Receptor Binding Domain Antigens Research Articles

Plant-Produced Glycosylated and In Vivo Deglycosylated Receptor Binding Domain Proteins of SARS-CoV-2 Induce Potent Neutralizing Responses in Mice.

The COVID-19 pandemic, caused by SARS-CoV-2, has rapidly spread to more than 222 countries and has put global public health at high risk. The world urgently needs cost-effective and safe SARS-CoV-2 vaccines, antiviral, and therapeutic drugs to control it. In this study, we engineered the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein and produced it in the plant Nicotiana benthamiana in a glycosylated and deglycosylated form. Expression levels of both glycosylated (gRBD) and deglycosylated (dRBD) RBD were greater than 45 mg/kg fresh weight. The purification yields were 22 mg of pure protein/kg of plant biomass for gRBD and 20 mg for dRBD, which would be sufficient for commercialization of these vaccine candidates. The purified plant-produced RBD protein was recognized by an S protein-specific monoclonal antibody, demonstrating specific reactivity of the antibody to the plant-produced RBD proteins. The SARS-CoV-2 RBD showed specific binding to angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 receptor. In mice, the plant-produced RBD antigens elicited high titers of antibodies with a potent virus-neutralizing activity. To our knowledge, this is the first report demonstrating that mice immunized with plant-produced deglycosylated RBD form elicited high titer of RBD-specific antibodies with potent neutralizing activity against SARS-CoV-2 infection. Thus, obtained data support that plant-produced glycosylated and in vivo deglycosylated RBD antigens, developed in this study, are promising vaccine candidates for the prevention of COVID-19.

New Impedimetric Sandwich Immunosensor for Ultrasensitive and Highly Specific Detection of Spike Receptor Binding Domain Protein of SARS-CoV-2.

An impedance sensing platform-combined conducting nanocomposite layer was fabricated to develop an effective and rapid method for detection of coronavirus infection (COVID-19) specific spike receptor binding domain (RBD) protein, a precious antigen marker of COVID-19 disease. Coronavirus infection has spread globally and swiftly with major impacts on health, economy, and quality of life of communities. Fast and reliable detection of COVID-19 is a very significant issue for the effective treatment of this bad illness. For this aim, first, an Epoxy functional group-substituted thiophene monomer was synthesized and electrodeposited on a single-use indium tin oxide (ITO) platform in the presence of acetylene black by employing a cyclic voltammetry technique; thus, a conducting nanocomposite (C-NC) layer with high conductivity was obtained. This composite was electrodeposited for the first time on the ITO surface to generate a facile and cost-effective impedimetric biosensor. In addition, this composite provided proper attachment points for antibody binding and also supported the biosensor construction. The immuno-specific biointeractions between anti-RBD and RBD proteins hampered the electron transfer between the ITO substrate surface and electrolyte, and this reaction caused variations in impedance signals, and these signals were proportional to the immobilized RBD antigen amounts. The as-prepared immunosensor showed a wide linear dynamic range (0.0012-120 pg/mL), an ultra-low detection limit of 0.58 fg/mL with added superiorities of great selectivity, suitable repeatability, multiple reusability, and excellent reproducibility.

Longitudinal Serology of SARS-CoV-2-Infected Individuals in India: A Prospective Cohort Study.

.Clinical and epidemiological characteristics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are now widely available, but there are few data regarding longitudinal serology in large cohorts, particularly those from low-income and middle-income countries. We established an ongoing prospective cohort of 3,840 SARS-CoV-2-positive individuals according to RT-PCR in the Delhi-National Capital Region of India to document clinical and immunological characteristics during illness and convalescence. The immunoglobulin G (IgG) responses to the receptor binding domain (RBD) and nucleocapsid were assessed at 0 to 7 days, 10 to 28 days, and 6 to 10 weeks after infection. The clinical predictors of seroconversion were identified by multivariable regression analysis. The seroconversion rates during the postinfection windows of 0 to 7 days, 10 to 28 days, and 6 to 10 weeks were 46%, 84.7%, and 85.3%, respectively (N = 743). The proportion with a serological response increased with the severity of coronavirus disease 2019 (COVID-19). All participants with severe disease, 89.6% with mild to moderate infection, and 77.3% of asymptomatic participants had IgG antibodies to the RBD antigen. The threshold values for the nasopharyngeal viral RNA RT-PCR of a subset of asymptomatic and symptomatic seroconverters were comparable (P = 0.48) to those of nonseroconverters (P = 0.16) (N = 169). This is the first report of longitudinal humoral immune responses to SARS-CoV-2 over a period of 10 weeks in South Asia. The low seropositivity of asymptomatic participants and differences between assays highlight the importance of contextualizing the understanding of population serosurveys.

