New perspectives on the induction and acceleration of immune-associated thrombosis by PF4 and VWF

Platelet factor 4 (PF4), also known as chemokine (C-X-C motif) ligand 4 (CXCL4), is a specific protein synthesized from platelet α particles. The combination of PF4 and heparin to form antigenic complexes is an important mechanism in the pathogenesis of heparin-induced thrombocytopenia (HIT), but vaccine-induced immune thrombotic thrombocytopenia (VITT) related to the COVID-19 vaccine makes PF4 a research hotspot again. Similar to HIT, vaccines, bacteria, and other non-heparin exposure, PF4 can interact with negatively charged polyanions to form immune complexes and participate in thrombosis. These anions include cell surface mucopolysaccharides, platelet polyphosphates, DNA from endothelial cells, or von Willebrand factor (VWF). Among them, PF4–VWF, as a new immune complex, may induce and promote the formation of immune-associated thrombosis and is expected to become a new target and therapeutic direction. For both HIT and VITT, there is no effective and targeted treatment except discontinuation of suspected drugs. The research and development of targeted drugs based on the mechanism of action have become an unmet clinical need. Here, this study systematically reviewed the characteristics and pathophysiological mechanisms of PF4 and VWF, elaborated the potential mechanism of action of PF4–VWF complex in immune-associated thrombosis, summarized the current status of new drug research and development for PF4 and VWF, and discussed the possibility of this complex as a potential biomarker for early immune-associated thrombosis events. Moreover, the key points of basic research and clinical evaluation are put forward in the study.

COVID-19 spike polypeptide vaccine reduces the pathogenesis and viral infection in a mouse model of SARS-CoV-2

The SARS-CoV-2 coronavirus, which causes a respiratory disease called COVID-19, has been declared a pandemic by the World Health Organization (WHO) and is still ongoing. Vaccination is the most important strategy to end the pandemic. Several vaccines have been approved, as evidenced by the ongoing global pandemic, but the pandemic is far from over and no fully effective vaccine is yet available. One of the most critical steps in vaccine development is the selection of appropriate antigens and their proper introduction into the immune system. Therefore, in this study, we developed and evaluated two proposed vaccines composed of single and multiple SARS-CoV-2 polypeptides derived from the spike protein, namely, vaccine A and vaccine B, respectively. The polypeptides were validated by the sera of COVID-19-vaccinated individuals and/or naturally infected COVID-19 patients to shortlist the starting pool of antigens followed by in vivo vaccination to hACE2 transgenic mice. The spike multiple polypeptide vaccine (vaccine B) was more potent to reduce the pathogenesis of organs, resulting in higher protection against the SARS-CoV-2 infection.

COVID-19 spike polypeptide vaccine reduces the pathogenesis and viral infection in a mouse model of SARS-CoV-2.

The SARS-CoV-2 coronavirus, which causes a respiratory disease called COVID-19, has been declared a pandemic by the World Health Organization (WHO) and is still ongoing. Vaccination is the most important strategy to end the pandemic. Several vaccines have been approved, as evidenced by the ongoing global pandemic, but the pandemic is far from over and no fully effective vaccine is yet available. One of the most critical steps in vaccine development is the selection of appropriate antigens and their proper introduction into the immune system. Therefore, in this study, we developed and evaluated two proposed vaccines composed of single and multiple SARS-CoV-2 polypeptides derived from the spike protein, namely, vaccine A and vaccine B, respectively. The polypeptides were validated by the sera of COVID-19-vaccinated individuals and/or naturally infected COVID-19 patients to shortlist the starting pool of antigens followed by in vivo vaccination to hACE2 transgenic mice. The spike multiple polypeptide vaccine (vaccine B) was more potent to reduce the pathogenesis of organs, resulting in higher protection against the SARS-CoV-2 infection.

New perspectives on the induction and acceleration of immune-associated thrombosis by PF4 and VWF.

