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1.
Nature ; 603(7902): 706-714, 2022 03.
Article in English | MEDLINE | ID: covidwho-1764186

ABSTRACT

The SARS-CoV-2 Omicron BA.1 variant emerged in 20211 and has multiple mutations in its spike protein2. Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron's evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways3 demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis.


Subject(s)
COVID-19/pathology , COVID-19/virology , Membrane Fusion , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Serine Endopeptidases/metabolism , Virus Internalization , Adult , Aged , Aged, 80 and over , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , COVID-19 Vaccines/immunology , Cell Line , Cell Membrane/metabolism , Cell Membrane/virology , Chlorocebus aethiops , Convalescence , Female , Humans , Immune Sera/immunology , Intestines/pathology , Intestines/virology , Lung/pathology , Lung/virology , Male , Middle Aged , Mutation , Nasal Mucosa/pathology , Nasal Mucosa/virology , SARS-CoV-2/drug effects , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Tissue Culture Techniques , Virulence , Virus Replication
2.
Viruses ; 14(2)2022 02 17.
Article in English | MEDLINE | ID: covidwho-1705787

ABSTRACT

In light of an increasing number of vaccinated and convalescent individuals, there is a major need for the development of robust methods for the quantification of neutralizing antibodies; although, a defined correlate of protection is still missing. Sera from hospitalized COVID-19 patients suffering or not suffering from acute respiratory distress syndrome (ARDS) were comparatively analyzed by plaque reduction neutralization test (PRNT) and pseudotype-based neutralization assays to quantify their neutralizing capacity. The two neutralization assays showed comparable data. In case of the non-ARDS sera, there was a distinct correlation between the data from the neutralization assays on the one hand, and enzyme-linked immune sorbent assay (ELISA), as well as biophysical analyses, on the other hand. As such, surface plasmon resonance (SPR)-based assays for quantification of binding antibodies or analysis of the stability of the antigen-antibody interaction and inhibition of syncytium formation, determined by cell fusion assays, were performed. In the case of ARDS sera, which are characterized by a significantly higher fraction of RBD-binding IgA antibodies, there is a clear correlation between the neutralization assays and the ELISA data. In contrast to this, a less clear correlation between the biophysical analyses on the one hand and ELISAs and neutralization assays on the other hand was observed, which might be explained by the heterogeneity of the antibodies. To conclude, for less complex immune sera-as in cases of non-ARDS sera-combinations of titer quantification by ELISA with inhibition of syncytium formation, SPR-based analysis of antibody binding, determination of the stability of the antigen-antibody complex, and competition of the RBD-ACE2 binding represent alternatives to the classic PRNT for analysis of the neutralizing potential of SARS-CoV-2-specific sera, without the requirement for a BSL3 facility.


Subject(s)
Antibodies, Viral/blood , Convalescence , Immune Sera/analysis , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/blood , Spike Glycoprotein, Coronavirus/immunology , Adult , Aged , Aged, 80 and over , Antibodies, Neutralizing/immunology , COVID-19/immunology , Enzyme-Linked Immunosorbent Assay , Female , Hospitalization/statistics & numerical data , Humans , Immune Sera/immunology , Immunity, Humoral , Male , Middle Aged , Neutralization Tests
3.
J Virol ; 96(1): e0096421, 2022 01 12.
Article in English | MEDLINE | ID: covidwho-1631789

