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    SARS-CoV-2 cysteine-like protease (Mpro) for serology assays


    To date, the vast majority of the serological test for SARS-CoV-2 specific antibodies are based on serum detection of antibodies to either the viral spike glycoproteins (the major target for neutralising antibodies) or the viral nucleocapsid protein that are known to be highly immunogenic in other coronaviruses. However, other non-structural viral proteins not incorporated into the infection viral particle, specifically the viral cysteine-like protease, have demonstrated to be potent immunogens.

    Mpro is a potent antigen in infected patients that can be used in serological tests.

    Although this protein is not exposed in the viral particle, Mpro carries out a critical role in viral replication. Like other beta-coronaviruses, SARS-CoV-2 is a positive-sense RNA virus that expresses all of its proteins as a single polypeptide chain and Mpro cleaves the 1ab polyprotein to yield the rest of the mature proteins of the virus. Since this activity is essential for the viral life cycle, Mpro structure and function has been studied intensively. In particular, Mpro has been suggested as a target for specific inhibitors that might act as potent anti-viral agents.

    Mpro-Specific antibodies can be detected in serum of COVID-19 patients with high specificity and sensitivity.

    According to the study carried out by CSIC in collaboration with the Immunostep, coronavirus-infected individuals generate an antibody response against the Cys-like protease (Mpro).
    In this study other SARs-CoV-2 proteins, commonly used in serology test were used for comparison and Mpro and NP were expressed in E coli and two different constructs of the Receptor Binding Domain (RBD) of the spike proteins also were used.

    36 covid-19 patients with positive PCR and 33 healthy donors were recruited and ELISA assays were performed to detect Mpro as well as RBD and Nucleocapsid specific antibodies of the IgG, IgA and IgM subclasses in sera. As a result of this research, it could be observed that the specificity and sensitivity was above 90% for detection of IgG antibodies of the three proteins tests, with values of sensitivity and specificity for Mpro of 97% and 100% respectively. Further analysis showed a trend for higher titre antibody responses in patients with more severe disease, being more pronounce for IgM against Mpro and RBD.

    Figure 1 : Infographic and heat map showing how ELISA Mpro Assay provides outstanding values of sensitivity and specificity enabling  to confirm patients with recent or prior infection and open new immunological questions about the heterogeneity of humoral response against the viral proteins between patients and the potential use of Mpro such as a prognostic indicator.

    An assay to detect IgA antibodies against the main SARS-CoV-2 protease “Mpro” by ELISA.

    There are five different classes of human antibodies, also called immunoglobulins. Of these immunoglobulins, three (IgG, IgM, and IgA) are commonly used in serological tests for the diagnosis of different diseases. IgA is present in high concentrations in the mucous membranes, particularly in the internal walls of the respiratory and gastrointestinal tracts, as well as in saliva and tears, and is in fact the most important immunoglobulin for fighting infectious pathogens in the respiratory and digestive system at the point of entry of the pathogen. The detection of specific IgA in the serum of patients infected with SARS-CoV-2 has been widely described, making its appearance earlier, stronger and more persistent than IgM. In general, the average time of IgA, IgM and IgG seroconversion has been reported to be 4-6, 4-6 and 5-10 days after the onset of symptoms, respectively6. These data suggest that IgA detection can improve the diagnostic outcome in the early stages of infection and make them good potential markers for identifying patients with COVID-19.

    As we described above, the vast majority of serological tests for specific antibodies to SARS-CoV-2 are based on serum detection of antibodies to either the S protein (the main target of neutralizing antibodies) or the N protein, which is known to be highly immunogenic protein in other coronaviruses and is the most widely used.  In this sense, in an attempt to increase the diagnostic possibilities of COVID-19 patients, at Immunostep we developed this assay that is based on the detection of specific antibodies against this main viral protease (Mpro or 3CLpro), which plays a critical role in viral replication. (Anti-SARS-CoV-2 Immunostep Mpro ELISA Assay)

    The kit contains microtiter plates upholstered with the recombinant protein Mpro. In a first step, the patient’s diluted sample is incubated in the wells, allowing specific antigen antibodies to bind to the Mpro protein. After washing the wells, to remove unbound antibodies, in a second incubation step, an anti-human immunoglobulin (IgA) antibody conjugated to horseradish peroxidase (HRP) is added. After a second washing step, the substrate tetramethylbenzidine (TMB) is added causing the enzyme-conjugated to the anti-IgA antibody catalyzes a color reaction. The color intensity of the reaction is proportional to the amount of antigen-specific antibodies present in the sample.

    Figure 2: Graphic representation of the principle and method of ELISA Assays.

    Mpro Can be detected to differentiate immune response profiles.

    According to another study carried out by CSIC in collaboration with Immunostep, the use of the antigens previously mentioned (RBD, S and N protein) in combination with the immunogenic Mpro can more fully describe the magnitude and duration of the immune response in SARS-CoV-2-infected patients. In order to facilitate comprehensive characterization of COVID-19 patients with a high throughput approach, a multi-antigen assay was developed with several viral antigens immobilised on fluorescent beads, to allow flow cytometry detection of the multiple antibodies generated during SARS-CoV-2 infections.

    As the pandemic has advanced, it has been established that not all the patients respond in the same manner to the infection by SARS-CoV-2. In fact, a large body of clinical manifestations have been described and it has been suggested that different types of immune response may contribute to these different presentations. Therefore, it will likely be relevant to characterise potentially biased antibody responses when exploring the association between SARS CoV 2 infection and different clinical manifestations.

    As a result of this research, we could observe differences in the specific Ig responses against the four antigens allows easy discrimination between vaccinated and naturally infected individuals (Anti-SARS-CoV-2 Immunostep Multiplex Assay). Further, machine learning analysis allowed classification of patients and healthy controls, without any error just using IgG data. Thus, a simple multiantigen serological test clearly discriminates between patients and healthy controls with a 100% confidence. Modification of the algorithm to take Ig isotype data into account also allowed high confidence discrimination between mild and severe presentations of COVID-19 disease, at least retrospectively.

    Figure 3: SARS-CoV-2 multiplex assay vaccinated samples data compared with positive and negative samples.

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