogens, including avian influenza virus (AIV) and Newcastle disease virus (NDV), cause enormous economic losses to the global poultry industry. The efficacy of vaccines can be improved by the introduction of effective adjuvants. This study evaluated a novel water-in-oil emulsion adjuvant, CAvant® WO-60, which effectively enhanced both the immunogenicity of conserved influenza antigen sM2HA2 and inactivated whole H9N2 antigen (iH9N2). CAvant® WO-60 induced both humoral and cell-mediated immunity in mice and provided 100% protection from challenge with 10 LD50 of A/Aquatic bird/Korea/W81/2005 (H5N2) and A/Chicken/Korea/116/2004 (H9N2) AIV. Importantly, immunization of chickens with iH9N2 plus inactivated NDV LaSota (iNDV) bivalent inactivated vaccine emulsified in CAvant® WO-60 induced seroprotective levels of antigen-specific antibody responses. Taken together, these results suggested that CAvant® WO-60 is a promising adjuvant for poultry vaccines.

The Potential Adjuvanticity of CAvant®SOE for Foot-and-Mouth Disease Vaccine.

Foot-and-mouth disease (FMD) is a notifiable contagious disease of cloven-hoofed mammals. A high potency vaccine that stimulates the host immune response is the foremost strategy used to prevent disease persistence in endemic regions. FMD vaccines comprise inactivated virus antigens whose immunogenicity is potentiated by immunogenic adjuvants. Oil-based adjuvants have clear advantages over traditional adjuvant vaccines; however, there is potential to develop novel adjuvants to increase the potency of FMD vaccines. Thus, we aimed to evaluate the efficacy of a novel water-in-oil emulsion, called CAvant®SOE, as a novel vaccine adjuvant for use with inactivated FMD vaccines. In this study, we found that inactivated A22 Iraq virus plus CAvant®SOE (iA22 Iraq-CAvant®SOE) induced effective antigen-specific humoral (IgG, IgG1, and IgG2a) and cell-mediated immune responses (IFN-γ and IL-4) in mice. Immunization of pigs with a single dose of iA22 Iraq-CAvant®SOE also elicited effective protection, with no detectable clinical symptoms against challenge with heterologous A/SKR/GP/2018 FMDV. Levels of protection are strongly in line with vaccine-induced neutralizing antibody titers. Collectively, these results indicate that CAvant®SOE-adjuvanted vaccine is a promising candidate for control of FMD in pigs.

Identification of novel immunogenic proteins against Streptococcus parauberis in a zebrafish model by reverse vaccinology.

Streptococcus parauberis is the major infectious agent of streptococcosis in the olive flounder (Paralichthys olivaceus), causing serious economic damage. In this study, we identified potential vaccine candidates against S. parauberis by reverse vaccinology. In total, the 2 out of 21 proteins were identified as vaccine candidates from two available S. parauberis genomes. The membrane-anchored protein SEC10/PgrA and the metal ABC transporter substrate-binding lipoprotein mtsA were potent antigenic proteins based on western blotting with mouse-derived antiserum against whole bacteria of S. parauberis serotypes I and II. In particular, metal ABC transporter substrate-binding lipoprotein (mtsA) showed similar protective immunity to that of whole-cell bacterins against S. parauberis in a zebrafish model. These results suggest that mtsA may be considered as a novel candidate in the development of vaccines against S. parauberis.

Hyper-enhanced production of foreign recombinant protein by fusion with the partial polyhedrin of nucleopolyhedrovirus.

To enhance the production efficiency of foreign protein in baculovirus expression systems, the effects of polyhedrin fragments were investigated by fusion expressing them with the enhanced green fluorescent protein (EGFP). Recombinant viruses were generated to express EGFP fused with polyhedrin fragments based on the previously reported minimal region for self-assembly and the KRKK nuclear localization signal (NLS). Fusion expressions with polyhedrin amino acids 19 to 110 and 32 to 110 lead to localization of recombinant protein into the nucleus and mediate its assembly. The marked increase of EGFP by these fusion expressions was confirmed through protein and fluorescence intensity analyses. The importance of nuclear localization for enhanced production was shown by the mutation of the NLS within the fused polyhedrin fragment. In addition, when the polyhedrin fragment fused with EGFP was not localized in the nucleus, some fragments increased the production of protein. Among these fragments, some degradation of only the fused polyhedrin was observed in the fusion of amino acids 19 to 85 and 32 to 85. The fusion of amino acids 32 to 85 may be more useful for the enhanced and intact production of recombinant protein. The production of E2 protein, which is a major antigen of classical swine fever virus, was dramatically increased by fusion expression with polyhedrin amino acids 19 to 110, and its preliminary immunogenicity was verified using experimental guinea pigs. This study suggests a new option for higher expression of useful foreign recombinant protein by using the partial polyhedrin in baculovirus.

