Construction of a potent pan-vaccine based on the evolutionary tendency of SARS-CoV-2 spike protein.

SARS-CoV-2 continued to spread globally along with different variants. Here, we systemically analyzed viral infectivity and immune-resistance of SARS-CoV-2 variants to explore the underlying rationale of viral mutagenesis. We found that the Beta variant harbors both high infectivity and strong immune resistance, while the Delta variant is the most infectious with only a mild immune-escape ability. Remarkably, the Omicron variant is even more immune-resistant than the Beta variant, but its infectivity increases only in Vero E6 cells implying a probable preference for the endocytic pathway. A comprehensive analysis revealed that SARS-CoV-2 spike protein evolved into distinct evolutionary paths of either high infectivity plus low immune resistance or low infectivity plus high immune resistance, resulting in a narrow spectrum of the current single-strain vaccine. In light of these findings and the phylogenetic analysis of 2674 SARS-CoV-2 S-protein sequences, we generated a consensus antigen (S6) taking the most frequent mutations as a pan-vaccine against heterogeneous variants. As compared to the ancestry SWT vaccine with significantly declined neutralizations to emerging variants, the S6 vaccine elicits broadly neutralizing antibodies and full protections to a wide range of variants. Our work highlights the importance and feasibility of a universal vaccine strategy to fight against antigen drift of SARS-CoV-2.

Self-assessment of COVID-19 vaccination efficacy using a simple POCT for SARS-CoV-2 S1 protein antibody IgG-IgM

BackgroundMore than ten novel COVID-19 vaccines have been approved with protections against SARS-CoV-2 infections ranges between 52-95%. It is of great interest to the vaccinees who have received the COVID-19 vaccines, vaccine developers and authorities to identify the non-responders in a timely manner so intervention can take place by either giving additional boosts of the same vaccine or switching to a different vaccine to improve the protection against the SARS-CoV-2 infections. A robust correlation was seen between binding antibody titer and efficacy (p=0.93) in the clinic studies of 7 COVID-19 vaccines, so it is of urgency to develop a simple POCT for vaccinees to self-assess their immune response at home. MethodsUsing CHO cell-expressed full length SARS-CoV2 S1 protein as coating antigen on colloidal gold particles, a SARS-CoV-2 S1 IgG-IgM antibody lateral flow test kit (POCT) was developed. The test was validated with negative human sera collected prior to the COVID-19 outbreaks, and blood samples from human subjects prior, during, and post-immunization of COVID-19 vaccines. ResultsThe specificity of the POCT was 99.0%, as examined against 947 normal human sera and 20 whole blood samples collected pre-immunization. The limit of detection was 50 IU/mL of pseudovirus neutralizing titer (PVNT) using human anti-SARS-2 neutralizing standards from convalescent sera. The sensitivity of POCT for SARS-CoV-2 S1 protein antibody IgG-IgM was compared with SARS-CoV-2 RBD antibody ELISA and determined to be 100% using 23 blood samples from vaccinated human subjects and 10 samples from non-vaccinated ones. Whole blood samples were collected from 119 human subjects (ages between 22-61 years) prior to, during, and post-vaccination of five different COVID-19 vaccines. Among them, 115 people tested positive for SARS-CoV-2 S1 antibodies (showing positive at least once) and 4 people tested negative (tested negative at least twice on different days), demonstrating 96.64% of seroconversion after full-vaccination. 92.3% (36/39) of the human subjects who were younger than 45 achieved seroconversion within 2 weeks while only 57.1% (4/7) of subjects older than 45 tested positive for S1 antibodies, suggesting that younger people develop protection much faster than older ones. Even though the S1 antibody level in 88% of human subjects vaccinated with inactivated virus dropped below 50 IU/mL two months later, one boost could quickly raise the S1 antibody titer above 50 IU/mL of PVNT, indicates that the initial vaccination was successful and immunization memory was developed. ConclusionUsing the lateral flow tests of SARS-CoV2 S1 IgG+IgM, vaccinated human subjects can easily self-assess the efficacy of their vaccination at home. The vaccine developer could quickly identify those non-responders and give them an additional boost to improve the efficacy of their vaccines. Vaccinees who failed in response could switch to different types of COVID-19 vaccines since there are more than 10 COVID-19 vaccines approved using three different platform technologies. HighlightsO_LIMore than ten novel COVID-19 vaccines have been approved with protections against SARS-CoV-2 infections ranges between 52-95%. It is of great interest to the vaccinees who have received the COVID-19 vaccines, vaccine developers and authorities to identify the non-responders in a timely manner. C_LIO_LIA highly specific and very simple lateral flow test kit for measurement of SARS-CoV-2 S1IgG+IgM antibodies post-immunization of COVID-19 vaccine using peripheral blood was developed as a home-test assay with a limit of detection (LOD) at 50 IU/mL of pseudovirus neutralizing titer (PVNT). C_LIO_LIAfter full vaccinations with COVID-19 vaccines, 96.6% of the volunteers successfully achieved the seroconversion of SARS-CoV-2 S1 IgG+IgM antibody. C_LIO_LI92.3% (36/39) of the human subjects who were younger than 45 achieved seroconversion within 2 weeks while only 57.1% (4/7) of subjects older than 45 tested positive for S1 antibodies, suggesting that younger people develop protection much faster than older ones. C_LIO_LIEven though the S1 antibody level in 88% of human subjects vaccinated with inactivated virus dropped below the detection 2-6 months later, one boost could quickly raise the S1 antibody titer above 50 IU/mL of PVNT, indicating that the initial vaccination was successful and immunization memory was developed. C_LI

