Haplotyping of Plasmodium falciparum Thrombospondin-Related Adhesive Protein Coding Gene in Isolates from Buea, Cameroon

Haplotyping of Plasmodium falciparum Thrombospondin-Related Adhesive Protein Coding Gene in Isolates from Buea, Cameroon

Loading document ...
Loading page ...


Author(s): Dieudonné L. Njimoh, Marcel M. Nyuylam, Daniel N. Bangwen, Brice T. Meulah, Alexandra L. Z. Djomkam, Stephen M. Ghogomu

Download Full PDF Read Complete Article

DOI: 10.18483/ijSci.1770 94 300 37-47 Volume 7 - Aug 2018


Knowledge of the genetic diversity of leading malaria vaccine candidate genes in various geographical regions is very essential for the development of a very successful malaria vaccine. This study aimed at assessing the haplotype variation of Plasmodium falciparum thrombospondin-related adhesive protein (PfTRAP) gene in malaria isolates from Buea, Cameroon. Parasitized blood samples were collected on whatmann filter papers by finger pricks from 291 participants. Plasmodium infection was assessed by thick and thin blood films. DNA samples were isolated from blood spots using the chelex method and used for falciparum speciation and PfTRAP gene amplification by polymerase chain reaction. PfTRAP amplicons were digested using four different restriction enzymes. The PfTRAP sequences were amplified from 83 of 172 falciparium positive DNA extracts. Digestion of the gene with the BglII gave two of four possible variants {(+-; 2.4%), (--; 100%)}, three with SspI {(+-; 1.2%), (-+; 15%), (--; 80.7%)} and all four with AflII {(+-; 71.1%), (++; 16.9%), (-+; 4.8%); (--; 7.2%)}. The single TaqαI site on the gene was present in 65% of the samples. Haplotype analysis showed that 14 of the 96 possible PfTRAP haplotypes were present in our isolates with haplotype 91 (----+-+) being the most prevalent (32%). We have demonstrated the various PfTRAP haplotypes circulating with low frequency in the malaria endemic region of Buea. These findings will add to the knowledge of the PfTRAP haplotypes circulating worldwide which is very vital for a TRAP-based/multi-antigen malaria vaccine development that will be globally effective.


