Examination of Immune Response to SARS-COV-2 in PBMCs and serum

Globally, there have been nearly 800 million confirmed cases of Covid-19 and 13 billion vaccine doses administered. These exposures create T cell and B cell immune memory responses in individuals that protect against future infections. B cells create antibodies that circulate in the body and attach to the virus. Memory T cells recognize and kill infected cells while also recruiting other immune cells. Not all individuals have robust immune memory responses and immunity against SARS-CoV-2 typically declines after 3-15 months. We measured cellular and humoral (antibody) immunity against SARS-COV-2 in four vaccinated individuals in order to investigate a potential correlation between the number of T-cells that are reactive to SARS-CoV-2 peptide and the serum concentration of Sars-CoV-2 antibodies. Peripheral Blood Mononuclear Cells (PBMCs) and serum samples were collected from 4 healthy adult females who had had 3 doses of the Pfizer mRNA COVID-19 vaccine (Comirnaty) and at least one confirmed SARS-COV-2 infection. The number of SARS-COV-2 reactive T cells per 100k PBMCs was determined using ImmunoSpot® Human INF-y Single-Color Enzymatic ELISPOT Assay. Thawed PBMCs were counted for viability and rested overnight (37°C; 5% CO2). The cells were plated at concentrations ranging from 5x10⁵ to 1x10⁵ cells/well, with SARS-COV-2 peptides (Miltenyi Biotech) or pos/neg controls. After an 18-hour incubation, plates were processed according to the manufacturer's protocol to determine the number of IFN-y producing cells. Spots were imaged and counted on a CTL ImmunoSpot® S6 Ultra analyzer, then adjusted to spots per 100k cells, correcting for background activation. The serum concentration of SARS-COV-2 IgG Antibodies was determined using RayBio® COVID-19 N and S1 TND protein Human IgG ELISA Kit. Thawed serum was diluted 1:1500. The ELISA was processed according to manufacturer’s protocol. Optical density (OD) at 450nm was measured using a Synergy 2 microplate and compared to a standard curve. PRISM was used to extrapolate the unknown values of the serum samples and reported in units/mL serum. Although we expected that antibody concentrations and T cell counts would be positively related to each other, in our small sample, there was no discernible relationship. Furthermore, we expected that the number of days since last exposure (either illness or vaccine shot) would be negatively related to measures of immune memory. Once again, there was no such relationship in our data. These results show that the immune memory response can vary between individuals and that one measurement cannot be used to predict the other. However, due to our small sample size, our conclusions cannot be said to represent the population at large. Both measures of adaptive immune memory are known to peak shortly after exposure and gradually decline over months and years.