Circumsporozoite Protein Gene Research Articles

Genetic polymorphism and evidence of signatures of selection in the Plasmodium falciparum circumsporozoite protein gene in Tanzanian regions with different malaria endemicity

BackgroundIn 2021 and 2023, the World Health Organization approved RTS,S/AS01 and R21/Matrix M malaria vaccines, respectively, for routine immunization of children in African countries with moderate to high transmission. These vaccines are made of Plasmodium falciparum circumsporozoite protein (PfCSP), but polymorphisms in the gene raise concerns regarding strain-specific responses and the long-term efficacy of these vaccines. This study assessed the Pfcsp genetic diversity, population structure and signatures of selection among parasites from areas of different malaria transmission intensities in Mainland Tanzania, to generate baseline data before the introduction of the malaria vaccines in the country.MethodsThe analysis involved 589 whole genome sequences generated by and as part of the MalariaGEN Community Project. The samples were collected between 2013 and January 2015 from five regions of Mainland Tanzania: Morogoro and Tanga (Muheza) (moderate transmission areas), and Kagera (Muleba), Lindi (Nachingwea), and Kigoma (Ujiji) (high transmission areas). Wright’s inbreeding coefficient (Fws), Wright’s fixation index (FST), principal component analysis, nucleotide diversity, and Tajima’s D were used to assess within-host parasite diversity, population structure and natural selection.ResultsBased on Fws (< 0.95), there was high polyclonality (ranging from 69.23% in Nachingwea to 56.9% in Muheza). No population structure was detected in the Pfcsp gene in the five regions (mean FST = 0.0068). The average nucleotide diversity (π), nucleotide differentiation (K) and haplotype diversity (Hd) in the five regions were 4.19, 0.973 and 0.0035, respectively. The C-terminal region of Pfcsp showed high nucleotide diversity at Th2R and Th3R regions. Positive values for the Tajima’s D were observed in the Th2R and Th3R regions consistent with balancing selection. The Pfcsp C-terminal sequences revealed 50 different haplotypes (H_1 to H_50), with only 2% of sequences matching the 3D7 strain haplotype (H_50). Conversely, with the NF54 strain, the Pfcsp C-terminal sequences revealed 49 different haplotypes (H_1 to H_49), with only 0.4% of the sequences matching the NF54 strain (Hap_49).ConclusionsThe findings demonstrate high diversity of the Pfcsp gene with limited population differentiation. The Pfcsp gene showed positive Tajima’s D values, consistent with balancing selection for variants within Th2R and Th3R regions. The study observed differences between the intended haplotypes incorporated into the design of RTS,S and R21 vaccines and those present in natural parasite populations. Therefore, additional research is warranted, incorporating other regions and more recent data to comprehensively assess trends in genetic diversity within this important gene. Such insights will inform the choice of alleles to be included in the future vaccines.

The first complete genome of the simian malaria parasite Plasmodium brasilianum

Naturally occurring human infections by zoonotic Plasmodium species have been documented for P. knowlesi, P. cynomolgi, P. simium, P. simiovale, P. inui, P. inui-like, P. coatneyi, and P. brasilianum. Accurate detection of each species is complicated by their morphological similarities with other Plasmodium species. PCR-based assays offer a solution but require prior knowledge of adequate genomic targets that can distinguish the species. While whole genomes have been published for P. knowlesi, P. cynomolgi, P. simium, and P. inui, no complete genome for P. brasilianum has been available. Previously, we reported a draft genome for P. brasilianum, and here we report the completed genome for P. brasilianum. The genome is 31.4 Mb in size and comprises 14 chromosomes, the mitochondrial genome, the apicoplast genome, and 29 unplaced contigs. The chromosomes consist of 98.4% nucleotide sites that are identical to the P. malariae genome, the closest evolutionarily related species hypothesized to be the same species as P. brasilianum, with 41,125 non-synonymous SNPs (0.0722% of genome) identified between the two genomes. Furthermore, P. brasilianum had 4864 (82.1%) genes that share 80% or higher sequence similarity with 4970 (75.5%) P. malariae genes. This was demonstrated by the nearly identical genomic organization and multiple sequence alignments for the merozoite surface proteins msp3 and msp7. We observed a distinction in the repeat lengths of the circumsporozoite protein (CSP) gene sequences between P. brasilianum and P. malariae. Our results demonstrate a 97.3% pairwise identity between the P. brasilianum and the P. malariae genomes. These findings highlight the phylogenetic proximity of these two species, suggesting that P. malariae and P. brasilianum are strains of the same species, but this could not be fully evaluated with only a single genomic sequence for each species.