Platelet factor 4 (PF4), also known as chemokine (C-X-C motif) ligand 4 (CXCL4), is a specific protein synthesized from platelet α particles. The combination of PF4 and heparin to form antigenic complexes is an important mechanism in the pathogenesis of heparin-induced thrombocytopenia (HIT), but vaccine-induced immune thrombotic thrombocytopenia (VITT) related to the COVID-19 vaccine makes PF4 a research hotspot again. Similar to HIT, vaccines, bacteria, and other non-heparin exposure, PF4 can interact with negatively charged polyanions to form immune complexes and participate in thrombosis. These anions include cell surface mucopolysaccharides, platelet polyphosphates, DNA from endothelial cells, or von Willebrand factor (VWF). Among them, PF4-VWF, as a new immune complex, may induce and promote the formation of immune-associated thrombosis and is expected to become a new target and therapeutic direction. For both HIT and VITT, there is no effective and targeted treatment except discontinuation of suspected drugs. The research and development of targeted drugs based on the mechanism of action have become an unmet clinical need. Here, this study systematically reviewed the characteristics and pathophysiological mechanisms of PF4 and VWF, elaborated the potential mechanism of action of PF4-VWF complex in immune-associated thrombosis, summarized the current status of new drug research and development for PF4 and VWF, and discussed the possibility of this complex as a potential biomarker for early immune-associated thrombosis events. Moreover, the key points of basic research and clinical evaluation are put forward in the study.

Establishment of multicenter COVID-19 therapeutics preclinical test system in Republic of Korea.

Throughout the recent COVID-19 pandemic, South Korea led national efforts to develop vaccines and therapeutics for SARS-CoV-2. The project proceeded as follows: 1) evaluation system setup (including Animal Biosafety Level 3 (ABSL3) facility alliance, standardized nonclinical evaluation protocol, and laboratory information management system), 2) application (including committee review and selection), and 3) evaluation (including expert judgment and reporting). After receiving 101 applications, the selection committee reviewed pharmacokinetics, toxicity, and efficacy data and selected 32 final candidates. In the nonclinical efficacy test, we used golden Syrian hamsters and human angiotensin-converting enzyme 2 transgenic mice under a cytokeratin 18 promoter to evaluate mortality, clinical signs, body weight, viral titer, neutralizing antibody presence, and histopathology. These data indicated eight new drugs and one repositioned drug having significant efficacy for COVID-19. Three vaccine and four antiviral drugs exerted significant protective activities against SARS-CoV-2 pathogenesis. Additionally, two anti-inflammatory drugs showed therapeutic effects on lung lesions and weight loss through their mechanism of action but did not affect viral replication. Along with systematic verification of COVID-19 animal models through large-scale studies, our findings suggest that ABSL3 multicenter alliance and nonclinical evaluation protocol standardization can promote reliable efficacy testing against COVID-19, thus expediting medical product development.

Immunogenicity and Durability of Antibody Responses to Homologous and Heterologous Vaccinations with BNT162b2 and ChAdOx1 Vaccines for COVID-19.

During the COVID-19 pandemic, vaccines were developed based on various platform technologies and were approved for emergency use. However, the comparative analysis of immunogenicity and durability of vaccine-induced antibody responses depending on vaccine platforms or vaccination regimens has not been thoroughly examined for mRNA- or viral vector-based vaccines. In this study, we assessed spike-binding IgG levels and neutralizing capacity in 66 vaccinated individuals prime-boost immunized either by homologous (BNT162b2-BNT162b2 or ChAdOx1-ChAdOx1) or heterologous (ChAdOx1-BNT162b2) vaccination for six months after the first vaccination. Despite the discrepancy in intervals for the prime-boost vaccination regimen of different COVID-19 vaccines, we found stronger induction and relatively rapid waning of antibody responses by homologous vaccination of the mRNA vaccine, while weaker boost effect and stable maintenance of humoral immune responses were observed in the viral vector vaccine group over 6 months. Heterologous vaccination with ChAdOx1 and BNT162b2 resulted in an effective boost effect with the highest remaining antibody responses at six months post-primary vaccination.

