COVID-ONE-humoral immune: The One-stop Database for COVID-19-specific Antibody Responses and Clinical Parameters

Coronavirus disease 2019 (COVID-19), which is caused by SARS-CoV-2, varies with regard to symptoms and mortality rates among populations. Humoral immunity plays critical roles in SARS-CoV-2 infection and recovery from COVID-19. However, differences in immune responses and clinical features among COVID-19 patients remain largely unknown. Here, we report a database for COVID-19-specific IgG/IgM immune responses and clinical parameters (COVID-ONE humoral immune). COVID-ONE humoral immunity is based on a dataset that contains the IgG/IgM responses to 21 of 28 known SARS-CoV-2 proteins and 197 spike protein peptides against 2,360 COVID-19 samples collected from 783 patients. In addition, 96 clinical parameters for the 2,360 samples and information for the 783 patients are integrated into the database. Furthermore, COVID-ONE humoral immune provides a dashboard for defining samples and a one-click analysis pipeline for a single group or paired groups. A set of samples of interest is easily defined by adjusting the scale bars of a variety of parameters. After the "START" button is clicked, one can readily obtain a comprehensive analysis report for further interpretation. COVID-ONE-humoral immune is freely available at www.COVID-ONE.cn.

COVID-ONE-humoral immune: The One-stop Database for COVID-19-specific Antibody Responses and Clinical Parameters

Coronavirus disease 2019 (COVID-19), which is caused by SARS-CoV-2, varies with regard to symptoms and mortality rates among populations. Humoral immunity plays critical roles in SARS-CoV-2 infection and recovery from COVID-19. However, differences in immune responses and clinical features among COVID-19 patients remain largely unknown. Here, we report a database for COVID-19-specific IgG/IgM immune responses and clinical parameters (COVID-ONE humoral immune). COVID-ONE humoral immunity is based on a dataset that contains the IgG/IgM responses to 21 of 28 known SARS-CoV-2 proteins and 197 spike protein peptides against 2,360 COVID-19 samples collected from 783 patients. In addition, 96 clinical parameters for the 2,360 samples and information for the 783 patients are integrated into the database. Furthermore, COVID-ONE humoral immune provides a dashboard for defining samples and a one-click analysis pipeline for a single group or paired groups. A set of samples of interest is easily defined by adjusting the scale bars of a variety of parameters. After the "START" button is clicked, one can readily obtain a comprehensive analysis report for further interpretation. COVID-ONE-humoral immune is freely available at www.COVID-ONE.cn.

Clinical characteristics of 95 COVID-19 patients with bacterial and fungal infections / 中华微生物学和免疫学杂志

Objective:To retrospectively analyze the clinical characteristics and drug resistance among COVID-19 patients with bacterial and fungal infections.Methods:Clinical data of COVID-19 patients whose blood, urine, sputum and alveolar lavage fluid samples were positive for bacteria and fungi were collected in Tongji Hospital from February 10 to March 31, 2020. WHONET5.6 software was used to analyze drug susceptibility test results.Results:A total of 95 COVID-19 patients positive for pathogenic bacteria were enrolled and among them, 23 were non-critical patients and 72 were critical patients. The main symptoms in these patients included fever, cough with sputum, fatigue and dyspnea. Male and female critical patients accounted for 63.89% and 36.11%, respectively. Most of the patients with bacterial and fungal infections were critical type, accounting for 23.61%. The mortality rates of non-critical and critical patients were 13.04% and 61.11%, respectively. A total of 179 strains of pathogenic bacteria were isolated. The positive rate of Escherichia coli in non-critical patients was 37.50%, which was higher than that in critical patients. However, the positive rates of Acinetobacter baumannii and Klebsiella pneumoniae in critical patients were both 29.87%, higher than those in non-critical patients. There was no significant difference in the positive rate of gram-positive bacteria or fungi between non-critical and critical patients. It was noteworthy that the positive rate of Candida parapsilosis in blood samples of critical patients was relatively high, reaching 36.40%. Drug susceptibility test results showed that no carbapenem-resistant Escherichia coli stains were detected and 60.87% of Klebsiella pneumoniae strains were resistant to carbapenems. Acinetobacter baumannii strains were 100% resistant to three antimicrobial drugs. Methicillin-resistant Staphylococcus aureus strains accounted for 71.43%, but no vancomycin-resistant gram-positive cocci were found. Conclusions:Critical COVID-19 patients were mostly male and prone to multiple bacterial and fungal infections. The mortality of critical patients was higher than that of non-critical patients. Critical COVID-19 was often complicated by hospital acquired infections caused by bacteria including Acinetobacter baumannii and Klebsiella pneumoniae with high drug resistance.