Molecular identification of vivax malaria relapse patients in the Yunnan Province based on homology analysis of the Plasmodium vivax circumsporozoite protein gene

More than 85% of the malaria burden in the Yunnan Province is caused by imported vivax malaria, and Yunnan is also where the majority of vivax malaria patients are diagnosed in China. Timely removal of the infection sources of Plasmodium vivax and its breeding environment remains the key to eliminating the secondary transmission of imported malaria. To that end, blood samples were collected from cases diagnosed and revalidated as single species infection with P. vivax in the Yunnan Province from 2013 to 2020. Specifically, samples from vivax malaria patients with suspected relapses episodes were subjected to PCR amplification, product sequencing, and analysis of the P. vivax circumsporozoite protein (pvcsp) gene. In total, 77 suspected relapse patients were identified out of 2484 cases infected with P. vivax, with a total of 81 recurrent episodes. A total of 156 CDS (coding DNA sequence) chains were obtained through PCR amplification and sequencing of the pvcsp gene from 159 blood samples, 121 of which can be matched to the paired sequences of 59 vivax malaria patients with both primary attack and recurrent experience. Of the 59 pairs of pvcsp gene sequences, every one of 31 pairs showed only one haplotype and no variant sites (VS), meaning every two paired sequence was completely homologous. Every one of the remaining 28 paired sequences had two haplotypes but no length polymorphism, indicating that the paired sequences was “weakly heterologous” with no fragment insertions (or deletions). All 59 vivax malaria patients with recurrences were caused by the activation of P. vivax hypnozoites originated from the same population as the primary infection. The paired analysis of the similarity between high variant genes allowed the identification of relapse episodes caused by P. vivax homologous hypnozoites and also demonstrated pvcsp gene as one of the candidate molecular markers for tracing infection origin.

Genetic polymorphism of circumsporozoite protein (CSP) in Plasmodium malariae isolates from Malaysia.

Information about Plasmodium malariae is scanty worldwide due to its "benign" nature and low infection rates. Consequently, studies on the genetic polymorphisms of P. malariae are lacking. Here, we report genetic polymorphisms of 28 P. malariae circumsporozoite protein (Pmcsp) isolates from Malaysia which were compared with those in other regions in Asia as well as those from Africa. Phylogenetic analysis revealed that most Malaysian P. malariae isolates clustered together but independently from other Asian isolates. Low nucleotide diversity was observed in Pmcsp non-repeat regions in contrast to high nucleotide diversity observed in non-repeat regions of Plasmodium knowlesi CSP gene, the current major cause of malaria in Malaysia. This study contributes to the characterisation of naturally occurring polymorphisms in the P. malariae CSP gene.

Plasmodium spp. in macaques, Macaca fascicularis, in Malaysia, and their potential role in zoonotic malaria transmission.

Macaques, Macaca fascicularis, are a known reservoir of Plasmodium knowlesi, the agent of simian malaria which is the predominant zoonotic species affecting humans in Malaysia and other Southeast Asian countries. Recently, a naturally acquired human infection of another simian malaria parasite, P. cynomolgi has been reported. Thus, it is crucial to study the distribution of simian Plasmodium infections with particular attention to the macaques. Four hundred and nineteen (419) long-tailed macaques (Macaca fascicularis) were trapped in selected areas where human cases of P. knowlesi and P. cynomolgi have been reported. Nested polymerase chain reaction (PCR) was conducted to identify the Plasmodium spp., and circumsporozoite protein (CSP) genes of P. knowlesi samples were sequenced. Plasmodium cynomolgi infection was shown to be the most prevalent among the macaque population (68.4%). Although 50.6% of analyzed samples contained single infections either with P. knowlesi, P. cynomolgi, P. inui, P. coatneyi, or P. fieldi, mixed infections with double, triple, quadruple, and all 5 species were also detected. Infection with P. cynomolgi and P. knowlesi were the highest among Malaysian macaques in areas where humans and macaques are in close contact. The risk of zoonotic infection in these areas needs to be addressed since the number of zoonotic malaria cases is on the rise. With the elimination of human malaria, the risk of humans being infected with simian malaria is very high and steps should be taken to mitigate this issue.

Spatiotemporal Dynamic of the RTS,S/AS01 Malaria Vaccine Target Antigens in Senegal.

