Development of a neutralization monoclonal antibody with a broad neutralizing effect against SARS-CoV-2 variants.

BACKGROUND: The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has challenged the effectiveness of current therapeutic regimens. Here, we aimed to develop a potent SARS-CoV-2 antibody with broad neutralizing effect by screening a scFv library with the spike protein receptor-binding domain (RBD) via phage display. METHODS: SKAI-DS84 was identified through phage display, and we performed pseudovirus neutralization assays, authentic virus neutralization assays, and in vivo neutralization efficacy evaluations. Furthermore, surface plasmon resonance (SPR) analysis was conducted to assess the physical characteristics of the antibody, including binding kinetics and measure its affinity for variant RBDs. RESULTS: The selected clones were converted to human IgG, and among them, SKAI-DS84 was selected for further analyses based on its binding affinity with the variant RBDs. Using pseudoviruses, we confirmed that SKAI-DS84 was strongly neutralizing against wild-type, B.1.617.2, B.1.1.529, and subvariants of SARS-CoV-2. We also tested the neutralizing effect of SKAI-DS84 on authentic viruses, in vivo and observed a reduction in viral replication and improved lung pathology. We performed binding and epitope mapping experiments to understand the mechanisms underlying neutralization and identified quaternary epitopes formed by the interaction between RBDs as the target of SKAI-DS84. CONCLUSIONS: We identified, produced, and tested the neutralizing effect of SKAI-DS84 antibody. Our results highlight that SKAI-DS84 could be a potential neutralizing antibody against SARS-CoV-2 and its variants.

Heterologous vaccination utilizing viral vector and protein platforms confers complete protection against SFTSV.

Severe fever with thrombocytopenia syndrome virus was first discovered in 2009 as the causative agent of severe fever with thrombocytopenia syndrome. Despite its potential threat to public health, no prophylactic vaccine is yet available. This study developed a heterologous prime-boost strategy comprising priming with recombinant replication-deficient human adenovirus type 5 (rAd5) expressing the surface glycoprotein, Gn, and boosting with Gn protein. This vaccination regimen induced balanced Th1/Th2 immune responses and resulted in potent humoral and T cell-mediated responses in mice. It elicited high neutralizing antibody titers in both mice and non-human primates. Transcriptome analysis revealed that rAd5 and Gn proteins induced adaptive and innate immune pathways, respectively. This study provides immunological and mechanistic insight into this heterologous regimen and paves the way for future strategies against emerging infectious diseases.

Recombinant measles virus encoding the spike protein of SARS-CoV-2 efficiently induces Th1 responses and neutralizing antibodies that block SARS-CoV-2 variants.

Owing to the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants, the development of effective and safe vaccines has become a priority. The measles virus (MeV) vaccine is an attractive vaccine platform as it has been administered to children for more than 40 years in over 100 countries. In this study, we developed a recombinant MeV expressing the full-length SARS-CoV-2 spike protein (rMeV-S) and tested its efficacy using mouse and hamster models. In hCD46Tg mice, two-dose rMeV-S vaccination induced higher Th1 secretion and humoral responses than one-dose vaccination. Interestingly, neutralizing antibodies induced by one-dose and two-dose rMeV-S immunization effectively blocked the entry of the α, ß, γ, and δ variants of SARS-CoV-2. Furthermore, two-dose rMeV-S immunization provided complete protection against SARS-CoV-2 in the hamster model. These results suggest the potential of rMeV-S as a vaccine candidate for targeting SARS-CoV-2 and its variants.

Effective inactivated influenza vaccine for the elderly using a single-stranded RNA-based adjuvant.