Antibody neutralization to SARS-CoV-2 and variants after one year in Wuhan

Most COVID-19 patients can build effective humoral immunity against SARS-CoV-2 after recovery(1, 2). However, it remains unknown how long the protection can maintain and how efficiently it can protect people from the reinfection of the emerging SARS-CoV-2 variants. Here we evaluated the sera from 248 COVID-19 convalescents around one year post-infection in Wuhan, the earliest epicenter of SARS-CoV-2. We demonstrated that the SARS-CoV-2 immunoglobulin G (IgG) maintains at a high level and potently neutralizes the infection of the original strain (WT) and the B.1.1.7 variant in most patients. However, they showed varying degrees of efficacy reduction against the other variants of concern (P.1, B.1.525, and especially B.1.351) in a patient-specific manner. Mutations in RBD including K417N, E484K, and E484Q/L452R (B.1.617) remarkably impair the neutralizing activity of the convalescents sera. Encouragingly, we found that a small fraction of patients sera showed broad neutralization potency to multiple variants and mutants, suggesting the existence of broadly neutralizing antibodies recognizing the epitopes beyond the mutation sites. Our results suggest that the SARS-CoV-2 vaccination effectiveness relies more on the timely re-administration of the epitope-updated vaccine than the durability of the neutralizing antibodies.

Antibody Cocktail Exhibits Broad Neutralization against SARS-CoV-2 and SARS-CoV-2 variants

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has precipitated multiple variants resistant to therapeutic antibodies. In this study, 12 high-affinity antibodies were generated from convalescent donors in early outbreaks using immune antibody phage display libraries. Of them, two RBD-binding antibodies (F61 and H121) showed high affinity neutralization against SARS-CoV-2, whereas three S2-target antibodies failed to neutralize SARS-CoV-2. Following structure analysis, F61 identified a linear epitope located in residues G446 -S494, which overlapped with angiotensin-converting enzyme 2 (ACE2) binding sites, while H121 recognized a conformational epitope located on the side face of RBD, outside from ACE2 binding domain. Hence the cocktail of the two antibodies achieved better performance of neutralization to SARS-CoV-2. Importantly, F61 and H121 exhibited efficient neutralizing activity against variants B.1.1.7 and B.1.351, those showed immune escape. Efficient neutralization of F61 and H121 against multiple mutations within RBD revealed a broad neutralizing activity against SARS-CoV-2 variants, which mitigated the risk of viral escape. Our findings defined the basis of therapeutic cocktails of F61 and H121 with broad neutralization and delivered a guideline for the current and future vaccine design, therapeutic antibody development, and antigen diagnosis of SARS-CoV-2 and its novel variants.