Accounting Transparency, Fear Sentiment and the COVID-19 Epidemic: For Public Health Security and the Construction of an Early Warning System

A central issue of public health security and the construction of an early warning system is to establish a set of responsibility-oriented incentives and restraint mechanisms. This is closely related to the accounting transparency of the institutional environment and the fear sentiment of the individual's predicament. This study analyses the relationship between accounting transparency, fear sentiment, and COVID-19 through a VAR model analysis. The results show a significant and negative relationship between accounting transparency and daily new COVID-19 patients. In particular, accounting transparency has a negative impact on the increase in the number of people infected with a two-period lag, while the three-period lag in the number of new epidemics has a negative impact on accounting information. Second, accounting transparency has a positive impact on the increase in the search volume on COVID-19 within a three-period lag. After the three-period lag, the number of new epidemics has a positive impact on accounting information. Third, an increase in fear sentiment can be driven by the fear of COVID-19. Fourth, in the public health early warning system, according to the abovementioned time characteristics, the system arranges the emotional counseling, early warning incentives, and institutional constraints to be dealt with in the first 4 days. In addition, in the early warning target-oriented system setting, the parallel system helps to improve the early warning efficiency.

Development of transgenic models susceptible and resistant to SARS-CoV-2 infection in FVB background mice.

Coronavirus disease (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is currently spreading globally. To overcome the COVID-19 pandemic, preclinical evaluations of vaccines and therapeutics using K18-hACE2 and CAG-hACE2 transgenic mice are ongoing. However, a comparative study on SARS-CoV-2 infection between K18-hACE2 and CAG-hACE2 mice has not been published. In this study, we compared the susceptibility and resistance to SARS-CoV-2 infection between two strains of transgenic mice, which were generated in FVB background mice. K18-hACE2 mice exhibited severe weight loss with definitive lethality, but CAG-hACE2 mice survived; and differences were observed in the lung, spleen, cerebrum, cerebellum, and small intestine. A higher viral titer was detected in the lungs, cerebrums, and cerebellums of K18-hACE2 mice than in the lungs of CAG-hACE2 mice. Severe pneumonia was observed in histopathological findings in K18-hACE2, and mild pneumonia was observed in CAG-hACE2. Atrophy of the splenic white pulp and reduction of spleen weight was observed, and hyperplasia of goblet cells with villi atrophy of the small intestine was observed in K18-hACE2 mice compared to CAG-hACE2 mice. These results indicate that K18-hACE2 mice are relatively susceptible to SARS-CoV-2 and that CAG-hACE2 mice are resistant to SARS-CoV-2. Based on these lineage-specific sensitivities, we suggest that K18-hACE2 mouse is suitable for highly susceptible model of SARS-CoV-2, and CAG-hACE2 mouse is suitable for mild susceptible model of SARS-CoV-2 infection.

Identification of coronaviruses in farmed wild animals reveals their evolutionary origins in Guangdong, southern China.

Coronavirus infections cause diseases that range from mild to severe in mammals and birds. In this study, we detected coronavirus infections in 748 farmed wild animals of 23 species in Guangdong, southern China, by RT-PCR and metagenomic analysis. We identified four coronaviruses in these wild animals and analysed their evolutionary origins. Coronaviruses detected in Rhizomys sinensis were genetically grouped into canine and rodent coronaviruses, which were likely recombinants of canine and rodent coronaviruses. The coronavirus found in Phasianus colchicus was a recombinant pheasant coronavirus of turkey coronavirus and infectious bronchitis virus. The coronavirus in Paguma larvata had a high nucleotide identity (94.6-98.5 per cent) with a coronavirus of bottlenose dolphin (Tursiops truncates). These findings suggested that the wildlife coronaviruses may have experienced homologous recombination and/or crossed the species barrier, likely resulting in the emergence of new coronaviruses. It is necessary to reduce human-animal interactions by prohibiting the eating and raising of wild animals, which may contribute to preventing the emergence of the next coronavirus pandemic.

