Establishment and evaluation of fluorescent RT-RAA assay for SARS-CoV-2 sgRNAs detection

Objective To establish a reverse-transcription recombinase-aided amplification assay（RT-RAA） to rapidly detect SARS-CoV-2 sub-genomic RNAs（sgRNAs）. Methods The primers and probe for isothermal nucleic acid amplification were designed based on the 5′-leader and 7a and N gene sequence of SARS-CoV-2, and the sgRNAs of SARS-COV-2 were rapidly detected within 30 min at 39 ℃.The sensitivity, specificity and consistency of the assay were evaluated. Results The detection limit of the method was 20 copies/μl and there were no cross-reactions with other respiratory pathogens, showing decent sensitivity and specificity.The results of the assay were concordant with that of real-time PCR, indicating a better consistency of two methods（κ=0.762,P<0.001）. Conclusions The fluorescence RT-RAA assay established in the study can be used for the rapid detection of SARS-CoV-2 sgRNAs, which is of great significance for the rapid diagnosis of COVID 19.

A survey on the safety of the SARS-CoV-2 vaccine among a population with stroke risk in China

Background The safety of the COVID-19 vaccine in patients at stroke risk is poorly understood. Methods A survey was conducted on risk factors related to stroke and adverse reactions to vaccines. The participants were divided into low-, medium-, and high-risk groups, according to the stroke risk scorecard recommended by the Stroke Prevention and Control Engineering Committee of the National Health and Family Planning Commission. Factors associated with adverse reactions were analyzed. Reasons for non-vaccination and the aggravation of underlying diseases after vaccination were investigated. Results 1747 participants participated (138 unvaccinated) and 36.8, 22.1, 41.1% of the vaccinated participants had low, medium, high risk of stroke, respectively. The incidence of adverse reactions after the first and second injection was 16.6, 13.7%, respectively. There was no difference in the incidence of adverse reactions among different risk groups. Sex, vaccine type, sleep quality, worry of adverse reactions, age, and education level were significantly related to adverse reactions to vaccination. The most popular reason for non-vaccination for medium- or high risk-participants was the aggravation of the existing disease. Only 0.3% of vaccinated participants reported slight changes in blood pressure, sugar levels, and lipid levels. No aggravation of stroke sequelae, atrial fibrillation, or transient ischemic attack was reported. Conclusions Vaccination against COVID-19 (inactive virus) is safe for people at risk of stroke when the existing disease condition is stable. It is suggested to strengthen vaccine knowledge and ensure good sleep before vaccination.

Antibody Persistence and Safety through 6 Months after Heterologous Orally Aerosolised Ad5-nCoV in individuals primed with two-dose CoronaVac previously (preprint)