Abnormal Upregulation of Cardiovascular Disease Biomarker PLA2G7 Induced by Proinflammatory Macrophages in COVID-19 patients

BACKGROUNDCoronavirus disease 2019 (COVID-19) triggers distinct patterns of pneumonia progression with multiorgan disease, calling for cell- and/or tissue-type specific host injury markers. METHODSAn integrated hypothesis-free single biomarker analysis framework was performed on nasal swabs (n = 484) from patients with COVID-19 in GSE152075. The origin of candidate biomarker was assessed in single-cell RNA data (GSE145926). The candidate biomarker was validated in a cross-sectional cohort (n = 564) at both nucleotide and protein levels. RESULTSPhospholipase A2 group VII (PLA2G7) was identified as a candidate biomarker in COVID-19. PLA2G7 was predominantly expressed by proinflammatory macrophages in lungs emerging with progression of COVID-19. In the validation stage, PLA2G7 was found in patients with COVID-19 and pneumonia, especially in severe pneumonia, rather than patients suffered mild H1N1 influenza infection. Up to 100% positive rates of PLA2G7 were positively correlated with not only viral loads in patients with COVID-19 but also severity of pneumonia in non-COVID-19 patients. Although Ct values of PLA2G7 in severe pneumonia was significantly lower than that in moderate pneumonia (P = 7.2e-11), no differences were observed in moderate pneumonia with COVID-19 between severe pneumonia without COVID-19 (P = 0.81). Serum protein levels of PLA2G7, also known as lipoprotein-associated phospholipase A2 (Lp-PLA2), were further found to be elevated and beyond the upper limit of normal in patients with COVID-19, especially among the re-positive patients. CONCLUSIONSWe firstly identified and validated PLA2G7, a biomarker for cardiovascular diseases (CVDs), was abnormally enhanced in COVID-19 patients at both nucleotide and protein aspects. These findings provided indications into the prevalence of cardiovascular involvements seen in COVID-19 patients. PLA2G7 could be a hallmark of COVID-19 for monitoring disease progress and therapeutic response. FUNDINGThis study was supported by grants from China Mega-Projects for Infectious Disease (2018ZX10711001), National Natural Science Foundation of China (82041023).

Distributions and risks of SARS-CoV-2 in hospital outdoor environment

The outbreak of coronavirus infectious disease-2019 (COVID-19) pandemic has rapidly spread throughout over 200 countries, posing a global threat to human health. Till 15th May 2020, there are over 4.5 million confirmed cases, with roughly 300,000 death1. To date, most studies focus on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in indoor environment owing to its main transmission routes via human respiratory droplets and direct contact2,3. It remains unclear whether SARS-CoV-2 can spill over and impose transmission risks to outdoor environments despite potential threats to people and communities. Here, we investigated the presence of SARS-CoV-2 by measuring viral RNA in 73 samples from outdoor environment of three hospitals in Wuhan. We detected SARS-CoV-2 in soils (205-550 copies/g), aerosols (285-1,130 copies/m3) and wastewaters (255 to 18,744 copies/L) in locations close to hospital departments receiving COVID-19 patients or in wastewater treatment sectors. These findings reveal significant viral spillover in hospital outdoor environments that was possibly caused by respiratory droplets from patients or aerosolized particles from wastewater containing SARS-CoV-2. In contrast, SARS-CoV-2 was not detected in other areas or on surfaces with regular disinfection implemented. Soils may behave as viral warehouse through deposition and serve as a secondary source spreading SARS-CoV-2 for a prolonged time. For the first time, our findings demonstrate that there are high-risk areas in hospital outdoor environments to spread SARS-CoV-2, calling for sealing of wastewater treatment unit and complete sanitation to prevent COVID-19 transmission risks.