ABSTRACT

A comprehensive analysis and characterization of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection model that mimics non-severe and severe coronavirus disease 2019 (COVID-19) in humans is warranted for understating the virus and developing preventive and therapeutic agents. Here, we characterized the K18-hACE2 mouse model expressing human (h)ACE2 in mice, controlled by the human keratin 18 (K18) promoter, in the epithelia, including airway epithelial cells where SARS-CoV-2 infections typically start. We found that intranasal inoculation with higher viral doses (2 × 103 and 2 × 104 PFU) of SARS-CoV-2 caused lethality of all mice and severe damage of various organs, including lung, liver, and kidney, while lower doses (2 × 101 and 2 × 102 PFU) led to less severe tissue damage and some mice recovered from the infection. In this hACE2 mouse model, SARS-CoV-2 infection damaged multiple tissues, with a dose-dependent effect in most tissues. Similar damage was observed in postmortem samples from COVID-19 patients. Finally, the mice that recovered from infection with a low dose of virus survived rechallenge with a high dose of virus. Compared to other existing models, the K18-hACE2 model seems to be the most sensitive COVID-19 model reported to date. Our work expands the information available about this model to include analysis of multiple infectious doses and various tissues with comparison to human postmortem samples from COVID-19 patients. In conclusion, the K18-hACE2 mouse model recapitulates both severe and non-severe COVID-19 in humans being dose-dependent and can provide insight into disease progression and the efficacy of therapeutics for preventing or treating COVID-19. IMPORTANCE The pandemic of coronavirus disease 2019 (COVID-19) has reached nearly 240 million cases, caused nearly 5 million deaths worldwide as of October 2021, and has raised an urgent need for the development of novel drugs and therapeutics to prevent the spread and pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To achieve this goal, an animal model that recapitulates the features of human COVID-19 disease progress and pathogenesis is greatly needed. In this study, we have comprehensively characterized a mouse model of SARS-CoV-2 infection using K18-hACE2 transgenic mice. We infected the mice with low and high doses of SARS-CoV-2 to study the pathogenesis and survival in response to different infection patterns. Moreover, we compared the pathogenesis of the K18-hACE2 transgenic mice with that of the COVID-19 patients to show that this model could be a useful tool for the development of antiviral drugs and therapeutics.


Subject(s)
COVID-19/pathology , Disease Models, Animal , SARS-CoV-2/pathogenicity , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/immunology , COVID-19/mortality , COVID-19/virology , Humans , Immune Sera/immunology , Keratin-18/genetics , Mice , Mice, Transgenic , Promoter Regions, Genetic , Reinfection/immunology , Reinfection/mortality , Reinfection/pathology , Reinfection/virology , SARS-CoV-2/immunology , Viral Proteins/genetics , Viral Proteins/metabolism
4.
Nature ; 602(7898): 682-688, 2022 02.
Article in English | MEDLINE | ID: covidwho-1616995

ABSTRACT

The Omicron (B.1.1.529) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was initially identified in November 2021 in South Africa and Botswana, as well as in a sample from a traveller from South Africa in Hong Kong1,2. Since then, Omicron has been detected globally. This variant appears to be at least as infectious as Delta (B.1.617.2), has already caused superspreader events3, and has outcompeted Delta within weeks in several countries and metropolitan areas. Omicron hosts an unprecedented number of mutations in its spike gene and early reports have provided evidence for extensive immune escape and reduced vaccine effectiveness2,4-6. Here we investigated the virus-neutralizing and spike protein-binding activity of sera from convalescent, double mRNA-vaccinated, mRNA-boosted, convalescent double-vaccinated and convalescent boosted individuals against wild-type, Beta (B.1.351) and Omicron SARS-CoV-2 isolates and spike proteins. Neutralizing activity of sera from convalescent and double-vaccinated participants was undetectable or very low against Omicron compared with the wild-type virus, whereas neutralizing activity of sera from individuals who had been exposed to spike three or four times through infection and vaccination was maintained, although at significantly reduced levels. Binding to the receptor-binding and N-terminal domains of the Omicron spike protein was reduced compared with binding to the wild type in convalescent unvaccinated individuals, but was mostly retained in vaccinated individuals.


Subject(s)
Antibodies, Neutralizing/immunology , COVID-19 Vaccines/immunology , COVID-19/immunology , COVID-19/virology , Convalescence , Immune Evasion/immunology , Immune Sera/immunology , SARS-CoV-2/immunology , 2019-nCoV Vaccine mRNA-1273/immunology , Adult , Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , BNT162 Vaccine/administration & dosage , BNT162 Vaccine/immunology , COVID-19/transmission , Female , Humans , Immunization, Secondary , Models, Molecular , Neutralization Tests , SARS-CoV-2/classification , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
5.
Nature ; 602(7898): 676-681, 2022 02.
Article in English | MEDLINE | ID: covidwho-1616993

