Mature dengue virus particles inactivated by a non-ionic detergent retain quaternary neutralizing epitopes and induce potent neutralizing antibodies

Wen-Yang Tsai¹Theodore C Pierson²Jens Wrammert³Wanwisa Dejnirattisai⁴Amonrat Jumnainsong⁵Juthathip Mongkolsapaya⁶Gavin Screaton⁷James E. Crowe, Jr.⁸Wei-Kung Wang^1*

• ¹University of Hawaii at Manoa, Honolulu, United States
• ²Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States
• ³Emory Vaccine Center, and Division of Infectious Disease, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, United States, Atlanta, United States
• ⁴Nuffield Department of Medicine, Wellcome Trust Centre for Human Genetics, University of Oxford,, Oxford, United Kingdom
• ⁵The Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand, Khon Kaen, Thailand
• ⁶Nuffield Department of Medicine, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom
• ⁷Division of Medical Sciences, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom
• ⁸Department of Pediatrics, Pathology, Microbiology and Immunology, and The Vanderbilt Center for Antibody Therapeutics, Vanderbilt University Medical Center, Vanderbilt University, Nashville, United States

Introduction: The four serotypes of dengue virus (DENV1-4) are the leading cause of arboviral diseases in humans. Currently, developing a safe and effective DENV vaccine remains a challenge. Previously we reported potently neutralizing human monoclonal antibodies (mAbs) preferentially recognize mature DENV particles, on which there is limited access to the fusion loop (FL) epitope and no premembrane (prM) protein. As FL and prM antibodies are weakly-or non-neutralizing and contribute to antibody-dependent enhancement, mature DENV particles represent an ideal DENV vaccine candidate. Several inactivated flavivirus vaccines, generated using formalin, ultraviolet or other inactivation methods, have progressed through preclinical and clinical studies. Little is known about how different inactivation methods affect viral epitopes and the quality of antibodies induced. Methods: We investigated epitopes on Tween 20-inactivated mature DENV1 particles by testing a panel of well-characterized human mAbs and membrane integrity by sucrose-gradient ultracentrifugation and protein K digestion. We examined the mechanisms of Tween 20 inactivation by measuring RNA copy numbers, virus binding to cells and acid exposure, and antibody responses induced by Tween 20-inactivated mature DENV1 particles in mice. Results: Tween 20 inactivation better preserved the epitopes recognized by potently neutralizing mAbs compared with other methods. Sucrose-gradient ultracentrifugation and protein K digestion revealed no disruption of membrane integrity by Tween 20. Mechanistically, Tween 20 treatment caused reduced virus binding to cells and RNA degradation, which was reverted by pre-treatment with RNAseOUT, suggesting the involvement of extracellular RNase, and prevented the envelope protein conformational changes induced by acid exposure. Moreover, Tween 20-inactivated mature DENV1 particles induced a neutralizing antibody response to all four DENV serotypes characterized by competition with several potently neutralizing mAbs and limited recognition of FL.Conclusion: Our results suggest that Tween 20-inactivated mature particles are a promising strategy for DENV vaccine development.