Background: Heterologous orally administered adenovirus type-5 vector-based COVID-19 vaccine (Ad5-nCoV) in individuals who were primed with two-dose CoronaVac (an inactivated SARS-CoV-2 vaccine, by Sinovac) previously, has been reported to be safe and highly immunogenic within 28 days post-boosting. However, antibody persistence and safety up to 6 months of this regimen are not been reported yet. Methods: This is a randomized, open label, single-center trial on safety and immunogenicity of heterologous boost immunization with an orally administered aerosolised Ad5-nCoV vs. homologous boost immunization with CoronaVac after two-dose priming with CoronaVac in Chinese adults aged 18 years and older (NCT05043259). We followed the participants in this trial, including 140 in the low-dose aerosolised Ad5-nCoV group, 139 in the high-dose aerosolised Ad5-nCoV group, and 140 in the CoronaVac group for 6 months. Neutralising antibodies (NAbs) against live wild-type SARS-CoV-2 virus and omicron variant, and receptor-binding domain (RBD)-specific IgG antibodies were detected in serum samples collected at 28 days, 3 months, and 6 months after the booster dose. Serious adverse events (SAEs) were documented till month 6. Results: The low-dose and high-dose heterologous boost immunisation groups had NAb GMTs against live wild-type SARS-CoV-2 of 1937.3 [95% CI 1466.9, 2558.4] and 1350.8 [95% CI 952.6, 1915.3], which were 26.4 folds and 18.4 folds higher than that the CoronaVac group did (73.5 [95%CI 52.3, 103.3]) at 28 days. The low-dose and high-dose heterologous boost immunisation groups had NAb GMTs against live wild-type SARS-CoV-2 of 530.1 (95% CI 412.5, 681.1) and 457.6 (95%CI 349.4, 599.2), which were 26.0 folds and 22.4 folds higher than that the CoronaVac group did (20.4 [95%CI 14.3, 29.1]) at 3 months, respectively. At 6 months, the low-dose and high-dose heterologous booster groups had NAb GMTs against live wild-type SARS-CoV-2 of 312.9 (95%CI 237.7, 411.8) and 251.1 (95%CI 178.2, 354.0), which were 30.1 folds and 24.1 folds higher than the CoronaVac group did (10.4 [95%CI 7.8, 14.0]), respectively. Additionally, the low-dose and high-dose heterologous booster groups had NAb GMTs against live omicron variant of 52.0 (95%CI 37.2, 72.6) and 23.1 (95%CI 15.7, 33.9) at 28 days, 27.9 (95% CI 18.8, 41.3) and 23.3 (95%CI 16.2, 33.3) at 3 months, 16.0 (95%CI 10.9, 23.5) and 12.0 (95%CI 8.5, 16.8) at 6 months, respectively. However, nearly all participants had no detectable NAbs for omicron variant in the CoronaVac group at either 28 days, 3 months, or 6 months. No vaccine-related SAEs were observed. Conclusions: These data suggested that heterologous aerosolised Ad5-nCoV following two-dose CoronaVac priming was safe and persistently more immunogenic than three-dose CoronaVac, although immune responses waned over time.

Heterologous CoronaVac plus Ad5-nCOV versus homologous CoronaVac vaccination among elderly: a phase 4, non-inferiority, randomized study (preprint)

Importance People over 60 developed less protection after two doses of inactivated COVID-19 vaccine than younger people. Heterologous vaccination might provide greater immunity and protection against variants of concern. Objective To assess the safety and immunogenicity of a heterologous immunization with an adenovirus type 5-vectored vaccine (Convidecia) among elderly who were primed with an inactivated vaccine (CoronaVac) previously. Design An observer-blind, randomized (1:1) trial, conducted from August 26 to November 13, 2021. Setting A single center in Jiangsu Province, China. Participants 299 participants aged 60 years and olderof them 199 primed with two doses of CoronaVac in the past 3-6 months and 100 primed with one dose of CoronaVac in the past 1-2 months. Intervention Convidecia or CoronaVac as boosting dose Main Outcomes and Measures Geometric mean titers (GMTs) of neutralizing antibodies against wild-type SARS-CoV-2, and Delta and Omicron variants 14 days post boosting, and adverse reactions within 28 days. Results In the three-dose regimen cohort (n=199; mean (SD) age, 66.7 (4.2) years; 74 (37.2%) female), 99 and 100 received a third dose of Convidecia (group A) and CoronaVac (group B), respectively. In the two-dose regimen cohort (n=100; mean (SD) age, 70.5 (6.0) years; 49 (49%) female), 50 and 50 received a second dose of Convidecia (group C) and CoronaVac (group D), respectively. GMTs of neutralizing antibodies against wild-type SARS-CoV-2 at day 14 were 286.4 (95% CI: 244.6, 335.2) in group A and 48.2 (95% CI: 39.5, 58.7) in group B, with GMT ratio of 6.2 (95% CI: 4.7, 8.1), and 70.9 (95% CI: 49.5, 101.7) in group C and 9.3 (95% CI: 6.2, 13.9) in group D, with GMT ratio of 7.6 (95% CI: 4.1, 14.1). There was a 6.3-fold (GMTs, 45.9 vs 7.3) and 7.5-fold (32.9 vs 4.4) increase in neutralizing antibodies against Delta and Omicron variants in group A, respectively, compared with group B. However, there was no significant difference between group C and group D. Both heterologous and homologous booster immunizations were safe and well tolerated. Conclusions and Relevance Heterologous prime-boost regimens with CoronaVac and Convidecia induced strong neutralizing antibodies in elderly, which was superior to that induced by the homologous boost, without increasing safety concerns. Trial Registration Clinical Trials.gov NCT04952727

