Degree of immunoglobulin kappa light chain glycosylation of anti-spike SARS CoV-2 antibodies correlates with COVID-19 severity. (preprint)

Glycosylation of antibodies and the effects this has on inflammatory responses has concentrated predominately on the study of glycosylation moieties found in the Fc region of heavy chains. Light chain glycosylation and their ratios are relatively understudied. Nevertheless, variable glycosylation and ratio of {kappa} and {lambda} light chains have been associated with worse prognosis in myeloma and in tissue deposition amyloidosis. The {kappa} {lambda} light chains, of antibodies binding to SARS-CoV2 nucleocapsid and spike protein were analysed, using MALDI-ToF MS, in respect to their intensity, ratios, glycosylation patterns and any pattern changes correlating with COVID-19 severity. The molecular masses and signal intensity of {kappa} and {lambda} glycosylated and non-glycosylated light chains were measured for immunoglobulins isolated from plasma of seropositive and seronegative health care workers (HCW), and convalescent patients who had suffered from acute respiratory distress syndrome (ARDS). Overall, there was no significant changes in {kappa} to {lambda} ratio of total IgG (via protein G capture) antibodies between the groups. A non-statistically significant trend towards {lambda} light chains was found in antibodies against SARS CoV-2 Nucleocapsid and Spike proteins. However, detailed analysis of the molecular forms found a significant increase and bias towards un-glycosylated light chains and in particular un-glycosylated {kappa} light chains, in antibodies against SAR-CoV-2 spike protein, from convalescent COVID-ARDS patients. Here we have demonstrated a bias towards un-glycosylated {kappa} chains in anti-spike antibodies in those who suffered from ARDS as a result of SARS-CoV2 infection 3 months after recovery. How this relates to the immunopathology of COVID-19 requires further study.

Mechanism Based Designing, Synthesis and Evaluation of Nitrogen and Non-nitrogen Derivatives of Biphosphonates as Anti-inflammatory Agents (preprint)

The inflammatory mechanisms are complex due to the involvement of multiple pathways. The messengers like PIP2 and IP3 are phosphonate containing biomolecules which are desirable for the delivery of biological responses. In the present study, we designed a novel series of nitrogen and non-nitrogen containing bisphosphonates fused with more lipophilic chalcone moiety. We assume to maintain the hydrophilic ratio of BPs with hydrophobic chalcone substitution. The synthesized compounds 1–11, demonstrated potential anti-inflammatory activity against carrageenan-induced rat paw edema model and in-vitro COX-2 assay. The modifications over the synthesized derivatives were supported by in silico studies and SAR of bisphosphonates. In vitro study was carried out at concentration of 1µM while in vivo study was performed using the carrageenan-induced rat paw edema model. From the study it was concluded that the binding pattern of synthesized derivatives was remarkably similar to the co-crystallized ligand, and also the anti-inflammatory effect of compound was based on the interactions between bisphosphonates and amino acids. The distance between the P-C-P linkage of bisphosphonates to nitrogen and free carbon attached to nitrogen were found to be important for the maximum interaction and biological response considering the co-crystalized ligand in another enzyme. An assessment of cyclooxygenase inhibitory activity (in vitro) of compounds (1, 9, and 11) revealed a structure activity relationship while in vivo inhibitory action of compound 9, 11, 1, 10, and 3 is indicative of its inhibitory action on the prostaglandin synthesis which may be mediated through the inhibition of COX-2 enzyme.

Determination of IgG1 and IgG3 SARS-CoV-2 spike protein and nucleocapsid binding – Who is binding who and why? (preprint)

The involvement of IgG3 in the humoral immune response to SARS-CoV2 infection has been implicated in the pathogenesis of ARDS in COVID-19. The exact molecular mechanism is unknown but may be due to the differential ability of IgG3 Fc region to fix complement and stimulate cytokine release. We examined convalescent patients’ antibodies binding to immobilised nucleocapsid and spike protein by MALDI-ToF mass spectrometry. IgG3 was a major immunoglobulin found in all samples. Differential analysis of the spectral signatures found for nucleocapsid versus spike protein demonstrated that the predominant humoral immune response to nucleocapsid was IgG3, whilst against spike it was IgG1. However, the spike protein displayed a strong affinity for IgG3 itself which it would bind from control plasma samples as well as from those previously infected with SARS-CoV2, much in the way Protein-G binds IgG1. Furthermore, detailed spectral analysis indicated a mass shift consistent with hyper-glycosylation or glycation was a characteristic of the IgG3 captured by the spike protein.