Potential spreading risks and disinfection challenges of medical wastewater by the presence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) viral RNA in septic tanks of fangcang hospital

The outbreak of coronavirus infectious disease-2019 (COVID-19) pneumonia raises the concerns of effective deactivation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in medical wastewater by disinfectants. In this study, we evaluated the presence of SARS-CoV-2 viral RNA in septic tanks of Wuchang Fangcang Hospital and found the high level of (0.05-1.87)x104 copies/L after disinfection with sodium hypochlorite. Embedded viruses in stool particles might be released in septic tanks, behaving as a source of SARS-CoV-2 and potentially contributing to its spread through drainage pipelines. Current recommended disinfection strategy (free chlorine above 6.5 mg/L after 1.5-hour contact) needs to be reevaluated to completely remove SARS-CoV-2 viral RNA in non-centralized disinfection system and effectively deactivate SARS-CoV-2. The effluents showed negative results for SARS-CoV-2 viral RNA when overdosed with sodium hypochlorite but had high a level of disinfection by-product residuals, possessing significant ecological risks.

Genome-wide data inferring the evolution and population demography of the novel pneumonia coronavirus (SARS-CoV-2)

As the highly risk and infectious diseases, the outbreak of coronavirus disease 2019 (COVID-19) poses unprecedent challenges to global health. Up to March 3, 2020, SARS-CoV-2 has infected more than 89,000 people in China and other 66 countries across six continents. In this study, we used 10 new sequenced genomes of SARS-CoV-2 and combined 136 genomes from GISAID database to investigate the genetic variation and population demography through different analysis approaches (e.g. Network, EBSP, Mismatch, and neutrality tests). The results showed that 80 haplotypes had 183 substitution sites, including 27 parsimony-informative and 156 singletons. Sliding window analyses of genetic diversity suggested a certain mutations abundance in the genomes of SARS-CoV-2, which may be explaining the existing widespread. Phylogenetic analysis showed that, compared with the coronavirus carried by pangolins (Pangolin-CoV), the virus carried by bats (bat-RaTG13-CoV) has a closer relationship with SARS-CoV-2. The network results showed that SARS-CoV-2 had diverse haplotypes around the world by February 11. Additionally, 16 genomes, collected from Huanan seafood market assigned to 10 haplotypes, indicated a circulating infection within the market in a short term. The EBSP results showed that the first estimated expansion date of SARS-CoV-2 began from 7 December 2019.

Impact of pooling of inactivated SARS-CoV-2 samples on nucleic acid detection / 公共卫生与预防医学

Objective To investigate the impact of pooling of inactivated samples on testing results of nucleic acid detection of SARS-CoV-2, and to provide a scientific detection scheme for the SARS-CoV-2 nucleic acid screening of large population samples. Methods The SARS-CoV-2 positive throat swab samples and the negative throat swab samples were inactivated at 56°C for 30 minutes, and mixed according to the ratio of positive samples to negative samples at 1:4, 1:9, and 1:19, respectively. Real-time fluorescent RT-PCR technology was used to detect the ORF-lab and N genes in the original solution and mixed solution. Results The nucleic acid test results of the 20 groups of inactivated samples were all positive. The nucleic acid test results of the 1:5 mixture and the 1:10 mixture were also positive. One group of samples of the 1:20 mixture was negative for ORF-lab and positive for the N gene. The Ct values of nucleic acid detection among all groups were significantly correlated. There was no statistically significant difference in the positive rate between different sample groups. Compared with the original solution, the Ct values of the ORF-lab gene of 1:5 mixture, 1:10 mixture, and 1:20 mixture samples increased by 1.73, 2.86, and 4.05, respectively, while the Ct values of the N gene of 1:5 mixture, 1:10 mixture and 1:20 mixture samples increased by 1.69, 2.79, and 3.25, respectively. Conclusion When conducting nucleic acid screening for SARS-CoV-2 in large population samples, a mixed test of less than 10 inactivated samples would not affect the qualitative results in most cases, but the results of weak positive samples may be affected.