Immunogenicity and safety of a recombinant adenovirus type-5 COVID 19 vaccine in adults: data from a randomised, double-blind, placebo-controlled, single-dose, phase 3 trial in Russia (preprint)

BackgroundTo determine the immunogenicity, efficacy, reactogenicity, and safety of a single dose of recombinant adenovirus type-5 vectored COVID-19 vaccine (Ad5-nCoV, 5 x 1010 viral particles per 0.5 mL dose), we conducted a single-dose, randomised, double-blind, placebo-controlled, parallel group (3:1 Ad5-nCoV:placebo), phase 3 trial (Prometheus). MethodsFrom 11-September-2020 to 05-May-2021, across six sites in the Russian Federation, 496 participants were injected with either placebo or Ad5-nCoV expressing the full-length spike (S) protein from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ResultsSeroconversion (the primary endpoint) rates of 78.5% (95% CI: 73.9; 82.6) against receptor binding domain (RBD), 90.6% (95% CI: 87.2; 93.4) against S protein and 59% (95% CI: 53.3; 64.6) against neutralising SARS-CoV2 antibodies 28 days post-vaccination. Geometric mean titres (GMTs) were also elevated for antibodies against the RBD (405.32 [95% CI: 361.58; 454.46]) and S protein (678.86 [95% CI: 607.44; 754.40]) compared to the GMT of neutralising antibodies against SARS-CoV-2 (16.73 [95% CI: 15.36; 18.22]). Using an IFN-{gamma} ELISpot assay after stimulating the cells with full-length S protein we showed that the Ad5-nCoV vaccine induced the most robust cellular immune response on Days 14 and 28. Up to Day 28, the primary and all secondary endpoints of the Ad5-nCoV vaccine were statistically superior to the placebo ([p] <0.001). Systemic reactions were reported in 113 of 496 (22.8%) participants (Ad5-nCoV, 26.9%; Placebo, 10.5%), and local reactions were reported in 108 (21.8%) participants (Ad5-nCoV, 28.5%; Placebo, 1.6%). These were generally mild and resolved within 7 days after vaccination. Of the six serious adverse events reported, none of the events were vaccine related. There were no deaths or premature withdrawals. ConclusionA single-dose of Ad5-nCoV vaccine induced a marked specific humoral and cellular immune response with a favourable safety profile. Trial RegistrationClinicalTrials.gov: NCT04540419

Aerosolized Ad5-nCoV booster vaccination elicited potent immune response against the SARS-CoV-2 Omicron variant after inactivated COVID-19 vaccine priming (preprint)

The SARS-CoV-2 Omicron variant has become the dominant SARS-CoV-2 variant around the world and exhibits immune escape to current COVID-19 vaccines to some extent due to its numerous spike mutations. Here, we evaluated the immune responses to booster vaccination with intramuscular adenovirus-vectored vaccine (Ad5-nCoV), aerosolized Ad5-nCoV, a recombinant protein subunit vaccine (ZF2001) or homologous inactivated vaccine (CoronaVac) in those who received two doses of inactivated COVID-19 vaccines 6 months prior. We found that the Ad5-nCoV booster induced potent neutralizing activity against the wild-type virus and Omicron variant, while aerosolized Ad5-nCoV generated the greatest neutralizing antibody responses against the Omicron variant at day 28 after booster vaccination, at 14.1-fold that of CoronaVac, 5.6-fold that of ZF2001 and 2.0-fold that of intramuscular Ad5-nCoV. Similarly, the aerosolized Ad5-nCoV booster produced the greatest IFNgamma T-cell response at day 14 after booster vaccination. The IFNgamma T-cell response to aerosolized Ad5-nCoV was 12.8-fold for CoronaVac, 16.5-fold for ZF2001, and 5.0-fold for intramuscular Ad5-nCoV. Aerosolized Ad5-nCoV booster also produced the greatest spike-specific B cell response. Our findings suggest that inactivated vaccine recipients should consider adenovirus-vectored vaccine boosters in China and that aerosolized Ad5-nCoV may provide a more efficient alternative in response to the spread of the Omicron variant.