There is an unmet need for new influenza vaccine strategies that compensate for impaired vaccine responses in elderly individuals. Here, we evaluated the effectiveness of a single-stranded RNA (ssRNA) as an adjuvant to enhance the efficacy of inactivated influenza vaccine (IIV) in mouse models. Immunization with the ssRNA along with IIV reduced viral titers as well as pathological and inflammatory scores in the lungs after influenza challenge in aged mice. ssRNA induced balanced Th1/Th2 responses with an increase in IgA titers. Moreover, the ssRNA adjuvant markedly increased the frequency of influenza HA-specific T cells and IFN-γ production along with the expression of genes related to innate and adaptive immune systems that could overcome immunosenescence in aged mice. Our findings indicate that ssRNA is an efficient vaccine adjuvant that boosts cellular and humoral immunity in aged mice, demonstrating its potential as a novel adjuvant for currently available influenza virus vaccines for elderly individuals.

Nanoformulated Single-Stranded RNA-Based Adjuvant with a Coordinative Amphiphile as an Effective Stabilizer: Inducing Humoral Immune Response by Activation of Antigen-Presenting Cells.

As agonists of TLR7/8, single-stranded RNAs (ssRNAs) are safe and promising adjuvants that do not cause off-target effects or innate immune overactivation. However, low stability prevents them from mounting sufficient immune responses. This study evaluates the adjuvant effects of ssRNA derived from the cricket paralysis virus intergenic region internal ribosome entry site, formulated as nanoparticles with a coordinative amphiphile, containing a zinc/dipicolylamine complex moiety as a coordinative phosphate binder, as a stabilizer for RNA-based adjuvants. The nanoformulated ssRNA adjuvant was resistant to enzymatic degradation in vitro and in vivo, and that with a coordinative amphiphile bearing an oleyl group (CA-O) was approximately 100 nm, promoted effective recognition, and improved activation of antigen-presenting cells, leading to better induction of neutralizing antibodies following single immunization. Hence, CA-O may increase the efficacy of ssRNA-based adjuvants, proving useful to meet the urgent need for vaccines during pathogen outbreaks.

Evaluation of glycoprotein E subunit and live attenuated varicella-zoster virus vaccines formulated with a single-strand RNA-based adjuvant.

INTRODUCTION: Varicella-zoster virus (VZV), a human alphaherpesvirus 3, elicits both chickenpox and shingles and/or postherpetic neuralgia. A live attenuated vaccine (LAV) and glycoprotein E (gE) subunit vaccine were developed to prevent VZV-induced diseases. We recently reported that single-strand RNA (ssRNA) based on the intergenic region of the internal ribosome entry site of cricket paralysis virus (CrPV) is an effective adjuvant for protein-based and virus-like particle-based vaccines. Here, Chinese hamster ovary expression system and an LAV from Oka/SK strains. METHODS: We appraised the adjuvant effect of the same CrPV ssRNA encoding the gE gene formulated in the two vaccines using VZV-primed C57BL/6 mice and guinea pigs. Humoral immunity and cell-mediated immunity were assessed by enzyme-linked immunosorbent assay (ELISA) and ELISPOT in gE subunit vaccine and by ELISA and fluorescent antibody to membrane antigen in LAV. RESULTS: The gE subunit vaccine-induced gE-specific antibodies and CD4+ T-cell responses (indicated by interferon-γ [IFN-γ] and interleukin-2 secretion) in the ssRNA-based adjuvant containing the VZV gE gene. Therefore, an ssRNA adjuvant combined with gE antigen can trigger the innate immune response and induce an adaptive immune response to ultimately activate humoral and cell-mediated responses. VZV LAV could also induce VZV-specific antibodies and IFN-γ stimulated by LAV, whereas the effect of ssRNA as a vaccine adjuvant could not be confirmed. However, the ssRNA adjuvant increased VZV-specific neutralizing antibody response. CONCLUSIONS: Taken together, these results highlight that the gE subunit vaccine and LAV developed in this study can be functional VZV vaccines, and ssRNAs appear to function better as adjuvants in a subunit vaccine than in an LAV.

Comprehensive Analysis of the Safety Profile of a Single-Stranded RNA Nano-Structure Adjuvant.

