title: Complex Minigene Library Vaccination for Discovery of Pre-Erythrocytic Plasmodium T Cell Antigens.
abstract: Development of a subunit vaccine targeting liver-stage Plasmodium parasites requires the identification of antigens capable of inducing protective T cell responses. However, traditional methods of antigen identification are incapable of evaluating T cell responses against large numbers of proteins expressed by these parasites. This bottleneck has limited development of subunit vaccines against Plasmodium and other complex intracellular pathogens. To address this bottleneck, we are developing a synthetic minigene technology for multi-antigen DNA vaccines. In an initial test of this approach, pools of long (150 bp) antigen-encoding oligonucleotides were synthesized and recombined into vectors by ligation-independent cloning to produce two DNA minigene library vaccines. Each vaccine encoded peptides derived from 36 (vaccine 1) and 53 (vaccine 2) secreted or transmembrane pre-erythrocytic P. yoelii proteins. BALB/cj mice were vaccinated three times with a single vaccine by biolistic particle delivery (gene gun) and screened for interferon-γ-producing T cell responses by ELISPOT. Library vaccination induced responses against four novel antigens. Naïve mice exposed to radiation-attenuated sporozoites mounted a response against only one of the four novel targets (PyMDH, malate dehydrogenase). The response to PyMDH could not be recalled by additional homologous sporozoite immunizations but could be partially recalled by heterologous cross-species sporozoite exposure. Vaccination against the dominant PyMDH epitope by DNA priming and recombinant Listeria boosting did not protect against sporozoite challenge. Improvements in library design and delivery, combined with methods promoting an increase in screening sensitivity, may enable complex minigene screening to serve as a high-throughput system for discovery of novel T cell antigens.
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, Washington, United States of America.|Center for Emerging and Re-emerging Infectious Diseases, University of Washington, Seattle, Washington, United States of America.
authors.forename: Brad C
authors.initials: BC
authors.lastname: Stone
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, Washington, United States of America.|Center for Emerging and Re-emerging Infectious Diseases, University of Washington, Seattle, Washington, United States of America.
authors.forename: Arnold
authors.initials: A
authors.lastname: Kas
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, Washington, United States of America.|Center for Emerging and Re-emerging Infectious Diseases, University of Washington, Seattle, Washington, United States of America.
authors.forename: Zachary P
authors.initials: ZP
authors.lastname: Billman
authors.affiliation: Department of Microbiology, University of Washington, Seattle, Washington, United States of America.
authors.forename: Deborah H
authors.initials: DH
authors.lastname: Fuller
authors.affiliation: Department of Microbiology, University of Washington, Seattle, Washington, United States of America.
authors.forename: James T
authors.initials: JT
authors.lastname: Fuller
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, Washington, United States of America.
authors.forename: Jay
authors.initials: J
authors.lastname: Shendure
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, Washington, United States of America.|Center for Emerging and Re-emerging Infectious Diseases, University of Washington, Seattle, Washington, United States of America.|Department of Microbiology, University of Washington, Seattle, Washington, United States of America.|Seattle Malaria Clinical Trials Center, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.|Human Challenge Center, Center for Infectious Disease Research, Seattle, Washington, United States of America.
authors.forename: Sean C
authors.initials: SC
authors.lastname: Murphy
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000737:chemistry / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D003429:Cross Reactions; D018604:Epitope Mapping; D005260:Female; D015723:Gene Library; D007371:Interferon-gamma / Q000096:biosynthesis; D017780:Malaria Vaccines / Q000235:genetics* / Q000276:immunology*; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D010967:Plasmodium yoelii / Q000276:immunology*; D034101:Sporozoites / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D014611:Vaccination
chemical_list: D000953:Antigens, Protozoan; D017780:Malaria Vaccines; D007371:Interferon-gamma; D008291:Malate Dehydrogenase