ABSTRACT

The B.1.1.529/Omicron variant of SARS-CoV-2 was only recently detected in southern Africa, but its subsequent spread has been extensive, both regionally and globally1. It is expected to become dominant in the coming weeks2, probably due to enhanced transmissibility. A striking feature of this variant is the large number of spike mutations3 that pose a threat to the efficacy of current COVID-19 vaccines and antibody therapies4. This concern is amplified by the findings of our study. Here we found that B.1.1.529 is markedly resistant to neutralization by serum not only from patients who recovered from COVID-19, but also from individuals who were vaccinated with one of the four widely used COVID-19 vaccines. Even serum from individuals who were vaccinated and received a booster dose of mRNA-based vaccines exhibited substantially diminished neutralizing activity against B.1.1.529. By evaluating a panel of monoclonal antibodies against all known epitope clusters on the spike protein, we noted that the activity of 17 out of the 19 antibodies tested were either abolished or impaired, including ones that are currently authorized or approved for use in patients. Moreover, we also identified four new spike mutations (S371L, N440K, G446S and Q493R) that confer greater antibody resistance on B.1.1.529. The Omicron variant presents a serious threat to many existing COVID-19 vaccines and therapies, compelling the development of new interventions that anticipate the evolutionary trajectory of SARS-CoV-2.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/virology , Immune Evasion/immunology , SARS-CoV-2/immunology , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19/blood , COVID-19/immunology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/immunology , Cell Line , Convalescence , Evolution, Molecular , Humans , Immune Sera/immunology , Inhibitory Concentration 50 , Models, Molecular , Mutation , Neutralization Tests , SARS-CoV-2/chemistry , SARS-CoV-2/classification , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
6.
Nature ; 602(7898): 657-663, 2022 02.
Article in English | MEDLINE | ID: covidwho-1616990

ABSTRACT

The SARS-CoV-2 B.1.1.529 (Omicron) variant contains 15 mutations of the receptor-binding domain (RBD). How Omicron evades RBD-targeted neutralizing antibodies requires immediate investigation. Here we use high-throughput yeast display screening1,2 to determine the profiles of RBD escaping mutations for 247 human anti-RBD neutralizing antibodies and show that the neutralizing antibodies can be classified by unsupervised clustering into six epitope groups (A-F)-a grouping that is highly concordant with knowledge-based structural classifications3-5. Various single mutations of Omicron can impair neutralizing antibodies of different epitope groups. Specifically, neutralizing antibodies in groups A-D, the epitopes of which overlap with the ACE2-binding motif, are largely escaped by K417N, G446S, E484A and Q493R. Antibodies in group E (for example, S309)6 and group F (for example, CR3022)7, which often exhibit broad sarbecovirus neutralizing activity, are less affected by Omicron, but a subset of neutralizing antibodies are still escaped by G339D, N440K and S371L. Furthermore, Omicron pseudovirus neutralization showed that neutralizing antibodies that sustained single mutations could also be escaped, owing to multiple synergetic mutations on their epitopes. In total, over 85% of the tested neutralizing antibodies were escaped by Omicron. With regard to neutralizing-antibody-based drugs, the neutralization potency of LY-CoV016, LY-CoV555, REGN10933, REGN10987, AZD1061, AZD8895 and BRII-196 was greatly undermined by Omicron, whereas VIR-7831 and DXP-604 still functioned at a reduced efficacy. Together, our data suggest that infection with Omicron would result in considerable humoral immune evasion, and that neutralizing antibodies targeting the sarbecovirus conserved region will remain most effective. Our results inform the development of antibody-based drugs and vaccines against Omicron and future variants.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Immune Evasion/immunology , Neutralization Tests , SARS-CoV-2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antibodies, Neutralizing/classification , Antibodies, Viral/classification , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/immunology , Cells, Cultured , Convalescence , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/immunology , Humans , Immune Sera/immunology , Models, Molecular , Mutation , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism
7.
Emerg Microbes Infect ; 11(1): 337-343, 2022 Dec.
Article in English | MEDLINE | ID: covidwho-1585241