Adjuvants enhance the efficacy of vaccines by stimulating immune response-related gene expression and pathways. Although some adjuvants have been approved for commercial use in human vaccines (e.g., Alum, MF59, and AS03), they might elicit adverse side effects, such as autoimmune diseases. Recently, we developed a novel single-stranded RNA (ssRNA) nano-structure adjuvant, which can stimulate both Th1 and Th2 responses. In this study, we evaluated the safety and toxicological profiles of this ssRNA nano-structure adjuvant in vitro and in vivo. Mice were intramuscularly immunized with the ssRNA nano-structure adjuvant three times, once every 2 weeks. The results indicate no significant differences in hematological and serum biochemistry parameters between the ssRNA-treated groups and the control group. From a histopathological perspective, no evidence of tissue damage was found in any group. The levels of IgE and anti-nuclear antibodies, which are markers of autoimmune disease, were not different between the ssRNA-treated groups and the control group. The findings of this study suggest that the ssRNA nano-structure can be used as a safe adjuvant to increase vaccine efficacies.

Cricket paralysis virus internal ribosome entry site-derived RNA promotes conventional vaccine efficacy by enhancing a balanced Th1/Th2 response.

An ideal adjuvant should increase vaccine efficacy through balanced Th1/Th2 responses and be safe to use. Recombinant protein-based vaccines are usually formulated with aluminum (alum)-based adjuvants to ensure an adequate immune response. However, use of alum triggers a Th2-biased immune induction, and hence is not optimal. Although the adjuvanticity of RNA has been reported, a systematic and overall investigation on its efficacy is lacking. We found that single strand RNA (termed RNA adjuvant) derived from cricket paralysis virus intergenic region internal ribosome entry site induced the expression of various adjuvant-function-related genes, such as type 1 and 2 interferon (IFN) and toll-like receptor (TLR), T cell activation, and leukocyte chemotaxis in human peripheral blood mononuclear cells; furthermore, its innate and IFN transcriptome profile patterns were similar to those of a live-attenuated yellow fever vaccine. This suggests that protein-based vaccines formulated using RNA adjuvant function as live-attenuated vaccines. Application of the RNA adjuvant in mouse enhanced the efficacy of Middle East respiratory syndrome spike protein, a protein-subunit vaccine and human papillomavirus L1 protein, a virus-like particle vaccine, by activating innate immune response through TLR7 and enhancing pAPC chemotaxis, leading to a balanced Th1/Th2 responses. Moreover, the combination of alum and the RNA adjuvant synergistically induced humoral and cellular immune responses and endowed long-term immunity. Therefore, RNA adjuvants have broad applicability and can be used with all conventional vaccines to improve vaccine efficacy qualitatively and quantitively.

Development of an RNA Expression Platform Controlled by Viral Internal Ribosome Entry Sites.

Since 1990, many nucleic acid expression platforms consisting of DNA or RNA have been developed. However, although RNA expression platforms have been relatively neglected, several such platforms capped at the 5' end of RNA by an anti-reverse cap analog have now been developed. At the same time, the capping reaction is a bottleneck in the production of such platforms, with high cost and low efficiency. Here, we investigated several viral and eukaryotic internal ribosome entry sites (IRESs) to develop an optimal RNA expression platform, because IRES-dependent translation does not require a capping step. RNA expression platforms constructed with IRESs from the 5' untranslated regions of the encephalomyocarditis virus (EMCV) and the intergenic region of the cricket paralysis virus (CrPV) showed sufficient expression efficiency compared with cap-dependent RNA expression platforms. However, eukaryotic IRESs exhibited a lower viral IRES expression efficiency. Interestingly, the addition of a poly(A) sequence to the 5' end of the coxsackievirus B3 (CVB3) IRES (pMA-CVB3) increased the expression level compared with the CVB3 IRES without poly(A) (pCVB3). Therefore, we developed two multiexpression platforms (termed pMA-CVB3-EMCV and pCrPV-EMCV) by combining the IRESs of CVB3, CrPV, and EMCV in a single-RNA backbone. The pMA-CVB3-EMCV-derived RNA platform showed the highest expression level. Moreover, it clearly exhibited expression in mouse muscles in vivo. These RNA expression platforms prepared using viral IRESs will be useful in developing potential RNA-based prophylactic or therapeutic vaccines, because they have better expression efficiency and do not need a capping step.

