Fisetin inhibits TNF-α/NF-κB-induced IL-8 expression by targeting PKCδ in human airway epithelial cells.

Fisetin (3,7,3',4'-tetrahydroxyflavone), a natural flavonoid, is a therapeutic agent for respiratory inflammatory diseases such as chronic obstructive pulmonary disease (COPD). However, detailed molecular mechanisms regarding the target protein of fisetin remain unknown. Fisetin significantly reduces tumour necrosis factor alpha (TNF-α)-induced interleukin (IL)-8 levels by inhibiting both nuclear factor kappa B (NF-κB) transcriptional activity and the phosphorylation of its upstream effectors. We show that fisetin prevents interactions between protein kinase C (PKC)δ and TNF receptor-associated factor 2 (TRAF2), thereby inhibiting the inhibitor of kappa B kinase (IKK)/NF-κB downstream signalling cascade. Furthermore, we found that fisetin directly binds to PKCδ in vitro. Our findings provide evidence that fisetin inhibits the TNF-α-activated IKK/NF-κB cascade by targeting PKCδ, thereby mediating inflammatory diseases such as COPD. These data suggest that fisetin is a good therapeutic drug for the treatment of inflammatory lung diseases, such as COPD, by inhibiting the TNF-α/NF-κB signalling pathway.

Verproside inhibits TNF-α-induced MUC5AC expression through suppression of the TNF-α/NF-κB pathway in human airway epithelial cells.

Airway mucus secretion is an essential innate immune response for host protection. However, overproduction and hypersecretion of mucus, mainly composed of MUC5AC, are significant risk factors in asthma and chronic obstructive pulmonary disease (COPD) patients. Previously, we reported that verproside, a catalpol derivative iridoid glycoside isolated from Pseudolysimachion rotundum var. subintegrum, is a potent anti-asthmatic candidate drug in vivo. However, the molecular mechanisms underlying the pharmacological actions of verproside remain unknown. Here, we found that verproside significantly reduces the expression levels of tumor necrosis factor alpha (TNF-α)-induced MUC5AC mRNA and protein by inhibiting both nuclear factor kappa B (NF-κB) transcriptional activity and the phosphorylation of its upstream effectors such as IκB kinase (IKK)ß, IκBα, and TGF-ß-activated kinase 1 (TAK1) in NCI-H292 cells. Moreover, verproside attenuated TNF-α-induced MUC5AC transcription more effectively when combined with an IKK (BAY11-7082) or a TAK1 (5z-7-oxozeaenol) inhibitor than when administered alone. Importantly, we demonstrated that verproside negatively modulates the formation of the TNF-α-receptor (TNFR) 1 signaling complex [TNF-RSC; TNFR1-recruited TNFR1-associated death domain protein (TRADD), TNFR-associated factor 2 (TRAF2), receptor-interacting protein kinase 1 (RIP1), and TAK1], the most upstream signaling factor of NF-κB signaling. In silico molecular docking studies show that verproside binds between TRADD and TRAF2 subunits. Altogether, these results suggest that verproside could be a good therapeutic candidate for treatment of inflammatory airway diseases such as asthma and COPD by blocking the TNF-α/NF-κB signaling pathway.

Timing of maternal vaccination against COVID-19 for effective protection of neonates: cohort study.

Introduction: Although the safety and effectiveness of COVID-19 vaccination during pregnancy have been proven, there is still little data explaining neonatal outcomes of maternal pre-pregnancy vaccination. Methods: Here, we investigated the impact of vaccination and SARS-CoV-2 infection on maternal-neonate immune response in a cohort study involving 141 pregnant individuals, and defined the importance of maternal COVID-19 vaccination timing for its effectiveness. Results and discussion: Our data indicate that vertically transferred maternal hybrid immunity provides significantly better antiviral protection for a neonate than either maternal post-infection or post-vaccination immunity alone. Higher neutralization potency among mothers immunized before pregnancy and their newborns highlights the promising role of pre-pregnancy vaccination in neonatal protection. A comparison of neutralizing antibody titers calculated for each dyad suggests that infection and pre-/during-pregnancy vaccination all support transplacental transfer, providing the offspring with strong passive immunity against SARS-CoV-2. Analysis of neutralizing antibody levels in maternal sera collected during pregnancy and later during delivery shows that immunization may exert a positive effect on maternal protection.

ß-Arrestin 2 mediates G protein-coupled receptor 43 signals to nuclear factor-κB.

G-protein coupled receptor 43 (GPR43) serves as a receptor for short-chain fatty acids (SCFAs), implicated in neutrophil migration and inflammatory cytokine production. However, the intracellular signaling pathway mediating GPR43 signaling remains unclear. Here, we show that ß-arrestin 2 mediates the internalization of GPR43 by agonist. Agonism of GPR43 reduced the phosphorylation and nuclear translocation of nuclear factor-κB (NF-κB), which was relieved by short interfering RNA (siRNA) of ß-arrestin 2. Subsequently, mRNA expression of proinflammatory cytokines, interleukin (IL)-6 and IL-1ß, was downregulated by activation of GPR43 and knockdown of ß-arrestin 2 recovered the expression of the cytokines. Taken together, these results suggest that GPR43 may be a plausible target for a variety of inflammatory diseases.