Laboratory information management system for COVID-19 non-clinical efficacy trial data.

BACKGROUND: As the number of large-scale studies involving multiple organizations producing data has steadily increased, an integrated system for a common interoperable format is needed. In response to the coronavirus disease 2019 (COVID-19) pandemic, a number of global efforts are underway to develop vaccines and therapeutics. We are therefore observing an explosion in the proliferation of COVID-19 data, and interoperability is highly requested in multiple institutions participating simultaneously in COVID-19 pandemic research. RESULTS: In this study, a laboratory information management system (LIMS) approach has been adopted to systemically manage various COVID-19 non-clinical trial data, including mortality, clinical signs, body weight, body temperature, organ weights, viral titer (viral replication and viral RNA), and multiorgan histopathology, from multiple institutions based on a web interface. The main aim of the implemented system is to integrate, standardize, and organize data collected from laboratories in multiple institutes for COVID-19 non-clinical efficacy testings. Six animal biosafety level 3 institutions proved the feasibility of our system. Substantial benefits were shown by maximizing collaborative high-quality non-clinical research. CONCLUSIONS: This LIMS platform can be used for future outbreaks, leading to accelerated medical product development through the systematic management of extensive data from non-clinical animal studies.

Screening Host Antiviral Proteins under the Enhanced Immune Responses Induced by a Variant Strain of Porcine Epidemic Diarrhea Virus.

While discussing the ideal candidates of viral restriction factor, the interferon (IFN) and interferon-stimulated genes (ISGs) could be considered potential targets. However, numerous viruses have evolved multiple strategies to modulate the host innate immune signaling for optimal infection, including the porcine epidemic diarrhea virus (PEDV), a coronavirus spreading widely around the world with high morbidity and mortality in piglets. The immunosuppression mediated by PEDV infection creates an impediment for studying the host-virus interactions and screening the antiviral ISGs. Here, the PEDV variant strain 85-7C40 was screened using the continuous passaging, which showed significantly attenuated viral replication compared with its parent on MARC-145 cells. The comparative transcriptome analysis (accession nos. SRR13154018 to SRR13154026) indicated that 85-7C40 infection led to enhanced immune response on MARC-145 cells, particularly to the IFN antiviral signaling, which mediated the stronger activation of numerous ISGs. Numerous ISGs activated by 85-7C40 showed antiviral effects against the wild-type strain infection, particularly the IFI44 (an ISG upregulated specifically by the 85-7C40 infection) and OASL (upregulated higher in 85-7C40 than 85-7-infected cells), exhibited powerful antiviral activity. IFI44 promoted the production of RIG-I, while the OASL interacted directly with RIG-I, and then they both activated the phosphorylation of STAT1, indicating that they restricted PEDV replication by positively regulating the type I IFN response. Our results provided insight into the ISGs with antiviral activity against PEDV infection and also expanded our understanding of the innate immune response to PEDV infection, which may promote the development of novel therapeutics. IMPORTANCE Host innate immune responses, particularly interferon (IFN) antiviral signaling, can activate diverse downstream ISGs to exert antiviral effects. However, porcine epidemic diarrhea virus (PEDV) infection has evolved multiple strategies to escape from this immune clearance. The immunosuppression mediated by PEDV infection creates an impediment for studying the host-virus interactions. We screened a PEDV variant strain, 85-7C40, which induced enhanced immune responses on MARC-145 cells and thus mediated the stronger activation of numerous ISGs. The laboratory-generated variant might induce inconsistent immune responses with a natural wild-type strain during infection, while numerous ISGs activated by 85-7C40 showed antiviral effects against the wild-type strain infection, particularly the IFI44 and OASL, restricted PEDV replication by positively regulating the type I IFN response. These findings were suggestive of the immune-enhanced variant being capable of using as an ideal viral model for screening the efficient antiviral proteins and elucidating the underlying mechanisms between PEDV and host innate immune responses.

