ABSTRACT
@#ObjectiveTo construct self-amplifying RNA(saRNA)vaccine of severe acute respiratory syndrome coronavirus2(SARS-CoV-2)Delta mutant strain(B.1.617.2)based on Coxsackievirus-A5(CV-A5)replicon and evaluate its immunogenicity.MethodsThe recombinant plasmids pDelta-S10,pDelta-S5 and pDelta-S1(10,5 and 1 amino acid residues at the upstream of S-VP1/2A cleavage site of the fusion polyprotein respectively)were constructed by In-fusion cloning of the plasmids containing the full-length genome sequence of CV-A5 and substituting the S protein gene of SARS-CoV-2 Delta mutant for the P1 structural protein gene of CV-A5 with different lengths.Three RNA molecules,Delta-S10,Delta-S5 and Delta-S1,were obtained by in vitro transcription of linearized recombinant plasmids and transfected into HEK-293T cells respectively,which were analyzed for the expression of S protein by Western blot.The RNA molecule with the highest expression of S protein was screened out and detected for the self-amplification in HEK-293T cells by qPCR.BALB/c mice(female,6 ～ 8 weeks old and five for each group)were immunized i.m.with two doses(0.5 and 2.5 μg)of the screened Delta-S packaged with lipid nanoparticles for once on day 1 and day 14 seperately.Blood samples were collected on days 14and 28,detected for serum binding antibody titers by ELISA,and detected for neutralizing antibody titers by micro neutralization method.The spleens were harvested on day 42 and detected for the level of IFNγ secreted by mouse spleen cells by enzyme linked enzyme linked immunospot assay(ELISPOT).ResultsThe recombinant RNA molecule Delta-S10showed the highest expression of S protein and self-amplified in HEK-293T cells,which of both high and low doses induced specific binding antibody against SARS-CoV-2 Delta S1 protein in mice with obvious dose effect and enhanced immune effect;The high dose of Delta-S10 induced neutralizing antibodies and cellular immune responses in mice.ConclusionThe SARS-CoV-2 Delta mutant(B.1.617.2)saRNA vaccine Delta-S10 based on CV-A5 replicon was successfully constructed,which induced humoral and cellular immune responses in mice,laying a foundation of the further study of the construction of SARS-CoV-2 saRNA vaccine by enterovirus replication elements.
ABSTRACT
@#Objective To optimize the culture conditions of four vaccine candidates of severe acute respiratory symptom coronavirus 2(SARS-CoV-2) Omicron variants BA.1,BA.1.1,BA.2 and BA.5 in Vero cells.Methods The harvest time(24,48,72 and 96 h) and MOI(0.01,0.001,0.0001 and 0.000 01) of four Omicron variants cultured in Vero cells were optimized by using cytopathic effect(CPE),viral nucleic acid copy number and viral titer as evaluation indexes.Results The optimum harvest time of the four Omicron variants BA.1,BA.1.1,BA.2 and BA.5 in Vero cells was 72 h,and the optimum MOI was 0.001～0.000 01,0.001～0.000 01,0.01～0.000 01 and 0.01～0.000 01,respectively.Conclusion The culture conditions of four Omicron variants in Vero cells were optimized,which laid a foundation of the development of SARS-CoV-2 Omicron variant inactivated vaccine based on Vero cells.
ABSTRACT
@#Objective To compare the sensitivity(dilution) of antigen-detecting rapid diagnostic cards for severe acute respiratory symptom coronavirus 2(SARS-CoV-2) at home and abroad to different strains.Methods Vaccine bulks of four SARS-CoV-2 strains(original strain,Beta,Delta and Omicron) produced by Wuhan Institute of Biological Products Co.,Ltd.were used as the sample panel for sensitivity assessment,of which a series of diluted samples were detected by using 21 batches of SARS-CoV-2 antigen-detecting rapid diagnostic cards from 17 domestic and foreign manufacturers and SARS-CoV-2 nucleic acid detection reagent from Shanghai GeneoDx Biotech Co.,Ltd,respectively.The sensitivity of antigendetecting rapid diagnostic cards and nucleic acid detection reagent was evaluated according to the dilutions.The results of SARS-CoV-2 antigen-detecting rapid diagnostic reagents and nucleic acid detection reagent were compared to determine the nucleic acid detection Ct value corresponding to the group of antigen-detecting rapid diagnostic reagent with the highest dilution,namely the highest sensitivity.Results The sensitivity of antigen detection cards for the vaccine bulks of original strain,Beta,Delta and Omicron was 1:10～1:8 × 10~4.1:10~3～1:2 × 10~5,1:10~2～1:4 × 10~4,and 1:10～1:4 × 10~5,respectively;The sensitivity of nucleic acid detection cards was 10~(-6),10~(-5),10~(-4) and 10~(-7),respectively.The Ct values of N gene which were reached by high sensitivity antigen-detecting rapid diagnostic cards were as follows:original strain(10~(-4)) of more than 31,Beta variant(10~(-5)) of more than 36,Delta variant(10~(-4)) of more than 34,Omicron variant(10~(-5)) of more than 33,meeting the requirements of domestic and European Union for SARS-CoV-2 antigen-detecting rapid diagnostic cards.Conclusion All the antigen-detecting rapid diagnostic cards detected the four virus strains,while the sensitivity of different reagents to different variants varies to some extent,among which the sensitivity to Omicron variant varies the most.