Humoral Immune Response of Heterologous ChAdOx1 nCoV-19 and mRNA-1273 Prime-Boost Vaccination against SARS-CoV-2 Variants in Korea.

The immunogenicity of a heterologous vaccination regimen consisting of ChAdOx1 nCoV-19 (a chimpanzee adenovirus-vectored vaccine) followed by mRNA-1273 (a lipid-nanoparticle-encapsulated mRNA-based vaccine) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), specifically the omicron variant (B.1.1.529), is poorly studied. The aim of this study was to evaluate the neutralizing antibody activity and immunogenicity of heterologous ChAdOx1 nCoV-19 and mRNA-1273 prime-boost vaccination against wild-type (BetaCoV/Korea/KCDC03/2020), alpha, beta, gamma, delta, and omicron variants of SARS-CoV-2 in Korea. A 50% neutralizing dilution (ND50) titer was determined in serum samples using the plaque reduction neutralization test. Antibody titer decreased significantly at 3 months compared with that at 2 weeks after the 2nd dose. On comparing the ND50 titers for the above-mentioned variants of concerns, it was observed that the ND50 titer for the omicron variant was the lowest. This study provides insights into cross-vaccination effects and can be useful for further vaccination strategies in Korea.

Optimization of Zika DNA vaccine by delivery systems.

In our previous study, we designed and evaluated the efficacy of six DNA vaccine candidates based on the E protein of Zika virus (ZIKV). To optimize the DNA vaccine, we inoculated C57BL/6 and IFNAR1- mice with the vaccine candidate expressing tandem repeated ZIKV envelope domain III (ED III × 3) doses; 50 µg by intramuscular (IM), jet injection (JET), or electroporation (EP) routes. Results showed that vaccination by all routes induced humoral and cellular immunity. Among them, EP induced robust ZIKV E specific-total IgG and neutralizing antibodies, as well as T cell responses. Additionally, EP showed superior protective efficacy against the ZIKV Brazil strain compared to the IM and JET routes. Finally, in the dose optimization test of EP route, cellular immunity of 50 µg was induced a significant level than other dose groups. These results showed that the EP delivery system enhanced the potential immunogenicity and protective efficacy of DNA vaccines.

Development of an attenuated smallpox vaccine candidate: The KVAC103 strain.

Smallpox, a disease caused by the variola virus, is one of the most dangerous diseases and had killed numerous people before it was eradicated in 1980. However, smallpox has emerged as the most threatening bio-terrorism agent; as the first- and second-generation smallpox vaccines have been controversial and have caused severe adverse reactions, new demands for safe smallpox vaccines have been raised and some attenuated smallpox vaccines have been developed. We have developed a cell culture-based highly attenuated third-generation smallpox vaccine candidate KVAC103 strain by 103 serial passages of the Lancy-Vaxina strain derived from the Lister in Vero cells. Several clones were selected, taking into consideration their shape, size, and growth rate in mammalian cells. The clones were then inoculated intracerebrally in suckling mice to test for neurovirulence by observing survival. Protective immune responses in adult mice were examined by measuring the levels of neutralization antibodies and IFN-γ expression. Among several clones, clone 7 was considered the best alternative candidate because there was no mortality in suckling mice against a lethal challenge. In addition, enhanced neutralizing antibodies and T-cell mediated IFN-γ production were observed in clone 7-immunized mice. Clone 7 was named "KVAC103" and was used for the skin toxicity test and full-genome analysis. KVAC103-inoculated rabbits showed reduced skin lesions compared to those inoculated with the Lister strain, Lancy-Vaxina. A whole genome analysis of KVAC103 revealed two major deleted regions that might contribute to the reduced virulence of KVAC103 compared to the Lister strain. Phylogenetic inference supported the close relationship with the Lister strain. Collectively, our data demonstrate that KVAC103 holds promise for use as a third-generation smallpox vaccine strain due to its enhanced safety and efficacy.

Enhanced effect of modified Zika virus E antigen on the immunogenicity of DNA vaccine.

It has been reported worldwide that the Zika virus (ZIKV) could be transmitted through placentas and sexual contact. ZIKV can also cause Guillain-Barre syndrome, microcephaly and neurological abnormalities. However, there are no approved vaccines available. We constructed six DNA vaccine candidates and tested the immunogenicity. Tandem repeated envelope domain Ⅲ (ED Ⅲ × 3) induced highly total IgG and neutralization antibody, as well as CD8+ T cell responses. Also, stem region-removed envelope (E ΔSTEM) elicited a robust production of IFN-γ in mice. To examine in vivo protection, we used mice treated with an IFNAR1 blocking antibody before and after the challenge. Vaccination with the two candidates led to a decline in the level of viral RNAs in organs. Moreover, the sera from the vaccinated mice did not enhance the infection of Dengue virus in K562 cells. These findings suggest the potential for the development of a novel ZIKV DNA vaccine.

