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Background: Plasmodium falciparum erythrocyte binding antigen-175 (PfEBA-175) is a candidate antigen for a blood-stage malaria vaccine, while various polymorphisms and dimorphism have prevented to development of effective vaccines based on this gene. This study aimed to investigate the dimorphism of PfEBA-175 on both the Bioko Island and continent of Equatorial Guinea, as well as the genetic polymorphism and natural selection of global PfEBA-175., Methods: The allelic dimorphism of PfEBA-175 region II of 297 bloods samples from Equatorial Guinea in 2018 and 2019 were investigated by nested polymerase chain reaction and sequencing. Polymorphic characteristics and the effect of natural selection were analyzed using MEGA 7.0, DnaSP 6.0 and PopART programs. Protein function prediction of new amino acid mutation sites was performed using PolyPhen-2 and Foldx program., Results: Both Bioko Island and Bata district populations, the frequency of the F-fragment was higher than that of the C-fragment of PfEBA-175 gene. The PfEBA-175 of Bioko Island and Bata district isolates showed a high degree of genetic variability and heterogeneity, with π values of 0.00407 & 0.00411 and Hd values of 0.958 & 0.976 for nucleotide diversity, respectively. The values of Tajima's D of PfEBA-175 on Bata district and Bioko Island were 0.56395 and - 0.27018, respectively. Globally, PfEBA-175 isolates from Asia were more diverse than those from Africa and South America, and genetic differentiation quantified by the fixation index between Asian and South American countries populations was significant (FST > 0.15, P < 0.05). A total of 310 global isolates clustered in 92 haplotypes, and only one cluster contained isolates from three continents. The mutations A34T, K109E, D278Y, K301N, L305V and D329N were predicted as probably damaging., Conclusions: This study demonstrated that the dimorphism of F-fragment PfEBA-175 was remarkably predominant in the study area. The distribution patterns and genetic diversity of PfEBA-175 in Equatorial Guinea isolates were similar another region isolates. And the levels of recombination events suggested that natural selection and intragenic recombination might be the main drivers of genetic diversity in global PfEBA-175. These results have important reference value for the development of blood-stage malaria vaccine based on this antigen., (© 2021. The Author(s).)

Background: Thrombospondin-related adhesive protein (TRAP) is a transmembrane protein that plays a crucial role during the invasion of Plasmodium falciparum into liver cells. As a potential malaria vaccine candidate, the genetic diversity and natural selection of PfTRAP was assessed and the global PfTRAP polymorphism pattern was described., Methods: 153 blood spot samples from Bioko malaria patients were collected during 2016-2018 and the target TRAP gene was amplified. Together with the sequences from database, nucleotide diversity and natural selection analysis, and the structural prediction were preformed using bioinformatical tools., Results: A total of 119 Bioko PfTRAP sequences were amplified successfully. On Bioko Island, PfTRAP shows its high degree of genetic diversity and heterogeneity, with π value for 0.01046 and Hd for 0.99. The value of dN-dS (6.2231, p < 0.05) hinted at natural selection of PfTRAP on Bioko Island. Globally, the African PfTRAPs showed more diverse than the Asian ones, and significant genetic differentiation was discovered by the fixation index between African and Asian countries (Fst > 0.15, p < 0.05). 667 Asian isolates clustered in 136 haplotypes and 739 African isolates clustered in 528 haplotypes by network analysis. The mutations I116T, L221I, Y128F, G228V and P299S were predicted as probably damaging by PolyPhen online service, while mutations L49V, R285G, R285S, P299S and K421N would lead to a significant increase of free energy difference (ΔΔG > 1) indicated a destabilization of protein structure., Conclusions: Evidences in the present investigation supported that PfTRAP gene from Bioko Island and other malaria endemic countries is highly polymorphic (especially at T cell epitopes), which provided the genetic information background for developing an PfTRAP-based universal effective vaccine. Moreover, some mutations have been shown to be detrimental to the protein structure or function and deserve further study and continuous monitoring.

Background: Plasmodium falciparum circumsporozoite protein (PfCSP) is a potential malaria vaccine candidate, but various polymorphisms of the pfcsp gene among global P. falciparum population become the major barrier to the effectiveness of vaccines. This study aimed to investigate the genetic polymorphisms and natural selection of pfcsp in Bioko and the comparison among global P. falciparum population., Methods: From January 2011 to December 2018, 148 blood samples were collected from P. falciparum infected Bioko patients and 96 monoclonal sequences of them were successfully acquired and analysed with 2200 global pfcsp sequences mined from MalariaGEN Pf3k Database and NCBI., Results: In Bioko, the N-terminus of pfcsp showed limited genetic variations and the numbers of repetitive sequences (NANP/NVDP) were mainly found as 40 (35%) and 41 (34%) in central region. Most polymorphic characters were found in Th2R/Th3R region, where natural selection (p > 0.05) and recombination occurred. The overall pattern of Bioko pfcsp gene had no obvious deviation from African mainland pfcsp (Fst = 0.00878, p < 0.05). The comparative analysis of Bioko and global pfcsp displayed the various mutation patterns and obvious geographic differentiation among populations from four continents (p < 0.05). The global pfcsp C-terminal sequences were clustered into 138 different haplotypes (H&#95;1 to H&#95;138). Only 3.35% of sequences matched 3D7 strain haplotype (H&#95;1)., Conclusions: The genetic polymorphism phenomena of pfcsp were found universal in Bioko and global isolates and the majority mutations located at T cell epitopes. Global genetic polymorphism and geographical characteristics were recommended to be considered for future improvement of malaria vaccine design.

Background: Malaria is still a serious public health problem on Bioko Island (Equatorial Guinea), although the number of annual cases has been greatly reduced since 2004 through the Bioko Island Malaria Control Project (BIMCP). A better understanding of malaria parasite population diversity and transmission dynamics is critical for assessing the effectiveness of malaria control measures. The objective of this study is to investigate the genetic diversity of Plasmodium falciparum populations and multiplicity of infection (MOI) on Bioko Island 7 years after BIMCP., Methods: A total of 181 patients with uncomplicated P. falciparum malaria diagnosed with microscopy were collected from Bioko Island from January 2011 to December 2014. Parasite DNA was extracted using chelex-100 and species were identified using a real-time PCR followed by high-resolution melting. Plasmodium falciparum msp1 and msp2 allelic families were determined using nested PCR., Results: Three msp1 alleles (K1, MAD20, and RO33) and two msp2 alleles (FC27 and 3D7) were analysed in all samples. In msp1, the MAD20 allelic family was predominant with 96.69% (175/178) followed respectively by the K1 allelic family with 96.07% (171/178) and R033 allelic family with 70.78% (126/178). In msp2, the FC27 allelic family was the most frequently detected with 97.69% (169/173) compared to 3D7 with 72.25% (125/173). Twenty-six different alleles were observed in msp1 with 9 alleles for K1, 9 alleles for MAD20 and 8 alleles for R033. In msp2, 25 individual alleles were detected with 5 alleles for FC27 and 20 alleles for 3D7. The overall MOI was 5.51 with respectively 3.5 and 2.01 for msp1 and msp2. A significant increase in overall MOI was correlated with the age group of the patients (P = 0.026) or parasite densities (P = 0.04)., Conclusions: The present data showed high genetic diversity and MOI values among the P. falciparum population in the study, reflecting both the high endemic level and malaria transmission on Bioko Island. These data provide valuable information for surveillance of P. falciparum infection and for assessing the appropriateness of the current malarial control strategies in the endemic area.