Heterologous prime-boost immunization with CoronaVac and Convidecia (preprint)

ABSTRACT Background The safety and immunogenicity of heterologous prime-boost COVID-19 vaccine regimens with one shot of a recombinant adenovirus type-5-vectored COVID-19 vaccine Convidecia has not been reported. Methods We conducted a randomized, controlled, observer-blinded trial of heterologous prime-boost immunization with CoronaVac and Convidecia in healthy adults 18-59 years of age. Eligible participants who were primed with one or two doses of CoronaVac were randomly assigned at a 1:1 ratio to receive a booster dose of Convidecia or CoronaVac. Participants were masked to the vaccine received but not to the three-dose or two-dose regimen. The occurrences of adverse reactions within 28 days after the vaccination were documented. The geometric mean titers of neutralizing antibodies against live SARS-CoV-2 virus were measured at 14 and 28 days after the booster vaccination. Results Between May 25 and 26, 2021, a total of 300 participants were enrolled. Participants who received a booster shot with a heterologous dose of Convidecia reported increased frequencies of solicited injection-site reactions than did those received a homogeneous dose of CoronaVac, but frequencies of systemic reactions. The adverse reactions were generally mild to moderate. The heterologous immunization with Convidecia induced higher live viral neutralizing antibodies than did the homogeneous immunization with CoronaVac (197.4[167.7, 232.4] vs. 33.6[28.3, 39.8] and 54.4[37. 9, 78.0] vs. 12.8[9.3, 17.5]) at day 14 in the three- and two-dose regimen cohort, respectively. Conclusions The heterologous prime-boost regimen with Convidecia after the priming with CoronaVac was safe and significantly immunogenic than a homogeneous boost with CoronaVac ( ClinicalTrials.gov , number NCT04892459 ).

The Effect of Different Ventilation Modes on the Outcomes of Patients Infected with Coronavirus Disease 2019 (COVID-19): a Protocol of Systematic Review and Network Meta-Analysis (preprint)

Background: Coronavirus disease 2019 (COVID-19) outbreak has spread around the world, the high mortality rate and strong infectious cause surging global patients diagnosed patients and death while in response to the outbreak, a myriad of scientific research workers and researchers have made unremitting efforts, but effective treatments are still limited, even may say that there is no specific treatment. So a large number of patients with severe patients need treatment for respiratory support, in particular, based on various realistic factors, different way of ventilation is widely used in clinical, which kind of ventilation way is the best one of the most effective ventilation strategy is not clear, so we planned a network meta-analysis to evaluate different ventilation methods on new crown the efficacy and safety of patients, expect to find an optimal ventilation strategy.Methods and analysis: Two authors will independently search the electronic databases, preprints databases, Clinical Study Registration website and COVID-19 research related project database from December 1, 2019 to November 5, 2020. The primary outcomes are 1) All-cause mortality; 2) Transmission of COVID-19 to health care workers and other people; 3) Length of hospital stay; 4) Length of ICU stay. A systemic review and a network meta-analysis based on Bayesian framework will be performed to assess the effect of different ventilation modes on the outcomes of patients infected with COVID-19. The Grading of Recommendations Assessment, Development and Evaluation System (GRADE) will be used to evaluate the quality of evidence.Discussion: COVID-19 has spread around the world and become a global public health security problem. With limited treatment available, a large number of critically ill patients need ventilator support treatment, and the demand for ventilators has increased sharply. To our knowledge, this study will be the first systematic review and NMA to analyze the efficacy and safety of different ventilation modes in patients with COVID-19. This study expected to obtain the best choice of ventilation mode for COVID-19 patients based on high quality evidence. Ethics and dissemination: Ethical approval is not required owning to it is a literature-based study. The final conclusion will be disseminated through peer-reviewed publication. PROSPERO registration number: CRD42020219581

Cancer Patient Management Strategy in a Cancer Center of Zhejiang, China During the COVID-19 Pandemic (preprint)