The immunogenicity and protection effect of an inactivated coxsackievirus A6, A10, and A16 vaccine against hand, foot, and mouth disease.

Coxsackievirus belongs to the Enterovirus genus of the Picornaviridae family and is one of the major pathogens associated with human hand, foot, and mouth disease (HFMD). Historically, outbreaks of HFMD have mainly been caused by enterovirus 71 and coxsackievirus A16. Recently, coxsackieviruses A6 and A10 have been associated with increased occurrences of sporadic HFMD cases and outbreak events globally. In this study, the immunogenicity of coxsackieviruses A6, A10, and A16 (CA6, CA10, and CA16), which were inactivated by formalin or ß-propiolactone (BPL) under different conditions, was evaluated as multivalent vaccine candidates. CA6 induced similar immune responses with both inactivation methods, and the immune efficacy of CA10 and CA16 was better following inactivation with BPL than with formalin. There was no sufficient cross-reactivity or cross-protectivity against heterologous strains in groups vaccinated with the BPL-inactivated (BI) monovalent vaccine. Sufficient neutralizing antibody and cell-mediated immune responses were induced in the BI-trivalent vaccinated group. These findings suggest that BI-CA6, CA10, and CA16 are potential multivalent vaccine candidates and that a multivalent vaccine is needed to control HFMD. The coxsackievirus multivalent vaccine could be useful for the development of effective HFMD vaccines.

Enhanced neutralizing antibody response induced by inactivated enterovirus 71 in cynomolgus monkeys.

Enterovirus 71 (EV71) is a major etiological agent of various public health issues, particularly in the Asia-Pacific region. EV71 causes hand-foot-and-mouth disease (HFMD) and is associated with serious neurological disorders in young children. A formalin-inactivated EV71 candidate vaccine (KCDC-HFMDV1-EV71) based on the C4 subgenotype was previously developed and confirmed to be a potential candidate vaccine for prevention of EV71 infection in mice. In this study, an inactivated EV71 vaccine was used for analysis of long-term immunogenicity and efficacy in cynomolgus monkeys, a common nonhuman primate model. The vaccine was immunized three times at 0, 4, and 8 weeks with either 20-µg doses of EV71 candidate vaccine formulated with aluminum hydroxide gel adjuvant or phosphate-buffered saline as a control. The group immunized with the inactivated EV71 showed significantly increased EV71-specific antibody and serum neutralizing antibody titers at 3 weeks after vaccination and maintained these elevated titers until the end of the experiment (54 weeks after vaccination). The sera from vaccinated cynomolgus monkeys showed a crossreactive neutralizing antibody response to the heterologous subtype of EV71 (B1-4, C1, and C2). These findings suggest that the inactivated EV71 candidate vaccine may be a potential vaccine candidate and valuable tool for the control of HFMD.

An inactivated hand-foot-and-mouth disease vaccine using the enterovirus 71 (C4a) strain isolated from a Korean patient induces a strong immunogenic response in mice.

Enterovirus 71 (EV71) is a major causative agent of hand-foot-and-mouth disease (HFMD) frequently occurring in children. HFMD induced by EV71 can cause serious health problems and has been reported worldwide, particularly in the Asia-Pacific region. In this study, we assessed the immunogenicity of a formalin-inactivated HFMD vaccine using an EV71 strain (FI-EV71 C4a) isolated from a Korean patient. The vaccine candidate was evaluated in mice to determine the vaccination doses and vaccine schedules. BALB/c mice were intramuscularly administered 5, 10, or 20 µg FI-EV71 vaccine, followed by a booster 2 weeks later. EV71-specific antibodies and neutralizing antibodies were induced and maintained until the end of the experimental period in all vaccinated groups. To determine the effectiveness of adjuvant for the EV71 vaccine, three adjuvants, i.e., aluminium hydroxide gel, monophosphoryl lipid A, and polyinosinic-polycytidylic acid, were administered separately with the FI-EV71 vaccine to mice via the intramuscular route. Mice administered the FI-EV71 vaccine formulated with all three adjuvants induced a significantly increased antibody response compared with that of the single adjuvant groups. The vaccinated group with triple adjuvants exhibited more rapid induction of EV71-specific and neutralizing antibodies than the other groups. These results suggested that the role of adjuvant in inactivated vaccine was important for eliciting effective immune responses against EV71. In conclusion, our results showed that FI-EV71 was a potential candidate vaccine for prevention of EV71 infection.