A screening study on the detection strain of Coxsackievirus A6: the key to evaluating neutralizing antibodies in vaccines.

The increasing incidence of diseases caused by Coxsackievirus A6 (CV-A6) and the presence of various mutants in the population present significant public health challenges. Given the concurrent development of multiple vaccines in China, it is challenging to objectively and accurately evaluate the level of neutralizing antibody response to different vaccines. The choice of the detection strain is a crucial factor that influences the detection of neutralizing antibodies. In this study, the National Institutes for Food and Drug Control collected a prototype strain (Gdula), one subgenotype D1, as well as 13 CV-A6 candidate vaccine strains and candidate detection strains (subgenotype D3) from various institutions and manufacturers involved in research and development. We evaluated cross-neutralization activity using plasma from naturally infected adults (n = 30) and serum from rats immunized with the aforementioned CV-A6 strains. Although there were differences between the geometric mean titer (GMT) ranges of human plasma and murine sera, the overall trends were similar. A significant effect of each strain on the neutralizing antibody test (MAX/MIN 48.0 ∼16410.3) was observed. Among all strains, neutralization of the S112 strain by 15 different sera resulted in higher neutralizing antibody titers (GMTS112 = 132.0) and more consistent responses across different genotypic immune sera (MAX/MIN = 48.0). Therefore, S112 may serve as a detection strain for NtAb testing in various vaccines, minimizing bias and making it suitable for evaluating the immunogenicity of the CV-A6 vaccine.

Binding of berberine to PEBP1 synergizes with sorafenib to induce the ferroptosis of hepatic stellate cells.

Hepatic stellate cell (HSC) activation is the key process in hepatic fibrosis (HF) development. Targeted death of HSCs could be effective in the prevention and treatment of HF. Phosphatidylethanolamine-binding protein (PEBP)1 can trigger ferroptosis by mediating peroxide production, but how it modulates HSC ferroptosis is not known. We screened natural small molecules that could bind with PEBP1, and investigated the mechanism by which it promotes HSC ferroptosis. The maximum binding energy of berberine with PEBP1 was - 8.51 kcal/mol, indicating that berberine could bind strongly with PEBP1. Berberine binding to PEBP1 could promote HSC ferroptosis via synergy of its actions with those of sorafenib, but it could not induce ferroptosis alone. Combined administration of berberine enhanced the ferroptotic effects of low-dose sorafenib upon HSCs. Herein, we revealed that PEBP1 might be a target that could enhance the effects of sorafenib, which could provide a new therapeutic approach for HF treatment.

SARS-CoV-2 Omicron (B.1.1.529) infection in rhesus macaques, hamsters, and BALB/c mice with severe lung histopathological damage.

Since the first SARS-CoV-2 outbreak in late 2019, the SARS-CoV-2 genome has harbored multiple mutations, especially spike protein mutations. The currently fast-spreading Omicron variant that manifests without symptoms or with upper respiratory diseases has been recognized as a serious global public health problem. However, its pathological mechanism is largely unknown. In this work, rhesus macaques, hamsters, and BALB/C mice were employed as animal models to explore the pathogenesis of Omicron (B.1.1.529). Notably, Omicron (B.1.1.529) infected the nasal turbinates, tracheae, bronchi, and lungs of hamsters and BALB/C mice with higher viral loads than in those of rhesus macaques. Severe histopathological damage and inflammatory responses were observed in the lungs of Omicron (B.1.1.529)-infected animals. In addition, viral replication was found in multiple extrapulmonary organs. Results indicated that hamsters and BALB/c mice are potential animal models for studies on the development of drugs/vaccines and therapies for Omicron (B.1.1.529).

Comparative analysis of the biological characteristics of three CV-A10 clones adaptively cultured on Vero cells.

Coxsackievirus A10 (CV-A10) is a major pathogen that causes hand, foot, and mouth disease. There are no effective therapeutic drugs for CV-A10 infection; therefore, CV-A10 vaccines should be developed. Previously, we isolated a CV-A10 strain (N25) that can be cultured on Vero cells. In this study, the N25 strain was plaque-purified three times from Vero cells, and three clones were selected for adaptive culture. The three clones of the 5th, 12th, and 19th generations were compared and analyzed in terms of viral titers, plaque morphology, pathogenicity in suckling mice, and nucleotide and amino acid sequences of the complete genome. The infectivity titers of the three clones (P2-P22) were maintained at 6.5-7.0 lgCCID50 /ml. The three clones began to proliferate at 6 h and peaked at 36 h; the corresponding CCID50 was in the range of 106.5 -106.875 /ml, which gradually decreased. The suckling mice in the challenged group exhibited clinical symptoms such as paralysis of the limbs, which gradually worsened until death. The inactivated vaccines prepared using the three clones efficiently induced antigen-specific serum antibodies in mice. There were eight nucleotide mutations in the three clones, which resulted in two and four amino acid substitutions in the VP3 and VP1 coding regions, respectively. The nucleotide and amino acid sequence homology between the three clones and N25 were 99.92%-100% and 99.78%-100%, respectively, indicating high genetic stability. Our findings provide a theoretical basis for screening CV-A10 vaccine candidate clones.