Investigation and analysis of the status of new coronavirus contamination in biosafety laboratories / 公共卫生与预防医学

Objective To understand the status of new coronavirus contamination in the biosafety laboratory environment, identify key areas prone to contamination, and provide evidence for disinfection of central objects. Methods surfaces of high-frequency contact environment and protective equipment were sampled with moistened sterile cotton swabs after experiment and before disinfection, the results of the one-step real-time reverse transcription polymerase chain reaction (RT-PCR) of open reading frame 1ab and N fragment were used to evaluate the pollution status. Results Environmental surveys found 4 of 217 samples of environmental objects to be positive for new coronavirus RNA, that positive rate was 1.84%. Among them, BSL-3, BSL-2, and BSL-1 were sampled 23, 184, and 10 respectively. The 3 positive samples were from surfaces of nucleic acid extraction room of BSL-2 and from the handles of pass-through box, laboratory door handles and the outer surface of the alcohol watering pot respectively. The 1 positive sample was from the forearm of the protective clothing in BSL-2 laboratory. Conclusion There was a certain degree of virus pollution in key areas of the new coronavirus laboratory. The BSL-2 laboratory has a higher risk of environmental pollution than the BSL-3 and BSL-1 laboratories.

Genetic Characteristics of 2009 Pandemic H1N1 Influenza A Viruses Isolated from Mainland China / 中国病毒学·英文版

A total of 100 HIN1 flu real-time-PCR positive throat swabs collected from fever patients in Zhejiang,Hubei and Guangdong between June and November 2009,were provided by local CDC laboratories.After MDCK cell culture,57 Influenza A Pandemic (H1N1) viruses were isolated and submitted for whole genome sequencing.A total of 39 HA sequences,52 NA sequences,36 PB2 sequences,31 PB1 sequences,40 PA sequences,48 NP sequences,51 MP sequences and 36 NS sequences were obtained,including 20 whole genome sequences.Sequence comparison revealed they shared a high degree of homology (96％～99％) with known epidemic strains (A/Califomia/04/2009(H1N1).Phylogenetic analysis showed that although the sequences were highly conserved,they clustered into a small number of groups with only a few distinct strains.Site analysis revealed three substitutions at loop 220 (221-228) of the HA receptor binding site in the 39 HA sequences:A/Hubei/86/2009 PKVRDQEG→PKVRDQEA,A/Zhejiang/08/2009 PKVRDQEG→PKVRDQER,A/Hubei/75/2009PKVRDQEG→PKVRDQGG,the A/Hubei/75/2009 was isolated from an acute case,while the other two were from patients with mild symptoms.Other key sites such as 119,274,292 and 294 amino acids of NA protein,627 of PB2 protein were conserved.Meanwhile,all the M2 protein sequences possessed the Ser32Asn mutation,suggesting that these viruses were resistant to adamantanes.Comparison of these sequences with other H1N1 viruses collected from the NCBI database provides insight into H1N1 transmission and circulation patterns.

A rapid method for molecular diagnosis of 22q11.2 microdeletion syndrome / 第二军医大学学报

Objective:To explore a rapid, economical and efficient approach for molecular detection of 22q11.2 micro-deletion syndrome. Methods: Fifty ventricular septal defect (VSD) patients (33 males and 17 females, age ranged from 1 month to 15 years), who were hospitalized in Nanjing Children's Hospital from Jan. 2004 to Jan. 2005, were randomly selected for this study. The peripheral blood of VSD patients and the buckle cells of their parents were obtained. Three short tandem-repeat polymorphism (STRP) markers, D22S944, 22D_4_2 and 22D_4_3, were used for fluorescent in situ hybridization(FISH)study and genotype analysis. Results: 22D_4_2 and 22D_4_3 produced clear electrophoresis band, and the detections were rapid and efficient. The 3 STRP markers were consistent with the Hardy-Weinberg equilibrium expectations, and their heterozygosity was high in the present population from Chinese Han nationality in Jiangsu province. FISH confirmed that 5 of the 50 VSD patients had a deletion within chromosome 22q11.2. Conclusion: Three STRP markers (D22S944, 22D_4_2 and 22D_4_3) (analysis) combined with FISH as a supplementary is an efficient and reliable approach for detection of (22q11.2 microdeletion.)