Combinations of alum and synthetic toll-like receptor agonists as adjuvants for CoVID-19 vaccines

Abstract Aluminum-based adjuvants (“alum”) are the most widely-used adjuvants for human vaccines, with an extensive history of safety and efficacy. Alum is known to elicit high antibody titers by driving a Th2-based immune response, which may not be optimal for many viral infections, such as SARS-CoV-2. Therefore, combining the strong safety and efficacy attributes of alum with a Th1-polarizing adjuvant could improve immunity against viral antigens. Here we test the combination of alum with synthetic TLR4- and TLR7/8 -ligands in vaccines against SARS-CoV-2. The combination of alum and TLR agonists resulted in efficient adjuvantation of both humoral and cell-mediated immunity. Alum adsorption studies showed low association between the small, positively-charged receptor binding domain (RBD) antigen and aluminum hydroxide (Alhydrogel) and high adsorption to negatively charged aluminum phosphate (Adju-phos). However, Adju-phos and Alhydrogel both enhanced immunity to the SARS-CoV-2 RBD antigen, suggesting antigen adsorption may not be required for the immune enhancing effects of alum. These data demonstrated that adjuvants combining alum with a TLR agonist result in an improved anti-viral immune response. However, the ability of alum to adsorb to an antigen may not predict its ability to effectively adjuvant.

Development of a quantitative COVID-19 Multiplex Assay and its use for serological surveillance in a low SARS-CoV-2 incidence community

Abstract A serological COVID-19 Multiplex Assay was developed and validated using serum sample sets from convalescent patients and those collected prior to the onset of the 2020 pandemic. After initial testing of multiple potential antigens expressed in a mammalian system, the SARS-CoV-2 nucleoprotein (NP) and receptor-binding domain (RBD) of the spike protein were selected for the final human COVID-19 Multiplex Assay. A comparison of synthesized and mammalian expressed RBD proteins revealed clear advantages of the mammalian protein. Antibodies directed against NP strongly correlated with SARS-CoV-2 virus neutralization assay titers (rsp = 0.726), while the anti-RBD correlation was moderate (rsp = 0.436). Pan-Ig, IgG, IgA, and IgM against the NP and RBD antigens were evaluated on the validation sample sets. Detection of NP and RBD specific IgG and IgA had outstanding performance (AUC>0.90) for distinguishing patients from controls, but the dynamic range of the IgG assay was substantially greater than for IgA. The COVID-19 Multiplex Assay was then utilized to identify seroprevalence to SARS-CoV-2 in people living in a low-incidence community in Ithaca NY. Samples were taken from a cohort of healthy volunteers (n=332) in early June 2020. Only two volunteers had a positive result on a COVID-19 PCR test performed prior to serum sampling. Serological testing revealed an exposure rate of at least 1.2% (NP) or as high as 5.1% (RBD), which was higher than the measured incidence rate of 0.16% in the county at that time. This highly sensitive and quantitative assay will be useful for continuing to monitor community exposure rates, duration of immune response following infection, and monitoring vaccination responses as vaccines become widely available.