ABSTRACT
@#Objective To analyze the correlation of different methods for the detection of antibody titer after immunization with severe acute respiratory symptom corona virus 2(SARS-CoV-2) vaccine,and provide a methodological basis for the specific antibody detection.Methods The seroconversion rate of IgG antibody and neutralizing antibody titer of SARS-CoV-2 inactivated vaccine clinical trial serum samples were detected by micro-cell neutralization assay and three ELISA methods(using S protein,N protein and inactivated whole virus particles as antigens respectively),and the correlation among the methods was analyzed.Results The seroconversion rates detected by neutralizing antibody,S antibody,N antibody and whole virus antibody were 84.3%,91.3%,65.2% and 46.6%,and the geometric mean titers were 16.6,945.9,72.7 and 18.8,respectively.The correlation coefficients(r) between the results of three ELISA methods(using S protein,N protein and inactivated whole virus particles as antigens) and micro-cell neutralization assay were 0.494,0.371 and 0.181respectively.Conclusion Detection of SARS-CoV-2 S antibody level reflected the activation of the humoral immune response characterized by elevated antibody level to a great extent.
ABSTRACT
@#ObjectiveTo construct and identify a recombinant adenovirus expressing S protein receptor binding domain(RBD)and N protein of severe acute respiratory symptom coronavirus 2(SARS-CoV-2)Delta variant.MethodsThe RBD and N gene fragments of SARS-CoV-2 were cloned into pcDNA3.0BA vector respectively to construct recombinant plasmid pcDNA3.0BA-RBD-N. The RBD-CMV-N fragment was amplified by PCR and inserted into shuttle vector pShuttle-CMV. The shuttle plasmid pShuttle-RBD-N was then homologously recombined with pAdeasy-1 to obtain recombinant plasmid pAdeasy-1-RBD-N,which was transfected into HEK293 cells for recombinant adenovirus Ad-RBD-N packaging. The transcription of RBD and N genes of recombinant adenovirus in HEK293 cells was detected by RT-PCR,while the expre-ssion of RBD and N proteins by Western blot and immunofluorescence assay. 12 female BALB/c mice were immunized with Ad-RBD-N by intramuscular injection at a dose of 5 × 109copies per mouse. Blood samples were collected 14 d after immunization,and the serum antibody titers were measured by ELISA.ResultsThe RBD and N genes of recombinant adenovirus were transcribed normally in HEK293 cells,and the RBD and N proteins were expressed normally in MA104 cells. Mice immunized with the recombinant adenovirus produced specific IgG antibodies against RBD and N proteins.ConclusionThe recombinant adenovirus expressing S protein RBD and N protein of SARS-CoV-2 Delta variant was succe-ssfully constructed,which laid a foundation of the follow-up research on Delta variant vaccines.