Sipjeondaebo-tang Alleviates Oxidative Stress-Mediated Liver Injury through Activation of the CaMKK2-AMPK Signaling Pathway.

Sipjeondaebo-tang (SDT) is used frequently as a herbal prescription to treat deficiency syndromes in traditional Korean medicine. We investigated the hepatoprotective effects of SDT against oxidative stress and attempted to clarify the underlying molecular mechanisms. SDT pretreatment reduced arachidonic acid (AA) plus iron-mediated cytotoxicity in a concentration-dependent manner and prevented changes in apoptosis-related protein expression. In addition, SDT pretreatment significantly reduced glutathione depletion, hydrogen peroxide production, and mitochondrial dysfunction via treatment with AA plus iron. SDT increased the phosphorylation of AMP-activated protein kinase (AMPK) in accordance with the phosphorylation of Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2). Experiments using an AMPK chemical inhibitor (Compound C) or CaMKK2 chemical inhibitor (STO-609) suggested that the CaMKK2-AMPK signaling pathway contributes to SDT-mediated protection of mitochondria and cells. Moreover, administration of SDT for 4 consecutive days to mice significantly reduced the alanine aminotransferase and aspartate aminotransferase activities induced by carbon tetrachloride, and the numbers of degenerated hepatocytes, infiltrated inflammatory cells, nitrotyrosine-positive cells, and 4-hydroxynonenal-positive cells in liver tissue. Therefore, SDT protects hepatocytes from oxidative stress via CaMKK2-dependent AMPK activation and has the therapeutic potential to prevent or treat oxidative stress-related liver injury.

Development of a diagnostic system for detection of specific antibodies and antigens against Middle East respiratory syndrome coronavirus.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a single-stranded RNA virus that causes severe respiratory disease in humans with a high fatality rate. Binding of the receptor binding domain (RBD) of the spike (S) glycoprotein to dipeptidyl peptidase 4 is the critical step in MERS-CoV infection of a host cell. No vaccines or clinically applicable treatments are currently available for MERS-CoV. Therefore, rapid diagnosis is important for improving patient outcomes through prompt treatment and protection against viral outbreaks. In this study, the aim was to establish two ELISA systems for detecting antigens and antibodies against MERS-CoV. Using a recombinant full-length S protein, an indirect ELISA was developed and found to detect MERS-CoV-specific antibodies in animal sera and sera of patient with MERS. Moreover, MAbs were induced with the recombinant S protein and RBD and used for sandwich ELISA to detect the MERS-CoV S protein. Neither ELISA system exhibited significant intra-assay or inter-assay variation, indicating good reproducibility. Moreover, the inter-day precision and sensitivity were adequate for use as a diagnostic kit. Thus, these ELISAs can be used clinically to diagnose MERS-CoV.

Epimedium koreanum Ameliorates Oxidative Stress-Mediated Liver Injury by Activating Nuclear Factor Erythroid 2-Related Factor 2.

