MSACL 2022 Abstract

The global COVID 19 pandemic prompted the accelerated development and worldwide utilisation of novel vaccines. With the emergence of variants and the natural process of waning immunity, it has become increasingly clear that there is a need for more flexible and comprehensive tests used for immunosurveillance and research on vaccine effectiveness. Although large scale and low-cost ELISA platform tests are commonly used in practice, they have limitations. Currently testing predominantly focuses on total gamma immunoglobulins (IgG). Antibody mediated immunity is a complex process that involves multiple factors beyond just IgG. Only a highly multiplex and targeted analytical platform can provide the necessary flexibility for a more comprehensive study of this important biological process.

To address this, we developed a simple "bait capture" assay with enhanced capability to detect and quantitate multiple immunoglobulins against SARS CoV2 Spike S1 protein in a single serum sample preparation. By applying targeted MRM-LC-MS/MS approach for detection of unique peptide sequences from the constant heavy chains of immunoglobulins, we were able to accurately identify and quantitate all major serum antibody isotypes and their subclasses. In addition to antibodies we also added key proteins from the classical complement pathway activation, an important player in host – virus interactions.

To assess the performance of our assay, we applied it to a well-defined serological cohort from the UK based COVIDsortium bioresource. This cohort comprised of longitudinal serum samples from healthcare workers, with and without confirmed previous exposure to natural Covid infection and who had received two doses of mRNA based vaccine (Pfizer/BioNTech). As well as using the original Wuhan Hu-1 spike protein as a bait, we also evaluated the samples serological response against alpha (B.1.1.7), beta (B.1.351) and delta (B.1.617.2) S1 spike proteins.

Our method showed a good correlation (r = 0.83) of IgG1 with Anti-S Elecsys (Roche) assay. We observed an increase in serum IgG1 levels after first exposure to antigen regardless of its origin, natural infection or vaccine, for all variants. Second antigen exposure showed the highest change with subject variability between 3 and 10 fold. Interestingly, subjects that have had 3rd exposure i.e. natural infection and 2 doses of vaccine showed no further increase. The IgG1 levels measured against the original Wuhan Hu-1 correlated well (r = 0.85) with live virus neutralization (IC50). Correlation with other variants was notably lower, with beta (B.1.351) being the lowest (r = 0.78). Natural infection as exposure had an interesting effect on the other immunoglobulins. As one example, we observed significantly higher levels of Immunoglobulin Alpha subclass 1 (IgA1) against all variant spike proteins at second exposure for those who had previous natural infection.
We observed strong correlation of spike – IgG1 to the complement activating component 1q (C1q), for all variants however, the ratio of IgG1 to C1q was altered against the delta VoC with reduction of C1q.

In summary we have developed an alternative platform to ELISA methodology for a comprehensive characterisation of antibody response to SARS CoV2 vaccination using mass spectrometry. The flexibility and multiplexity offered by targeted mass spectrometry make this platform applicable not only to vaccines to SARS CoV2 but to any other pathogen, current or future. We have developed modifications to the assay that allows us to use it on dried blood spots, saliva and dried saliva spots.