Poster Presentation 12th Australasian Virology Society Meeting 2024

Understanding the factors that influence SARS-CoV-2 neutralising antibody titres (#213)

Francesca Mordant 1 , Eva Stadler 2 , Siddhartha Mahanty 3 , Miles Davenport 2 , David Khoury 2 , Kanta Subbarao 1
  1. Department of Microbiology and Infectious Diseases, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia
  2. Infection Analytics Program, Kirby Institute, UNSW Sydney, Sydney, NSW, Australia
  3. Department of Infectious Diseases, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia

Neutralising antibodies are an important correlate of protection for SARS-CoV-2 vaccines and can be assessed by a variety of laboratory assays; however, interpreting results from different assays remains challenging. Here, we investigate the correlation between neutralising antibody assays, and the factors that drive differences in absolute titre values, including: virus (live/pseudotyped), assay readout (cytopathic effect (CPE)/focus-forming units (FFU)/luciferase expression), and format (what the neutralising antibody titre/ND50 measures).

 A panel of sera from monovalent ancestral COVID-19 vaccinees was tested against the ancestral strain of SARS-CoV-2 in a head-to-head comparison of three neutralisation assays: 1) a CPE-based live virus microneutralisation assay, 2) a focus-forming (“ImmunoSpot”) live virus assay, and 3) a ‘pseudovirus’ assay using lentiviral particles pseudotyped with SARS-CoV-2 Spike expressing luciferase. ND50 values were estimated by fitting a logistic function to the data.

 There is a strong correlation between titres obtained from all three assays. ND50 values were on average 15.4- and 12.0-fold lower in the CPE assay (median=226) than the ImmunoSpot (median=3381) and pseudovirus (median=2091) assays, respectively. The CPE method gave the lowest absolute titres, and titres from the ImmunoSpot and pseudovirus assays were highly similar, suggesting the use of live vs. pseudotyped virus, and assay readout (CPE/FFU/luminescence) did not strongly affect absolute titres. The format of the assay had the greatest influence on absolute titre values; the ImmunoSpot and pseudovirus assays measure the dilution of serum required to neutralise 50% of virus (“per virion neutralisation”), while the CPE-based microneutralisation assay measures the dilution of serum that completely neutralises infectivity in 50% of wells of a microtitre plate (“per well neutralisation”), resulting in lower titres.

 Overall, the results obtained were highly correlated across methods. The format is crucial for interpreting results from different assay types, and clear terminology is essential for describing what and how different assays measure neutralising antibodies.