Antibody landscape against SARS-CoV-2 proteome revealed significant differences between non-structural/ accessory proteins and structural proteins

The immunogenicity of SARS-CoV-2 proteome is largely unknown, especially for non-structural proteins and accessory proteins. Here we collected 2,360 COVID-19 sera and 601 control sera. We analyzed these sera on a protein microarray with 20 proteins of SARS-CoV-2, built an antibody response landscape for IgG and IgM. We found that non-structural proteins and accessory proteins NSP1, NSP7, NSP8, RdRp, ORF3b and ORF9b elicit prevalent IgG responses. The IgG patterns and dynamic of non-structural/ accessory proteins are different from that of S and N protein. The IgG responses against these 6 proteins are associated with disease severity and clinical outcome and declined sharply about 20 days after symptom onset. In non-survivors, sharp decrease of IgG antibodies against S1 and N protein before death was observed. The global antibody responses to non-structural/ accessory proteins revealed here may facilitate deeper understanding of SARS-CoV-2 immunology. HighlightsO_LIAn antibody response landscape against SARS-CoV-2 proteome was constructed C_LIO_LINon-structural/accessory proteins elicit prevalent antibody responses but likely through a different mechanism to that of structural proteins C_LIO_LIIgG antibodies against non-structural/accessory proteins are more associated with disease severity and clinical outcome C_LIO_LIFor non-survivors, the levels of IgG antibodies against S1 and N decline significantly before death C_LI

SARS-CoV-2 antibody signatures for predicting the outcome of COVID-19

The COVID-19 global pandemic is far from ending. There is an urgent need to identify applicable biomarkers for early predicting the outcome of COVID-19. Growing evidences have revealed that SARS-CoV-2 specific antibodies evolved with disease progression and severity in COIVD-19 patients. We assumed that antibodies may serve as biomarkers for predicting disease outcome. By taking advantage of a newly developed SARS-CoV-2 proteome microarray, we surveyed IgG responses against 20 proteins of SARS-CoV-2 in 1,034 hospitalized COVID-19 patients on admission and followed till 66 days. The microarray results were further correlated with clinical information, laboratory test results and patient outcomes. Cox proportional hazards model was used to explore the association between SARS-CoV-2 specific antibodies and COVID-19 mortality. We found that nonsurvivors induced higher levels of IgG responses against most of non-structural proteins than survivors on admission. In particular, the magnitude of IgG antibodies against 8 non-structural proteins (NSP1, NSP4, NSP7, NSP8, NSP9, NSP10, RdRp, and NSP14) and 2 accessory proteins (ORF3b and ORF9b) possessed significant predictive power for patient death, even after further adjustments for demographics, comorbidities, and common laboratory biomarkers for disease severity (all with p trend < 0.05). Additionally, IgG responses to all of these 10 non-structural/accessory proteins were also associated with the severity of disease, and differential kinetics and serum positive rate of these IgG responses were confirmed in COVID-19 patients of varying severities within 20 days after symptoms onset. The AUCs for these IgG responses, determined by computational cross-validations, were between 0.62 and 0.71. Our findings have important implications for improving clinical management, and especially for developing medical interventions and vaccines.

Systematic evaluation of SARS-CoV-2 spike protein derived peptides for diagnosis of COVID-19 patients

Serological test plays an essential role in monitoring and combating COVID-19 pandemic. Recombinant spike protein (S protein), especially S1 protein is one of the major reagents for serological tests. However, the high cost in production of S protein, and the possible cross-reactivity with other human coronaviruses poses unneglectable challenges. Taking advantage of a peptide microarray of full spike protein coverage, we analyzed 2,434 sera from 858 COVID-19 patients, sera from 63 asymptomatic patients and 610 controls collected from multiple clinical centers. Based on the results of the peptide microarray, we identified several S protein derived 12-mer peptides that have high diagnosis performance. Particularly, for monitoring IgG response, one peptide (aa 1148-1159 or S2-78) has a comparable sensitivity (95.5%, 95% CI 93.7-96.9%) and specificity (96.7%, 95% CI 94.8-98.0%) to that of S1 protein for detection of both COVID-19 patients and asymptomatic infections. Furthermore, the performance of S2-78 IgG for diagnosis was successfully validated by ELISA with an independent sample cohort. By combining S2-78/ S1 with other peptides, a two-step strategy was proposed to ensure both the sensitivity and specificity of S protein based serological assay. The peptide/s identified in this study could be applied independently or in combination with S1 protein for accurate, affordable, and accessible COVID-19 diagnosis. One Sentence SummaryEight S protein-derived peptides, particularly S2-78 (aa 1148-1159), are of high performance for diagnosis of COVID-19 as well as discrimination of other coronaviruses.

