Our progress in fight against COVID-19 pandemic
Summary of available products
In response to COVID-19 pandemia, we have produced and purified recombinant SARS-CoV-2 antigens from CHO cells for diagnostic use in gram quantities. Multiple grams of N, S1, S2 and RBD proteins will be produced with stable CHO cell lines that are currently under development. These viral proteins are ideal for use as antigens in antibody discovery or as immunological tools to measure the concentration of virus-specific IgG, IgM or IgA antibodies in patient’s blood samples.
Table 1. Antigens currently available for purchase
|Nucleoprotein||Spike S1||RBD||RBD2||Spike S2||ACE2||ACE2-Fc|
|Production cell line||CHO||CHO||CHO||CHO||CHO||CHO||CHO|
|Molecular Weight (kDa)||46.9||76.4||23||26.4||58.9||84.9||110|
|Purity||>90%||> 90%||>95%||>95%||> 90%||>80%||>95%|
|Amino acids||Full length||14-681||331-524||319-541||693-1218||20-742||18-740|
|Binding to ACE2||NA||Yes||Yes||Yes||NA||NA||NA|
|Tested on clinical samples||Yes||Yes||Yes||Yes||Yes||NA||NA|
SARS-CoV-2 is a positive-sense single-stranded RNA virus that can cause severe respiratory illness COVID-19. It is a strain of severe acute respiratory syndrome-related coronavirus, and its 30 kb genome encodes four structural proteins - S (Spike), E (Envelope), M (Membrane) and N (nucleocapsid).
Figure 1. Structure of SARS-CoV-2.
Spike (S) protein is a trimeric viral surface protein consisting of two subunits, S1 and S2. The RBD (receptor binding domain) of S1 subunit binds to the ACE2 receptor. Neutralizing antibody response to the S protein plays an important role in immunity. Homotrimers of the S protein make up the distinctive spike-like structure that is heavily N-glycosylated.
The nucleocapsid protein (N) is responsible for packaging and protecting coronavirus genomic RNA. It is a good target for virus detection as nucleocapsid protein is the most abundant protein of coronavirus and it is highly immunogenic.
Infection cycle of SARS-CoV-2
Figure 2. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) receptor to enter the host cell, where the virus replicates. Virus inserts its RNA into the host cell, where it forces the cell to produce new virus proteins and replicates the RNA genome. Viral proteins are glycosylated and transported to the host cell membrane, where the assembled particles bud from the host cell.
Proteins available, expression system, functional binding and glycosylation
Using our patented QMCF technology and vast experience in protein production, we have produced and purified functional recombinant SARS-CoV-2 antigens from CHO cells for diagnostic use. All proteins go through rigorous testing to ensure the highest quality and similarity to natural protein forms. For Spike S1, RBD and RBD2 binding to ACE-2 has been confirmed using the Octet platform.
Figure 3. ACE2 and different domains of Spike protein available for purchase.
All the antigens are produced in CHO cells, which enables proper glycosylation pattern and thus contributes to protein’s antigenic properties. PNGase F treatment displays the extent of N-glycosylation, where a large mobility shift of N and S proteins is seen when comparing deglycosylated and native forms, indicating that these proteins are N-glycosylated.
Figure 4. Mobility shift between produced antigens before and after deglycosylation treatment with PNGase F.
Figure 5. Comparison of different diagnostic methods.
Diagnostic tests for detecting SARS-CoV-2 infection can be divided into three- nucleic acid analysis, serological antibody identification and virus detection with diagnostic antibodies. We are focused on developing the two latter named diagnostic tools. Nucleic acid analysis is based on identifying RNA of the virus during infection from nasopharyngeal swab samples, while serological tests look for antibodies generated against virus proteins (antigens). Diagnostic antibodies are used to detect viral proteins from nasopharyngeal swab samples. Nucleic acid analysis and diagnostic antibodies are used to detect active infection while serological tests show us if patient has already had the virus.Figure 5. Comparison of different diagnostic methods (JOONIS)
Figure 6. Two types of immunoassay diagnostic tests.We are closely collaborating with Estonian health system to gather clinical samples from patients recovered from COVID-19. Our serological tests are functional and we are in the process of validating with mentioned clinical samples. Clinical samples also provide vital information about the characteristics of immune response against SARS-CoV-2 infection, showing which antigens are targeted more often and which of IgG, IgA and IgM are more common. From patients’ blood samples we have extracted antibodies binding to viral antigens and are currently screening for most effective diagnostic and therapeutic antibodies.
Figure 7. N, S1, S2 and RBD proteins’ ability to capture specific IgG antibodies from clinical samples has been experimentally confirmed with ELISA assay.