The RTS,S/AS01 malaria vaccine confers only moderate protection against malaria. Evidence suggests that the effectiveness of the RTS,S/AS01 vaccine depends upon the parasite population genetics, specifically regarding the circumsporozoite protein haplotypes in the population. We investigated Plasmodium falciparum circumsporozoite protein (PfCSP) gene sequences from two endemic sites in 2018 in Senegal. The PfCSP sequences were compared with those retrieved from the Pf3k genome database. In the central repeat region of PfCSP, the distribution of haplotypes differed significantly between the two study sites (Fisher's exact test, P < 0.001). No 3D7 vaccine strain haplotype was observed in this locus. In the C-terminal region, there was no significant difference in haplotypes distribution between Kedougou and Diourbel (Fischer's exact test, P = 0.122). The 3D7 haplotype frequency was 8.4% in early samples (2001-2011), but then it contracted in the subsequent years. The extensive plasticity of the P. falciparum genes coding the RTS,S/AS01 vaccine target antigens may influence the immune responses to circulating alleles. Monitoring the genetic diversity baseline and its dynamics over time and space would be instrumental in rationally improving the malaria RTS,S/AS01 vaccine and/or its implementation schedule.

Identification of immunodominant epitopes in allelic variants VK210 and VK247 of Plasmodium Vivax Circumsporozoite immunogen.

Plasmodium vivax-induced malaria is among the leading causes of morbidity and mortality in sub-tropical and tropical regions and infect 2.85 billion people globally. The continual rise and propagation of resistance against anti-malarial drugs is a prerequisite to develop a potent vaccine candidate for Plasmodium vivax (P. vivax). Circumsporozoite protein (CSP) is an important immunogen of malaria parasite that has the conserved CSP structure as an immune dominant B-cell epitope. In current study, we focused on designing multi-epitope vaccines (MEVs) using various immunoinformatics tools against Pakistani based allelic variants VK210 and VK247 of P. vivax CSP (PvCSP) gene. Antigenicity, allergic potential and physicochemical parameters of both PvCSP variants were assessed for the designed MEVs and they were within acceptable range suitable for post experimental investigations. The three-dimensional structures of both MEVs have been predicted ab initio, optimized, and validated by using different online servers. The both MEVs candidates were stable and free from aggregation-prone regions. The stability of both MEVs had been improved by a disulfide engineering approach. To estimate the binding energy and stability of the MEVs, molecular docking simulation and binding free energy calculations with TLR-4 immune receptor have been conducted. The docking score of PvCSP210 and PvCSP247 for TLR-4 was -6.34kJ/mol and-2.3kJ/mol, respectively. For PvCSP210-TLR4 system, mean RMSD was 4.96Å while PvCSP247-TLR4 system, average RMSD was 4.49Å. The binding free energy of PvCSP210-TLR4 complex and PvCSP247-TLR4 complex was -50.49/-117.15kcal/mol (MMGBSA/MMPSA) and -52.94/-96.26kcal/mol (MMGBSA/MMPSA), respectively. The expression of both MEVs produced in Escherichia coli K12 expression system by in silico cloning was significant. Immune simulation revealed that the proposed MEVs induce strong humoral and cellular immunological responses, in addition to significant production of interleukins and cytokines. In conclusions, we believed that the MEVs proposed in current research, using combine approach of immunoinformatics, structural biology and biophysical approaches, could induce protective and effective immune responses against P. vivax and the experimental validation of our findings could contribute to the development of potential malaria vaccine.

Natural malaria infection in anophelines vectors and their incrimination in local malaria transmission in Darién, Panama.

BackgroundMore than 85% of the malaria cases in Panama occur in poor, rural and indigenous regions like Darien Province. Vector diversity, infection rate and spatial distribution are important entomological parameters of malaria transmission dynamics. Their understanding is crucial for the development of effective disease control strategies. The objective of this study was to determine the composition of Anopheles species, their natural infection rate and their geographic distribution to better understand the malaria transmission dynamics in Darién, Panama.MethodsAnophelines mosquitoes were captured during the rainy and dry season of 2016. We selected five communities where adult anophelines were collected using CDC light-traps, and through protective human-baited traps. Detection of natural infection and Plasmodium genotype were detected via nested PCR through the amplification of ssrRNA and the circumsporozoite protein gene (csp), respectively.ResultsA total of 1,063 mosquitoes were collected mosquitoes were collected for the detection of natural infection with Plasmodium spp. Nine Anophelines species were identified, with the predominant species being: An. (Nys.) darlingi (45.0%) and An. (Nys.) albimanus (42.6%). Natural infection in An. (Nys.) albimanus with P. vivax was detected in one mosquito pool from the community Pueblo Tortuga (0.6%), three from Marraganti (1.7%), two from Bajo Chiquito (1.1%) and three pools from Alto Playona 3 (1.7%). For An. (Nys.) darlingi mosquitoes, we detected seven positive pools from the community Bajo Chiquito (4.0%), two pools from Marraganti (1.1%) and two pools from Alto Playona (1.1%). The P. vivax allelic variant VK210 was detected in infected mosquitoes.ConclusionThe results from this study provide new information on the transmission dynamics associated with anophelines vectors in the Darién region. This is the first report of natural P. vivax infection in An. (Nys.) darlingi and its incrimination as a potential malaria vector in this region of Panama. Additional studies are necessary to expand our knowledge and determine crucial parameters in malaria transmission in Darién, which in turn will aid the National Malaria Program in attaining an adequate malaria control strategy towards malaria elimination.