Plasmodium falciparum, Malaria, Vaccine Candidate, Haplotypes, Polymorphism


  1. WHO: World malaria Report 2017. Geneva: WHO; 2017. Available: www.who.int/malaria/publications/world-malaria-report-2017/en/
  2. Murray, CJL., Rosenfeld, LC., Lim, SS., Andrews, KG., Foreman, KJ., Haring, D., Fullman, N., Naghavi, M., Lozano, R., Lopez, AD. Global malaria mortality between 1980 and 2010: a systematic analysis. LANCET. 2012; 379 (9814): 413-431.
  3. Dondorp AM, Nosten F, Yi P, Das D, Phyo AP, Tarning J, Lwin KM, et al. Artemisinin Resistance in Plasmodium falciparum Malaria. The New England Journal of Medicine. 2009; 361(5): 455-467.
  4. WHO: World Malaria Report 2016: Geneva. WHO;2016. Available: www.who.int/malaria/publications/world-malaria-report-2016/report/en/
  5. Trape, JF. The public health impact of chloroquine resistance in Africa. Am J Trop Med Hyg. 2001; 64:127
  6. Menard D, Khim N, Beghain J, Adegnika AA, Shafiul-Alam M, Amodu O et al. A worldwide map of Plasmodium falciparum K13-Propeller Polymorphisms. N Engl J Med. 2016; 374:2453-2464.
  7. 7.Chattopadhyay R. and Kumar S. Malaria vaccine: Latest update and challenges ahead. Indian Journal of Experimental Biology. 2009; 47: 527-536.
  8. Hyde J. E. Targeting purine and pyrimidine metabolism in Human Apicomplexans parasites. Current drug targets. 2007; 8:31-47.
  9. Conway, DJ. Paths to a malaria vaccine illuminated by parasite genomics. Trends in Genetics. 2015; 31(2): 97-107.
  10. Tamminga C, Sedegah M, Regis D, Chuang I, Epstein JE, Spring M, et al. Adenovirus-5-Vectored P. falciparum Vaccine Expressing CSP and AMA1. Part B: Safety, Immunogenicity and Protective Efficacy of the CSP component. PLoS ONE. 2011; 6(10): e25868.
  11. Oguru BR, Apollo OJ, Mckinney D, Okoth W, Siangla J, Dubovsky F, et al. For the MSP-1 Malaria Vaccine Working Group: Blood stage malaria vaccine eliciting high antigen-specific antibody concentrations confers no protection to young children in western Kenya. PLoS One. 2009; 4:e4708.
  12. Sagara I, Ellis RD, Dicko A, Niambele MB, Kamate B, Guindo O, et al. A randomized and controlled Phase 1 study of the safety and immunogenicity of the AMA-1C1/Alhydrogel + CPG7909 vaccine for Plasmodium falciparum malaria in semi-immune Malian adults. Vaccjne, 2009; 27:7292-7298.
  13. EMA. First malaria vaccine receives positive scientific opinion from EMA, European Medicines Agency. 2015, Press release: EMA/CHMP/488348/2015.
  14. RTS,S Clinical Trials Partnership. Efficacy and safety of the RTS,S/ASO1 malaria vaccine during 18 months after vaccination: a randomized, controlled trial in children and young infants at 11 African sites. PLoS Med. 2014; 11: e1001685.
  15. Ewer k, Collins K, O’Hara G, Duncan C, Rowland R, Reyes-Sandoval A et al. Protection from malaria sporozoite challenge correlates with frequency of TRAP-specific CD8+ T cells secreting IFNγ. Malaria: New Approaches to Understanding Host-Parasite Interactions. 2010; s86.
  16. Cowan G, Krishna S, Crisanti A, Robson K,. Expression of thrombospondin-related anonymous protein in Plasmodium falciparum sporozoites. Lancet. 1992; 339: 1412–1413.
  17. Rogers WO, Malik A, Mellouk S, Nakamura K, Rogers MD, Szarfman A, Gordon DM, Nussler AK, Aikawa M, Hoffman SL. Characterization of Plasmodium falciparum sporozoite surface protein 2. Proc Natl Acad Sci USA. 1992; 89: 9176–9180.
  18. Khusmith S, Sedegah M, Hoffman SL. Complete protection against Plasmodium yoelli by adoptive transfer of a CD8+ cytotoxic T cell clone recognizing sporozoite surface protein 2. Infect. Immune. 1994; 62: 2979-2983.
  19. Alloueche A, Milligan P, Conway DJ, Pinder M, Bojang K, Doherty T, Tornieporth N, Cohen J, Greenwood BM. Protective efficacy of the RTS,S/AS02 Plasmodium falciparum malaria vaccine is not strain specific. Am J Trop Med Hyg. 2003; 68:97-101.
  20. Wizel B, Houghten RA, Parker KC, Coligan JE, Church P, Gordon DM, et al. Irradiated sporozoite vaccine induces HLA-B8-restricted cytotoxic T lymphocyte responses against two overlapping epitopes of the Plasmodium falciparum sporozoite surface protein 2. J. Exp. Med. 1995; 182: 1435–1445.
  21. Ogwang C, Afolabi M, Kimani D, Jagne YJ, Sheehy SH, et al. Safety and immunogenicity of heterologous prime-boost immunization with Plasmodium falciparum malaria candidate vaccines, ChAd63 ME-TRAP and MVA ME-TRAP, in healthy Gambian and Kenyan Adults. PLOS ONE. 2013; 8(3): e57726. Doi: 10.1371/journal.pone.0057726.
  22. Afolabi MO,Tiono AB, Adetifa UJ, Yaro JB, Drammeh A, Nebie I, Bliss C, et al. Safety and immunogenicity of ChAd63 and MVA ME-TRAP in West African children and infants. Molecular Therapy. 2016; 24(8): 1470-1477.
  23. Mensah VA, Gueye A, Ndiaye M, Edwards NJ, Wright D, Anagnostou NA, et al. Safety, Immunogenicity and Efficacy of Prime-Boost vaccination with ChAd63 and MVA encoding ME-TRAP against Plasmodium falciparum infection in adults in Senegal. PLOS ONE. 2016 ; 11(12): e0167951. Doi: 10.1371/journal.pone.0167951.