Production of high‐quality SARS‐CoV‐2 antigens: Impact of bioprocess and storage on glycosylation, biophysical attributes, and ELISA serologic tests performance

Serological assays are valuable tools to study SARS‐CoV‐2 spread and, importantly, to identify individuals that were already infected and would be potentially immune to a virus reinfection. SARS‐CoV‐2 Spike protein and its receptor binding domain (RBD) are the antigens with higher potential to develop SARS‐CoV‐2 serological assays. Moreover, structural studies of these antigens are key to understand the molecular basis for Spike interaction with angiotensin converting enzyme 2 receptor, hopefully enabling the development of COVID‐19 therapeutics. Thus, it is urgent that significant amounts of this protein became available at the highest quality. In this study, we produced Spike and RBD in two human derived cell hosts: HEK293‐E6 and Expi293F™. We evaluated the impact of different and scalable bioprocessing approaches on Spike and RBD production yields and, more importantly, on these antigens' quality attributes. Using negative and positive sera collected from human donors, we show an excellent performance of the produced antigens, assessed in serologic enzyme‐linked immunosorbent assay (ELISA) tests, as denoted by the high specificity and sensitivity of the test. We show robust Spike productions with final yields of approx. 2 mg/L of culture that were maintained independently of the production scale or cell culture strategy. To the best of our knowledge, the final yield of 90 mg/L of culture obtained for RBD production, was the highest reported to date. An in‐depth characterization of SARS‐CoV‐2 Spike and RBD proteins was performed, namely the antigen's oligomeric state, glycosylation profiles, and thermal stability during storage. The correlation of these quality attributes with ELISA performance show equivalent reactivity to SARS‐CoV‐2 positive serum, for all Spike and RBD produced, and for all storage conditions tested. Overall, we provide straightforward protocols to produce high‐quality SARS‐CoV‐2 Spike and RBD antigens, that can be easily adapted to both academic and industrial settings; and integrate, for the first time, studies on the impact of bioprocess with an in‐depth characterization of these proteins, correlating antigen's glycosylation and biophysical attributes to performance of COVID‐19 serologic tests.

Accurate SARS-CoV-2 seroprevalence surveys require robust multi-antigen assays

There is a plethora of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) serological tests based either on nucleocapsid phosphoprotein (N), S1-subunit of spike glycoprotein (S1) or receptor binding domain (RBD). Although these single-antigen based tests demonstrate high clinical performance, there is growing evidence regarding their limitations in epidemiological serosurveys. To address this, we developed a Luminex-based multiplex immunoassay that detects total antibodies (IgG/IgM/IgA) against the N, S1 and RBD antigens and used it to compare antibody responses in 1225 blood donors across Greece. Seroprevalence based on single-antigen readouts was strongly influenced by both the antigen type and cut-off value and ranged widely [0.8% (95% CI 0.4–1.5%)–7.5% (95% CI 6.0–8.9%)]. A multi-antigen approach requiring partial agreement between RBD and N or S1 readouts (RBD&N|S1 rule) was less affected by cut-off selection, resulting in robust seroprevalence estimation [0.6% (95% CI 0.3–1.1%)–1.2% (95% CI 0.7–2.0%)] and accurate identification of seroconverted individuals.

Engineering a self-navigated MnARK nanovaccine for inducing potent protective immunity against novel coronavirus.

Effective vaccines are vital to fight against the COVID-19 global pandemic. As a critical component of a subunit vaccine, the adjuvant is responsible for strengthening the antigen-induced immune responses. Here, we present a new nanovaccine that comprising the Receptor-Binding Domain (RBD) of spike protein and the manganese nanoadjuvant (MnARK), which induces humoral and cellular responses. Notably, even at a 5-fold lower antigen dose and with fewer injections, the MnARK vaccine immunized mice showed stronger neutralizing abilities against the infection of the pseudovirus (~270-fold) and live coronavirus (>8-fold) in vitro than that of Alum-adsorbed RBD vaccine (Alu-RBD). Furthermore, we found that the effective co-delivery of RBD antigen and MnARK to lymph nodes (LNs) elicited an increased cellular internalization and the activation of immune cells, including DCs, CD4+ and CD8+ T lymphocytes. Our findings highlight the importance of MnARK adjuvant in the design of novel coronavirus vaccines and provide a rationale strategy to design protective vaccines through promoting cellular internalization and the activation of immune-related pathways.