ABSTRACT
@#Objective To prepare the second generation internal control reference(B2)for Ig G antibody against severe acute respiratory symptom coronavirus 2(SARS-CoV-2)and evaluate its applicability in ELISA detection method. Methods Among the volunteers vaccinated with SARS-CoV-2 inactivated vaccine(BBIBP-Cor V)produced by Beijing Institute of Biological Products Co.,Ltd.,19 Ig G antibody positive plasma samples with ELISA-Ig G dilution ratio of 20 ～ 60 were screened,and the Ig G antibody,IgM antibody and neutralizing antibody were detected by ELISA,B2 was prepared from nonlipid plasma with ELISA-Ig G dilution ratio of 32 ～ 45,IgM negative and similar neutralizing antibody inhibition rate. The neutralizing antibody potency of the first generation internal control reference(B1)and B2 detected by ELISA was calibrated with the first generation WHO international standard of anti-SARS-CoV-2 immunoglobulin(NIBSC 20/136),and the accelerated stability(storage at 2 ～ 8 ℃ for 5,8,14,20,and 30 d respectively),the service stability(storage at 18 ～25 ℃ for 1,2,and 3 h respectively),the freeze-thaw stability(1,2 and 3 times)and the long-term stability(storage at-25 ℃ for10 months)of B2 were tested. B2 was used as standard to detect plasma after single vaccine immunization and mixed plasma was prepared according to different ELISA-Ig G dilution ratio. The correlation and linear regression analysis between ELISA-Ig G dilution ratio and neutralizing antibody potency of pseudovirus in mixed plasma were carried out. Results Among 19 plasma samples,5 samples were non-lipid plasma with ELISA-Ig G dilution ratio of 32 ～ 45,IgM negative and similar neutralizing antibody inhibition rate. B2 was prepared by mixing every plasma in equal volume fraction,and the dilution ratio of ELISA-Ig G was assigned to 32. The neutralizing antibody potency of B1 calibrated with NIBSC 20/136 was 133. 38 EIU/m L and that of B2 was 122. 14 EIU/m L. The recovery rates of accelerated stability,service stability,freeze-thaw stability and long-term stability of B2 were all in the range of(100 ± 15)%. The ELISA-Ig G dilution ratio of the mixed plasma from the same source was significantly correlated with the neutralizing antibody potency of pseudovirus.(each R~2> 0. 99,each P < 0. 000 1).Conclusion B2 prepared from plasma immunized with SARS-CoV-2 inactivated vaccine can replace B1 prepared from plasma of COVID-19 convalescent patients.
ABSTRACT
@#Objective To explore the distribution pattern of respiratory symptoms and relevant factors in patients with pulmonary nodules. Methods Demographic and clinical information were collected from patients who visited the Thoracic Surgery Outpatient Clinic of Guangdong Provincial People’s Hospital from January 2021 to January 2022. Hospital Anxiety and Depression Scale (HADS) was used to assess their anxiety and depression level. Results A total of 1 173 patients were enrolled, including 449 males and 724 females, with an average age of 46.94±11.43 years. Among the patients with pulmonary nodules, 37.7% of them had at least one respiratory symptom; 24.4% had cough, 14.0% had expectoration, 1.3% had hemoptysis and 14.9% had chest pain. Old age, male, exposure to second-hand smoking or environmental smoke, hair coloring and history of tuberculosis were major risk factors for respiratory symptoms (P<0.05). Middle age, old age, male, exposure to environmental smoke were major risk factors for cough (P<0.05); old age, smoking, larger maximum nodules diameters, exposure to environmental smoke and history of pneumonia were major risk factors for expectoration (P<0.05); male, multiple nodules, hair coloring, exposure to second-hand smoking and history of tuberculosis were major risk factors for chest pain (P<0.05). Symptomatic patients showed generally higher HADS scores than asymptomatic patients (P<0.001). Conclusion Cough, expectoration and chest pain are the predominant respiratory symptoms for patients with pulmonary nodules. The presentation of respiratory symptoms increases patients' anxiety and depression.
ABSTRACT
Children less than 6 years old are the most vulnerable group to get harm from Environmental Tobacco Smoke (ETS) exposure, especially their respiratory system. To determine the prevalence of ETS exposure and its association with asthma like symptoms among children less than 6 years old. A cross sectional study was conducted in between January till April 2014 among parents with children less than 6 years old in Selangor, Malaysia. A total of 142 parents were selected by simple random sampling and data were collected using guided questionnaire on ETS exposure and asthma like symptoms (cough and wheeze) among their children. A total of 52.8% of children found to be exposed to ETS, and 43.7% were exposed from their father. ETS exposure significantly associated with cough without cold (adjusted OR=5.46, 95% CI 2.37, 12.61), dry cough at night (adjusted OR=3.74, 95%CI 1.41, 9.95) and cough upon physical activity (adjusted OR=3.08, 95% 1.07, 8.89) among these children. Children less than 6 years old are vulnerable to respiratory problem due to ETS exposure especially on cough symptoms. More strategies should be developing to reduce the exposure to ETS.