Three doses of prototypic SARS-CoV-2 inactivated vaccine induce cross-protection against its variants of concern.

Variants are globally emerging very quickly following pandemic prototypic SARS-CoV-2. To evaluate the cross-protection of prototypic SARS-CoV-2 vaccine against its variants, we vaccinated rhesus monkeys with three doses of prototypic SARS-CoV-2 inactivated vaccine, followed by challenging with emerging SARS-CoV-2 variants of concern (VOCs). These vaccinated animals produced neutralizing antibodies against Alpha, Beta, Delta, and Omicron variants, although there were certain declinations of geometric mean titer (GMT) as compared with prototypic SARS-CoV-2. Of note, in vivo this prototypic vaccine not only reduced the viral loads in nasal, throat and anal swabs, pulmonary tissues, but also improved the pathological changes in the lung infected by variants of Alpha, Beta, and Delta. In summary, the prototypic SARS-CoV-2 inactivated vaccine in this study protected against VOCs to certain extension, which is of great significance for prevention and control of COVID-19.

Proliferation characteristics of coxsackievirus A10 in mice and immune protection ability of experimental inactivated vaccine.

Coxsackievirus A10 (CVA10) is the main pathogen of hand, foot, and mouth disease in China. However, there are no CVA10-specific drugs and vaccines, and the pathogenesis and effects of this virus in the body are unknown. We investigated the effect of a clinically isolated CVA10 virus strain (CVA10-25) to investigate its effect in suckling mice through different infection routes. We observed the dynamic distribution and proliferation of the virus in mouse tissues by infecting suckling mice with different doses of the virus and mice of different ages with the same dose of the virus. We also analysed the pathological characteristics after infection. A formaldehyde-inactivated experimental vaccine was prepared to immunise 5-week-old BALB/c female mice three times, and newborn suckling mice were tested for the presence of maternally transmitted antibodies. The viral load in each organ after intracerebral administration was higher than that after intraperitoneal administration; the peroral administration route did not cause disease in mice. Mouse paralysis and death after infection were related to age. The skeletal muscles, heart, and lung showed histopathological changes after infection. We established a 2-day-old BALB/c suckling mouse model that could be infected intracranially to study the pathogenesis and pathology of CVA10. Maternally transmitted antibodies protected the mice against the virus. This study provides a reference for CVA10-related pathogenesis and vaccine research.

Immunological evaluation of an inactivated SARS-CoV-2 vaccine in rhesus macaques.

Because of the relatively limited understanding of coronavirus disease 2019 (COVID-19) pathogenesis, immunological analysis for vaccine development is needed. Mice and macaques were immunized with an inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine prepared by two inactivators. Various immunological indexes were tested, and viral challenges were performed on day 7 or 150 after booster immunization in monkeys. This inactivated SARS-CoV-2 vaccine was produced by sequential inactivation with formaldehyde followed by propiolactone. The various antibody responses and specific T cell responses to different viral antigens elicited in immunized animals were maintained for longer than 150 days. This comprehensive immune response could effectively protect vaccinated macaques by inhibiting viral replication in macaques and substantially alleviating immunopathological damage, and no clinical manifestation of immunopathogenicity was observed in immunized individuals during viral challenge. This candidate inactivated vaccine was identified as being effective against SARS-CoV-2 challenge in rhesus macaques.

Preclinical safety assessment of a combined vaccine against Hepatitis a virus and enterovirus 71.