Abstract P23: Detection of humoral and cellular responses to SARS-CoV-2 in COVID-19 convalescents

Abstract Introduction: The emergence of SARS-CoV-2 virus, which causes COVID-19, is a major global health hazard. Therefore, a comprehensive characterization of the humoral and cellular immune responses to this virus is essential to combat the COVID-19 pandemic. Our goal was to develop reliable methods and tools for the analysis of humoral and cellular B- and T- cell responses, which will facilitate scientific research for prediction of disease progression, long-term immunity and will support vaccine development. Methods: Plasma samples and PBMCs of COVID-19 convalescent and healthy donors were obtained. For the detection of SARS-CoV-2 specific antibodies and identification of antigen-specific B cells, we manufactured recombinant mono-biotinylated protein variants of the Spike (S), Receptor Binding Domain (RBD) and Nucleoprotein (N). To identify antigen-reactive T cells, SARS-CoV-2 peptide pools were synthetized for the S, N and Membrane (M) antigens and used for stimulation. The peptide pools consist of mainly 15-mer peptides having an 11-mer amino acid overlap and thereby overspan a whole protein sequence. Results: To determine the presence of SARS-CoV-2 reactive antibodies a flow-based bead assay using recombinant, mono-biotinylated SARS-CoV-2 antigens loaded onto Streptavidin (SAV)-coated-PMMA beads was set up. The beads were incubated with plasma samples and fluorochrome conjugated anti-human isotype specific antibodies for flow cytometric analysis. All the antigens tested were shown to be suitable for the detection of antibodies to SARS-CoV-2 in COVID-19 convalescent plasma. To assess the feasibility of recombinant antigens for the detection and isolation of antigen-specific B cells, the mono-biotinylated Spike and RBD antigens were tetramerized on fluorochrome-conjugated SAV. These tetramers were used for staining, magnetic enrichment and flow cytometric sorting of B cells specific to SARS-CoV-2 antigens. We were able to demonstrate that our recombinant antigens can be used to assess the presence and enable the phenotyping and isolation of rare antigen-specific B cells. For further characterization of the SARS-CoV-2 reactive T cell immunity PBMCs were short term stimulated with the S, M and N peptide pools. After intracellular staining of IFNg, TNFa, IL-2 and CD154, reactive T cells were detected using flow cytometry. We could demonstrate T cell reactivity towards each peptide pool. However, strengths of T cell responses towards the S, M and N peptide pools were heterogeneous between different COVID-19 convalescent individuals. Conclusion: To support and improve current research activities for the identification and characterization of SARS-CoV-2 reactive humoral and cellular B- and T- cell responses, potent tools and assays were developed. Described here research solutions offer the opportunity to successfully address and contribute to the investigation on healthy and dysfunctional immune reactions towards SARS-CoV-2. Citation Format: Anna Baranska, Marc Schuster, Marek Wieczorek, Sebastian Flade, Lennart Wessels, Burhan Karaca, Verena Traska, Mario Assenmacher, Gregor Winkels. Detection of humoral and cellular responses to SARS-CoV-2 in COVID-19 convalescents [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2021 Feb 3-5. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(6_Suppl):Abstract nr P23.

Capsid-like particles decorated with the SARS-CoV-2 receptor-binding domain elicit strong virus neutralization activity

The rapid development of a SARS-CoV-2 vaccine is a global priority. Here, we develop two capsid-like particle (CLP)-based vaccines displaying the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. RBD antigens are displayed on AP205 CLPs through a split-protein Tag/Catcher, ensuring unidirectional and high-density display of RBD. Both soluble recombinant RBD and RBD displayed on CLPs bind the ACE2 receptor with nanomolar affinity. Mice are vaccinated with soluble RBD or CLP-displayed RBD, formulated in Squalene-Water-Emulsion. The RBD-CLP vaccines induce higher levels of serum anti-spike antibodies than the soluble RBD vaccines. Remarkably, one injection with our lead RBD-CLP vaccine in mice elicits virus neutralization antibody titers comparable to those found in patients that had recovered from COVID-19. Following booster vaccinations, the virus neutralization titers exceed those measured after natural infection, at serum dilutions above 1:10,000. Thus, the RBD-CLP vaccine is a highly promising candidate for preventing COVID-19.

