{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,21]],"date-time":"2026-05-21T12:16:10Z","timestamp":1779365770302,"version":"3.53.0"},"reference-count":51,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2021,7,7]],"date-time":"2021-07-07T00:00:00Z","timestamp":1625616000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000200","name":"United States Agency for International Development","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100000200","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:p>The hurdles to effective blood stage malaria vaccine design include immune evasion tactics used by the parasite such as redundant invasion pathways and antigen variation among circulating parasite strains. While blood stage malaria vaccine development primarily focuses on eliciting optimal humoral responses capable of blocking erythrocyte invasion, clinically-tested<jats:italic>Plasmodium falciparum<\/jats:italic>(Pf) vaccines have not elicited sterile protection, in part due to the dramatically high levels of antibody needed. Recent development efforts with non-redundant, conserved blood stage antigens suggest both high antibody titer and rapid antibody binding kinetics are important efficacy factors. Based on the central role of helper CD4 T cells in development of strong, protective immune responses, we systematically analyzed the class II epitope content in five leading Pf blood stage antigens (RH5, CyRPA, RIPR, AMA1 and EBA175) using<jats:italic>in silico<\/jats:italic>,<jats:italic>in vitro<\/jats:italic>, and<jats:italic>ex vivo<\/jats:italic>methodologies. We employed<jats:italic>in silico<\/jats:italic>T cell epitope analysis to enable identification of 67 HLA-restricted class II epitope clusters predicted to bind a panel of nine HLA-DRB1 alleles. We assessed a subset of these for HLA-DRB1 allele binding<jats:italic>in vitro<\/jats:italic>, to verify the<jats:italic>in silico<\/jats:italic>predictions. All clusters assessed (40 clusters represented by 46 peptides) bound at least two HLA-DR alleles<jats:italic>in vitro<\/jats:italic>. The overall epitope prediction to<jats:italic>in vitro<\/jats:italic>HLA-DRB1 allele binding accuracy was 71%. Utilizing the set of RH5 class II epitope clusters (10 clusters represented by 12 peptides), we assessed stimulation of T cells collected from HLA-matched RH5 vaccinees using an IFN-\u03b3 T cell recall assay. All clusters demonstrated positive recall responses, with the highest responses \u2013 by percentage of responders and response magnitude \u2013 associated with clusters located in the N-terminal region of RH5. Finally, a statistically significant correlation between<jats:italic>in silico<\/jats:italic>epitope predictions and<jats:italic>ex vivo<\/jats:italic>IFN-\u03b3 recall response was found when accounting for HLA-DR matches between the epitope predictions and donor HLA phenotypes. This is the first comprehensive analysis of class II epitope content in RH5, CyRPA, RIPR, AMA1 and EBA175 accompanied by<jats:italic>in vitro<\/jats:italic>HLA binding validation for all five proteins and<jats:italic>ex vivo<\/jats:italic>T cell response confirmation for RH5.<\/jats:p>","DOI":"10.3389\/fimmu.2021.690348","type":"journal-article","created":{"date-parts":[[2021,7,7]],"date-time":"2021-07-07T07:54:13Z","timestamp":1625644453000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":11,"title":["Identification and Immune Assessment of T Cell Epitopes in Five Plasmodium falciparum Blood Stage Antigens to Facilitate Vaccine Candidate Selection and Optimization"],"prefix":"10.3389","volume":"12","author":[{"given":"Vinayaka","family":"Kotraiah","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Timothy W.","family":"Phares","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Frances E.","family":"Terry","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pooja","family":"Hindocha","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarah E.","family":"Silk","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carolyn