The Trans-omics Landscape of COVID-19

System-wide molecular characteristics of COVID-19, especially in those patients without comorbidities, have not been fully investigated. We compared extensive molecular profiles of blood samples from 231 COVID-19 patients, ranging from asymptomatic to critically ill, importantly excluding those with any comorbidities. Amongst the major findings, asymptomatic patients were characterized by highly activated anti-virus interferon, T/natural killer (NK) cell activation, and transcriptional upregulation of inflammatory cytokine mRNAs. However, given very abundant RNA binding proteins (RBPs), these cytokine mRNAs could be effectively destabilized hence preserving normal cytokine levels. In contrast, in critically ill patients, cytokine storm due to RBPs inhibition and tryptophan metabolites accumulation contributed to T/NK cell dysfunction. A machine-learning model was constructed which accurately stratified the COVID-19 severities based on their multi-omics features. Overall, our analysis provides insights into COVID-19 pathogenesis and identifies targets for intervening in treatment.

Antibody dynamics to SARS-CoV-2 in Asymptomatic and Mild COVID-19 patients

ImportanceAsymptomatic COVID-19 infections have a long duration of viral shedding and contribute substantially to disease transmission. However, the missing asymptomatic cases have been significantly overlooked because of imperfect sensitivity of nucleic acid testing. We aimed to investigate the humoral immunity in asymptomatics, which will help us develop serological tests and improve early identification, understand the humoral immunity to COVID-19, and provide more rational control strategies for the pandemic. ObjectiveTo better control the pandemic of COVID-19, dynamics of IgM and IgG responses to 23 proteins of SARS-CoV-2 and neutralizing antibody in asymptomatic COVID-19 infections after exposure time were investigated. Design, setting, and participants63 asymptomatic individuals were screened by RT-qPCR and ELISA for IgM and IgG from 11,776 personnel returning to work, and close contacts with the confirmed cases in different communities of Wuhan by investigation of clusters and tracing infectious sources. 63 healthy contacts with both negative results for NAT and antibodies were selected as negative controls. 51 mild patients without any preexisting conditions were also screened as controls from 1056 patients during hospitalization in Tongji Hospital. A total of 177 participants were enrolled in this study and serial serum samples (n=213) were collected. The research was conducted between 17 February 2020 and 28 April 2020. Serum IgM and IgG profiles of 177 participants were further probed using a SARS-CoV-2 proteome microarray. Neutralizing antibody responses in different population were detected by a pseudotyped virus neutralization assay system. The dynamics of IgM and IgG antibodies and neutralizing antibodies were analyzed with exposure time or symptoms onset. ResultsAsymptomatics were classified into four subgroups based on NAT and serological tests. In particular, only 19% had positive NAT results while approximately 81% detected positive IgM/IgG responses. Comparative SARS-CoV-2 proteome microarray further demonstrated that there was a significantly difference of antibody dynamics responding to S1 or N proteins among three populations, although IgM and IgG profiles could not be used to differentiate them. S1 specific IgM responses were elicited in asymptomatic individuals as early to the seventh day after exposure and peaked on days from 17d to 25d, which might be used as an early diagnostic biomarker and give an additional 36.5% seropositivity. Mild patients produced stronger both S1 specific IgM and neutralizing antibody responses than asymptomatic individuals. Most importantly, S1 specific IgM/IgG responses and the titers of neutralizing antibody in asymptomatic individuals gradually vanished in two months. Conclusions and relevanceOur findings might have important implications for the definition of asymptomatic COVID-19 infections, diagnosis, serological survey, public health and immunization strategies.

Molecular epidemiology and antimicrobial resistance of diarrheagic Escherichia coli among children with acute diarrhea / 中华检验医学杂志