Genetic characterization of Plasmodium vivax isolates from Pakistan using circumsporozoite protein (pvcsp) and merozoite surface protein-1 (pvmsp-1) genes as genetic markers

BackgroundPlasmodium vivax contributes to over 70% malaria burden in Pakistan, but limited data exists on various aspects including genetic diversity of the parasite as compared to other parts of the world. Since the information about the genetic diversity of P. vivax assists to understand the population dynamics of the parasite, the current study was designed to understand population divergence of P. vivax in Pakistan using circumsporozoite protein (pvcsp) and merozoite surface protein-1 (pvmsp-1) genes as molecular markers.MethodsThe PCR for pvcsp and pvmsp-1 genes was carried out for 150 P. vivax isolates, followed by DNA sequencing of 35 and 30, respectively. Genetic diversity and polymorphism were analysed using ChromasPro, ClustalW, MEGA7, DnaSP v.5 and WebLogo programs.ResultsThe PCR for pvcsp and pvmsp-1 genes was carried out for 150 P. vivax isolates and resulting the PCR products of 1100 bp for pvcsp and ~ 400 bp for pvmsp-1 genes, respectively. In the central-repeat region (CRR) of pvcsp gene, sequences comprised of four variable repeats of PRMs, out of which GDRADGQPA (PRM1), GDRAAGQPA (PRM2) were more extensively dispersed among the P. vivax isolates. Partial sequences (~ 400 bp) of block 2 of pvmsp-1 gene depicted high level of diversity.ConclusionThe results revealed the polymorphism and genetic diversity especially at the CRR of pvcsp and block 2 of pvmsp-1 genes, respectively. The base-line data presented here warrants future studies to investigate more into the genetic diversity of P. vivax with large sample size from across the country for better understanding of population dynamics of P. vivax that will help to control malaria at individual and community level.

Generation of a Genetically Modified Chimeric Plasmodium falciparum ParasiteExpressing Plasmodium vivax Circumsporozoite Protein for Malaria Vaccine Development.

Chimeric rodent malaria parasites with the endogenous circumsporozoite protein (csp) gene replaced with csp from the human parasites Plasmodium falciparum (Pf) and P. vivax (Pv) are used in preclinical evaluation of CSP vaccines. Chimeric rodent parasites expressing PfCSP have also been assessed as whole sporozoite (WSP) vaccines. Comparable chimeric P. falciparum parasites expressing CSP of P. vivax could be used both for clinical evaluation of vaccines targeting PvCSP in controlled human P. falciparum infections and in WSP vaccines targeting P. vivax and P. falciparum. We generated chimeric P. falciparum parasites expressing both PfCSP and PvCSP. These Pf-PvCSP parasites produced sporozoite comparable to wild type P. falciparum parasites and expressed PfCSP and PvCSP on the sporozoite surface. Pf-PvCSP sporozoites infected human hepatocytes and induced antibodies to the repeats of both PfCSP and PvCSP after immunization of mice. These results support the use of Pf-PvCSP sporozoites in studies optimizing vaccines targeting PvCSP.