M.","family":"Nielsen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Leonard","family":"Moise","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kenneth D.","family":"Tucker","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rebecca","family":"Ashfield","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"William D.","family":"Martin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anne S.","family":"De Groot","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Simon J.","family":"Draper","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gabriel M.","family":"Gutierrez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amy R.","family":"Noe","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2021,7,7]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0008138","article-title":"High Antibody Titer Against Apical Membrane Antigen-1 Is Required to Protect Against Malaria in the Aotus Model","volume":"4","author":"Dutta","year":"2009","journal-title":"PloS One"},{"key":"B2","doi-asserted-by":"publisher","first-page":"189","DOI":"10.1179\/136485910X12647085215534","article-title":"Blood-Stage Malaria Vaccines - Recent Progress and Future Challenges","volume":"104","author":"Goodman","year":"2010","journal-title":"Ann Trop Med Parasitol"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa1008115","article-title":"A Field Trial to Assess a Blood-Stage Malaria Vaccine","volume":"365","author":"Thera","year":"2011","journal-title":"N Engl J Med"},{"key":"B4","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1003943","article-title":"Plasmodium Falciparum Erythrocyte Invasion: Combining Function With Immune Evasion","volume":"10","author":"Wright","year":"2014","journal-title":"PloS Pathog"},{"key":"B5","doi-asserted-by":"publisher","first-page":"216","DOI":"10.1016\/j.cell.2019.05.025","article-title":"Human Antibodies That Slow Erythrocyte Invasion Potentiate Malaria-Neutralizing Antibodies","volume":"178","author":"Alanine","year":"2019","journal-title":"Cell"},{"key":"B6","doi-asserted-by":"publisher","first-page":"1953","DOI":"10.1038\/s41467-019-09894-4","article-title":"A Defined Mechanistic Correlate of Protection Against Plasmodium Falciparum Malaria in Non-Human Primates","volume":"10","author":"Douglas","year":"2019","journal-title":"Nat Commun"},{"key":"B7","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.1302045","article-title":"Neutralization of Plasmodium Falciparum Merozoites by Antibodies Against Pfrh5","volume":"192","author":"Douglas","year":"2014","journal-title":"J Immunol"},{"key":"B8","doi-asserted-by":"publisher","DOI":"10.1016\/j.isci.2020.101310","article-title":"T Helper Plasticity Is Orchestrated by STAT3, Bcl6, and Blimp-1 Balancing Pathology and Protection in Malaria","volume":"23","author":"Carpio","year":"2020","journal-title":"iScience"},{"key":"B9","doi-asserted-by":"publisher","DOI":"10.1182\/blood-2004-10-4047","article-title":"Malaria-Specific Transgenic CD4(+) T Cells Protect Immunodeficient Mice From Lethal Infection and Demonstrate Requirement for a Protective Threshold of Antibody Production for Parasite Clearance","volume":"106","author":"Stephens","year":"2005","journal-title":"Blood"},{"key":"B10","doi-asserted-by":"publisher","DOI":"10.1016\/j.pt.2008.08.008","article-title":"Recent Insights Into Humoral and Cellular Immune Responses Again St Malaria","volume":"24","author":"Beeson","year":"2008","journal-title":"Trends Parasitol"},{"key":"B11","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1708383114","article-title":"Atypical Activation of Dendritic Cells by Plasmodium Falciparum","volume":"114","author":"Gotz","year":"2017","journal-title":"Proc Natl Acad Sci USA"},{"key":"B12","doi-asserted-by":"publisher","DOI":"10.1016\/j.xcrm.2021.100207","article-title":"Protein\/AS01B Vaccination Elicits Stronger, More Th2-Skewed Antigen-Specific Human T Follicular Helper Cell Responses Than Heterologous Viral