Gabriela Alvarado1, Khalil Ettayebi2, Robert L Atmar3, Robin G Bombardi4, Nurgun Kose4, Mary K Estes3, James E Crowe5. 1. Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee. 2. Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas. 3. Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas; Department of Medicine, Baylor College of Medicine, Houston, Texas. 4. Vanderbilt Vaccine Center, Vanderbilt University Medical Center, Nashville, Tennessee. 5. Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee; Vanderbilt Vaccine Center, Vanderbilt University Medical Center, Nashville, Tennessee; Department of Pediatrics, Vanderbilt University Medical Center, Nashville, Tennessee. Electronic address: james.crowe@vanderbilt.edu.
Abstract
BACKGROUND & AIMS: Human noroviruses are responsible for approximately 200,000 deaths worldwide each year. In 2012, the GII.4 Sydney strain emerged and became the major circulating norovirus strain associated with human disease. Our understanding of the human norovirus-specific antibody response is limited because few human monoclonal antibodies (mAbs) to noroviruses have been described, and there are no functional assays to measure virus neutralization. We studied the antibody-mediated response to the genogroup (G) II.4 strain by isolating mAbs to GII.4 from infected patients and developing virus neutralization assays. METHODS: We used a robust human hybridoma technique to isolate mAbs from patients previously infected with norovirus and identified mAbs that blocked virus binding to cell receptors, using virus-like particles to test blockade ability. We tested the ability of select mAbs to neutralize live human noroviruses using stem cell-derived human enteroids. RESULTS: We isolated a panel of 25 IgG or IgA human mAbs that recognized norovirus GII.4 Sydney 2012 and determined their potential to block virus binding to cell receptors. In competition binding studies, most antibodies recognized 3 major antigenic sites on the GII.4 Sydney 2012 protruding (P) domain. CONCLUSIONS: We isolated and characterized human mAbs that neutralize live human norovirus GII.4 Sydney 2012-the predominant strain responsible for recent outbreaks. Analyses of these antibodies identified neutralizing epitopes; further studies will provide insight into the human immune response to this deadly virus.
BACKGROUND & AIMS:Human noroviruses are responsible for approximately 200,000 deaths worldwide each year. In 2012, the GII.4 Sydney strain emerged and became the major circulating norovirus strain associated with human disease. Our understanding of the humannorovirus-specific antibody response is limited because few human monoclonal antibodies (mAbs) to noroviruses have been described, and there are no functional assays to measure virus neutralization. We studied the antibody-mediated response to the genogroup (G) II.4 strain by isolating mAbs to GII.4 from infected patients and developing virus neutralization assays. METHODS: We used a robust human hybridoma technique to isolate mAbs from patients previously infected with norovirus and identified mAbs that blocked virus binding to cell receptors, using virus-like particles to test blockade ability. We tested the ability of select mAbs to neutralize live human noroviruses using stem cell-derived human enteroids. RESULTS: We isolated a panel of 25 IgG or IgAhumanmAbs that recognized norovirus GII.4 Sydney 2012 and determined their potential to block virus binding to cell receptors. In competition binding studies, most antibodies recognized 3 major antigenic sites on the GII.4 Sydney 2012 protruding (P) domain. CONCLUSIONS: We isolated and characterized humanmAbs that neutralize live humannorovirus GII.4 Sydney 2012-the predominant strain responsible for recent outbreaks. Analyses of these antibodies identified neutralizing epitopes; further studies will provide insight into the human immune response to this deadly virus.
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