Synthesis of deuterated S-217622 (Ensitrelvir) with antiviral activity against coronaviruses including SARS-CoV-2.

S-217622 (Ensitrelvir) is a reversible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3-chymotrypsin-like protease (3CLpro) inhibitor which obtained emergency regulatory approval in Japan for the treatment of SARS-CoV-2 infection on Nov 22, 2022. Herein, analogs of S-271622 with deuterium-for-hydrogen replacement were synthesized for comparison of the antiviral activities and pharmacokinetic (PK) profiles. Compared to the parent compound, C11-d2-S-217622 compound YY-278 retained in vitro activity against 3CLpro and SARS-CoV-2. X-ray crystal structural studies showed similar interactions of SARS-CoV-2 3CLpro with YY-278 and S-271622. The PK profiling revealed the relatively favorable bioavailability and plasma exposure of YY-278. In addition, YY-278, as well as S-217622, displayed broadly anti-coronaviral activities against 6 other coronaviruses that infect humans and animals. These results laid the foundation for further research on the therapeutic potential of YY-278 against COVID-19 and other coronaviral diseases.

Preparation of a new monoclonal antibody against nucleocapsid protein of swine acute diarrhea syndrome coronavirus and identification of its linear antigenic epitope.

Swine acute diarrhea syndrome coronavirus (SADS-CoV), which causes severe diarrhea in newborn piglets, was first identified in Southern China in 2017. Since the Nucleocapsid (N) protein in SADS-CoV is highly conserved and plays a key role in virus replication, it is often used as a target protein in scientific research. In this study, the N protein of SADS-CoV was successfully expressed, and a new monoclonal antibody (mAb), 5G12, against the protein was generated successfully. The mAb 5G12 can be used to detect SADS-CoV strains by indirect immunofluorescence assay (IFA) and western blotting. The mAb 5G12 epitope was located to amino acids 11 EQAESRGRK 19 by evaluating the antibody for reactivity with a series of truncated N protein segments. The biological information analysis showed that the antigenic epitope had a high antigenic index and conservation. This study will help further understand the protein structure and function of SADS-CoV and in the establishment of specific SADS-CoV detection methods.

Remdesivir Metabolite GS-441524 Effectively Inhibits SARS-CoV-2 Infection in Mouse Models.

The outbreak of coronavirus disease 2019 (COVID-19) has resulted in a global pandemic due to the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At the time of this manuscript's publication, remdesivir is the only COVID-19 treatment approved by the United States Food and Drug Administration. However, its effectiveness is still under question due to the results of the large Solidarity Trial conducted by the World Health Organization. Herein, we report that the parent nucleoside of remdesivir, GS-441524, potently inhibits the replication of SARS-CoV-2 in Vero E6 and other cell lines. Challenge studies in both an AAV-hACE2 mouse model of SARS-CoV-2 and in mice infected with murine hepatitis virus, a closely related coronavirus, showed that GS-441524 was highly efficacious in reducing the viral titers in CoV-infected organs without notable toxicity. Our results support that GS-441524 is a promising and inexpensive drug candidate for treating of COVID-19 and other CoV diseases.

Nonsteroidal anti-inflammatory drugs (NSAIDs) and nucleotide analog GS-441524 conjugates with potent in vivo efficacy against coronaviruses.

Coronaviruses (CoVs) infect a broad range of hosts, including humans and various animals, with a tendency to cross the species barrier, causing severe harm to human society and fostering the need for effective anti-coronaviral drugs. GS-441524 is a broad-spectrum antiviral nucleoside with potent anti-CoVs activities. However, its application is limited by poor oral bioavailability. Herein, we designed and synthesized several conjugates via covalently binding NSAIDs to 5'-OH of GS-441524 through ester bonds. The ibuprofen conjugate, ATV041, exhibited potent in vitro anti-coronaviral efficacy against four zoonotic coronaviruses in the alpha- and beta-genera. Oral-dosed ATV041 resulted in favorable bioavailability and rapid tissue distribution of GS-441524 and ibuprofen. In MHV-A59 infected mice, ATV041 dose-dependently decreased viral RNA replication and significantly reduced the proinflammatory cytokines in the liver and the lung at 3 dpi. As a result, the MHV-A59-induced lung and liver inflammatory injury was significantly alleviated. Taken together, this work provides a novel drug conjugate strategy to improve oral PK and offers a potent anti-coronaviral lead compound for further studies.