Genotyping and Phylogenetic Analysis of Plasmodium vivax Circumsporozoite Protein (pvcsp) Gene of Clinical Isolates in South-Eastern Iran

Circumsporozoite protein (CSP) is one of the most important surface sporozoite antigens in malaria, recently considered as a candidate for vaccination. Considering the importance of CSP, this study was conducted to investigate the polymorphism and genetic diversity of Plasmodium vivax Circumsporozoite Protein (Pvcsp) in the southeastern region of Iran during 2015-2016. To investigate polymorphism and genetic diversity, 20 blood samples were collected from patients with P. vivax, then DNA was extracted and amplified using partial sequence of CSP gene. Polymerase chain reaction (PCR) products were sequenced and compared to sequences from genomic databases using BLAST. Genetic evaluation and phylogenic analysis were performed using MEGA7 and DnaSP5 software's on 38 sequences include 20 sequences of our study and 18 sequences of Gene Bank. Eleven isolates were VK210 genotype and 9 isolates contained VK247. The result of variable segregation nucleotide site indicated that the differentiation of sequences in CSP were 25.67% in our 20 samples which are less than the 38 samples with a value of 26.67%. Comparing the ratio of dN/dS regions in the CSP gene indicates that the CSP varies more synonymously and amino acid has lower variation. Out of 38 samples, 35 unique haplotypes were identified based on 1042 nucleotide sequences in CSP, showing a variation percentage of 99.4%. The Tajima D analyses showed that CSP gene in P. vivax had a positive number in the total analyzed sequences, which means that the P. vivax mutations are in order to select positive evolution.

Human infection with Plasmodium knowlesi on the Laos-Vietnam border

BackgroundBorder malaria in the Greater Mekong region of Southeast Asia poses a serious threat to the health of the ethnic minority populations of the region. Traditionally thought to be caused primarily by the malaria parasites Plasmodium falciparum and Plasmodium vivax, recently a zoonotic parasite, Plasmodium knowlesi, has been identified in some countries of the region. The presence of this parasite poses a challenge to malaria control programmes, as it is maintained in a zoonotic reservoir of forest-dwelling macaque monkeys.MethodsA cross-sectional malaria parasite species prevalence survey was conducted along the Laos-Vietnam border in the central part of the two countries. Human blood samples were collected from Savannakhet in Laos and Quang Tri in Vietnam between August and October 2010 and assayed for the presence of human malaria parasite species and P. knowlesi. A PCR targeting the 18S small subunit ribosomal RNA gene and circumsporozoite protein gene was used for Plasmodium species identification.ResultsNine cases of P. knowlesi were detected by PCR in blood samples from the Laos side and three from the Vietnam side. All P. knowlesi infections were found in co-infection with P. vivax, with some triple infections of P. knowlesi, P. vivax and P. falciparum detected in Laos. Phylogenetic analysis of these parasites suggests that P. knowlesi is circulating in the Laos-Vietnam border region.ConclusionThis report shows that P. knowlesi is transmited on both sides of the Vietnam-Laos border. Continued monitoring of the range and prevalence of P. knowlesi on both the sides of Laos-Vietnam border is of importance to the National Malaria Control Programmes of both countries.

Genetic polymorphisms in the circumsporozoite protein of Plasmodium malariae show a geographical bias

BackgroundPlasmodium malariae is characterized by its long asymptomatic persistence in the human host. The epidemiology of P. malariae is incompletely understood and is hampered by the limited knowledge of genetic polymorphisms. Previous reports from Africa have shown heterogeneity within the P. malariae circumsporozoite protein (pmcsp) gene. However, comparative studies from Asian countries are lacking. Here, the genetic polymorphisms in pmcsp of Asian isolates have been characterized.MethodsBlood samples from 89 symptomatic P. malariae-infected patients were collected, from Thailand (n = 43), Myanmar (n = 40), Lao PDR (n = 5), and Bangladesh (n = 1). pmcsp was amplified using semi-nested PCR before sequencing. The resulting 89 pmcsp sequences were analysed together with 58 previously published pmcsp sequences representing African countries using BioEdit, MEGA6, and DnaSP.ResultsPolymorphisms identified in pmcsp were grouped into 3 populations: Thailand, Myanmar, and Kenya. The nucleotide diversity and the ratio of nonsynonymous to synonymous substitutions (dN/dS) in Thailand and Myanmar were higher compared with that in Kenya. Phylogenetic analysis showed clustering of pmcsp sequences according to the origin of isolates (Asia vs. Africa). High genetic differentiation (Fst = 0.404) was observed between P. malariae isolates from Asian and African countries. Sequence analysis of pmcsp showed the presence of tetrapeptide repeat units of NAAG, NDAG, and NAPG in the central repeat region of the gene. Plasmodium malariae isolates from Asian countries carried fewer copies of NAAG compared with that from African countries. The NAPG repeat was only observed in Asian isolates. Additional analysis of 2 T-cell epitopes, Th2R and Th3R, showed limited heterogeneity in P. malariae populations.ConclusionsThis study provides valuable information on the genetic polymorphisms in pmcsp isolates from Asia and advances our understanding of P. malariae population in Asia and Africa. Polymorphisms in the central repeat region of pmcsp showed association with the geographical origin of P. malariae isolates and can be potentially used as a marker for genetic epidemiology of P. malariae population.