ABSTRACT

ABSTRACTThe emerging new VOC B.1.1.529 (Omicron) variant has raised serious concerns due to multiple mutations, reported significant immune escape, and unprecedented rapid spreading speed. Currently, studies describing the neutralization ability of different homologous and heterologous booster vaccination against Omicron are still lacking. In this study, we explored the immunogenicity of COVID-19 breakthrough patients, BBIBP-CorV homologous booster group and BBIBP-CorV/ZF2001 heterologous booster group against SARS-CoV-2 pseudotypes corresponding to the prototype, Beta, Delta, and the emergent Omicron variant.Notably, at 14 days post two-dose inactivated vaccines, pVNT titre increased to 67.4 GMTs against prototype, 8.85 against Beta and 35.07 against Delta, while neutralization activity against Omicron was below the lower limit of quantitation in 80% of the samples. At day 14 post BBIBP-CorV homologous booster vaccination, GMTs of pVNT significantly increased to 285.6, 215.7, 250.8, 48.73 against prototype, Beta, Delta, and Omicron, while at day 14 post ZF2001 heterologous booster vaccination, GMTs of pVNT significantly increased to 1436.00, 789.6, 1501.00, 95.86, respectively. Post booster vaccination, 100% samples showed positive neutralization activity against Omicron, albeit illustrated a significant reduction (5.86- to 14.98-fold) of pVNT against Omicron compared to prototype at 14 days after the homologous or heterologous vaccine boosters.Overall, our study demonstrates that vaccine-induced immune protection might more likely be escaped by Omicron compared to prototypes and other VOCs. After two doses of inactivated whole-virion vaccines as the "priming" shot, a third heterologous protein subunit vaccine and a homologous inactivated vaccine booster could improve neutralization against Omicron.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19 Vaccines/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Adult , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19/prevention & control , COVID-19/virology , COVID-19 Vaccines/administration & dosage , Female , Humans , Immune Sera/immunology , Immunization, Secondary , Immunogenicity, Vaccine , Middle Aged , SARS-CoV-2/genetics , Vaccination
8.
Viruses ; 13(12)2021 12 11.
Article in English | MEDLINE | ID: covidwho-1572660

ABSTRACT

Patients with COVID-19 generally raise antibodies against SARS-CoV-2 following infection, and the antibody level is positively correlated to the severity of disease. Whether the viral antibodies exacerbate COVID-19 through antibody-dependent enhancement (ADE) is still not fully understood. Here, we conducted in vitro assessment of whether convalescent serum enhanced SARS-CoV-2 infection or induced excessive immune responses in immune cells. Our data revealed that SARS-CoV-2 infection of primary B cells, macrophages and monocytes, which express variable levels of FcγR, could be enhanced by convalescent serum from COVID-19 patients. We also determined the factors associated with ADE, and found which showed a time-dependent but not viral-dose dependent manner. Furthermore, the ADE effect is not associated with the neutralizing titer or RBD antibody level when testing serum samples collected from different patients. However, it is higher in a medium level than low or high dilutions in a given sample that showed ADE effect, which is similar to dengue. Finally, we demonstrated more viral genes or dysregulated host immune gene expression under ADE conditions compared to the no-serum infection group. Collectively, our study provides insight into the understanding of an association of high viral antibody titer and severe lung pathology in severe patients with COVID-19.


Subject(s)
Antibody-Dependent Enhancement/immunology , Leukocytes/virology , SARS-CoV-2/pathogenicity , COVID-19/immunology , Cells, Cultured , Gene Expression Profiling , Humans , Immune Sera/immunology , Leukocytes/metabolism , Receptors, IgG/metabolism , Virus Replication/immunology
9.
Nature ; 600(7889): 512-516, 2021 12.
Article in English | MEDLINE | ID: covidwho-1428879

ABSTRACT

The number and variability of the neutralizing epitopes targeted by polyclonal antibodies in individuals who are SARS-CoV-2 convalescent and vaccinated are key determinants of neutralization breadth and the genetic barrier to viral escape1-4. Using HIV-1 pseudotypes and plasma selection experiments with vesicular stomatitis virus/SARS-CoV-2 chimaeras5, here we show that multiple neutralizing epitopes, within and outside the receptor-binding domain, are variably targeted by human polyclonal antibodies. Antibody targets coincide with spike sequences that are enriched for diversity in natural SARS-CoV-2 populations. By combining plasma-selected spike substitutions, we generated synthetic 'polymutant' spike protein pseudotypes that resisted polyclonal antibody neutralization to a similar degree as circulating variants of concern. By aggregating variant of concern-associated and antibody-selected spike substitutions into a single polymutant spike protein, we show that 20 naturally occurring mutations in the SARS-CoV-2 spike protein are sufficient to generate pseudotypes with near-complete resistance to the polyclonal neutralizing antibodies generated by individuals who are convalescent or recipients who received an mRNA vaccine. However, plasma from individuals who had been infected and subsequently received mRNA vaccination neutralized pseudotypes bearing this highly resistant SARS-CoV-2 polymutant spike, or diverse sarbecovirus spike proteins. Thus, optimally elicited human polyclonal antibodies against SARS-CoV-2 should be resilient to substantial future SARS-CoV-2 variation and may confer protection against potential future sarbecovirus pandemics.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Immune Evasion , Immune Sera/immunology , Mutation , SARS-CoV-2/genetics , SARS-CoV-2/immunology , COVID-19/immunology , COVID-19/prevention & control , COVID-19/virology , Convalescence , Cross Reactions , Humans , Neutralization Tests , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
10.
Nat Commun ; 12(1): 4598, 2021 07 26.
Article in English | MEDLINE | ID: covidwho-1327197

