Abstract Title
Optimization and miniaturization of a Human Intestinal Enteroid (HIE) neutralization assay for evaluating homotypic human norovirus (HuNoV) antibody responses
Presenter
KHALIL ETTAYEBI, BAYLOR COLLEGE OF MEDICINE
Co-Author(s)
Khalil Ettayebi1, Mary K. Estes1,3, Sasirekha Ramani1, Astrid Borkowski2, Michael E. Abram2, Robert L. Atmar1,3
1 Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA; 2 HilleVax, Inc., Boston, MA, USA; 3 Department of Medicine, Baylor College of Medicine, Houston, TX, USA.
Abstract Category
Vaccines and immunity
Abstract
Background: HuNoVs are the principal cause of acute viral gastroenteritis worldwide, with the GII.4/Sydney/2012 being the dominant contemporary variant for the last two decades. Recent advances in HIE culture have enabled robust in vitro cultivation and quantification of neutralizing antibody (nAb) responses to HuNoVs, providing a physiologically relevant model for vaccine and therapeutic assessment. To support high-throughput, cost-effective serological screening in clinical trials, we optimized and miniaturized a HIE neutralization assay in 384-well plates. This serum-sparing approach allows for the evaluation of true nAb responses using minimal serum sample volumes, in contrast to traditional ligand-blocking assays. Additionally, our assay expands coverage to both GI and GII norovirus genotypes, supporting comprehensive, heterotypic immune profiling in vaccine and antiviral studies.
Methods: Established and optimized conditions for J4FUT2-KI HIE plating and GII.4/Sydney/2012 infection in 384-well format, comparing performance to conventional 96-well assays. Evaluated and compared anti-GII.4/Sydney nAb levels in reference standards and 24 paired sera samples of a bivalent GI.1/GII.4c virus-like particle norovirus vaccine candidate in adults (NOR-211 study).
Results: This miniaturized assay demonstrated strong correlation with our established 96-well assays. The pilot evaluation of 24-paired sera demonstrated homotypic GII.4/Sydney/2012 nAb seroresponses in vaccinated adults using J4FUT2-KI HIEs. Results showed consistency with prior neutralization data from J2 HIEs and histo blood antigen (HBGA)-blocking antibody assays (correlation coefficients r > 0.90), indicating comparable reliability across platforms.
Conclusion: This optimized assay offers enhanced scalability and sensitivity, facilitating efficient screening of candidate therapeutics and comprehensive assessment of humoral immunity in future norovirus vaccine trials.
Methods: Established and optimized conditions for J4FUT2-KI HIE plating and GII.4/Sydney/2012 infection in 384-well format, comparing performance to conventional 96-well assays. Evaluated and compared anti-GII.4/Sydney nAb levels in reference standards and 24 paired sera samples of a bivalent GI.1/GII.4c virus-like particle norovirus vaccine candidate in adults (NOR-211 study).
Results: This miniaturized assay demonstrated strong correlation with our established 96-well assays. The pilot evaluation of 24-paired sera demonstrated homotypic GII.4/Sydney/2012 nAb seroresponses in vaccinated adults using J4FUT2-KI HIEs. Results showed consistency with prior neutralization data from J2 HIEs and histo blood antigen (HBGA)-blocking antibody assays (correlation coefficients r > 0.90), indicating comparable reliability across platforms.
Conclusion: This optimized assay offers enhanced scalability and sensitivity, facilitating efficient screening of candidate therapeutics and comprehensive assessment of humoral immunity in future norovirus vaccine trials.