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

Abstract

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.

Keywords: Immunogenicity; Malaria vaccines; Plasmodium falciparum; Polymorphisms.

Fig. 1

Distribution of parasitemia across parishes. Abedi had the highest percentage of participants with parasitemia > 5000 parasites/µL of blood, while Aere had the least

Fig. 2

The age dependent distribution of parasitemia in a study population in Apac District, a region of intense malaria transmission in Uganda. It was observed that parasitemia decreased with age and with density of parasites per microliter of blood. There were more older participants with < 5000 parasites/µL, while younger participants had > 5000 parasites/µL

Fig. 3

Graph showing distribution of parasitemia with clinicaloutcomes in the study population. There were more participants with parasitemia of < 5000 parasites/µL in the symptomatic group compared to the asymptomatic group. Interestingly, there were more asymptomatic cases in the > 5000 parasites/µL group. This distribution shows that people in Apac district are able to harbour high parasitemia and yet show no symptoms. A function of immunity acquired after repeated exposures

Fig. 4

SERA amplification, M is 100 bp molecular weight marker, 1–7 are field samples, K1, HB3 are laboratory isolates of Plasmodium falciparum used as positive controls, −ve is the negative control. The expected product size is at 175 bp (ORI) and 199 bp (ORII). b EBA-175 amplification, M, 100 bp molecular weight marker, lane 1 and K1-mixed infections with both 795 bp and 714 bp alleles. Lanes 2, 6, 8, 10, Dd2 and 3D7 infection with the F-allele at 795 bp, while lanes 3, 5 and 7 represent the C-allele at 714 bp. −VE and −N are the negative and nested negative controls. c A gel picture of CSP amplification, M, 100 bp molecular weight marker, lanes 1–7 are field isolates, K1 and 3D7 are positive controls, −ve is a negative control. The expected product size is at 321 bp

Fig. 5

The age dependent distribution of SERA5 (a) and EBA-175 (b) alleles in a study population in Apac district, a region of intense malaria transmission in Uganda ORII was more prevalent among the < 5 year participants as compared to the those > 10 years. Similarly, FCR fragment was more prevalent in the under 5 population. This could mean that these two figments from different genes are a target of immunity

Fig. 6

a SERA5, ORII allele was more prevalent among participants with a parasitemia > 5000 parasites/µL of blood, while CAMP allele was higher in the group with a parasitemia of < 5000 parasites/µL of blood, b EBA-175 FCR allele was observed more in the group with a parasitemia of > 5000 parasites/µL of blood, compared to ORI which was observed more in the group with < 5000 parasites/µL of blood

Fig. 7

a Shows that allele ORII was more prevalent in the symptomatic group compared to the asymptomatic group, b shows distribution of EBA-175 alleles with clinical outcomes in the study population. The FCR allele was more prevalent in the symptomatic group compared to the asymptomatic group

Fig. 8

CSP haplotypes observed in Ugandan, Sierra Leonean, Asian, and Gabonese P. falciparum isolates. X- Represents the variable site immediately after the central NANP repeats (amino acid residues 308–317). Th2R and Th3R are the non-variable regions in the C-terminal portion of the CSP gene (amino acid residues 326–342 and 356–378 respectively). Haplotype E12, observed in Sierra Leone was similar to one haplotype from two Ugandan samples (U40028 and U20123); these haplotypes are asterisked

Fig. 9

MOI with clinical outcomes (a) and parasitemia (b). In a, MOI was high in the symptomatic group as compared to the asymptomatic group. In b, MOI increased with parasitemia