Production of classical swine fever virus envelope glycoprotein E2 as recombinant polyhedra in baculovirus-infected silkworm larvae.

Although, classical swine fever virus (CSFV) envelope glycoprotein E2 subunit vaccine has been developed using the baculovirus expression system, the expression of viral antigens in baculovirus-infected insect cells is often ineffective. Therefore, an alternative strategy to the traditional baculovirus expression system is needed that is more productive and effective. Here, we report a novel strategy for the large-scale production of a CSFV E2 in the larvae of a baculovirus-infected silkworm, Bombyx mori. We constructed a recombinant B. mori nucleopolyhedrovirus (BmNPV) that expressed recombinant polyhedra together with the N-terminal 179 amino acids of CSFV E2 (E2ΔC). BmNPV-E2ΔC-infected silkworm larvae expressed native polyhedrin and approximately 44-kDa fusion protein that was detected using both anti-polyhedrin and anti-CSFV E2 antibodies. Electron and confocal microscopy both demonstrated that the recombinant polyhedra contained both the fusion protein and native polyhedrin were morphologically normal and contained CSFV E2ΔC. The CSFV E2ΔC antigen produced in BmNPV-E2ΔC-infected silkworm larvae reached 0.68 mg/ml of hemolymph and 0.53 mg/larva at 6-days post-infection. Six-week-old female BALB/c mice that were immunized with the E2ΔC protein purified from solubilized recombinant polyhedra elicited CSFV E2 antibodies, which indicated that the CSFV E2ΔC protein from recombinant polyhedra was immunogenic. The virus neutralization test showed that the serum from mice that were treated with E2ΔC protein from recombinant polyhedra contained significant levels of virus neutralization activity. These results demonstrate that this strategy can be used for the large-scale production of CSFV E2 antigen.

C-Methylated Flavonoids from Cleistocalyx operculatus and Their Inhibitory Effects on Novel Influenza A (H1N1) Neuraminidase.

As part of an ongoing study focused on the discovery of anti-influenza agents from plants, four new (1-4) and 10 known (5-14) C-methylated flavonoids were isolated from a methanol extract of Cleistocalyx operculatus buds using an influenza H1N1 neuraminidase inhibition assay. Compounds 4, 7, 8, and 14, with a chalcone skeleton, showed significant inhibitory effects on the viral neuraminidases from two influenza viral strains, H1N1 and H9N2. Compound 4 showed the strongest inhibitory activity against the neuraminidases from novel influenza H1N1 (WT) and oseltamivir-resistant novel H1N1 (H274Y mutant) expressed in 293T cells with IC50 values of 8.15 ± 1.05 and 3.31 ± 1.34 µM, respectively. Compounds 4, 7, 8, and 14 behaved as noncompetitive inhibitors in the kinetic studies. These results indicate that C-methylated flavonoids from C. operculatus have the potential to be developed as neuraminidase inhibitors for novel influenza H1N1.

Molecular characterization of ORFs 2 to 7 of Korean porcine reproductive and respiratory syndrome virus (CA) and its protein expression by recombinant baculoviruses.

To determine the characteristics of the Korean porcine reproductive and respiratory syndrome virus (PRRSV), CA, which was isolated from the serum of an infected pig in 2006, we investigated the nucleotide sequence and expression of the structural ORFs (ORFs 2 to 7) using the bApGOZA system. We found that the structural ORFs 2 to 7 of CA consisted of 3188 nucleotides that were the same as those formed from VR-2332. Comparison of the CA with the other strains revealed nucleotide sequence identity ranging from 89.8 to 99.5%. To better understand the genetic relationships between other strains, phylogenetic analyses were performed. The CA strain was closely related to the other North American genotype strains but formed a distinct branch with high bootstrap support. Additionally, expression levels of the PRRSV proteins in insect cells were strong or partially weak. The results of this study have implications for both the taxonomy of PRRSV and vaccine development.