RH5.1-CyRPA-Ripr antigen combination vaccine shows little improvement over RH5.1 in a preclinical setting Julie Healer 1,2 , Jennifer K. Thompson 1 , Karen L. Mackwell 1 , Cecille D. Browne 3 , Benjamin A. Seager 1,2 , Anna Ngo 1 , Kym N. Lowes 1,2 , Sarah E. Silk 4 , David Pulido 4 , Lloyd D. W. King 4 , Jayne M. Christen 3 , Amy R. Noe 3 , Vinayaka Kotraiah 3 , Paul J. Masendycz 1 , Rajkannan Rajagopalan 5 , Leanne Lucas 5 , Marianne M. Stanford 5† , Lorraine Soisson 6 , Carter Diggs 6 , Robin Miller 6 , Susan Youll 6 , Kaye Wycherley 1 , Simon J. Draper 4 and Alan F. Cowman 1,2 * 1 The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia, 2 University of Melbourne, Melbourne, VIC, Australia, 3 Leidos Life Sciences, Frederick, MD, United States, 4 Department of Biochemistry, University of Oxford, Oxford, United Kingdom, 5 IMV, Inc., Dartmouth, NS, Canada, 6 Malaria Vaccine Development Program, United States Agency for International Development (USAID), Washington, DC, United States Background: RH5 is the leading vaccine candidate for the Plasmodium falciparum blood stage and has shown impact on parasite growth in the blood in a human clinical trial. RH5 binds to Ripr and CyRPA at the apical end of the invasive merozoite form, and this complex, designated RCR, is essential for entry into human erythrocytes. RH5 has advanced to human clinical trials, and the impact on parasite growth in the blood was encouraging but modest. This study assessed the potential of a protein-in-adjuvant blood stage malaria vaccine based on a combination of RH5, Ripr and CyRPA to provide improved neutralizing activity against P. falciparum in vitro. Methods: Mice were immunized with the individual RCR antigens to down select the best performing adjuvant formulation and rats were immunized with the individual RCR antigens to select the correct antigen dose. A second cohort of rats were immunized with single, double and triple antigen combinations to assess immunogenicity and parasite neutralizing activity in growth inhibition assays. Results: The DPX® platform was identified as the best performing formulation in potentiating P. falciparum inhibitory antibody responses to these antigens. The three antigens derived from RH5, Ripr and CyRPA proteins formulated with DPX induced highly inhibitory parasite neutralising antibodies. Notably, RH5 either as a single antigen or in combination with Ripr and/or CyRPA, induced inhibitory antibodies that outperformed CyRPA, Ripr. Frontiers in Cellular and Infection Microbiology frontiersin.org 01 OPEN ACCESS EDITED BY Kai Matuschewski, Humboldt University of Berlin, Germany REVIEWED BY Anja Scholzen, Byondis, Netherlands Matthijs Miklas Jore, Radboud University Medical Centre, Netherlands *CORRESPONDENCE Alan F. Cowman cowman@wehi.edu.au † PRESENT ADDRESS Dalhousie University, Halifax, NS, Canada SPECIALTY SECTION This article was submitted to Parasite and Host, a section of the journal Frontiers in Cellular and Infection Microbiology RECEIVED 20 September 2022 ACCEPTED 05 December 2022 PUBLISHED 20 December 2022 CITATION Healer J, Thompson JK, Mackwell KL, Browne CD, Seager BA, Ngo A, Lowes KN, Silk SE, Pulido D, King LDW, Christen JM, Noe AR, Kotraiah V, Masendycz PJ, Rajagopalan R, Lucas L, Stanford MM, Soisson L, Diggs C, Miller R, Youll S, Wycherley K, Draper SJ and Cowman AF (2022) RH5.1-CyRPA-Ripr antigen combination vaccine shows little improvement over RH5.1 in a preclinical setting. Front. Cell. Infect. Microbiol. 12:1049065. doi: 10.3389/fcimb.2022.1049065 TYPE Original Research PUBLISHED 20 December 2022 DOI 10.3389/fcimb.2022.1049065