ABSTRACT
We evaluated the association between respiratory symptoms and hydration volume during last 1 week of life in terminal cancer patients using retrospective study. The subjects were 138 terminally patients with malignancies. Patients were classified into two groups: the low hydration group (group L, n=85) who received 1,000 ml or less of artificial hydration per day in 1 week before death and high hydration group (group H, n=53) who received over 1,000 ml per day. We compared appearance of dyspnea and bronchial secretion on group L with group H. 64.1% of group H had dyspnea, and 52.8% had bronchial secretion. These fractions are significantly higher than group L (32.9%, 15.3%). In the results of multiple regression analysis, lung involvement (odds ratio: 3.55), hydration over 1,000 ml per day (3.54), and administration of opioid (0.40) were significantly related dyspnea. Lung involvement (7.29), hydration over 1,000 ml per day (4.43), and oral intake (0.31) were significantly related bronchial secretion. Our results provide preliminary evidence that excessive artificial hydration therapies influence the respiratory symptoms in terminal cancer patients. 1,000 ml of hydration may be used as a rough indication in terminal stage.
ABSTRACT
PURPOSE: The aim of this study was to evaluate the significance of pH monitoring at two levels, hypopharynx and esophagus in gastroesophageal reflux. METHODS: 29 patients with pathological gastroesophageal reflux were classified into two groups: Group I had recurrent respiratory symptoms and Group II had not recurrent respiratory symptoms. The ambulatory pH monitoring was performed at the hypopharynx and the esophagus simultaneously with two channel catheter for 18~24 hr. The pathological reflux was defined when the percent of time that pH was below 4.0 exceeding the 95th percentile of normal value. Hypopharyngeal reflux was defined as the pharyngeal pH drops below 4. RESULTS: 39 patients were performed pH monitoring at the level of hypopharynx and esophagus for 24 hours. Among 7 patients with chronic respiratory symptom, 6 patients (85.7%) have pathological esophageal reflux. Among 32 patients without chronic respiratory symptom, 23 patients (71.8%) have pathological esophageal reflux. Thus 29 out of 39 patients (74%) have pathological esophageal reflux. In the Group I, all parameters except the longest episode showed significant differences between hypopharyngeal and esophageal pH monitoring. None of parameters showed significant differences between group I and II in the pharyngeal pH monitoring. CONCLUSION: Regardless of presence of respiratory symptoms, the pH monitoring at the pharyngeal level in patients with gastroesophageal reflux did not showed any differences compared with the esophageal pH monitoring. Therefore we may reconsider the usefulness of hypopharyngeal pH monitoring in patients with chronic respiratory symptoms.
Subject(s)
Humans , Catheters , Esophageal pH Monitoring , Esophagus , Gastroesophageal Reflux , Hydrogen-Ion Concentration , Hypopharynx , Reference ValuesABSTRACT
BACKGROUND: It is known that air borne particulates are closely related with the respiratory symptoms and peak expiratory flow (PEF) in patients with respiratory diseases. In Korea, concentrations of airborne dust and inhalable particulates are dramatically increased by the Yellow sand phenomenon, especially in springtime. OBJECTIVE: This study was performed to evaluate the effect of Yellow sand-related airborne dust on the respiratory symptoms and pulmonary function of patients with bronchial asthma. METHOD: 21 non-smoking, tree pollen non-sensitized subjects with bronchial asthma in Seoul were included in this study. For each subject, asthma and rhinoconjunctivitis symptoms were recorded by self-administered standardized questionnaire and by monitoring PEF between April and May 2000. Symptom score and diurnal variability of PEF were compared with the levels of inhalable particulate matter (PM10) checked in that time at 25 points in Seoul. RESULTS: 15 (71.4%) of the enrolled subjects successfully finished this study. There was no statistically significant relationship between the increment of Yellow sand-related PM10 and respiratory symptom or PEF variability. Of the 15 subjects, there were increased symptoms for the eye in 2 (13.3%) subjects, nose in 3 (20.0%), and sinus in 1 (6.7%), which were associated with Yellow sand-related increase of airborne dust levels. 5 (33.3%) subjects showed changes of PEF variability proportional to the change of PM10. CONCLUSION: Meteorological phenomenon related with Yellow sand has a potential risk of aggravating the upper respiratory tract symptoms and of deteriorating pulmonary functions in patients with asthma.
