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Pooled Amplicon Deep Sequencing of Candidate Plasmodium falciparum Transmission-Blocking Vaccine Antigens
Polymorphisms within Plasmodium falciparum vaccine candidate antigens have the potential to compromise vaccine efficacy. Understanding the allele frequencies of polymorphisms in critical binding regions of antigens can help in the designing of strain-transcendent vaccines. Here, we adopt a pooled deep-sequencing approach, originally designed to study P. falciparum drug resistance mutations, to study the diversity of two leading transmission-blocking vaccine candidates, Pfs25 and Pfs48/45. We sequenced 329 P. falciparum field isolates from six different geographic regions. Pfs25 showed little diversity, with only one known polymorphism identified in the region associated with binding of transmission-blocking antibodies among our isolates. However, we identified four new mutations among eight non-synonymous mutations within the presumed antibody-binding region of Pfs48/45. Pooled deep sequencing provides a scalable and cost-effective approach for the targeted study of allele frequencies of P. falciparum candidate vaccine antigens.
Author Notes
Financial support: This project was funded by the National Institute for Allergy and Infectious Diseases (NIAID) grant R01AI089819 (JJJ), R21AI101427 (NK), R21 AI103466 (NK), and R01AI107949 (SRM). Christian M. Parobek was supported by the National Institute of General Medicine Sciences T32GM008719, T32GM007092, and F30AI109979. Nicholas F. Brazeau was funded by a grant from the IDSA Medical Scholars Program. David L. Saunders and Chanthap Lon were supported by the U.S. Department of Defense Global Emerging Surveillance Program. Billy Ngasala and Andreas Mårtensson were supported by The Swedish Development Cooperation Agency (SIDA) (Bil-Tz 16/9875007059). Andreas Mårtensson was also supported by The Swedish Medical Research Council (2013-6594). Sample collection in Sainte Marie was financially supported by Institut Pasteur de Madagascar.
Authors' addresses: Jonathan J. Juliano, Nicholas F. Brazeau, and Irving Hoffman, Department of Medicine, Division of Infectious Diseases, University of North Carolina at Chapel Hill, Chapel Hill, NC, E-mails: jonathan_juliano@med.unc.edu, nbrazeau1@gmail.com, and irving_hoffman@med.unc.edu. Christian M. Parobek, Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC, E-mail: christian_parobek@med.unc.edu. Billy Ngasala, Department of Parasitology, Muhimbili University of Health and Allied Sciences, Dar el Salam, United Republic of Tanzania, E-mail: bngasala70@yahoo.co.uk. Milijaona Randrianarivelojosia, Department of Malariology, Institut Pasteur de Madagascar, Antananarivo, Madagascar, E-mail: milijaon@pasteur.mg. Chanthap Lon and David L. Saunders, Department of Immunology and Medicine, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand, E-mails: chanthapl.ca@afrims.org and david.saunders@afrims.org. Kashamuka Mwandagalirwa and Antoinette Tshefu, School of Public Health, University of Kinshasa, Kinshasa, The Democratic Republic of the Congo, E-mails: mkashamuka@yahoo.com and antotshe@yahoo.com. Ravi Dhar, Sushant Lok, Gurgaon, India, E-mail: rdhar_in@yahoo.com. Bidyut K. Das, Department of Medicine, SCB Medical College, Odisha, India, E-mail: bidyutdas@hotmail.com. Francis Martinson, University of North Carolina Project, Lilongwe, Malawi, E-mail: francis_martinson@med.unc.edu. Andreas Mårtensson, Infectious Diseases Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden, E-mail: andreas.martensson@ki.se. Nirbhay Kumar, Department of Tropical Medicine, School of Public Health and Tropical Medicine, Tulane University, New Orleans, LA, E-mail: nkumar@tulane.edu. Steven R. Meshnick, Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, E-mail: meshnick@email.unc.edu.