Vectors","volume":"2","author":"Nielsen","year":"2021","journal-title":"Cell Rep Med"},{"key":"B13","doi-asserted-by":"publisher","first-page":"e93683","DOI":"10.1172\/jci.insight.93683","article-title":"Human Vaccination Against Plasmodium Vivax Duffy-binding Protein Induces Strain-Transcending Antibodies","volume":"2","author":"Payne","year":"2017","journal-title":"JCI Insight"},{"key":"B14","doi-asserted-by":"publisher","first-page":"e96381","DOI":"10.1172\/jci.insight.96381","article-title":"Human Vaccination Against RH5 Induces Neutralizing Antimalarial Antibodies That Inhibit RH5 Invasion Complex Interactions","volume":"2","author":"Payne","year":"2017","journal-title":"JCI Insight"},{"key":"B15","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0031208","article-title":"Phase Ia Clinical Evaluation of the Safety and Immunogenicity of the Plasmodium Falciparum Blood-Stage Antigen AMA1 in ChAd63 and MVA Vaccine Vectors","volume":"7","author":"Sheehy","year":"2012","journal-title":"PloS One"},{"key":"B16","doi-asserted-by":"publisher","DOI":"10.1038\/mt.2012.223","article-title":"ChAd63-MVA-vectored Blood-Stage Malaria Vaccines Targeting MSP1 and AMA1: Assessment of Efficacy Against Mosquito Bite Challenge in Humans","volume":"20","author":"Sheehy","year":"2012","journal-title":"Mol Ther"},{"key":"B17","doi-asserted-by":"publisher","DOI":"10.1038\/mt.2011.176","article-title":"Phase Ia Clinical Evaluation of the Plasmodium Falciparum Blood-Stage Antigen MSP1 in ChAd63 and MVA Vaccine Vectors","volume":"19","author":"Sheehy","year":"2011","journal-title":"Mol Ther"},{"key":"B18","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.1502137","article-title":"Vaccine Induction of Lymph Node-Resident Simian Immunodeficiency Virus Env-Specific T Follicular Helper Cells in Rhesus Macaques","volume":"196","author":"Vargas-Inchaustegui","year":"2016","journal-title":"J Immunol"},{"key":"B19","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.1502472","article-title":"Germinal Center B Cell and T Follicular Helper Cell Responses to Viral Vector and Protein-in-Adjuvant Vaccines","volume":"197","author":"Wang","year":"2016","journal-title":"J Immunol"},{"key":"B20","doi-asserted-by":"publisher","first-page":"176ra132","DOI":"10.1126\/scitranslmed.3005191","article-title":"Induction of ICOS+CXCR3+CXCR5+ TH Cells Correlates With Antibody Responses to Influenza Vaccination","volume":"5","author":"Bentebibel","year":"2013","journal-title":"Sci Transl Med"},{"key":"B21","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.1800986","article-title":"With a Little Help From T Follicular Helper Friends: Humoral Immunity to Influenza Vaccination","volume":"202","author":"Koutsakos","year":"2019","journal-title":"J Immunol"},{"key":"B22","doi-asserted-by":"publisher","DOI":"10.1128\/CVI.00082-10","article-title":"Safety and Immunogenicity of a Recombinant Nonglycosylated Erythrocyte Binding Antigen 175 Region II Malaria Vaccine in Healthy Adults Living in an Area Where Malaria Is Not Endemic","volume":"17","author":"El Sahly","year":"2010","journal-title":"Clin Vaccine Immunol"},{"key":"B23","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0163066","article-title":"Safety and Immunogenicity of EBA-175 Rii-Ng Malaria Vaccine Administered Intramuscularly in Semi-Immune Adults: A Phase 1, Double-Blinded Placebo Controlled Dosage Escalation Study","volume":"11","author":"Koram","year":"2016","journal-title":"PloS One"},{"key":"B24","doi-asserted-by":"publisher","DOI":"10.1016\/j.vaccine.2017.09.027","article-title":"Safety and Immunogenicity of a Recombinant Plasmodium Falciparum AMA1-DiCo Malaria Vaccine Adjuvanted With GLA-SE or Alhydrogel(R) in European and African Adults: A Phase 1a\/1b, Randomized, Double-Blind Multi-Centre Trial","volume":"35","author":"Sirima","year":"2017","journal-title":"Vaccine"},{"key":"B25","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gky1006","article-title":"The Immune Epitope Database (IEDB): 2018 Update","volume":"47","author":"Vita","year":"2019","journal-title":"Nucleic