Immunogenicity after Second ChAdOx1 nCoV-19 (AZD1222) Vaccination According to the Individual Reactogenicity, Health Status and Lifestyle.

The immune-acquired responses after vaccination vary depending on the type of vaccine and the individual. The purpose of this study was to investigate the relationship between the acquisition of immunity and the side effects, health status, and lifestyle after completion of the second dose of AZD1222. Blood samples were collected after a second dose of AZD1222. The Euroimmun Anti-SARS-CoV-2 ELISA (IgG) for anti-S1 antibody, the cPASS SARS-CoV-2 neutralizing antibody detection kit for the surrogate virus neutralization test, and the T-spot Discovery SARS-CoV-2 kit were used to identify cellular immunogenicity. Patient experience of adverse effects was investigated using questionnaires. Information on health status and lifestyle were collected from the most recent health checkup data. Generally, females experience more reactogenicity in both intensity and duration. The rash of the first shot and chills of the second shot were associated with humoral immunity. However, comprehensive adverse effects had no correlation with humoral and cellular immunity. The T-spot-positive group had a higher creatinine level, which reflects muscle mass, than the T-spot-negative group. Males presented a higher level of T-spot assays. Body mass index and age were negatively correlated with the T-spot assay and anti-S1 antibody, respectively. Immune acquisition after the second AZD1222 shot was not associated with reactogenicity. However, individuals' sex, age, and BMI were found to be associated with immunogenicity after vaccination.

Indels in SARS-CoV-2 occur at template-switching hotspots.

The evolutionary dynamics of SARS-CoV-2 have been carefully monitored since the COVID-19 pandemic began in December 2019. However, analysis has focused primarily on single nucleotide polymorphisms and largely ignored the role of insertions and deletions (indels) as well as recombination in SARS-CoV-2 evolution. Using sequences from the GISAID database, we catalogue over 100 insertions and deletions in the SARS-CoV-2 consensus sequences. We hypothesize that these indels are artifacts of recombination events between SARS-CoV-2 replicates whereby RNA-dependent RNA polymerase (RdRp) re-associates with a homologous template at a different loci ("imperfect homologous recombination"). We provide several independent pieces of evidence that suggest this. (1) The indels from the GISAID consensus sequences are clustered at specific regions of the genome. (2) These regions are also enriched for 5' and 3' breakpoints in the transcription regulatory site (TRS) independent transcriptome, presumably sites of RNA-dependent RNA polymerase (RdRp) template-switching. (3) Within raw reads, these indel hotspots have cases of both high intra-host heterogeneity and intra-host homogeneity, suggesting that these indels are both consequences of de novo recombination events within a host and artifacts of previous recombination. We briefly analyze the indels in the context of RNA secondary structure, noting that indels preferentially occur in "arms" and loop structures of the predicted folded RNA, suggesting that secondary structure may be a mechanism for TRS-independent template-switching in SARS-CoV-2 or other coronaviruses. These insights into the relationship between structural variation and recombination in SARS-CoV-2 can improve our reconstructions of the SARS-CoV-2 evolutionary history as well as our understanding of the process of RdRp template-switching in RNA viruses.

Comparison of Serologic Response of Hospitalized COVID-19 Patients Using 8 Immunoassays.

BACKGROUND: In Korea, there were issues regarding the use of immunoassays for anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies to detect infection. So, we compared antibody results of eight kinds of commercial immunoassays using clinical remnant specimens. METHODS: We compared the results of several immunoassay kits tested on 40 serum samples from 15 confirmed patients and 86 remnant serum samples from clinical laboratory. Eight kinds of IVD kits-four enzyme-linked immunosorbent assay, two lateral flow rapid immunochromatographic assays, and two chemiluminescent immunoassays with one RUO kit were tested. RESULTS: Among 40 serum samples from 15 coronavirus disease 2019 (COVID-19) patients, 35 yielded at least one positive result for detecting antibodies in the combined assessment. There were inconsistent results in 12 (28%) samples by single immunoassay. Forty samples collected in 2019 before the first COVID-19 Korean case showed negative results except for one equivocal result. CONCLUSION: The discrepant results obtained with different immunoassay kits in this study show that serological assessment of SARS-CoV-2 by a single immunoassay requires caution not only in detecting infection but also in assessing immunologic status.