Analysis of polymorphisms in the circumsporozoite protein gene of Plasmodium vivax isolates from Henan Province, China

BackgroundPlasmodium vivax malaria has historically been a major source of disease in Henan, China. In the 1970s, the morbidity of malaria was highest in the country. With support from the government and the efforts of healthcare personnel, the reported malaria cases have declined dramatically and a national elimination programme was launched in 2010. To achieve the goal, it is essential to study the diversity of autochthonous malaria and transmission of Plasmodium parasites, which will provide baseline data for disease control and management.MethodsThirty-two P. vivax isolates from Henan province were collected from 2008 to 2011, and circumsporozoite protein (csp) genes were analysed to estimate the genetic diversity of this parasite.ResultsThe assessment of csp sequences indicated that all the isolates were the VK210 type, however, none of them was identical to the VK210 strain. The sequences displayed variations in the central region, and eight sub-types were observed. Among the sub-types, HN7 was the most prevalent (37.5%), followed by HN3 (34.4%). A total of 653 repeat units were discovered in 32 Henan isolates. Nucleotide sequences were grouped in 13 unique repeat nucleotide sequence allotypes that coded for 7 different repeated amino acid allotypes. B (GNGAGGQAA) and D (GDRAAGQPA) were more frequent based on the results; they represented 53.9% (352/653) of the total. In comparison to the basic repeat units of VK210, more than 75% of the central repeat units had at least one non-synonymous nucleotide change.ConclusionsRecent P. vivax populations in Henan province showed some degree of genetic diversity in csp, with 8 sub-types among 32 samples. Meantime, the results also suggested its relative conserved parasite populations. This could provide interesting baseline data that allow identifying whether potential new cases differ from the parasites already circulating in the area.

37 NANP ve 4 NVDP Epitoplu Plasmodium falciparum Rekombinant Circumsporozoite Proteinin Ekspresyonu ve Moleküler Karakterizasyonu

Malaria is caused by the protozoan parasite Plasmodium, the leading cause of death amongst the parasitic diseases. The disease is transmitted to human by the bites of female Anopheles mosquitoes. According to the World Health Organization (WHO) data, there were an estimated 214 million malaria cases and estimated 438.000 deaths occurred worldwide, in 2015. It is observed that 90% of all the deaths due to malaria occur in Africa. 78% of these cases were children who are under five years old. Intensive malaria interventions helped to reduce malaria incidence by 37% between 2000 and 2015. Malaria is a curable disease if diagnosed and treated promptly and correctly. Drug resistance has developed against almost all anti-malarial drugs and an effective vaccine against malaria has not been developed yet. Vaccine studies initiated 40 years ago by sterile immunity against falciparum malaria through immunization by exposure to 1000 irradiated mosquitoes. Complex structures, complicated life cycles and various antigenic structures of Plasmodium species make vaccination studies difficult. Circumsporozoite protein (CSP), the most extensively studied protein is also present in the content of the vaccine candidate RTS,S which is currently closest to get license. CSP was the first described Plasmodium antigen because of its important role during initiation of the parasitic infection. CSP is the major surface coat protein of Plasmodium parasite. CSP is a soluble protein and recombinant form of the CSP can be produced in Escherichia coli. NANP repeat region is a target site for host antibodies. Recently many DNA, RNA and protein vaccine candidates are being developed against malaria. According to WHO, in the next 20 years period, malaria vaccine can be developed. In this study we aimed to produce recombinant CSP (rCSP). Initially, P.falciparum CSP gene was amplified by PCR. CSP gene was cloned in to the pJET cloning vector. The gene subcloned to the pET100 protein expression vector. E.coli cells were used for protein expression. After this process, purification and endotoxin removal protocols were performed. As a result, 1182 bp CSP gene was obtained from P.falciparum genomic DNA. Accuracy of cloning and DNA sequence of the CSP gene was determined with DNA sequence analysis. The gene sequence was recorded to the GenBank with a registration no KT315396. rCSP was expressed in E.coli cells. The existence of rCSP was verifiedwith Western Blot method and was purified and removed from endotoxins. rCSP aminoacid sequence and 3D shape was obtained.We believe that the production of recombinant CSP will enable us to contribute to the further malaria vaccine studies in our laboratory and country.

