CHA Vaccine Institute (South Korea)

The N-terminal S1 subunit of the spike protein (S1 protein) of porcine epidemic diarrhea virus (PEDV) is recognized as a potential diagnostic antigen for detecting anti-PEDV immunoglobulin A (IgA) levels in colostrum, a key indicator for assessing passive immunity against PEDV infection. Given the advantages of producing Fc-fusion proteins in mammalian cells, it is important to investigate how the addition of the Fc region affects the diagnostic performance of the PEDV-S1 protein. In this study, we successfully produced full-length PEDV-S1 protein fused with an Fc region (PEDV-S1-Fc protein) via transient gene expression in recombinant Chinese hamster ovary (rCHO) cells adapted to serum-free suspension culture. The resulting expression showed significantly higher volumetric productivity than previously reported. The aglycosylated form of PEDV-S1-Fc protein was also generated using PNGase F treatment of the purified protein, as well as tunicamycin treatment to inhibit glycosylation during cell culture. Notably, neither the addition of the Fc region nor the removal of N-glycans markedly affected the diagnostic function, as demonstrated by indirect ELISA using PEDV-positive and PEDV-negative colostrum samples. Moreover, the diagnostic performance of the PEDV-S1-Fc protein was further validated using total IgA purified from PEDV-positive colostrum. In summary, the full-length Fc-fused PEDV-S1 protein produced in rCHO cell culture exhibited high productivity and purity, making it a promising antigenic candidate for indirect ELISA-based detection of anti-PEDV IgA in colostrum.

ABSTRACT African swine fever (ASF), a contagious and lethal haemorrhagic disease of domestic pigs and wild boars, poses a significant threat to the global pig industry. Although experimental vaccine candidates derived from naturally attenuated, genetically engineered, or cell culture-adapted ASF virus have been tested, no commercial vaccine is accepted globally. We developed a safe and effective cell-adapted live attenuated vaccine candidate (ASFV-MEC-01) by serial passage of a field isolate in CA-CAS-01-A cells. ASFV-MEC-01, isolated via repeated plaque purification using next-generation sequencing analysis, was obtained at passage 18 and showed significant attenuation in 4- and 6-week-old pigs. ASFV-MEC-01 conferred 100% protection against challenge with lethal parental ASFV, which correlated with high ASFV-specific humoral and cellular immune responses. Additionally, ASFV-MEC-01 was not detected in blood until 28 days post-inoculation. Global transcriptome analysis showed that ASFV-MEC-01 lacking 12 genes triggered stronger innate antiviral responses than the parental virus, as exemplified by high levels of mRNA encoding interferon regulatory and inflammatory genes in PAM cells. Ectopic expression of most deleted genes increased replication of DNA viruses by suppressing production of interferons and pro-inflammatory cytokines. Among the genes deleted from ASFV-MEC-01, MGF100-1R interacted specifically with the scaffold dimerization domain of TBK1, thereby preventing TBK1 dimerization and impairing TBK1-mediated type I IFN and NF-κB signalling. These results suggest that attenuation of ASFV-MEC-01 may be mediated by induction of stronger type I IFN and NF-κB signalling within the host innate immune system. Thus, ASFV-MEC-01 could be a safe and effective live attenuated ASFV vaccine candidate with commercial potential.

TLR agonists have emerged as an efficient cancer vaccine adjuvant system that induces robust immune responses. L-pampo™, a proprietary vaccine adjuvant of TLR2 and TLR3 agonists, promotes strong humoral and cellular immune responses against infectious diseases. In this study, we demonstrate that vaccines formulated with L-pampo™ affect the recruitment and activation of dendritic cells (DCs) in draining lymph nodes (dLNs) and leading to antigen-specific T-cell responses and anti-tumor efficacy. We analyzed DC maturation and T-cell proliferation using flow cytometry and ELISA. We determined the effect of L-pampo™ on DCs in dLNs and antigen-specific T-cell responses using flow cytometric analysis and the ELISPOT assay. We employed murine tumor models and analyzed the anti-tumor effect of L-pampo™. We found that L-pampo™ directly enhanced the maturation and cytokine production of DCs and, consequently, T-cell proliferation. OVA or OVA peptide formulated with L-pampo™ promoted DC migration into dLNs and increased activation markers and specific DC subsets within dLNs. In addition, vaccines admixed with L-pampo™ promoted antigen-specific T-cell responses and anti-tumor efficacy. Moreover, the combination of L-pampo™ with an immune checkpoint inhibitor synergistically improved the anti-tumor effect. This study suggests that L-pampo™ can be a potent cancer vaccine adjuvant and a suitable candidate for combination immunotherapy.