Background: Due to the increased risk of viral infection and the severe shortage of medical resources during the pandemic of COVID-19, most hospitals in the epidemic areas significantly reduced non-emergency admissions and services, if not closed. As a result, it has been difficult to treat cancer patients on time, which adversely affects their prognosis. To address this problem, cancer centers must develop a strategic plan to manage both inpatients and outpatients during the pandemic, provide them with the necessary treatment, and at the same time prevent the spread of the virus among patients, visitors and medical staff. Methods: : Based upon the epidemic situation in Zhejiang Province, China, the number of running non-emergency medical wards in the Zhejiang Cancer Hospital was gradually increased in a controlled manner. All staff of the hospital received COVID-19 preventive training and was provided with three different levels of protection according to the risks of their services. Only patients without a known history of SARS-CoV-2 contact were eligible to schedule an appointment. Body temperature was measured on all patients upon their arrival at the hospital. Chest CT image, blood cell counting and travel/contact history were investigated in patients with fever. Respiratory tract samples, such as sputum and throat swabs, from all patients, including those clinically suspected of SARS-CoV-2 infection, were collected for nucleic acid detection of SARS-CoV-2 before treatment. Results: : A total of 3697 inpatients and 416 outpatients seeking cancer treatment were enrolled from February 1 to April 3, 2020, in compliance with the hospital’s infection-control interventions. The clinicopathological parameters of the patients were summarized herein. 4237 samples from 4101 patients produced negative RNA testing results. Four clinically suspected patients all presented negative RNA test results and were excluded from the SARS-CoV-2 infection through follow-up retesting and monitoring. Seven patients with only N-gene positive results were retested, followed by CT scan and SARS-CoV-2 contact history investigation. All of them were finally diagnosed as non-infected patients. There was one outpatient who was confirmed positive by virus RNA test and then followed up. She might be an asymptomatic laboratory-confirmed case. During the study period, there was no SARS-CoV-2 infection among staff, patients and escorts of patients in the Zhejiang Cancer Hospital. Conclusion: This study suggested our infection-control interventions, including viral nucleic acid test, could be used as a reliable method to screen cancer patients in the area with moderate COVID-19 prevalence. Cancer may not be a high-risk factor of SARS-CoV-2 infection.

Highly accurate and sensitive diagnostic detection of SARS-CoV-2 by digital PCR (preprint)

BACKGROUND: The outbreak of COVID-19 caused by a novel Coronavirus (termed SARS-CoV-2) has spread to over 140 countries around the world. Currently, reverse transcription quantitative qPCR (RT-qPCR) is used as the gold standard for diagnostics of SARS-CoV-2. However, the positive rate of RT-qPCR assay of pharyngeal swab samples are reported to vary from 30~60%. More accurate and sensitive methods are urgently needed to support the quality assurance of the RT-qPCR or as an alternative diagnostic approach. METHODSWe established a reverse transcription digital PCR (RT-dPCR) protocol to detect SARS-CoV-2 on 194 clinical pharyngeal swab samples, including 103 suspected patients, 75 close contacts and 16 supposed convalescents. RESULTS: The limit of blanks (LoBs) of the RT-dPCR assays were ~1.6, ~1.6 and ~0.8 copies/reaction for ORF 1ab, N and E genes, respectively. The limit of detection (LoD) was 2 copies/reaction. For the 103 fever suspected patients, the sensitivity of SARS-CoV-2 detection was significantly improved from 28.2% by RT-qPCR to 87.4% by RT-dPCR. For close contacts, the suspect rate was greatly decreased from 21% down to 1%. The overall sensitivity, specificity and diagnostic accuracy of RT-dPCR were 90%, 100% and 93 %, respectively. In addition, quantification of the viral load for convalescents by RT-dPCR showed that a longer observation period was needed in the hospital for elderly patients. CONCLUSION: RT-dPCR could be a confirmatory method for suspected patients diagnosed by RT-qPCR. Furthermore, RT-dPCR was more sensitive and suitable for low viral load specimens from the both patients under isolation and those under observation who may not be exhibiting clinical symptoms.