A highly infectious Plasmodium yoelii parasite, bearing Plasmodium falciparum circumsporozoite protein.

BackgroundPlasmodium circumsporozoite protein (CSP) is a major surface antigen present in the sporozoite (Spz) stage of a malaria parasite. RTS, S vaccine, the most clinically advanced malaria vaccine, consists of a large portion of Plasmodium falciparum CSP (PfCSP). A highly infectious, recombinant rodent malaria, Plasmodium yoelii parasite bearing a full-length PfCSP, PfCSP/Py Spz, was needed as a tool to evaluate the role of PfCSP in mediating, protective, anti-malaria immunity in a mouse model.MethodsA transgenic parasite, PfCSP/Py Spz, was generated by inserting a construct expressing the PfCSP at the locus of the P. yoelii CSP gene by double cross-over homologous recombination. Then the biological and protective properties of PfCSP/Py Spz were determined.ResultsThis PfCSP/Py parasite produced up to 30,000 Spz in mosquito salivary glands, which is equal or even higher than the number of Spz produced by wild-type P. yoelii parasites. Five bites of PfCSP/Py-infected mosquitoes could induce blood infection in BALB/c mice.ConclusionsThe current study has demonstrated a successful establishment of a transgenic P. yoelii parasite clone that is able to express a full-length PfCSP, PfCSP/Py parasite. Importantly, this PfCSP/Py parasite can be as infectious as the wild-type P. yoelii parasite both in mosquito vector and in mouse, a mammalian host. A new transgenic parasite that expresses a full-length PfCSP may become a useful tool for researchers to investigate immunity against PfCSP in a mouse model.

Population genomic structure and adaptation in the zoonotic malaria parasite Plasmodium knowlesi

Malaria cases caused by the zoonotic parasite Plasmodium knowlesi are being increasingly reported throughout Southeast Asia and in travelers returning from the region. To test for evidence of signatures of selection or unusual population structure in this parasite, we surveyed genome sequence diversity in 48 clinical isolates recently sampled from Malaysian Borneo and in five lines maintained in laboratory rhesus macaques after isolation in the 1960s from Peninsular Malaysia and the Philippines. Overall genomewide nucleotide diversity (π = 6.03 × 10(-3)) was much higher than has been seen in worldwide samples of either of the major endemic malaria parasite species Plasmodium falciparum and Plasmodium vivax. A remarkable substructure is revealed within P. knowlesi, consisting of two major sympatric clusters of the clinical isolates and a third cluster comprising the laboratory isolates. There was deep differentiation between the two clusters of clinical isolates [mean genomewide fixation index (FST) = 0.21, with 9,293 SNPs having fixed differences of FST = 1.0]. This differentiation showed marked heterogeneity across the genome, with mean FST values of different chromosomes ranging from 0.08 to 0.34 and with further significant variation across regions within several chromosomes. Analysis of the largest cluster (cluster 1, 38 isolates) indicated long-term population growth, with negatively skewed allele frequency distributions (genomewide average Tajima's D = -1.35). Against this background there was evidence of balancing selection on particular genes, including the circumsporozoite protein (csp) gene, which had the top Tajima's D value (1.57), and scans of haplotype homozygosity implicate several genomic regions as being under recent positive selection.

Genetic Diversity and Natural Selection of the Plasmodium knowlesi Circumsporozoite Protein Nonrepeat Regions.

Background Plasmodium knowlesi is a simian malaria parasite that has been identified to cause malaria in humans. To date, several thousand cases of human knowlesi malaria have been reported around Southeast Asia. Thus far, there is no detailed study on genetic diversity and natural selection of P. knowlesi circumsporozoite protein (CSP), a prominent surface antigen on the sporozoite of the parasite. In the present study, the genetic diversity and natural selection acting on the nonrepeat regions of the gene encoding P. knowlesi CSP were investigated, focusing on the T-cell epitope regions at the C-terminal of the protein.MethodsBlood samples from 32 knowlesi malaria patients and 2 wild monkeys (Macaca fascicularis) were used. The CSP of the P. knowlesi isolates was amplified by PCR, cloned into Escherichia coli, and sequenced. The nonrepeat regions of the CSP gene were analysed for genetic diversity, natural selection and haplotypic grouping using MEGA5 and DnaSP version 5.10.00 programmes. A haplotype network was constructed based on the C-terminal (Th2R/Th3R) T-cell epitope regions using the Median-Joining method in the NETWORK version 4.6.1.2 programme. Previously published sequences from other regions (Malaysia Borneo, Singapore) were also included in the analysis.ResultsA total of 123 P. knowlesi CSP sequences were analysed. Multiple sequence alignment revealed 58 amino acid changes, and 42 novel amino acid haplotypes were identified. Polymorphism was higher in the C-terminal Th2R/Th3R epitope (π = 0.0293, n = 123) region compared to the overall combined nonrepeat regions (π = 0.0120, n = 123). Negative natural selection was observed within the nonrepeat regions of the CSP gene. Within the C-terminal Th2R/Th3R epitope regions, there was evidence of slight positive selection. Based on haplotype network analysis of the Th2R/Th3R regions, five abundant haplotypes were identified. Sharing of haplotypes between humans and macaques were observed.ConclusionThis study contributes to the understanding of the type and distribution of naturally occurring polymorphism in the P. knowlesi CSP gene. This study also provides a measurement of the genetic diversity of P. knowlesi and identifies the predominant haplotypes within Malaysia based on the C-terminal Th2R/Th3R regions.