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected at least 180 million people since its identification as the cause of the current COVID-19 pandemic. The rapid pace of vaccine development has resulted in multiple vaccines already in use worldwide. The contemporaneous emergence of SARS-CoV-2 'variants of concern' (VOC) across diverse geographic locales underscores the need to monitor the efficacy of vaccines being administered globally. All WHO designated VOC carry spike (S) polymorphisms thought to enable escape from neutralizing antibodies. Here, we characterize the neutralizing activity of post-Sputnik V vaccination sera against the ensemble of S mutations present in alpha (B.1.1.7) and beta (B.1.351) VOC. Using de novo generated replication-competent vesicular stomatitis virus expressing various SARS-CoV-2-S in place of VSV-G (rcVSV-CoV2-S), coupled with a clonal 293T-ACE2 + TMPRSS2 + cell line optimized for highly efficient S-mediated infection, we determine that only 1 out of 12 post-vaccination serum samples shows effective neutralization (IC90) of rcVSV-CoV2-S: B.1.351 at full serum strength. The same set of sera efficiently neutralize S from B.1.1.7 and exhibit only moderately reduced activity against S carrying the E484K substitution alone. Taken together, our data suggest that control of some emergent SARS-CoV-2 variants may benefit from updated vaccines.


Subject(s)
Antibodies, Neutralizing/immunology , COVID-19 Vaccines/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Adult , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/genetics , Female , HEK293 Cells , Humans , Immune Sera/immunology , Male , Middle Aged , Mutation , Neutralization Tests , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/genetics , Vaccination/methods , Vesicular stomatitis Indiana virus/genetics , Vesicular stomatitis Indiana virus/immunology , Virus Internalization/drug effects , Virus Replication/drug effects , Virus Replication/genetics , Virus Replication/immunology
11.
Transfusion ; 61(7): 1998-2007, 2021 07.
Article in English | MEDLINE | ID: covidwho-1258988

ABSTRACT

BACKGROUND: In order to tackle the COVID-19 pandemic, a COVID-19 convalescent plasma (CCP) procurement program was initiated in Japan in April 2020. The program was a collaboration between a government-managed national hospital, an infectious disease research institute, and a blood banking organization. Each party assumed different responsibilities: recruitment, SARS-CoV-2 antibody profiling, and plasmapheresis; conduction of screening tests; and SARS-CoV-2 blood testing, respectively. METHODS: We adopted a two-point screening approach before the collected CCP was labeled as a CCP product for investigational use, for which we mainly tested anti-SARS-CoV-2 antibody eligibility and blood product eligibility. Anti-SARS-CoV-2 spike protein titer was measured using enzyme-linked immunosorbent assay, and the IC50 value was denoted as the neutralizing activity. Blood donor eligibility was extended beyond the normal blood donation guidelines to include a broader range of participants. After both eligibility criteria were confirmed, participants were asked to revisit the hospital for blood donation, which is a unique aspect of the Japanese CCP program, as most donations are taking place in normal blood donation venues in other countries. Some donors were re-scheduled for repeat plasma donations. As public interest in anti-SARS-CoV-2 antibodies increased, test results were given to the participants. RESULTS: As of September 17, 2020, our collection of CCP products was sufficient to treat more than 100 patients. As a result, projects for administration and distribution are also being conducted. CONCLUSIONS: We successfully implemented a CCP procurement scheme with the goal to expand to other parts of the country to improve treatment options for COVID-19.