The Prevalence of Antibodies to SARS-CoV-2 Among Blood Donors in China

BACKGROUND: The prevalence of antibodies to SARS-CoV-2 among blood donors in China remains unknown. To reveal the missing information, we investigated the seroprevalence of SARS-CoV-2 antibodies among blood donors in the cities of Wuhan, Shenzhen and Shijiazhuang of China. STUDY DESIGN AND METHODS: During January to April 2020, 42,794 samples from 38,144 healthy blood donors in the three cities were tested for total antibody (TAb) against SARS-CoV-2. Pseudotype SARS-CoV-2 neutralization tests (ppNAT) were further performed for all TAb-positive samples to confirm the antibody presence. The IgG against viral nucleocapsid protein (IgG-N) and receptor-binding domain (RBD) of the spike protein (IgG-RBD), and IgM against RBD antigen were also evaluated. Risk factors associating with SARS-CoV-2 seropositivity were analyzed. RESULTS: A total of 519 samples from 410 donors were confirmed by neutralization tests. The SARS-CoV-2 seroprevalence among blood donors was 2·29% (407/17,794, 95%CI: 2·08%-2·52%) in Wuhan, 0·029% (2/6,810, 0·0081%-0·11% in Shenzhen, and 0·0074% (1/13,540, 95%CI: 0·0013%-0·042%) in Shijiazhuang, respectively. The earliest emergence of SARS-CoV-2 seropositivity in blood donors was identified on January 20, 2020 in Wuhan. The weekly prevalence of SARS-CoV-2 antibodies in Wuhan’s blood donors changed dynamically, and were 0·08% (95%CI: 0·02%-0·28%) during January 15-22 (before city lockdown), 3·08% (95%CI: 2·67%-3·55%) during January 23 to April 7 (city quarantine period) and 2·33% (95%CI: 2·06%-2·63%) during April 8-30 (after lockdown easing). Female and older-age were identified to be independent risk factors for SARS-CoV-2 seropositivity among donors in Wuhan. CONCLUSION: The prevalence of antibodies to SARS-CoV-2 among blood donors in China was low, even in Wuhan city. According to our data, the earliest emergence of SARS-CoV-2 in Wuhan’s donors should not earlier than January, 2020. As most of population of China remained uninfected during the early wave of COVID-19 pandemic, effective public health measures are still certainly required to block viral spread before a vaccine is widely available.FUNDING STATEMENT: None. The research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors.DECLARATION OF INTERESTS: The authors declare no competing interests. ETHICS APPROVAL STATEMENT: This study was reviewed and approved by the Medical Ethical Committee of Beijing Hospital (2020BJYYEC‐070‐01) and written informed consent was obtained from each enrolled donor before donation.

The receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in SARS-CoV-2 patients.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that first emerged in late 2019 is responsible for a pandemic of severe respiratory illness. People infected with this highly contagious virus can present with clinically inapparent, mild, or severe disease. Currently, the virus infection in individuals and at the population level is being monitored by PCR testing of symptomatic patients for the presence of viral RNA. There is an urgent need for SARS-CoV-2 serologic tests to identify all infected individuals, irrespective of clinical symptoms, to conduct surveillance and implement strategies to contain spread. As the receptor binding domain (RBD) of the spike protein is poorly conserved between SARS-CoVs and other pathogenic human coronaviruses, the RBD represents a promising antigen for detecting CoV-specific antibodies in people. Here we use a large panel of human sera (63 SARS-CoV-2 patients and 71 control subjects) and hyperimmune sera from animals exposed to zoonotic CoVs to evaluate RBD's performance as an antigen for reliable detection of SARS-CoV-2-specific antibodies. By day 9 after the onset of symptoms, the recombinant SARS-CoV-2 RBD antigen was highly sensitive (98%) and specific (100%) for antibodies induced by SARS-CoVs. We observed a strong correlation between levels of RBD binding antibodies and SARS-CoV-2 neutralizing antibodies in patients. Our results, which reveal the early kinetics of SARS-CoV-2 antibody responses, support using the RBD antigen in serological diagnostic assays and RBD-specific antibody levels as a correlate of SARS-CoV-2 neutralizing antibodies in people.