  24. Mensah VA, Roetynck S, Kanteh EK, Bowyer G, Ndaw A, Oko F, Bliss CM, et al. Safety and immunogenicity of malaria vectored vaccines given with routine expanded program on immunization vaccines in Gambian infants and neonates: A randomized controlled trial. Front. Immunol. 2017; 8:1551. Doi: 10.3389/fimmu.2017.01551.
  25. Robson KJH, Hall JRS, Davies LC, Crisanti A, Hill AVS, Wellems TE. Polymorphism of the TRAP gene of Plasmodium falciparum. Proc R Soc [Lond] B. 1990; 242: 205–216.
  26. Robson KJH, Dolo A, Hackford IR, Doumbo O, Richards MB, Keita MM, Sidibe T, Bosman A, Modiano D, Crisanti A. Natural polymorphism in the Thrombospondin-Related Adhesive Protein of Plasmodium falciparum. Am. J. Trop. Med. Hyg. 1998; 58(1): 81-89.
  27. Ellis RD, Wu Y, Martin LB, Shaffer D, Miura K, Aebig J, et al. Phase I study in malaria naïve adults of BSAM2/Alhydrogen1(R)+CPG 7909, a blood stage vaccine against P. falciparum malaria. PLoS ONE. 2012; 7(10): e46094
  28. McCarthy JS, Marjason J, Elliott S, Fahey P, Bang G, Malkin E, et al. A phase I trial of MSP2-C1, a blood-stage malaria vaccine containing 2 isoforms of MSP2 formulated with Montanide(R) ISA 720. PLoS ONE. 2011; 6(9): e24413.
  29. Elias SC, Collins KA, Halstead FD, Choudhary P, Bliss CM, Ewer KJ, Sheehy SH, Duncan CJ, Biswas S, Hill AV, Draper SJ. Assessment of immune interference, antagonism and diversion following human immunization with biallelic blood-stage malaria viral-vectored vaccines and controlled malaria infection. J. Immunol. 2013; 190:1135-1147.
  30. Amambua-Ngwa A, Park DJ, Volkman SK, Barnes KG, Bei AK et al. SNP genotyping identifies new signatures of selection in a deep sample of West African Plasmodium falciparum malaria parasites. Mol. Biol. Evol. 2012; 29: 3249-3253.
  31. Miotto O, Almagro-Garcia J, Manske M, Macinnis B, Campino S, Rockett KA et al. Multiple populations of artemisinin resistant P. falciparum in Cambodia. Nat. Genet. 2013; 45(6): 648-655.
  32. Ouattara A, Takala-Harrison S, Thera MA, Coulibaly D, Niangaly A, Saye R, et al. Molecular basis of allele-specific efficacy of a blood-stage malaria vaccine: vaccine development implications. J. Infect. Dis. 2013; 207:511-519.
  33. Thera MA, Doumbo OK,Coulibaly D, Laurens MB, Ouattara A, Kone AK et al. A field trial to assess a blood-stage malaria vaccine. N. Engl. J. Med. 2011; 365:1004-1013.
  34. McConkey SJ, Reece WH, Moorthy VS, Webster D, Dunachie S, Butcher G, et al. Enhanced T-cell immunogenicity of plasmid DNA vaccines boosted by recombinant modified vaccinia virus Ankara in humans. Nat. med. 2003; 9(6): 729-735.
  35. Weedall GD, Preston BMJ, Thomas AW, Sutherland CJ, Conway DJ. Differential evidence of natural selection on two leading sporozoite stage malaria vaccine candidate antigens. International Journal of Parasitol. 2006; 37:77-85.
  36. Nazeri S, Zakeri S, AbouieMehrizi A, Djadid ND. Naturally acquired immune responses to thrombospondin-related adhesion protein (TRAP) of Plasmodium vivax in patients from areas of unstable malaria transmission. Acta Tropica. 2017; 173:45-54.
  37. 37.Takem NE, Achidi AE, Ndumbe PM. An update of malaria infection and anemia in adults in Buea, Cameroon. BioMed central. 2010; 3:121.
  38. 38. Bigoga JD, Manga L, Titanji VPK, Coetzee M, Leke RGF. Malaria vectors and transmission dynamics in coastal south-western Cameroon. Malar J. 2007; 6:5. doi: 10.1186/1475-2875-6-5.
  39. 39. Miguel RB, Coura JR, Samudio F, Suarez-Mutis MC. Evaluation of three different DNA extraction methods from blood samples collected in dried filter paper in Plasmodium subpatent infactions from the Amazon Region in Brazil. Rev. Inst. Med. Trop. Sao Paulo. 2013 ; 55(3):205-208.
  40. 40. Snounou G, Viriyakosol S, Zhu XP, Jarra W, Pinheiro L, Rosario VE, et al. High sensitivity of detection of human malaria parasites by the use of nested polymerase chain reaction. Molecular and Biochemical Parasitology. 1993; 61: 315-320.
  41. 41. Barry AE, Schultz L, Buckee CO, Reeder JC. Contrasting population structures of the genes encoding ten leading vaccine-candidate antigens of the human malaria parasite, Plasmodium falciparum. PLOS ONE. 2009; 4:e8497.
  42. 42. Ohashi J, Suzuki Y, Naka I, Hananantachai H, Patarapotikul J. Selection on the Thrombospondin-Related Adhesive Protein (TRAP) gene polymorphism of Plasmodium falciparum in Thailand. PLOS ONE. 2014; 9(2): e90522. Doi: 10.1371/journal.pone.0090522.
  43. 43. Chenet SM, Branch OH, Escalante AA, Lucas CM, Bacon DJ. Genetic diversity of vaccine candidate antigens in Plasmodium falciparum isolates from the Amazon basin of Peru. Malaria journal. 2008; 7:93. Doi: 10.1186/1475-2875-7-93.

Cite this Article:

International Journal of Sciences is Open Access Journal.
This article is licensed under a Creative Commons Attribution 4.0 International (CC BY 4.0) License.
Author(s) retain the copyrights of this article, though, publication rights are with Alkhaer Publications.

Search Articles

Issue June 2023

Volume 12, June 2023

Table of Contents

World-wide Delivery is FREE

Share this Issue with Friends:

Submit your Paper