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Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria
Published on: November 10, 2015
Next generation sequencing to detect variation in the Plasmodium falciparum circumsporozoite protein
Kavita Gandhi1, Mahamadou A Thera, Drissa Coulibaly
1Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA. Kavita.Gandhi@som.umaryland.edu
Insights
Next-generation sequencing (454) identified more genetic diversity in malaria vaccine targets (circumsporozoite protein T-cell epitopes) than older methods. However, it could not sequence B-cell epitopes.
Area of Science:
- Immunology
- Parasitology
- Genetics
Background:
- The RTS,S/AS01 malaria vaccine targets the Plasmodium falciparum circumsporozoite protein (CSP).
- Understanding CSP sequence diversity is crucial for improving malaria vaccine efficacy.
- Previous sequencing technologies had limitations in evaluating diversity within CSP's immunogenic regions.
Purpose of the Study:
- To evaluate 454 next-generation sequencing for assessing genetic diversity in CSP immunogenic regions.
- To compare 454 sequencing with Sanger sequencing for malaria vaccine target diversity analysis.
Main Methods:
- Sequencing of Plasmodium falciparum circumsporozoite gene (cs) portions using 454 and Sanger technologies.
- Analysis of samples from a study conducted in Bandiagara, Mali.
- Comparison of single nucleotide polymorphisms (SNPs) and haplotype detection between sequencing methods.
Main Results:
- 454 sequencing detected more SNPs and haplotypes in CSP T-cell epitopes compared to Sanger sequencing.
- 454 sequencing demonstrated superior ability to resolve genetic diversity in samples with multiple infections.
- 454 sequencing was unable to generate sequence data for CSP B-cell epitopes.
Conclusions:
- Next-generation sequencing (454) offers enhanced capabilities for analyzing genetic diversity in malaria vaccine targets, particularly T-cell epitopes.
- Further methodological development is needed to overcome limitations in sequencing B-cell epitopes using this technology.
- Improved understanding of CSP diversity can inform the development of more effective malaria vaccines.
Abstract:
The malaria vaccine RTS,S/AS01, based on immunogenic regions of the Plasmodium falciparum circumsporozoite protein (CSP), has partial efficacy against clinical malaria in African children. Understanding how sequence diversity in CSP T- and B-cell epitopes relates to naturally acquired and vaccine-induced immunity may be useful in efforts to improve the efficacy of CSP-based vaccines. However, limitations in sequencing technology have precluded thorough evaluation of diversity in the immunogenic regions of this protein. In this study, 454, a next generation sequencing technology, was evaluated as a method for assessing diversity in these regions. Portions of the circumsporozoite gene (cs) were sequenced both by 454 and Sanger sequencing from samples collected in a study in Bandiagara, Mali. 454 detected more single nucleotide polymorphisms and haplotypes in the T-cell epitopes than Sanger sequencing, and it was better able to resolve genetic diversity in samples with multiple infections; however, it failed to generate sequence for the B-cell epitopes.
