MSACL 2023 Abstract

Introduction
There is growing evidence that SARS-CoV2 exposure in some people can weaken our immune response to other seasonal winter viruses such as coronavirus, respiratory syncytial virus (RSV), and influenza (Inf). Also, cross reactivity of anti-viral immune responses from other viruses to SARS-CoV2 have been implicated in long COVID. Better methods for in depth antibody measurement are needed to further study these effects. Recent advances to clinical mass spectrometric assays have seen the development of a “spike-binding” test that determines immune response to SARS-CoV2 infections. Coupling a bait-and-capture extraction with multiplexed liquid chromatography tandem mass spectrometry (LC-MS/MS), in vitro viral antigens are used to capture specific/non-specific antibodies from plasma. Circulating complement proteins form complexes with the bound antibodies. Thus, entire immune-complexes can be captured prior to tryptic digestion and targeted LC-MS/MS analysis. These assays are currently useful for assessing response to recent infections, give insight on the maturation of the immune response and the degree of recognition antibodies may have to viral variants which conventional ELISA based tests cannot provide. They also have potential to be adapted for use in determining the need for booster vaccinations, or even used to screen suitable vectors in adeno-associated virus (AAV) gene therapy.

Objectives
This study sought to adapt a bait-and-capture LC-MS/MS assay for a panel of winter viruses and test its applicability on a small cohort of post-pandemic plasma samples (N=20).

Methods
Influenza A (InfA; hemagglutinin), influenza B (InfB; neuraminidase), human rhinovirus type 16 (HRV16; viral protein 0), human respiratory syncytial virus A (HRSVA; fusion), and 4 human coronavirus (229E, HKU1, NL63, OC43; spike) viral proteins were bound separately to 96-well plate (0.5 µg/well). Horse myoglobin (0.1 mg/mL) was used to block non-specific binding before the addition of 10 µL plasma. The samples were incubated for 1 hour. Wells were washed thrice with phosphate buffer solution (200 µL). Bound immunocomplexes were reduced and alkylated before tryptic digestion (45 oC, 1 h). Digestion was acidified and centrifuged (20 mins, 4500 rpm). The supernatant was analysed using reversed phase chromatography (ACQUITY Premier BEH C18 Column, 2.1 x 50 mm) coupled to a Waters Xevo TQ-XS triple quadrupole MS operating in multiple reaction monitoring (MRM) mode. Immunoglobulin (Ig) and complement peptides were normalised to the viral peptides.

Results
IgM was found in all samples (N=20/20) for each viral antigen (N=8). The mean highest and lowest IgM responses were seen in OC43 (response: 2.8x10^5) and HRV16 (1.5x10^5), respectively. IgG2 was detected in all samples and viruses, except in InfB (N=19/20). IgG1 was detected in all cases for OC43 and HRV16 (N=20/20). The mean highest and lowest IgG1 responses were OC43 (1.9x10^5) and HKU1 (1.6x104), respectively. One RSVA2 case saw elevated IgG1 (15.4-fold) and IgG2 (14.8-fold) responses compared to the mean, indicating recent exposure. IgG4 response was across viruses was low, detected between N=3/20 cases (229E) and N=10/20 cases (NL63). The complement protein C1qc was detected variably between viruses, ranging from N=2/20 cases (InfB) to N=7/20 cases (RSVA2). Similarly, C9 detection ranged between N=2/20 (NL63) and N=6/20 (RSVA2). No significance of IgG, IgM, or complement response was found on ANOVA testing between all viruses. On regression of C1qc against IgG1 z-scores, RSVA2 (R^2 0.99), NL63 (R^2 0.99), OC43 (R^2 0.83), and InfA (R^2 0.92) all correlated. Slopes for these viruses varied (1/slope: 1.28 - 1.92) indicating differential initiation of the complement pathways.

Conclusion
This proof-of-concept study demonstrated bait-and-capture assays may be readily adapted for the determination of immune response for different infections by LC-MS/MS. Variable IgG responses were seen across other coronaviruses, suggesting SARS-CoV2 exposure may have led to high-affinity immune response to some (OC43) but not others (HKU1). The assay also likely determined a recent exposure to RSVA2 for one case. Given larger clinical studies, thresholds could be set to determine recent or current infections. This test may also be adapted to pre-screen suitability to AAV gene therapy i.e., elevated Ig may predict rejection to viral vectors. A low IgG response to SARS-CoV-2 spike protein could also be used to determine need for a booster vaccination.