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Background  The development of malaria vaccines is constrained by genetic polymorphisms exhibited by  Plasmodium falciparum  antigens. The project the age-dependent distribution of alleles or haplotypes of three  P. falciparum  malaria vaccine candidates, Circumsporozoite Protein ( csp ), Erythrocyte Binding Antigen 175 (eba-175) and Serine Repeat Antigen 5 (sera5) in a region of intense malaria transmission in Uganda.    Methods  A cross-sectional study was carried out between August and November 2009 in which 250 study participants were selected from a population of 600. Finger prick blood samples were collected after informed consent from participants below 5 years, 5–10 years, and above 10 years of age. Blood was used for microscopy, RDT and dried blood spots.  Plasmodium falciparum  DNA was extracted by chelex method. Alleles of  sera5  and  eba-175  were determined by polymerase chain reaction (PCR) amplification followed by resolution of products by agarose gel electrophoresis. Allele calling was done using gel photographs from ethiduim bromide stained gels. Haplotypes of  csp  were identified by sequencing 63 PCR products using the  P. falciparum  7G8 laboratory strain sequence as a reference. The data were analysed using SPSS 16, EQX for windows and Chi-square test was used to calculate associations (P-values), Excel was used to generate graphs. The BioEdit and NCBI blast software programs were used to analyse the sequences from which  csp  haplotypes map was constructed.    Results   Eba-175  FCR3 (48/178) and CAMP (16/178) alleles were observed, the FCR3 (24/67) allele being predominant among children aged below 5 years old while the CAMP (12/67) allele was predominant among older participants.  Sera5  alleles ORI (6/204) and ORII (103/204) were observed in the population, ORII was more prevalent and was significantly associated with age (P values < 0.0001), parasite density (P-value < 0.0001) and clinical outcomes (P value = 0.018). There was marked  csp  diversity in the Th2/Th3 region. Out of 63 sequences, 16 conformed to the reference strain and one (1/16) was similar to a West African haplotype and the majority (14/16) of the haplotypes were unique to this study region. There was an age-dependent distribution of  csp  haplotypes with more haplotypes being harbored by children < 5-year of age, (10/16) compared to adults (2/16). Interestingly, the  csp  haplotype corresponding to 3D7 whose prototypical sequence is identical to the sequence of the leading malaria vaccine candidate RTS, S was not observed.    Conclusion  This data suggest that the  eba-175  FCR3 allele , sera5  ORII allele ,  and  csp  haplotypes are targets of host immunity and under immune selection pressure in Apac District. These molecules could provide alternative malaria vaccine candidates as sub-unit vaccines .

malaria journal

Age-dependent carriage of alleles and haplotypes of Plasmodium falciparum sera5, eba-175, and csp in a region of intense malaria transmission in Uganda