Effect of SARS-CoV-2 Spike protein glycosylation on the activation of the complement system.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) causes severe pandemic COVID-19. When activation of
the innate immune system fails to eliminate pathogen or to induce adequate adaptive response, persistent
inflammation, manifesting by cytokine storm, ARDS and organ failure may develop. The Spike glycoprotein (S-gp) is a
glycosylated trimeric protein protruding from the viral surface, highly homologous with that of SARS-CoV-1. The
complement system is a major branch of immune response, cross-talking with various mechanisms of immunity, both
innate and adaptive. We hypothesize that SARS-CoV-2 S-gp exposed N-glycans may be recognized by complement lectin
pathway (LP)-activating collectins and/or ficolins – pattern recognition molecules with affinity to carbohydrates. We
based our hypothesis on published data, concerning SARS-CoV-1 and SARS-CoV-2/COVID-19: 1. SARS-CoV-1 S-gp is as
target for mannose-binding lectin (MBL) and surfactant protein D (SP-D) 2. Glycosylation site of S-gp at Asn343 is
essential for MBL binding 3. Data from murine SARS-CoV-1 model showed a crucial role of the complement in the
development of cytokine shock and ARDS 4. Complement activation contributes to the COVID-19 and higher MBL serum
levels are associated with severe adverse effects in patients 5. Inhibition of complement (also with LP-inhibitors gives
promising effects of treatment 6. High expression of MBL and deposition of MASP-2 in lung tissues from COVID-19
patients.
Interaction of complement with S-gp may be essential for clearance of infection but on the other hand, it may hinder
the access of neutralizing antibodies to the viral epitopes. Furthermore, complement over-activation may lead to lifethreatening
events. To determine the role of S-gp glycosylation pattern in the interaction with these factors, we intend
to test the binding of recombinant and natural (complexed with MASP serine proteases, able to activate complement)
lectins MBL, collectin liver-1 (CL-L1), collectin kidney-1 (CL-K1) and ficolins: ficolin-1, -2, -3. to recombinant SARS-CoV-2
S-gp and its glycosylation variants, as well as investigate whether lectin-glycoprotein interaction leads to complement
activation. Furthermore, complement non-activating collectin SP-D will be included. We plan to express five
glycoproteins: 1. wild-type SARS-CoV-2 S-gp; 2. S-gp only with high-mannose N-glycans; In addition, to examine the role
of particular N-glycans, we will express 3 variants substitution at N-glycosylation sites: Asn343; Asn234; Asn801 as well
as 3. wild-type SARS-CoV-1 S-gp (control). These products will be used for analysis of glycopeptides and glycans derived
from the S-gp and its glycosylation variants, and for evaluation of the role of N-glycans in SARS-CoV-2 recognition by
selected lectins and SARS-CoV-2 specific antibodies.
Although recombinant antigens are widely used in vaccines/diagnostics, to confirm the functionality of our preparations,
we will test their reactivity with commercially available anti-S-gp antibodies as well as with antibodies present in sera
from patients diagnosed with paediatric inflammatory multisystem syndrome – temporally associated with SARS-CoV-2
(PIMS-TS). The usage of such sera will allow also determination of S-gp-specific antibody profile in patients and to
investigate an interplay between complement and antibodies. We intend to answer the following questions: 1. Which
PRM, specific for the lectin pathway (or SP-D) bind SARS-CoV-2 S-gp glycans? 2. What is the molecular basis of such
interaction? 3. Does it lead to complement activation? 4. Does it inhibit specific/cross-reacting antibody binding? 5. Do
antibodies raised in response to SARS-CoV-2 infection modify interaction of lectins with S-gp? 6. Are investigated factors
involved in pathogenesis of PIMS-TS?
The use of defined S-gp glycosylation variants may shed more light on complement activation and reveal the underlying
molecular mechanisms. The project should contribute to better understanding of the COVID-19 and PIMS-TS
pathogenesis by exploring early immune response: interaction of N-glycans of S-gp with the complement. The outcome
of the project may significantly influence the development of disease diagnostics (based on patients’ genotype related
to the complement), treatment (supplementation/inhibition therapy regarding the complement system) and prevention
(vaccination strategies based on the identified glycans).