Oxidative stress induced by reactive oxygen species is the main cause of various liver diseases. This study investigated the hepatoprotective effect of Epimedium koreanum Nakai water extract (EKE) against arachidonic acid (AA)[Formula: see text][Formula: see text][Formula: see text]iron-mediated cytotoxicity in HepG2 cells and carbon tetrachloride (CCl4-)-mediated acute liver injury in mice. Pretreatment with EKE (30 and 100[Formula: see text][Formula: see text]g/mL) significantly inhibited AA[Formula: see text][Formula: see text][Formula: see text]iron-mediated cytotoxicity in HepG2 cells by preventing changes in the expression of cleaved caspase-3 and poly(ADP-ribose) polymerase. EKE attenuated hydrogen peroxide production, glutathione depletion, and mitochondrial membrane dysfunction. EKE also increased the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2), transactivated anti-oxidant response element harboring luciferase activity, and induced the expression of anti-oxidant genes. Furthermore, the cytoprotective effect of EKE against AA[Formula: see text][Formula: see text][Formula: see text]iron was blocked in Nrf2 knockout cells. Ultra-performance liquid chromatography analysis showed that EKE contained icariin, icaritin, and quercetin; icaritin and quercetin were both found to protect HepG2 cells from AA[Formula: see text][Formula: see text][Formula: see text]iron via Nrf2 activation. In a CCl4-induced mouse model of liver injury, pretreatment with EKE (300[Formula: see text]mg/kg) for four consecutive days ameliorated CCl4-mediated increases in serum aspartate aminotransferase activity, histological activity index, hepatic parenchyma degeneration, and inflammatory cell infiltration. EKE also decreased the number of nitrotyrosine-, 4-hydroxynonenal-, cleaved caspase-3-, and cleaved poly(ADP-ribose) polymerase-positive cells in hepatic tissues. These results suggest EKE is a promising candidate for the prevention or treatment of oxidative stress-related liver diseases via Nrf2 activation.

Tryptanthrin prevents oxidative stress-mediated apoptosis through AMP-activated protein kinase-dependent p38 mitogen-activated protein kinase activation.

Tryptanthrin (6,12-dihydro-6,12-dioxoindolo-(2,1-b)-quinazoline) has been reported to have a variety of pharmacological activities. Present study investigated the cytoprotective effects of tryptanthrin on arachidonic acid (AA) + iron-mediated oxidative stress and the molecular mechanisms responsible. In HepG2 cells, pretreatment with tryptanthrin inhibited the cytotoxic effect of AA + iron in a concentration-dependent manner. In addition, tryptanthrin prevented the changes in the levels of apoptosis-related proteins, and attenuated reactive oxygen species production, glutathione depletion, and mitochondrial membrane impairment induced by AA + iron. Mechanistic investigations showed that tryptanthrin increased the phosphorylations of AMP-activated protein kinase (AMPK) and of p38 mitogen-activated protein kinase (p38). Furthermore, inhibition of AMPK or p38 reduced the ability of tryptanthrin to prevent AA + iron-induced cell death and mitochondrial dysfunction. Transfection experiments using AMPK mutants indicated that p38 phosphorylation by tryptanthrin was dependent on AMPK activation. In a phenylhydrazine-induced acute liver injury model, tryptanthrin decreased serum levels of alanine aminotransferase, aspartate aminotransferase, and bilirubin in mice. Additionally, tryptanthrin reduced numbers of degenerating hepatocytes, infiltrating inflammatory cells, 4-hydroxynonenal-, and nitrotyrosine-positive cells in hepatic tissues. Thus, these results suggest tryptanthrin has therapeutic potential to protect cells from oxidative injury via AMPK-dependent p38 activation.

Sublingual immunization with Japanese encephalitis virus vaccine effectively induces immunity through both cellular and humoral immune responses in mice.

The Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis. Although there are four classes of vaccines against JEV, all of them are administered by s.c or i.m injection. Here, the effectiveness of sublingual (s.l.) administration of a JEV live-attenuated vaccine or recombinant modified vaccinia virus Ankara (MVA) vaccine, including JEV prM/E, was investigated. The mice were immunized three times i.m. or s.c. One week after the final immunization by both s.l. and i.m. routes, the titers of IgG1 induced by the recombinant MVA vaccine were higher than those induced by the live-attenuated vaccine, whereas the titers of IgG2a induced by the live-attenuated vaccine were higher than those induced by the recombinant MVA vaccine. However, both vaccines induced neutralizing antibodies when given by either s.l. or i.m. routes, indicating that both vaccines induce appropriate Th1 and Th2 cell responses through the s.l. and i.m. routes. Moreover, both vaccines protected against induction of proinflammatory cytokines and focal spleen white pulp hyperplasia after viral challenge. Virus-specific IFN-γ+ CD4+ and CD8+ T cells appeared to increase in mice immunized via both s.l. and i.m. routes. Interestingly, virus-specific IL-17+ CD4+ T cells increased significantly only in the mice immunized via the s.l. route; however, the increased IL-17 did not affect pathogenicity after viral challenge. These results suggest that s.l. immunization may be as useful as i.m. injection for induction of protective immune responses against JEV by both live-attenuated and recombinant MVA vaccines.

Eupatilin induces Sestrin2-dependent autophagy to prevent oxidative stress.

Eupatilin (5,7-dihydroxy-3,4,6-trimethoxyflavone) has many pharmacological activities including anti-inflammation, anti-oxidant and anti-cancer effects. Autophagy is the basic cellular machinery involving the digestion of damaged cellular components. In the present study, we investigated the protection effects of eupatilin against arachidonic acid (AA) and iron-induced oxidative stress in HepG2 cells and tried to elucidate the molecular mechanisms responsible. Eupatilin increased cell viability against AA + iron in a concentration-dependent manner and prevented mitochondrial dysfunction and reactive oxygen species (ROS) production. In addition, AA + iron increased the levels of pro-apoptotic proteins and these changes were prevented by eupatilin. Eupatilin also induced autophagy, as evidenced by the accumulation of microtubule-associated protein 1 light chain3-II and the detection of autophagic vacuoles. Furthermore, the protective effects of eupatilin on mitochondrial dysfunction and ROS production were significantly abolished by autophagy inhibitors. Eupatilin also increased the mRNA level of sestrin-2 and its promoter-driven reporter gene activity, which resulted in the up-regulation of sestrin-2 protein. Finally, gene silencing using sestrin-2 siRNA and the ectopic expression of recombinant adenoviral sestrin-2 indicated that sestrin-2 induction by eupatilin was required for autophagy-mediated cytoprotection against AA + iron. Our results suggest that eupatilin activates sestrin-2-dependent autophagy, thereby preventing oxidative stress induced by AA + iron.

The tegmental-accumbal dopaminergic system mediates the anxiolytic effect of acupuncture during ethanol withdrawal.

This study investigated the involvement of the mesolimbic dopamine (DA) system in the anxiolytic effects of acupuncture during ethanol withdrawal (EW). Rats were intraperitoneally treated with 3g/kg/day of ethanol for 28 days and experienced 3 days of withdrawal. During EW, the rats were bilaterally treated with acupuncture at acupoints HT7 (Shenmen) or PC6 (Neiguan) or at a non-acupoint (tail) once daily for 1min over 3 days. High-performance liquid chromatographic (HPLC) analysis showed that EW significantly decreased both DA and 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the nucleus accumbens shell (NaccSh); however, these processes were inhibited by acupuncture at HT7 but not at PC6. Real-time polymerase chain reaction and western blot assays also revealed that acupuncture at HT7 prevented the EW-induced reductions in tyrosine hydroxylase mRNA expression in the ventral tegmental area (VTA) and tyrosine hydroxylase protein expression in the NaccSh. A prior intra-NaccSh infusion of a cocktail of the selective DA1 receptor antagonist SCH23390 and the selective DA2 receptor antagonist eticlopride blocked the anxiolytic effect of acupuncture at HT7 in elevated plus maze tests. In addition, acupuncture at HT7 suppressed EW-induced increased BDNF levels in the VTA. These findings suggest that acupuncture at HT7 improves the VTA-Nacc DAergic function via inhibition of BDNF expression in the VTA, thereby exerting anxiolytic effects during EW.