Subject(s)
Blood Donors , COVID-19/immunology , COVID-19/virology , Convalescence , Immune Sera/immunology , SARS-CoV-2/immunology , Adult , Aged , Antibodies, Viral/blood , Antibodies, Viral/immunology , Blood Preservation , COVID-19/diagnosis , COVID-19/epidemiology , Female , Humans , Immunization, Passive/methods , Japan , Male , Mass Screening , Middle Aged , Pandemics , Plasmapheresis , Young Adult
12.
Mol Cell Proteomics ; 20: 100059, 2021.
Article in English | MEDLINE | ID: covidwho-1087559

ABSTRACT

Antibodies play essential roles in both diagnostics and therapeutics. Epitope mapping is essential to understand how an antibody works and to protect intellectual property. Given the millions of antibodies for which epitope information is lacking, there is a need for high-throughput epitope mapping. To address this, we developed a strategy, Antibody binding epitope Mapping (AbMap), by combining a phage displayed peptide library with next-generation sequencing. Using AbMap, profiles of the peptides bound by 202 antibodies were determined in a single test, and linear epitopes were identified for >50% of the antibodies. Using spike protein (S1 and S2)-enriched antibodies from the convalescent serum of one COVID-19 patient as the input, both linear and potentially conformational epitopes of spike protein specific antibodies were identified. We defined peptide-binding profile of an antibody as the binding capacity (BiC). Conceptually, the BiC could serve as a systematic and functional descriptor of any antibody. Requiring at least one order of magnitude less time and money to map linear epitopes than traditional technologies, AbMap allows for high-throughput epitope mapping and creates many possibilities.


Subject(s)
COVID-19/immunology , Epitope Mapping/methods , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Antibodies, Viral/metabolism , Enzyme-Linked Immunosorbent Assay , Epitopes/metabolism , Escherichia coli Proteins/immunology , High-Throughput Nucleotide Sequencing , Humans , Immune Sera/blood , Immune Sera/immunology , Peptide Library
13.
Science ; 371(6530): 735-741, 2021 02 12.
Article in English | MEDLINE | ID: covidwho-1066809

ABSTRACT

Protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS-related emergent zoonotic coronaviruses is urgently needed. We made homotypic nanoparticles displaying the receptor binding domain (RBD) of SARS-CoV-2 or co-displaying SARS-CoV-2 RBD along with RBDs from animal betacoronaviruses that represent threats to humans (mosaic nanoparticles with four to eight distinct RBDs). Mice immunized with RBD nanoparticles, but not soluble antigen, elicited cross-reactive binding and neutralization responses. Mosaic RBD nanoparticles elicited antibodies with superior cross-reactive recognition of heterologous RBDs relative to sera from immunizations with homotypic SARS-CoV-2-RBD nanoparticles or COVID-19 convalescent human plasmas. Moreover, after priming, sera from mosaic RBD-immunized mice neutralized heterologous pseudotyped coronaviruses as well as or better than sera from homotypic SARS-CoV-2-RBD nanoparticle immunizations, demonstrating no loss of immunogenicity against particular RBDs resulting from co-display. A single immunization with mosaic RBD nanoparticles provides a potential strategy to simultaneously protect against SARS-CoV-2 and emerging zoonotic coronaviruses.


Subject(s)
Antibodies, Viral/immunology , Betacoronavirus/immunology , COVID-19 Vaccines/immunology , Nanoparticles , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , COVID-19/immunology , Coronavirus Infections/immunology , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Female , Immune Sera/immunology , Immunization , Immunoglobulin G/blood , Immunoglobulin G/immunology , Mice , Mice, Inbred BALB C , Neutralization Tests , Protein Domains , Receptors, Antigen, B-Cell/immunology , Spike Glycoprotein, Coronavirus/chemistry , Viral Zoonoses/immunology , Viral Zoonoses/virology
14.
Biochem Biophys Res Commun ; 543: 45-49, 2021 03 05.
Article in English | MEDLINE | ID: covidwho-1053221

ABSTRACT

In order to control the COVID-19 pandemic caused by SARS-CoV-2 infection, serious progress has been made to identify infected patients and to detect patients with a positive immune response against the virus. Currently, attempts to generate a vaccine against the coronavirus are ongoing. To understand SARS-CoV-2 immunoreactivity, we compared the IgG antibody response against SARS-CoV-2 in infected versus control patients by dot blot using recombinant viral particle proteins: N (Nucleocapsid), M (Membrane) and S (Spike). In addition, we used different protein fragments of the N and S protein to map immune epitopes. Most of the COVID-19 patients presented a specific immune response against the full length and fragments of the N protein and, to lesser extent, against a fragment containing amino acids 300-685 of the S protein. In contrast, immunoreactivity against other S protein fragments or the M protein was low. This response is specific for COVID-19 patients as very few of the control patients displayed immunoreactivity, likely reflecting an immune response against other coronaviruses. Altogether, our results may help develop method(s) for measuring COVID-19 antibody response, selectivity of methods detecting such SARS-CoV-2 antibodies and vaccine development.