Progress and Concept for COVID-19 Vaccine Development.

The recent outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), previously known by the provisional name 2019 novel coronavirus (2019-nCoV), in the city of Wuhan in China's Hubei province in 2019–2020 has been causing significant numbers of mortality and morbility in humans with the coronavirus infection diseases (COVID-19) with fever, severe respiratory illness, and pneumonia.[1-3] Till April 8, 2020, there have been over 1 431 973 confirmed cases globally, leading to at least 82 085 deaths. These SARS-CoV-2 isolates belong to the Betacoronavirus genus of the Coronaviradae family which is an enveloped single-stranded RNA virus containing a 30 kb genome with 14 open reading frames including four major viral structure proteins: spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins.[4-7] The S gene sequences of SARS-CoV-2 isolates have a 93.1% nucleotide sequence identity to the Rhinolophus affinis bat coronavirus RaTG13, but only less than 75% nucleotide sequence identity to the severe acute respiratory syndrome coronavirus (SARS-CoV). The viral S sequences of SARS-CoV-2 compared to SARS-CoV have three additional short insertions in the N-terminal domain, and four out of five key residues changes in the receptor-binding motif of S protein receptor binding domain (RBD).[6, 7] Although both SARS-CoV-2 and SARS-CoV share the same human cellular receptor-angiotensin converting enzyme II, SARS-CoV-2 appears to be more readily transmitted from human to human.[1, 8, 9] The S protein is the major target for COVID-19 vaccine development, mainly based on the elicitation of virus neutralizing antibodies as the immune correlates to vaccine protection. The current status of COVID-19 vaccine development includes, i) three phase I vaccine candidates, ii) 11 preclinical vaccine candidates, and iii) 26 research-stage vaccine candidates (Table 1; [https://www.raps.org/news-and-articles/news-articles/2020/3/covid-19-vaccine-tracker?feed=Regulatory-Focus?utm_source=Facebook&utm_medium=social&utm_campaign=Regulatory-Focus]). Most of these vaccine candidates are based on the S antigen either as inactivated vaccines, subunit vaccines, viral vectored vaccines, and nucleic acid-based DNA or mRNA vaccines. Among these vaccine candidates, the Coalition for Epidemic Preparedness Innovations (CEPI) has provided funding to develop COVID-19 vaccines using the following platform technology: a) Curevac Inc. (mRNA), b) Inovio Pharmaceuticals Inc. (DNA), c) Moderna, Inc. (mRNA), d) University of Queensland (molecular clam), e) Novavax (nanoparticles), f) University of Oxford (adenovirus vector), g) University of Hong Kong (live-attenuated influenza virus), and h) Institute of Pasteur (measles vector) to accelerate the development of vaccines and enable equitable access to these vaccines for people during outbreaks [https://cepi.net/covid-19/]. Phase I NCT04283461 Phase I ChiCTR2000030906 Phase I NCT04336410 Re-purposed SARS vaccine; S1 or RBD protein vaccine To date, many previous studies of SARS-CoV, Middle East respiratory syndrome-related coronavirus (MERS-CoV), and other coronavirus vaccines revealed several safety concerns associated with the use of coronavirus S-based vaccines, including inflammatory and immunopathological effects such as pulmonary eosinophilic infiltration and antibody-dependent disease enhancement (ADE) following subsequent viral challenge of vaccinated animals.[10-21] The anti-S antibodies for ADE may facilitate uptake by macrophage expressing FcR, leading to macrophage stimulation and the production of proinflammatory cytokines (IL-6, IL-8, and MCP1) and loss of tissue-repaired cytokine (TGFβ).[22] Moreover, the Th2-associated immunopathology has been documented for the inactivated vaccines of respiratory syncytial virus after viral challenge[23-25] and the inactivated vaccines of MERS-CoV after virus challenge.[20] Thus, the safety and the potentially harmful responses in vaccines to develop ADE antibodies against any coronaviruses should be carefully assessed in human trials.[26] It has been proposed that the neutralizing epitope-rich S1 region, or the RBD region, instead of the entire full-length S protein as an alternative target for MERS-CoV vaccine development.[27] Whether the use of S1 or RBD antigen of SARS-CoV-2, or the selection of Th1-skewed adjuvants rather than alum adjuvant, can avoid the inflammatory, immunopathological, and ADE effects, requires further studies from animal models and human trials. These findings are particularly important for developing a safe and effective COVID-19 vaccine. Suh-Chin Wu This work was supported by the Ministry of Science and Technology, Taiwan (MOST108-2321-B-007-001, MOST108-2321-B-002-006), and National Tsing Hua University (109R2807E1). The author declares no conflict of interest.