Since 2007, Hepatitis A (HAV) vaccination has been a part of the National Immunization Program of China. Recognizing enterovirus 71 (EV71) as the most important pathogen in severe hand, foot and mouth disease, an inactivated EV71 vaccine was successfully marketed in 2015. Based on the concept of one vaccine preventing two diseases and owing to similarities in vaccine preparation and the overlap of the eligible population, a combination of the inactivated HAV vaccine and inactivated EV71 vaccine is theoretically feasible and desirable. However, the optimal vaccinationschedule for this combination vaccine has yet to be optimized. Use of this combined vaccine would not only decrease the number of vaccinations, but also lower associated cost. This study aimed to investigate the toxicity and adverse reactions of the combined HAV-EV71 vaccine under Good Laboratory Practice conditions to provide a reference for clinical studies/applications in the future. CD®(Sprague Dawley) IGS rats were employed for single-dose toxicity testing using a high dose, and repeated-dose toxicity testing using high, as well as low doses. Animals that received only a single dose showed no obvious clinical symptoms nor abnormal body weight, and no significant gross pathological change at the experimental endpoint at necropsy. In the rats injected with three doses, phagocytosis of basophilic granules by macrophages was observed in the inguinal, mesenteric, and local lymph nodes, besides irritation at the administration site. At 56 days after the last dose, no significant histopathological change was observed in the lymph nodes, and local irritation gradually faded. Further, systematic allergy testing was performed in guinea pigs. After systemic sensitization and challenge with the HAV-EV71 vaccine, animals showed normal weight gain and no allergic reactions. This study, therefore, confirmed a good safety profile of the inactivated HAV and EV71 combined vaccine.

The safety and immunogenicity of an inactivated SARS-CoV-2 vaccine in Chinese adults aged 18-59 years: A phase I randomized, double-blinded, controlled trial.

BACKGROUND: This study examined the safety and immunogenicity of an inactivated SARS-CoV-2 vaccine. METHOD: In a phase I randomized, double-blinded, placebo-controlled trial involving 192 healthy adults 18-59 years old, two injections of three doses (50 EU, 100 EU, 150 EU) of an inactivated SARS-CoV-2 vaccine or placebo were administered intramuscularly at a 2- or 4-week interval. The safety and immunogenicity of the vaccine were evaluated. RESULTS: Vaccination was completed in 191 subjects. Forty-four adverse reactions occurred within 28 days, most commonly mild pain and redness at the injection site or slight fatigue. At days 14 and 28, the seroconversion rates were 87.5% and 79.2% (50 EU), 100% and 95.8% (100 EU), and 95.8% and 87.5% (150 EU), respectively, with geometric mean titers (GMTs) of 18.1 and 10.6, 54.5 and 15.4, and 37.1 and 18.5, respectively, for the schedules with 2-week and 4-week intervals. Seroconversion was associated with synchronous upregulation of antibodies against the S protein, N protein and virion and a cytotoxic T lymphocyte (CTL) response. No cytokines and immune cells related to immunopathology were observed. Transcriptome analysis revealed the genetic diversity of immune responses induced by the vaccine. INTERPRETATION: In a population aged 18-59 years in this trial, this inactivated SARS-CoV-2 vaccine was safe and immunogenic. TRIAL REGISTRATION: CTR20200943 and NCT04412538.

A comparative study on biological characteristics of ten coxsackievirus A10 virus strains.

In this study, we analyzed ten CVA10 strains were genotyped and cultured for 10 generations to detect plaque morphology, pathogenicity, growth and other characteristics. Mice were injected with live and inactivated virus to detect neutralizing antibody titers. The results suggested that all CVA10 strains fell into Genotype C. Each strain cultured on KMB17 and Vero cells, increased from 1st generation onwards to peak in the 3rd and 4th, and the titer at which each became infectious ranged from 5.0 to 6.5 and 6.0 to 7.0 lgCCID50/ml, respectively. Two-day-old BALB/c mice were selected and inoculated intracerebral with the CVA10 strains, Limb paralysis was significant as early as 3 d; paralysis of all limbs for 50% of affected mice. LT50 was approximately 6 d, all died within 8 d. Ten strains induced good immune response, the GMT value of booster immunizations was higher than that of initial immunization. This provide reference points for selecting CVA10 vaccine candidates.

N-terminally truncated nucleocapsid protein of SARS-CoV-2 as a better serological marker than whole nucleocapsid protein in evaluating the immunogenicity of inactivated SARS-CoV-2.