β‐arrestin 2 (ARRB2) is functionally implicated in cancer progression via various signaling pathways. However, its role in lung cancer remains unclear. To obtain clinical insight on its function in lung cancer, microarray data from lung tumor tissues (LTTs) and matched lung normal tissues (mLNTs) of primary non-small cell lung cancer (NSCLC) patients (n = 37) were utilized. ARRB2 expression levels were markedly decreased in all 37 LTTs compared to those in matched LNTs of NSCLC patients. They were significantly co-related to enrichment gene sets associated with oncogenic and cancer genes. Importantly, Gene Set Enrichment Analysis (GSEA) between three LTTs with highly down-regulated ARRB2 and three LTTs with lowly down-regulated ARRB2 revealed significant enrichments related to toll-like receptor (TLR) signaling and autophagy genes in three LTTs with highly down-regulated ARRB2, suggesting that ARRB2 was negatively involved in TLR-mediated signals for autophagy induction in lung cancer. Biochemical studies for elucidating the molecular mechanism revealed that ARRB2 interacted with TNF receptor-associated factor 6 (TRAF6) and Beclin 1 (BECN1), thereby inhibiting the ubiquitination of TRAF6-TAB2 to activate NF-κB and TRAF6-BECN1 for autophagy stimulated by TLR3 and TLR4, suggesting that ARRB2 could inhibit the TRAF6-TAB2 signaling axis for NF-κB activation and TRAF6-BECN1 signaling axis for autophagy in response to TLR3 and TLR4. Notably, ARRB2-knockout (ARRB2KO) lung cancer cells exhibited marked enhancements of cancer migration, invasion, colony formation, and proliferation in response to TLR3 and TLR4 stimulation. Altogether, our current data suggest that ARRB2 can negatively regulate lung cancer progression by inhibiting TLR3- and TLR4-induced autophagy.

BackgroundPorcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae (MHP) are economically significant pathogens in the pig industry. The use of combined vaccines against PCV2 and MHP is one of the most effective ways of protecting pigs from both diseases, and it has become a part of general management.ObjectivesThis study evaluated the efficacy of two new bivalent vaccines of PCV2 and MHP (Myco-X and Myco-XD) in SPF pigs. Myco-X and Myco-XD are a combined vaccine of MHP with PCV2b and PCV2d, respectively.MethodsSixteen pigs were divided into four groups: Myco-X-vaccinated challenged, Myco-XD-vaccinated challenged, unvaccinated challenged, and unvaccinated unchallenged. Two milliliters of Myco-X were administered intramuscularly, and 0.5 mL of Myco-XD was injected intradermally at 3 wk of age. The pigs were challenged with virulent PCV2d via the intramuscular and intranasal route 4 wk post-vaccination.ResultsAll vaccinated pigs showed effective reduction of the clinical signs, the PCV2d load in the blood and nasal swab samples, as well as lung and lymphoid tissue lesions in the challenge test. Compared to unvaccinated challenged animals, the vaccinated challenged animals showed significantly higher (p < 0.05) levels of anti-PCV2 IgG, PCV2d-specific interferon-γ (IFN-γ), and anti-MHP IgG.ConclusionsBased on clinical, microbiological, serological, and pathological assessments, this study confirmed that both combined vaccines could protect pigs against PCV2 infection caused by PCV2d. On the other hand, further research on the efficacy evaluation of these new vaccines against the MHP challenge and PCV2d/MHP co-challenge is needed.

The development of COVID-19 vaccines is critical in controlling global health issues under the COVID-19 pandemic. The subunit vaccines are the safest and most widely used vaccine platform and highly effective against a multitude of infectious diseases. An adjuvant is essential for subunit vaccines to enhance the magnitude and durability of immune responses. In this study, we determined whether a combination of toll-like receptor (TLR)1/2 and TLR3 agonists (L-pampo) can be a potent adjuvant for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subunit vaccine. We measured a neutralizing antibody (nAb) and an angiotensin-converting enzyme 2 (ACE2) receptor-blocking antibody against SARS-CoV-2 receptor-binding domain (RBD). We also detected interferon-gamma (IFN-γ) production by using ELISPOT and ELISA assays. By employing a ferret model, we detected nAbs and IFN-γ producing cells and measured viral load in nasal wash after the challenge of SARS-CoV-2. We found that SARS-CoV-2 antigens with L-pampo stimulated robust humoral and cellular immune responses. The efficacy of L-pampo was higher than the other adjuvants. Furthermore, in the ferret model, SARS-CoV-2 antigens with L-pampo elicited nAb response and antigen-specific cellular immune response against SARS-CoV-2, resulting in substantially decreased viral load in their nasal wash. Our study suggests that SARS-CoV-2 antigens formulated with TLR agonists, L-pampo, can be a potent subunit vaccine to promote sufficient protective immunity against SARS-CoV-2.