Aloe polymeric acemannan inhibits the cytokine storm in mouse pneumonia models by modulating macrophage metabolism.

The cytokine storm is highly associated with inflammatory-type disease severity and patients' survival. Plant polysaccharides, the main natural phytomedicine source, have a great potential to be an effective drug to treat cytokine storm. Herein we found that a polymeric acemannan (ABPA1) isolated from Aloe Vera Barbadensis extract C (AVBEC) exerted prominent inhibitory effects on inflammation-induced cytokine storm. The results displayed that ABPA1 effectively suppressed LPS-induced proinflammatory cytokines release in vitro. Moreover, ABPA1 treatment alleviated the cytokine storm and tissue damage in LPS- and IAV-induced mouse pneumonia models, and altered the phenotypic balance of macrophages in lung tissues. Functionally, ABPA1 enhanced macrophage M2 polarization and phagocytosis in RAW264.7 cells and inhibited LPS-induced M1 polarization. Mechanistically, ABPA1 enhanced mitochondrial metabolism and OXPHOS through activated PI3K/Akt/GSK-3ß signalling pathway. Overall, our findings suggest that ABPA1 may modulate macrophage activation and mitochondrial metabolism by targeting PI3K/Akt/GSK-3ß signalling pathway, thereby alleviating cytokine storm and inflammation.

Molecular Characterization and Phylogenetic Analysis of a Variant Recombinant Porcine Epidemic Diarrhea Virus Strain in China.

Since 2010, a variant of porcine epidemic diarrhea virus (PEDV) has re-emerged in several provinces of China, resulting in severe economic losses for the pork industry. Here, we isolated and identified a variant PEDV strain, SC-YB73, in Guangdong Province, China. The pathological observations of jejunum showed atrophy of villi and edema in the lamina propria. The sequence analysis of the viral genome identified a six-nucleotide insertion in the E gene, which has not previously been detected in PEDV strains. Furthermore, 50 nucleotide sites were unique in SC-YB73 compared with 27 other PEDV strains. The phylogenetic analysis based on the complete genome showed that SC-YB73 was clustered in variant subgroup GII-a, which is widely prevalent in the Chinese pig population. The recombination analysis suggested that SC-YB73 originated from the recombination of GDS47, US PEDV prototype-like strains TW/Yunlin550/2018, and COL/Cundinamarca/2014. In the present study, we isolated and genetically characterized a variant PEDV strain, thus providing essential information for the control of PED outbreaks in China.

The adenosine analog prodrug ATV006 is orally bioavailable and has preclinical efficacy against parental SARS-CoV-2 and variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus driving the ongoing coronavirus disease 2019 (COVID-19) pandemic, continues to rapidly evolve. Because of the limited efficacy of vaccination in prevention of SARS-CoV-2 transmission and continuous emergence of variants of concern (VOCs), orally bioavailable and broadly efficacious antiviral drugs are urgently needed. Previously, we showed that the parent nucleoside of remdesivir, GS-441524, has potent anti-SARS-CoV-2 activity. Here, we report that esterification of the 5'-hydroxyl moieties of GS-441524 markedly improved antiviral potency. This 5'-hydroxyl-isobutyryl prodrug, ATV006, demonstrated excellent oral bioavailability in rats and cynomolgus monkeys and exhibited potent antiviral efficacy against different SARS-CoV-2 VOCs in vitro and in three mouse models. Oral administration of ATV006 reduced viral loads and alleviated lung damage when administered prophylactically and therapeutically to K18-hACE2 mice challenged with the Delta variant of SARS-CoV-2. These data indicate that ATV006 represents a promising oral antiviral drug candidate for SARS-CoV-2.

Phylogeography and evolutionary dynamics analysis of porcine delta-coronavirus with host expansion to humans.