The coronavirus disease 2019 pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) had led to a serious public health crisis, and no specific treatments or vaccines are available yet. A nucleocapsid protein (NP)-based enzyme-linked immunosorbent assay (ELISA) detection method is not only important in disease diagnosis, but is required for the evaluation of vaccine efficacy during the development of an inactivated SARS-CoV-2 vaccine. In this study, we expressed both the NP and N-terminally truncated NP (ΔN-NP) of SARS-CoV-2 in an Escherichia coli expression system and described the purification of the soluble recombinant NP and ΔN-NP in details. The identities of the NP and ΔN-NP were confirmed with mass spectrometry. We then used immunoglobulin G detection ELISAs to compare the sensitivity of NP and ΔN-NP in detecting anti-SARS-CoV-2 antibodies. ΔN-NP showed greater sensitivity than NP in the analysis of serially diluted sera from mice and rabbits vaccinated with inactive SARS-CoV-2 and in human sera diluted 1:400. ΔN-NP showed a positive detection rate similar to that of the SARS-CoV-2 S protein in human sera. We conclude that ΔN-NP is a better serological marker than NP for evaluating the immunogenicity of inactivated SARS-CoV-2.

Randomized, Double-Blinded, Placebo-Controlled Phase 2 Trial of an Inactivated Severe Acute Respiratory Syndrome Coronavirus 2 Vaccine in Healthy Adults.

BACKGROUND: We evaluated an inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine for immunogenicity and safety in adults aged 18-59 years. METHODS: In this randomized, double-blinded, controlled trial, healthy adults received a medium dose (MD) or a high dose (HD) of the vaccine at an interval of either 14 days or 28 days. Neutralizing antibody (NAb) and anti-S and anti-N antibodies were detected at different times, and adverse reactions were monitored for 28 days after full immunization. RESULTS: A total of 742 adults were enrolled in the immunogenicity and safety analysis. Among subjects in the 0, 14 procedure, the seroconversion rates of NAb in MD and HD groups were 89% and 96% with geometric mean titers (GMTs) of 23 and 30, respectively, at day 14 and 92% and 96% with GMTs of 19 and 21, respectively, at day 28 after immunization. Anti-S antibodies had GMTs of 1883 and 2370 in the MD group and 2295 and 2432 in the HD group. Anti-N antibodies had GMTs of 387 and 434 in the MD group and 342 and 380 in the HD group. Among subjects in the 0, 28 procedure, seroconversion rates for NAb at both doses were both 95% with GMTs of 19 at day 28 after immunization. Anti-S antibodies had GMTs of 937 and 929 for the MD and HD groups, and anti-N antibodies had GMTs of 570 and 494 for the MD and HD groups, respectively. No serious adverse events were observed during the study period. CONCLUSIONS: Adults vaccinated with inactivated SARS-CoV-2 vaccine had NAb as well as anti-S/N antibody and had a low rate of adverse reactions. CLINICAL TRIALS REGISTRATION: NCT04412538.

Immune responses of a CV-A16 live attenuated candidate strain and its protective effects in rhesus monkeys.

Coxsackievirus A16 (CV-A16) is a major causative pathogen of hand, foot, and mouth diseases (HFMDs). The licensed HFMD vaccine targets EV-A71 without cross-protection against CV-A16. Thus, a CV-A16 vaccine is needed. In this study, the immunogenicity and protective efficacy of a live attenuated CV-A16 candidate, K168-8Ac, were evaluated in a rhesus monkey model. Four passages of this strain (P35, P50, P60, and P70) were administered to monkeys, and its protective effect was identified. The immunized monkeys were clinically asymptomatic, except for slight fever. Weak viraemia was observed, and two doses of vaccination were found to significantly reduce virus shedding. High levels of antibody responses were observed (1:1024-1:2048), along with a significant increase in plasma IL-8. The I.M. group showed a much stronger humoural immunity. Pathological damage was detected mainly in lung tissues, although thalamus, spinal cord, lymph nodes, and livers were involved. After the viral challenge, it was found that two doses of vaccine reduced virus shedding, and the degree of lung damage and the number of organs involved decreased as the passage number increased. Overall, a robust immune response and partial protection against CV-A16, triggered by the K168-8Ac strain, were demonstrated. This study provides valuable data for CV-A16 vaccine development.

PTP1B negatively regulates STAT1-independent Pseudomonas aeruginosa killing by macrophages.

Pseudomonas aeruginosa is the main conditional pathogen of immunodeficiency individuals. The mechanisms governing immune response to P. aeruginosa infection by macrophages remain incompletely defined. Herein, we demonstrate that protein tyrosine phosphatase-1B (PTP1B) is a critical negative regulator of P. aeruginosa infection response by macrophages. PTP1B-deficient macrophages display greatly enhanced bacterial phagocytosis and killing, accompanied by increased lysosome formation during P. aeruginosa infection. We also found that PTP1B repressed nitric oxide (NO) production and nitric oxide synthase (iNOS) induction following P. aeruginosa infection. PTP1B deficiency tended to upregulate the production of TRIF-interferon (IFN) pathway cytokines and chemokines, including IFN-ß and interferon γ-inducible protein 10 (CXCL10, IP-10). Unexpectedly, the phosphorylation level of STAT1 was not regulated by PTP1B. In vivo experiments also confirmed that the regulatory function of PTP1B was not dependent on STAT1. These findings demonstrate that STAT1 is dispensable for negative regulation of P. aeruginosa clearance by macrophages.