Subject(s)
COVID-19/immunology , Coronavirus Nucleocapsid Proteins/immunology , SARS-CoV-2/immunology , Coronavirus M Proteins/genetics , Coronavirus M Proteins/immunology , Coronavirus Nucleocapsid Proteins/genetics , Humans , Immune Sera/immunology , Immunity, Humoral , Immunoblotting , Immunoglobulin G/blood , Phosphoproteins/genetics , Phosphoproteins/immunology , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Virion/immunology
15.
Virology ; 556: 73-78, 2021 04.
Article in English | MEDLINE | ID: covidwho-1049897

ABSTRACT

The need to stem the current outbreak of SARS-CoV-2 responsible for COVID-19 is driving the search for inhibitors that will block coronavirus replication and pathogenesis. The coronavirus 3C-like protease (3CLpro) encoded in the replicase polyprotein is an attractive target for antiviral drug development because protease activity is required for generating a functional replication complex. Reagents that can be used to screen for protease inhibitors and for identifying the replicase products of SARS-CoV-2 are urgently needed. Here we describe a luminescence-based biosensor assay for evaluating small molecule inhibitors of SARS-CoV-2 3CLpro/main protease. We also document that a polyclonal rabbit antiserum developed against SARS-CoV 3CLpro cross reacts with the highly conserved 3CLpro of SARS-CoV-2. These reagents will facilitate the pre-clinical evaluation of SARS-CoV-2 protease inhibitors.


Subject(s)
Biosensing Techniques/methods , Coronavirus 3C Proteases/metabolism , Immune Sera/immunology , Luciferases/metabolism , SARS-CoV-2/metabolism , Animals , Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/genetics , Coronavirus 3C Proteases/immunology , Cross Reactions , Luciferases/genetics , Protease Inhibitors/pharmacology , Rabbits , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , SARS Virus/immunology , SARS Virus/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/immunology , Viral Nonstructural Proteins/metabolism , Virus Replication/drug effects
16.
J Clin Microbiol ; 59(2)2021 01 21.
Article in English | MEDLINE | ID: covidwho-1045660

ABSTRACT

Accurate serological assays to detect antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are needed to characterize the epidemiology of SARS-CoV-2 infection and identify potential candidates for coronavirus disease 2019 (COVID-19) convalescent plasma (CCP) donation. This study compared the performances of commercial enzyme immunoassays (EIAs) with respect to detection of IgG or total antibodies to SARS-CoV-2 and neutralizing antibodies (nAbs). The diagnostic accuracy of five commercially available EIAs (Abbott, Euroimmun, EDI, ImmunoDiagnostics, and Roche) for detection of IgG or total antibodies to SARS-CoV-2 was evaluated using cross-sectional samples from potential CCP donors who had prior molecular confirmation of SARS-CoV-2 infection (n = 214) and samples from prepandemic emergency department patients without SARS-CoV-2 infection (n = 1,099). Of the 214 potential CCP donors, all were sampled >14 days since symptom onset and only a minority (n = 16 [7.5%]) had been hospitalized due to COVID-19; 140 potential CCP donors were tested by all five EIAs and a microneutralization assay. Performed according to the protocols of the manufacturers to detect IgG or total antibodies to SARS-CoV-2, the sensitivity of each EIA ranged from 76.4% to 93.9%, and the specificity of each EIA ranged from 87.0% to 99.6%. Using a nAb titer cutoff value of ≥160 as the reference representing a positive test result (n = 140 CCP donors), the empirical area under the receiver operating curve for each EIA ranged from 0.66 (Roche) to 0.90 (Euroimmun). Commercial EIAs with high diagnostic accuracy to detect SARS-CoV-2 antibodies did not necessarily have high diagnostic accuracy to detect high nAb titers. Some but not all commercial EIAs may be useful in the identification of individuals with high nAb titers among convalescent individuals.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19 Serological Testing/methods , COVID-19/diagnosis , SARS-CoV-2/isolation & purification , COVID-19/blood , Cross-Sectional Studies , Humans , Immune Sera/immunology , Immunoenzyme Techniques , Immunoglobulin G/blood , Neutralization Tests , SARS-CoV-2/immunology , Sensitivity and Specificity
17.
J Clin Microbiol ; 59(2)2021 01 21.
Article in English | MEDLINE | ID: covidwho-1043639