New Obligations of Health Insurance Review and Assessment Service: Taking Full-fledged Action Against the COVID-19 Pandemic.

In 2020, the coronavirus disease 2019 (COVID-19) pandemic has caused unprecedented disruptions to global health systems. The Korea has taken full-fledged actions against this novel infectious disease, swiftly implementing a testing-tracing-treatment strategy. New obligations have therefore been given to the Health Insurance Review and Assessment Service (HIRA) to devote the utmost effort towards tackling this global health crisis. Thanks to the universal national health insurance and state-of-the-art information communications technology (ICT) of the Korea, HIRA has conducted far-reaching countermeasures to detect and treat cases early, prevent the spread of COVID-19, respond quickly to surging demand for the healthcare services, and translate evidence into policy. Three main factors have enabled HIRA to undertake pandemic control preemptively and systematically: nationwide data aggregated from all healthcare providers and patients, pre-existing ICT network systems, and real-time data exchanges. HIRA has maximized the use of data and pre-existing network systems to conduct rapid and responsive measures in a centralized way, both of which have been the most critical tactics and strategies used by the Korean healthcare system. In the face of new obligations, our promise is to strive for a more responsive and resilient health system during this prolonged crisis.

Clinical characteristics and fatal outcomes of hypertension in patients with severe COVID-19.

The aim of this study is to investigate clinical characteristics and fatal outcomes of hypertension as well as the role of angiotensin-converting enzyme inhibitors/angiotensin receptor blockers (ACEI/ARB) use in patients with severe coronavirus disease 2019 (COVID-19). A total of 220 (female: 51.8%) patients with severe COVID-19 were included. The mean age of included patients was 59.5 years and 70 (31.8%) patients had a history of hypertension. There were 23 patients (32.9%) receiving ACEI/ARB therapy. Patients with hypertension were older and had more comorbidities, and were more likely to suffer from severe inflammatory response and acute cardiac injury. Moreover, patients with hypertension were associated with significantly higher risk of in-hospital mortality than patients without hypertension. After adjustment of potential confounders, the independent correlation was still observed. In addition, ACEI/ARB users were associated with lower level of high-sensitivity cardiac troponin I and creatinine kinase-myocardial band, and lower risk of acute cardiac injury than ACEI/ARB non-users. In conclusion, patients with hypertension were more likely to suffer from severe inflammatory response, acute cardiac injury and had high risk of in-hospital mortality in severe COVID-19. The use of ACEI/ARB may protect patients with COVID-19 from acute cardiac injury.

Prognostic value of NT-proBNP in patients with severe COVID-19.

BACKGROUND: The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in China has been declared a public health emergency of international concern. The cardiac injury is a common condition among the hospitalized patients with COVID-19. However, whether N terminal pro B type natriuretic peptide (NT-proBNP) predicted outcome of severe COVID-19 patients was unknown. METHODS: The study initially enrolled 102 patients with severe COVID-19 from a continuous sample. After screening out the ineligible cases, 54 patients were analyzed in this study. The primary outcome was in-hospital death defined as the case fatality rate. Research information and following-up data were obtained from their medical records. RESULTS: The best cut-off value of NT-proBNP for predicting in-hospital death was 88.64 pg/mL with the sensitivity for 100% and the specificity for 66.67%. Patients with high NT-proBNP values (> 88.64 pg/mL) had a significantly increased risk of death during the days of following-up compared with those with low values (≤88.64 pg/mL). After adjustment for potential risk factors, NT-proBNP was independently correlated with in-hospital death. CONCLUSION: NT-proBNP might be an independent risk factor for in-hospital death in patients with severe COVID-19. TRIAL REGISTRATION: ClinicalTrials, NCT04292964. Registered 03 March 2020.