Distinct infection process of SARS-CoV-2 in human bronchial epithelial cell lines.

Coronavirus disease 2019, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), leads to a series of clinical symptoms of respiratory and pulmonary inflammatory reactions via unknown pathologic mechanisms related to the viral infection process in tracheal or bronchial epithelial cells. Investigation of this viral infection in the human bronchial epithelial cell line (16HBE) suggests that SARS-CoV-2 can enter these cells through interaction between its membrane-localized S protein with the angiotensin-converting enzyme 2 molecule on the host cell membrane. Further observation indicates distinct viral replication with a dynamic and moderate increase, whereby viral replication does not lead to a specific cytopathic effect but maintains a continuous release of progeny virions from infected cells. Although messenger RNA expression of various innate immune signaling molecules is altered in the cells, transcription of interferons-α (IFN-α), IFN-ß, and IFN-γ is unchanged. Furthermore, expression of some interleukins (IL) related to inflammatory reactions, such as IL-6, IL-2, and IL-8, is maintained at low levels, whereas that of ILs involved in immune regulation is upregulated. Interestingly, IL-22, an IL that functions mainly in tissue repair, shows very high expression. Collectively, these data suggest a distinct infection process for this virus in respiratory epithelial cells, which may be linked to its clinicopathological mechanism.

Safety and immunogenicity of an experimental live combination vaccine against enterovirus 71 and coxsackievirus A16 in rhesus monkeys.

Enterovirus 71 (EV-A71) and Coxsackievirus A16 (CV-A16) are the two most common pathogens causing hand, foot, and mouth disease (HFMD). Previously, we obtained one candidate live attenuated strain each for EV-A71 and CV-A16; here, we evaluated the safety and immunogenicity of a combinedlive vaccine against EV-A71 and CV-A16 generated from these two candidate strains. Rhesus monkeys were intramuscularly treated with a live combinationvaccine against both EV-A71 and CV-A16 or with either vaccine alone. No fever or atypical clinical signs were observed in any animals. Monkeys vaccinated with the combinationlive vaccine presented no notable pathological changes in the brain, spinal cord, lung, and liver; in contrast, these regions showed inflammatory cell infiltration in monkeys treated with EV-A71 alone or CV-A16 alone. Weak viremia was detected in plasma after inoculation with the combinationvaccine; however, the duration of viral shedding in feces was increased. Biochemical studies revealed a slight increase in aspartate aminotransferase levels in monkeys inoculated with the live combination vaccine; however, histopathological findings did not attribute this change to liver damage. We also found that the live combinationvaccine induced a dual humoral immune response. Cytokine analysis indicated that the combined EV-A71/CV-A16 vaccine significantly down-regulated interleukin-8 production. Here, we have demonstrated that the live attenuated EV-A71/CV-A16 vaccine was safe and could trigger a dual specific immune response. However, its immune protection efficacy requires further investigation.

PTP1B negatively regulates nitric oxide-mediated Pseudomonas aeruginosa killing by neutrophils.

Neutrophils play a critical role in host defense against Pseudomonas aeruginosa infection. Mechanisms underlying the negative regulation of neutrophil function in bacterial clearance remain incompletely defined. Here, we demonstrate that protein tyrosine phosphatase-1B (PTP1B) is a negative regulator of P. aeruginosa clearance by neutrophils. PTP1B-deficient neutrophils display greatly enhanced bacterial phagocytosis and killing, which are accompanied by increased Toll-like receptor 4 (TLR4) signaling activation and nitric oxide (NO) production following P. aeruginosa infection. Interestingly, PTP1B deficiency mainly upregulates the production of IL-6 and IFN-ß, leads to enhanced TLR4-dependent STAT1 activation and iNOS expression by neutrophils following P. aeruginosa infection. Further studies reveal that PTP1B and STAT1 are physically associated. These findings demonstrate a negative regulatory mechanism in neutrophil underlying the elimination of P. aeruginosa infection though a PTP1B-STAT1 interaction.