Acids Res"},{"key":"B26","doi-asserted-by":"publisher","first-page":"82","DOI":"10.6026\/97320630012082","article-title":"Implications From Predicted B-Cell and T-Cell Epitopes of Plasmodium Falciparum Merozoite Proteins EBA175-RII and Rh5","volume":"12","author":"Wamae","year":"2016","journal-title":"Bioinformation"},{"key":"B27","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2020.00442","article-title":"Better Epitope Discovery, Precision Immune Engineering, and Accelerated Vaccine Design Using Immunoinformatics Tools","volume":"11","author":"De Groot","year":"2020","journal-title":"Front Immunol"},{"key":"B28","doi-asserted-by":"publisher","DOI":"10.1007\/s002510050594","article-title":"Nine Major HLA Class I Supertypes Account for the Vast Preponderance of HLA-A and -B Polymorphism","volume":"50","author":"Sette","year":"1999","journal-title":"Immunogenetics"},{"key":"B29","doi-asserted-by":"crossref","DOI":"10.4049\/jimmunol.160.7.3363","article-title":"Several Common HLA-DR Types Share Largely Overlapping Peptide Binding Repertoires","volume":"160","author":"Southwood","year":"1998","journal-title":"J Immunol"},{"key":"B30","doi-asserted-by":"publisher","DOI":"10.1080\/21645515.2015.1061159","article-title":"Ivax: An Integrated Toolkit for the Selection and Optimization of Antigens and the Design of Epitope-Driven Vaccines","volume":"11","author":"Moise","year":"2015","journal-title":"Hum Vaccin Immunother"},{"key":"B31","doi-asserted-by":"publisher","DOI":"10.1016\/j.addr.2009.07.001","article-title":"T Cell Epitope: Friend or Foe? Immunogenicity of Biologics in Context","volume":"61","author":"Weber","year":"2009","journal-title":"Adv Drug Delivery Rev"},{"key":"B32","doi-asserted-by":"publisher","DOI":"10.4161\/hv.24615","article-title":"The Two-Faced T Cell Epitope: Examining the Host-Microbe Interface With Janusmatrix","volume":"9","author":"Moise","year":"2013","journal-title":"Hum Vaccin Immunother"},{"key":"B33","doi-asserted-by":"publisher","DOI":"10.1080\/21645515.2018.1560772","article-title":"Immune Escape and Immune Camouflage May Reduce the Efficacy of RTS,S Vaccine in Malawi","volume":"16","author":"Khan","year":"2020","journal-title":"Hum Vaccin Immunother"},{"key":"B34","doi-asserted-by":"publisher","DOI":"10.1038\/nature16141","article-title":"A Mechanism for Expansion of Regulatory T-Cell Repertoire and Its Role in Self-Tolerance","volume":"528","author":"Feng","year":"2015","journal-title":"Nature"},{"key":"B35","doi-asserted-by":"publisher","DOI":"10.1084\/jem.20052471","article-title":"Antibiotic-Refractory Lyme Arthritis Is Associated With HLA-DR Molecules That Bind a Borrelia Burgdorferi Peptide","volume":"203","author":"Steere","year":"2006","journal-title":"J Exp Med"},{"key":"B36","doi-asserted-by":"publisher","DOI":"10.1016\/j.medj.2021.03.014","article-title":"Reduced Blood-Stage Malaria Growth and Immune Correlates in Humans Following RH5 Vaccination","volume":"2","author":"Minassian","year":"2021","journal-title":"Med"},{"key":"B37","doi-asserted-by":"publisher","DOI":"10.1016\/j.vaccine.2011.02.019","article-title":"Coupling Sensitive In Vitro and In Silico Techniques to Assess Cross-Reactive CD4(+) T Cells Against the Swine-Origin H1N1 Influenza Virus","volume":"29","author":"Schanen","year":"2011","journal-title":"Vaccine"},{"key":"B38","doi-asserted-by":"publisher","first-page":"797","DOI":"10.1007\/s00251-004-0647-4","article-title":"Definition of Supertypes for HLA Molecules Using Clustering of Specificity Matrices","volume":"55","author":"Lund","year":"2004","journal-title":"Immunogenetics"},{"key":"B39","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gki357","article-title":"PEPVAC: A Web Server for Multi-Epitope Vaccine Development Based on the Prediction of Supertypic MHC Ligands","volume":"33","author":"Reche","year":"2005","journal-title":"Nucleic Acids Res"},{"key":"B40","doi-asserted-by":"publisher","DOI":"10.4161\/hv.25598","article-title":"Universal