Objective To learn the distribution, epidemiology and antimicrobial susceptibility of diarrheagenic Escherichia coli (DEC) isolated from patients with acute diarrhea among children less than 5 years old. Methods Totally 684 stool samples collected from Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between August 1, 2015 and September 30, 2016 were tested by culture and identified the common pathogens. PCR was applied to detect the virulence genes of DEC. Meanwhile, serotyping of enteropathogenic E. coli (EPEC) was performed by slide agglutination tests for all the isolates of EPEC. An antimicrobial sensitivity test was performed using the agar dilution method. Results A total of 149 (21.7％) enteric bacteria pathogens were isolated from 684 specimens. DEC was found in 54 cases, ranked 2nd among the pathogenic bacteria. DEC tended to occur in summer/autumn periods. EPEC was the most frequent DEC genotype, accounted for 50％ (27/54). Among EPEC, atypical EPEC was dominant, accounted for 77.8％ (21/27) and typical EPEC only accounted for 22.2％ (6/27). Followed by enteroaggregative E. coli 20.4％(11/54), enterotoxigenic E. coli 14.8％(8/54), enteroinvasive E. coli and Shiga toxin-producing E. coli 3.7％(2/54), 7.4％(4/54) cases were co-infected with more than one DEC genotypes. About 17/18 of suspicious DEC isolates can get the same genotypes by commercial multiplex PCR kit and single PCR test. Among the 27 EPEC strains, only 11(40.7％) strains can be detected by the slide agglutination serotyping method. More than 50％ (27/54) of DEC isolates were resistant to conventional first-line antibiotics (ampicillin, trimethoprim-sulfamethoxazole) and cefazolin, cefuroxime, cefotaxime, but relatively low resistance to cefoxitin, amikacin, piperacillin/tazobactam, imipenem and meropenem. However, there was still a 9.2％ (5/54) resistance rate to carbapenems. Conclusions DEC strains exhibited a high frequency of resistance to many antibiotics. Empirical antimicrobial therapy of severe DEC infection faces the challenge from the high resistance to ampicillin, trimethoprim-sulfamethoxazole. Even worse, some strains were resistant to relatively efficient drugs imipenem and meropenem. It is necessary to strengthen the epidemiological survey and antimicrobial resistance of DEC.

Pediatric primary nephrotic syndrome patients comprehensive detection of plasma protein Z and coagulation factor Ⅷ activity analysis / 国际检验医学杂志

Objective To investigate the changes of plasma protein Z and coagulation factor Ⅷ activity in children with primary nephrotic syndrome.Methods 94 children with primary nephrotic syndrome were se-lected as the observation group,and 63 healthy children were selected as the control group.The blood samples of peripheral blood were collected from the study group,and plasma protein Z and coagulation factor Ⅷ were measured by enzyme linked immunosorbent assay.Enzyme linked immunosorbent assay was used to measure plasma protein Z and coagulation factor Ⅷ.The changes of plasma protein Z and coagulation factor Ⅷ in the two groups were compared,and the changes of plasma protein Z and coagulation factor Ⅷ in the acute and re-covery phase,and the correlation between plasma protein Z and coagulation factor Ⅷ were observed.Results The observation group of plasma protein Z level is lower than the control group,blood coagulation factor Ⅷlevels higher than the control group,and the difference was statistically significant(P<0.05);plasma protein Z level in acute stage is lower than the recovery period,and coagulation factor Ⅷ level is higher than the recov-ery period,the differences were statistically significant(P<0.05);plasma protein Z and coagulation factor Ⅷwas negatively correlated.Conclusion The plasma protein Z level in children with primary nephrotic syn-drome is significantly reduced,and the activity of coagulation factor Ⅷ is significantly increased.Detection of plasma protein Z and coagulation factor Ⅷ level can predict primary nephrotic syndrome in children.

Analysis on risk factors for severe community acquired pneumonia in the elderly / 中国综合临床

Objective To evaluate the risk clinic factors of severe community-acquired pneumonia in older adults,and to provide examples for its clinic application.Methods Sixty-four case patients who were admitted for a diagnosis of severe community-acquired pneumonia in older adults in Beijing Haidian Hospital from January 2013 to July 2015 were selected as observation group.One hundred cases patients also admitted for a diagnosis of community-acquired pneumonia were selected as control group.Within 24 h of admission,the clinical data were collected,medical history were collected,and the serum biochemistry,coagulation index fibrin degradation product,D-dimer and inflammatory factor procalcitonin were detected.The data were analyzed by univariate analysis,and logistic regression analysis was used to analyze the variables with significant difference in single factor analysis.Results Single factor analysis showed that the percentage of severe pneumonia with central nervous system disorders,severe pneumonia with aspiration,severe pneumonia with two or more comorbidities,aspiration and severe pneumonia with bedridden patient in observation group were obviously higher than the control group,the difference between two groups have remarkable statistical significance((82.21％ (53/64) vs.32％(32/100),60.93％(39/64) vs.1％(1/100),(84.37％,54/64) vs.54％(54/100),90.62％(58/ 64) vs.28％ (28/100),59.37％ (38/64) vs.11％ (11/100);P＜0.01 or P＜0.05).The mortality of observation group was higher than the control group,the difference between two groups have remarkable statistical significance(56.62％ (36/64)] vs.3％ (3/100),P=0.001).Albumin of severe pneumonia was obviously lower than the control group,the difference between two groups have remarkable statistical significance ((27.79 ±8.53) g/L vs.(33.66±9.63) g/L,P=0.011).Fibrin degradation product of severe pneumonia was obviously lower than the control group,the difference between two groups have remarkable statistical significance ((i0.98 ± ± 1.32) ng/L vs.(3.61±0.98) ng/L,P=0.002).D-dimer of severe pneumonia was obviously higher than the control group,the difference between two groups have remarkable statistical significance ((2.68± 0.56) mg/L vs.(0.42±0.12) mg/L,P=0.001).Procalcitonin of severe pneumonia was obviously higher than the control group,the difference between two groups have remarkable statistical significance ((1.63±0.32) ng/L vs.(0.13 ± ±0.21) ng/L,P =0.015).The result of multiple factors logistic regression analysis showed the aspiration,hypoproteinemia,D-dimer were severe community-acquired pneumonia in older adults's independent risks (OR =1.782,1.208,1.356,P＜0.05).Conclusion Aspiration,hypoproteinemia,D-dimer are the factors of risking suffering severe community-acquired pneumonia in older adults.D-dimer could be the detection index of severe community-acquired pneumonia in older adults.