Subject(s)
Humans , Asthma , Dust , Korea , Nose , Particulate Matter , Pollen , Respiratory System , Seoul , Silicon Dioxide , Surveys and QuestionnairesABSTRACT
Objective To observe the type and prevalence of esophageal motility abnormality in patients with respiratory symptom.And to investigate the effect and clinical significance of ineffective esophageal motility(IEM)in the pathogenesis of GERD with respiratory symptom.Methods In the thirty-four GERD patients with respiratory symptoms of asthma,chronic cough,complaint of laryngeal pharynges,the esophageal manometry and 24 hour esophageal pH monitoring were performed by Multiple Functional Gastrointestinal Motility Monitor made by MMS,Holland.The peristaltic velocity,amplitude and duration of esophageal body and lower esophageal sphincter(LES)pressure was detected.Computer software was used to analyze pH monitoring result,including the percentage of pH0.05)between GERD patients with respiratory symptom group and normal control group.In the group of GERD patients with respiratory symptom,main abnormal esophageal motility type was IEM,the prevalance of IEM(41.2%)being significantly higher than that in group of typical reflux symptom GERD(18.5%)and normal control group(0).In 34 GERD patients with respiratory symptom,24 hour esophageal pH monitoring result of IEM group and normal esophageal motility group was analyzed:the total percentage of pH
ABSTRACT
Disease risk among elderly smokers is considered to be doubled due to their smoking habits and age as compared with elderly non-smokers. The investigators conducted risk assessments of smoking for respiratory symptoms among elderly people.A questionnaire survey on smoking habits and respiratory symptoms was conducted among 3, 000 persons of 56 years of age and over who were randomly selected from suburban residents in a prefecture in Japan in October, 1997. A total 1, 954 or 65.1% of individuals responded, consisting of 42.8% for men and 57.2% for women, with an average age of 73.6 years.In addition to descriptive analysis, multiple logistic regression analysis was conducted. The results are summarized as follows:Smokers accounted for 28.1% of men and 3.6% of women. Among all age-groups, the highest rate of smokers was observed in men of 56-69 years old (34.7%) which was lower than the national average rate for the 60-69 year-old group (56.1% of men and 14.5% of women in ‘97). The odds ratios and 95 percent confidence interval (95%CI) for “having phlegm every day” and “having phlegm for more than 4 days a week” among smokers were 2.06 (95%CI=1.41-3.01) and 2.77(95%CI=1.80-4.27). Significantly higher odds ratios among smokers were also observed for “wheezing” and “shortness of breath when hurrying”.Odds ratios for some respiratory symptoms including “having phlegm for more than 4 days per week” among inhalers were significantly high compared with non-smokers, whereas those among non-inhalers were not significantly different from 1.0.Odds ratios for symptoms of phlegm and wheezing were significantly higher (Odds ratio ≥2.0) among heavy smokers (Brinkman Index [B. I.] >900) compared to non-smokers, while odds ratios of the same symptoms were not different from 1.0 among light smokers (B.I. ≤500).
Subject(s)
Smoking , AgedABSTRACT
The prevalence of respiratory symptoms and impairments of ventilatory capacity were studied in a group of 74 taxi drivers in Inchon area. As a reference group, 94 office workers of same area, matched with cigarette smoking, were also included in this study. Taxi drivers complained of respiratory symptoms much more than reference group. Those were cough, sputum production, chest tightness and shortness of breath. The ventilatory capacities, FVC (forced vital capacity), FEV(1.0) (forced expiratory volume 1 second), MMF (maximal mid flow) and PFR (peak flow rate), were measured. All measured values of ventilatory capacities of taxi driver were significantly decreased than those of reference group. The rate (56.8%) of ventilatory capacity impairment in taxi drivers was significantly larger than those (14.9%) of reference group, and the most of them were restrictive type impairments. Also, Job variable, prefessional driver or not, was selected as important determinant for ventilatory capacity impairment.
Subject(s)
Cough , Dyspnea , Prevalence , Smoking , Sputum , ThoraxABSTRACT
Immotile cilia syndrome is a genetically determined disorder characterized by immotility or poor motility of the cilia in the airways and elsewhere. Since its first description in 1976 determination of a ciliary abnormality has now clarified its variable expression and pleiotropism. Certain specific defects in the ciliary axoneme can be found and are pathognmoic of the syndrome. These defects include missing dynein arms, abnormally short dynein, arms, spokes with no central sheath, missing central microtubules, and displacement of one of the nine peripheral doublets. We have studied 80 cases of bronchial or nasal mucosal biopsies that were performed with the suspicion of immotile cilia syndrome. Of 80 cases only 17 cases were sampled optimally to be able to observe under transmission and scanning electron microscopes. All 17 cases had certain abnormality of the cilia. They consisted of Ia(3 cases), Ib(3 cases), Id only(3 cases) and Id+other types(6 cases) a according to Sturgess classification. Seven cases consisted of 1 solitary and 6 combined form; II+Id(1 case) and II+Id+III(5 case). All 5 cases of type III were combined with Id and II. Clinically most pronounced manifestations were cough(82%), sputum(59%), rhinorrhea(41%) and nasal stuffiness(35%), All the patients were below the age of 15 years, and there were 6 boys and 11 girls.