ABSTRACT

Surrogate neutralization assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that can be done without biosafety level 3 containment and in multiple species are desirable. We evaluate a recently developed surrogate virus neutralization test (sVNT) in comparison to 90% plaque reduction neutralization tests (PRNT90) in human, canine, cat, and hamster sera. With PRNT90 as the reference, sVNT had sensitivity of 98.9% and specificity of 98.8%. Using a panel of immune sera corresponding to other coronaviruses, we confirm the lack of cross-reactivity to other coronaviruses in SARS-CoV-2 sVNT and PRNT90, except for cross-reactivity to SARS-CoV-1 in sVNT.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19/diagnosis , Neutralization Tests/methods , SARS-CoV-2/isolation & purification , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19/blood , COVID-19/pathology , Cats , Cricetinae , Cross Reactions , Dogs , Female , Humans , Immune Sera/immunology , Male , Neutralization Tests/standards , SARS-CoV-2/immunology , Sensitivity and Specificity
19.
Vaccine ; 38(50): 7892-7896, 2020 11 25.
Article in English | MEDLINE | ID: covidwho-899644

ABSTRACT

There is an urgent need for a safe and protective vaccine to control the global spread of SARS-CoV-2 and prevent COVID-19. Here, we report the immunogenicity and protective efficacy of a SARS-CoV-2 subunit vaccine (NVX-CoV2373) produced from the full-length SARS-CoV-2 spike (S) glycoprotein stabilized in the prefusion conformation. Cynomolgus macaques (Macaca fascicularis) immunized with NVX-CoV2373 and the saponin-based Matrix-M™ adjuvant induced anti-S antibody that was neutralizing and blocked binding to the human angiotensin-converting enzyme 2 (hACE2) receptor. Following intranasal and intratracheal challenge with SARS-CoV-2, immunized macaques were protected against upper and lower infection and pulmonary disease. These results support ongoing phase 1/2 clinical studies of the safety and immunogenicity of NVX-CoV2327 vaccine (NCT04368988).


Subject(s)
COVID-19 Vaccines/pharmacology , COVID-19/prevention & control , SARS-CoV-2/immunology , Adjuvants, Immunologic/pharmacology , Adolescent , Adult , Aged , Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Neutralizing , COVID-19/immunology , COVID-19 Vaccines/genetics , COVID-19 Vaccines/immunology , Chlorocebus aethiops , Female , Humans , Immune Sera/drug effects , Immune Sera/immunology , Macaca fascicularis , Male , Middle Aged , Spike Glycoprotein, Coronavirus/genetics , Vaccines, Synthetic/immunology , Vaccines, Synthetic/pharmacology , Vero Cells , Viral Load , Young Adult
20.
PLoS One ; 15(10): e0241471, 2020.
Article in English | MEDLINE | ID: covidwho-895078

ABSTRACT

Anecdotal evidence showed a negative correlation between Bacille Calmette-Guérin (BCG) vaccination and incidence of COVID-19. Incidence of the disease in children is much lower than in adults. It is hypothesized that BCG and other childhood vaccinations may provide some protection against SARS-CoV-2 infection through trained or adaptive immune responses. Here, we tested whether BCG, Pneumococcal, Rotavirus, Diphtheria, Tetanus, Pertussis, Hepatitis B, Haemophilus influenzae, Hepatitis B, Meningococcal, Measles, Mumps, and Rubella vaccines provide cross-reactive neutralizing antibodies against SARS-CoV-2 in BALB/c mice. Results indicated that none of these vaccines provided antibodies capable of neutralizing SARS-CoV-2 up to seven weeks post vaccination. We conclude that if such vaccines have any role in COVID-19 immunity, this role is not antibody-mediated.


Subject(s)
Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , Betacoronavirus/immunology , Coronavirus Infections/prevention & control , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Vaccines/immunology , Adolescent , Adult , Aged , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19 , Child , Child, Preschool , Coronavirus Infections/immunology , Cross Reactions , Female , Humans , Immune Sera/immunology , Immunogenicity, Vaccine , Mice , Mice, Inbred BALB C , Middle Aged , Neutralization Tests , Pneumonia, Viral/immunology , SARS-CoV-2 , Vaccination , Vaccines, Inactivated/immunology , Viral Vaccines/immunology , Young Adult
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