The inhibiting effect of pseudomonas aeruginosa to the growth of pathogenic fungi / 重庆医学

Objective To explore the inhibitory effect of 24 pseudomonas aeruginosa(PA) on pathogenic fungi ,such as candida albicans ,candida tropicalis ,candida glabrata ,candida parapsilosis ,candida krusei ,mucous spore bacterium (MSB) etc .Methods 24 PA isolates were collected from clinical specimens and identified by Gram′s stain ,oxidase production and the API 20NE system(bi-oMerieux ,France) .Cross-streaking method and sterilizing filter paper-disk method and co-cultured method were applied to observe the inhibitory effect of PA .Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) analyzed the difference of bacte-rial proteins of PA .Results The results showed that some strains of 24 PA had strong inhibitory effect against pathogenic fungi , some strain had partial effect and others had no effect .Co-cultured test showed that PA could inhibit the growth of fungal hyphae . SDS-PAGE displayed the significant difference in secretive proteins between the PA strains which had strong effect and no effect . Conclusion PA have inhibitory effect upon common pathogenic fungi and and this might be related to inhibit fungal hyphae forma-tion ,various protein secretion and inhibit the growth of fungi .

Construction and immunogenicity of recombinant porcine parvovirus-like particles with somatostatin / 生物工程学报

In order to obtain a virus-like particle vaccine both for porcine parvovirus (PPV) prevention and growth-promotion, VP2 gene of PPV NJ-a strain was amplified with PCR, and four copies of synthetic somatostatin gene were fused to the N-terminal of VP2 gene. The fused gene was cloned into pFast-HT A to construct the recombinant plasmid pFast-SS4-VP2, then the pFast-SS4-VP2 was transformed into DH10Bac competent cells and recombined with shuttle vector Bacmid, followed by identification with blue-white screening and PCR analysis for three cycles, and the positive recombinant was named as rBacmid-SS4-VP2. The positive Sf-9 cells were transfected with rBacmid-SS4-VP2 by Lipofectamine to produce recombinant baculovirus. When the cytopathic effect (CPE) was obvious, the transfected Sf-9 cell was harvested, and the positive recombinant virus was named as rBac-SS4-VP2. The insertion for the target gene into baculovirus genome was confirmed with PCR. SDS-PAGE and Western blotting revealed that the calculated protein of approximately 68 kDa was in the expressed in the insect cells. The Sf-9 cells infected with rBac-SS4-VP2 were stained positive against PPV antibody using the indirect immunofluorescence assay (IFA). Moreover, the virus particle self-assembly was observed under electron microscopy. 90 four-week-old mice were immunized by the recombinant protein coupled with different adjuvants alhydrogel, IMS and oil. VP2-specific ELISA antibodies, PPV-specific neutralizing antibody, somatostatin antibody and growth hormone levels were examined to evaluate the immunogenicity of this virus like particle. Results indicated that mice groups immunized rSS4-VP2 protein with alhydrogel and IMS developed similar humoral immune response comparing with inactived PPV vaccine. Mice group immunized with rSS4-VP2 generated higher level of SS antibody and growth hormone comparing with negative control, mice receiving rSS4-VP2 with alhydrogel developed the highest antibody titre than all other groups, while the oil group developed the lowest antibody level. This study provides not only a new rout for production of safe and effective virus like particle subunit vaccine, but also the foundations for peptide presentation and multivalent subunit vaccine design.