H1N1 Influenza Vaccine Development: Identification of Consensus Class II Hemagglutinin and Neuraminidase Epitopes Derived From Strains Circulating Between 1980 and 2011","volume":"9","author":"Moise","year":"2013","journal-title":"Hum Vaccin Immunother"},{"key":"B41","doi-asserted-by":"publisher","DOI":"10.1111\/cmi.13030","article-title":"Neutralising Antibodies Block the Function of Rh5\/Ripr\/CyRPA Complex During Invasion of Plasmodium Falciparum Into Human Erythrocytes","volume":"21","author":"Healer","year":"2019","journal-title":"Cell Microbiol"},{"key":"B42","doi-asserted-by":"publisher","first-page":"32","DOI":"10.1038\/s41541-018-0071-7","article-title":"Production, Quality Control, Stability, and Potency of Cgmp-Produced Plasmodium Falciparum RH5.1 Protein Vaccine Expressed in Drosophila S2 Cells","volume":"3","author":"Jin","year":"2018","journal-title":"NPJ Vaccines"},{"key":"B43","doi-asserted-by":"publisher","DOI":"10.1016\/j.pt.2020.04.003","article-title":"The RH5-CyRPA-Ripr Complex as a Malaria Vaccine Target","volume":"36","author":"Ragotte","year":"2020","journal-title":"Trends Parasitol"},{"key":"B44","doi-asserted-by":"publisher","DOI":"10.1080\/21645515.2015.1052197","article-title":"H7n9 T-Cell Epitopes That Mimic Human Sequences Are Less Immunogenic and may Induce Treg-Mediated Tolerance","volume":"11","author":"Liu","year":"2015","journal-title":"Hum Vaccin Immunother"},{"key":"B45","doi-asserted-by":"publisher","first-page":"48","DOI":"10.1016\/j.jhep.2014.08.026","article-title":"HCV Epitope, Homologous to Multiple Human Protein Sequences, Induces a Regulatory T Cell Response in Infected Patients","volume":"62","author":"Losikoff","year":"2015","journal-title":"J Hepatol"},{"key":"B46","doi-asserted-by":"publisher","first-page":"1283","DOI":"10.1038\/s41598-017-01372-5","article-title":"A Humanized Mouse Model Identifies Key Amino Acids for Low Immunogenicity of H7N9 Vaccines","volume":"7","author":"Wada","year":"2017","journal-title":"Sci Rep"},{"key":"B47","doi-asserted-by":"publisher","DOI":"10.1002\/1521-4141(200106)31:6<1837::aid-immu1837>3.0.co;2-h","article-title":"Complementarity and Redundancy of the Binding Specificity of HLA-DRB1, -DRB3, -DRB4 and -DRB5 Molecules","volume":"31","author":"Texier","year":"2001","journal-title":"Eur J Immunol"},{"key":"B48","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2013.11.002","article-title":"PD-1 Dependent Exhaustion of CD8+ T Cells Drives Chronic Malaria","volume":"5","author":"Horne-Debets","year":"2013","journal-title":"Cell Rep"},{"key":"B49","doi-asserted-by":"publisher","DOI":"10.1038\/srep26210","article-title":"Mice Lacking Programmed Cell Death-1 Show a Role for CD8(+) T Cells in Long-Term Immunity Against Blood-Stage Malaria","volume":"6","author":"Horne-Debets","year":"2016","journal-title":"Sci Rep"},{"key":"B50","doi-asserted-by":"publisher","DOI":"10.1038\/s41591-018-0117-4","article-title":"Cytotoxic CD8(+) T Cells Recognize and Kill Plasmodium Vivax-Infected Reticulocytes","volume":"24","author":"Junqueira","year":"2018","journal-title":"Nat Med"},{"key":"B51","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M311331200","article-title":"A Role for Apical Membrane Antigen 1 During Invasion of Hepatocytes by Plasmodium Falciparum Sporozoites","volume":"279","author":"Silvie","year":"2004","journal-title":"J Biol Chem"}],"container-title":["Frontiers in Immunology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2021.690348\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,1,3]],"date-time":"2023-01-03T04:34:14Z","timestamp":1672720454000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2021.690348\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,7,7]]},"references-count":51,"alternative-id":["10.3389\/fimmu.2021.690348"],"URL":"https:\/\/doi.org\/10.3389\/fimmu.2021.690348","relation":{},"ISSN":["1664-3224"],"issn-type":[{"value":"1664-3224","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,7,7]]},"article-number":"690348"}}