From 2003 onwards, three pandemics have been caused by coronaviruses: severe acute respiratory syndrome coronavirus (SARS-CoV); middle east respiratory syndrome coronavirus (MERS-CoV); and, most recently, SARS-CoV-2. Notably, all three were transmitted from animals to humans. This would suggest that animals are potential sources of epidemics for humans. The emerging porcine delta-coronavirus was reported to infect children. This is a red flag that marks the ability of PDCoV to break barriers of cross-species transmission to humans. Therefore, we conducted molecular genetic analysis of global clade PDCoV to characterize spatiotemporal patterns of viral diffusion and genetic diversity. PDCoV was classified into three major lineages, according to distribution and phylogenetic analysis of PDCoV. It can be inferred based on the analysis results of the currently known PDCoV strains that PDCoV might originate in Asia. We also selected six special spike amino acid sequences to align and analyze to find seven significant mutation sites. The accumulation of these mutations may enhance dynamic movements, accelerating spike protein membrane fusion events and transmission. Altogether, our study offers a novel insight into the diversification, evolution, and interspecies transmission and origin of PDCoV and emphasizes the need to study the zoonotic potential of the PDCoV and comprehensive surveillance and enhanced biosecurity precautions for PDCoV.

Kinetics of IgM and IgG antibody specific to SARS-CoV-2 nucleocapsid and spike proteins

：Objective To study the kinetics of IgM and IgG antibodies based on nucleocapsid（N） and spike（S） protein of SARS-Co V2-in COVID-19 patients. Methods Immunofluorescent kits were used to detect N and S protein specific IgM and IgG antibodies from Jan.21 to Feb.11, 2020 for the 60 hospitalized COVID-19 patients（48 mild, 12 severe cases） with a total of 290 plasma samples collected 9 weeks after the onset of the disease. Results The level of antibodies specific for S protein varied significantly with the course of disease（Ig M from 27.32 to 110.10 TU/ml, IgG from 56.85 to 135.00 TU/ml）, but not for N protein.Higher level of Ig M/Ig G antibodies specific to S protein was observed during the 2-7 week than that to N protein.The seropositive rate of antibodies gradually increased during the early stage of disease.IgM/IgG antibodies specific to N protein changed from 12.50% at the first week to peak level（51.72% and 86.21% respectively） at the 4 th week and those for S protein from 25.00% and 14.58% to 100.00%, and then declined.The seropositive rate of Ig M antibody specific to S protein was higher than that for N protein during 2-8 th week and that for Ig G antibody at 2, 3, 4, 6 and 7 th week.The seropositive rate of Ig G antibody specific to N protein in severe patients at the third week was higher than that in mild patients（100.00% vs 59.52%,χ2=9.67, P=0.001 9）, and the same as to Ig G antibody for S protein at the second week after disease onset（80.00% vs 46.58%, χ2=5.57, P=0.018 2）. Conclusions SARS-Co V2-S protein can induc stronger antibody response than N protein, and the antibody level was related to the severity of the disease.

Subunit Nanovaccine with Potent Cellular and Mucosal Immunity for COVID-19.

To combat the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, we formulated the S1 subunit of the virus with two adjuvants, amphiphilic adjuvant monophosphoryl lipid A for Toll-like receptor 4 and CpG oligodeoxynucleotide for Toll-like receptor 9, into cationic liposomes to produce a potent, safer, and translatable nanovaccine. The nanovaccine can efficiently elicit a humoral immune response and strong IgA antibodies in mice. The sera from the vaccinated mice significantly inhibit SARS-CoV-2 from infecting Vero cells. Moreover, relative to the free S1 with a traditional Alum adjuvant, the nanovaccine can elicit strong T-cell immunity by activating both CD4+ and CD8+ cells.

Preliminary evaluation of the safety and efficacy of oral human antimicrobial peptide LL-37 in the treatment of patients of COVID-19, a small-scale, single-arm, exploratory safety study (preprint)