Connecting clusters of COVID-19: an epidemiological and serological investigation

BackgroundElucidation of the chain of disease transmission and identification of the source of coronavirus disease 2019 (COVID-19) infections are crucial for effective disease containment. We describe an epidemiological investigation that, with use of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological assays, established links between three clusters of COVID-19.MethodsIn Singapore, active case-finding and contact tracing were undertaken for all COVID-19 cases. Diagnosis for acute disease was confirmed with RT-PCR testing. When epidemiological information suggested that people might have been nodes of disease transmission but had recovered from illness, SARS-CoV-2 IgG serology testing was used to establish past infection.FindingsThree clusters of COVID-19, comprising 28 locally transmitted cases, were identified in Singapore; these clusters were from two churches (Church A and Church B) and a family gathering. The clusters in Church A and Church B were linked by an individual from Church A (A2), who transmitted SARS-CoV-2 infection to the primary case from Church B (F1) at a family gathering they both attended on Jan 25, 2020. All cases were confirmed by RT-PCR testing because they had active disease, except for A2, who at the time of testing had recovered from their illness and tested negative. This individual was eventually diagnosed with past infection by serological testing. ELISA assays showed an optical density of more than 1·4 for SARS-CoV-2 nucleoprotein and receptor binding domain antigens in titres up to 1/400, and viral neutralisation was noted in titres up to 1/320.InterpretationDevelopment and application of a serological assay has helped to establish connections between COVID-19 clusters in Singapore. Serological testing can have a crucial role in identifying convalescent cases or people with milder disease who might have been missed by other surveillance methods.FundingNational Research Foundation (Singapore), National Natural Science Foundation (China), and National Medical Research Council (Singapore).

Establishment and evaluation of receptor binding domain (RBD)-based ELISA for Middle East respiratory syndrome coronavirus (MERS-CoV) antibody detection

Objective To establish an indirect enzyme-linked immunosorbent assay (ELISA)and to compare the efficiency of receptor binding domain (RBD) proteins in different forms for Middle East respiratory syndrome coronavirus (MERS-CoV) antibody detection. Methods The monomeric and trimeric forms of MERS-CoV RBD were expressed in Bac-insect cells, 293T cells and ExpiCHO-S™ expression system and then purified. The purified RBD proteins were identified with native gel electrophoresis and Western blot. Then, an equal amount of each RBD protein was used as coating antigen to establish an ELISA for detecting MERS-CoV IgG titer. For comparison, the newly developed ELISA and the commercial MERS-CoV IgG antibody detection kit (Euroimmune with S1 as the coating antigen) were used to measure the MERS-CoV antibody reference panel supplied by World Health Organization (WHO). Results The purified monomeric and trimeric MERS-CoV RBD were successfully prepared using 293T cells and ExpiCHO-S™ system. RBD antigens of different forms and from different systems could recognize MERS-CoV specific antibody without having any cross reaction with the sera from healthy adults. The in-house RBD-based ELISA had good detection consistency with the Euroimmune commercial kit. The positive samples showed higher and more concentrated values based on the RBD trimer than the monomer. Conclusions Novel indirect ELISA methods based on the monomeric and trimeric forms of RBD protein were established. The trimetric form-based ELISA achieved higher detection efficiency than the one using the monomer antigen, suggesting that it could be uses as a competent alternative to the commercial kit. Key words: Middle East respiratory syndrome coronavirus (MERS-CoV); Receptor binding domain (RBD); Antibody; Enzyme-linked immunosorbent assay (ELISA)