Background& Aims: The Coronavirus Disease 2019 (COVID-19) has become a global epidemic and has caused a lasting and huge loss of life security, economic development and social stability in more than 180 countries around the world. Unfortunately, there is still no specific treatment for COVID-19 till now, therefore, at this point, all potential therapies need to be critically considered. LL-37 is one of the best-studied human antimicrobial peptide (AMPs) that has a broad-spectrum activity against bacteria and viruses. The use of living, genetically modified organisms (GMOs) is an effective approach for delivery of therapeutic proteins. The aim of this study was to determine the safety and efficacy of the Lactococcus lactis which has been genetically modified to produce the therapeutic human antimicrobial peptide LL-37 (herein after referred to cas001) in the patients of COVID-19. Methods: Firstly we constructed genetically modified food-grade probiotic, Lactococcus lactis, with sequence of seven tandem repeats of mature human LL-37 under control of the nisin-inducible nisA promoter to produce the cas001. A total of 20 healthy SD rats, half male and half female (There were five male and five female in the control group, the same in treatment group) were used to observe the acute toxic reaction and death after daily administration of cas001 for three weeks, which helps to provide necessary reference basis for clinical dose selection, verificaition of toxic reaction and possible target organs. According to the estimated clinical dosage of 1 x 108CFU /kg/day, considering the conversion of body surface area, the dose for rats should be multiplied by 6.17 to 6 x 108 CFU/kg/day. We administrated 100 times higher dose at 6 x 1010 CFU/kg/day to rats. In order to investigate the pharmacokinetics of cas001, male SD rats (body weight 250-300g, 1 x 1010 /animal, n=3) were given oral administration of LL-37 bacteria powder. The concentration of LL-37 in the blood before and after gavage was detected by ELISA kit (Hycult biotechnology Cat# HK321). Human clinical study was approved by Ethics committee of Chinese PLA General Hospital (S2020-074-04) and a total of 11 patients with mild symptoms were enrolled in Wuhan hankou hospital and Huoshenshan hospital. They were enrolled voluntarily and all patients signed informed consent. Among them, there were 5 males and 6 females, aged 55 {+/-} 12 (36-70) years old, and the duration from onset to medication enrollment was 35 {+/-} 19 (5-68) days. 6 patients were nucleic acid positive and 5 patients were nucleic acid negative when they were enrolled. All patients received the oral drug cas001 treatment according to requirement(1 x 109 CFU/capsule, 3 capsules/time, three times a day for 3weeks), with an average follow-up time of 33 {+/-} 15 days (see table 1 for the results). Findings: Western blot analysis shows that reasonable amount of LL-37 were induced by different concentrations of nisin, which means we have successfully constructed cas001. In the pre-clinical safety evaluation test, after three weeks administration of cas001, no adverse effects were observed on the rat's body weight, food and water intake, hematological or serum biochemical parameters. The results showed that the LD50 of cas001 was higher than that of the 100 times of the expected clinical dose of 6 x 1010 CFU/day. These results showed that cas001 could be safe in animal experiments. In addition, rat pharmacokinetics results showed that the serum concentration of LL-37 reached peak 2 hours after gavage of cas001 and returned to basal level 6 hours after gavage. During study period, the volunteers did not feel any discomfort while taking the cas001 capsules, and two hours after oral administration, the concentration of LL-37 were increased in healthy volunteers. cas001 shows definite effect in the improvement of gastrointestinal symptoms and is possible to have effects in improving the systemic symptoms and respiratory symptoms and may play a role in the improvement of results of nucleic acid test and lung CT test. 11 patients enrolled showed good compliance, tolerance, subjective feeling and actively interacted with the doctors. None of the patients had any adverse reactions. Conclusions Based on above observations, we conclude here that as an oral anti-viral agent, cas001 displayed good safety profiles. It is very hard to reach conclusion of clinical outcomes related to the cas001, although changes of several symptoms indicate encouraging findings.

A Novel Triage Tool of Artificial Intelligence Assisted Diagnosis Aid System for Suspected COVID-19 pneumonia In Fever Clinics (preprint)

Currently, the prevention and control of COVID-19 outside Hubei province in China, and other countries has become more and more critically serious. We developed and validated a diagnosis aid model without CT images for early identification of suspected COVID-19 pneumonia (S-COVID-19-P) on admission in adult fever patients and made the validated model available via an online triage calculator. Patients admitted from Jan 14 to Feb 26, 2020 with the epidemiological history of exposure to COVID-19 were included [Model development (n = 132) and validation (n = 32)]. Candidate features included clinical symptoms, routine laboratory tests and other clinical information on admission. Features selection and model development were based on Lasso regression. The primary outcome is the development and validation of a diagnosis aid model for S-COVID-19-P early identification on admission. The development cohort contains 26 S-COVID-19-P and 7 confirmed COVID-19 pneumonia cases. The model performance in held-out testing set and validation cohort resulted in AUCs of 0.841 and 0.938, F-1 score of 0.571 and 0.667, recall of 1.000 and 1.000, specificity of 0.727 and 0.778, and the precision of 0.400 and 0.500. Based on this model, an optimized strategy for S-COVID-19-P early identification in fever clinics has also been designed. S-COVID-19-P could be identified early by a machine-learning model only used collected clinical information without CT images on admission in fever clinics with 100% recall score. The well performed and validated model has been deployed as an online triage tool, which is available at: https://intensivecare.shinyapps.io/COVID19/.