Distinct genetic difference between the Duffy binding protein (PkDBPαII) of Plasmodium knowlesi clinical isolates from North Borneo and Peninsular Malaysia.

BackgroundPlasmodium knowlesi is one of the monkey malaria parasites that can cause human malaria. The Duffy binding protein of P. knowlesi (PkDBPαII) is essential for the parasite’s invasion into human and monkey erythrocytes. A previous study on P. knowlesi clinical isolates from Peninsular Malaysia reported high level of genetic diversity in the PkDBPαII. Furthermore, 36 amino acid haplotypes were identified and these haplotypes could be separated into allele group I and allele group II. In the present study, the PkDBPαII of clinical isolates from the Malaysian states of Sarawak and Sabah in North Borneo was investigated, and compared with the PkDBPαII of Peninsular Malaysia isolates.MethodsBlood samples from 28 knowlesi malaria patients were used. These samples were collected between 2011 and 2013 from hospitals in North Borneo. The PkDBPαII region of the isolates was amplified by PCR, cloned into Escherichia coli, and sequenced. The genetic diversity, natural selection and phylogenetics of PkDBPαII haplotypes were analysed using MEGA5 and DnaSP ver. 5.10.00 programmes.ResultsForty-nine PkDBPαII sequences were obtained. Comparison at the nucleotide level against P. knowlesi strain H as reference sequence revealed 58 synonymous and 102 non-synonymous mutations. Analysis on these mutations showed that PkDBPαII was under purifying (negative) selection. At the amino acid level, 38 different PkDBPαII haplotypes were identified. Twelve of the 28 blood samples had mixed haplotype infections. Phylogenetic analysis revealed that all the haplotypes were in allele group I, but they formed a sub-group that was distinct from those of Peninsular Malaysia. Wright’s FST fixation index indicated high genetic differentiation between the North Borneo and Peninsular Malaysia haplotypes.ConclusionsThis study is the first to report the genetic diversity and natural selection of PkDBPαII of P. knowlesi from Borneo Island. The PkDBPαII haplotypes found in this study were distinct from those from Peninsular Malaysia. This difference may not be attributed to geographical separation because other genetic markers studied thus far such as the P. knowlesi circumsporozoite protein gene and small subunit ribosomal RNA do not display such differentiation. Immune evasion may possibly be the reason for the differentiation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0610-x) contains supplementary material, which is available to authorized users.

Expression and purification of recombinant circumsporozoite proteins of Plasmodium falciparum and evaluation of their binding activities to hepatocytes

Objective To amplify four fragments of circumsporozoite （ CSP） protein gene from Plasmodium falciparum FCC1/HN strain and express recombinant CSP proteins in prokaryotic vector pET28 a/EGFP for further evaluation of their binding activities to hepatic cells .Methods Four pairs of primers were designed according to the cDNA sequence of CSP protein from Plasmodium falciparum FCC1/HN strain and used to amplify the gene fragments by PCR .The cloned gene fragments were inserted into pET28a/EGFP to construct the recombinant expression plasmids .The transformed E.coli BL21 strains carry-ing expression plasmids were induced by IPTG to express CPS proteins .The recombinant CSP proteins were purified with Ni2＋chelating HiTrap HP column and detected by SDS-PAGE.The binding activities of the ex-pressed proteins to different tissues were also detected .Results Four gene fragments encoding CSP protein were successfully amplified and expressed in E.coil BL21 strain as fusion protein CSR1a-EGFP, CSR1b-EGFP, CSR2a-EGFP and CSR2b-EGFP with a relative molecular mass of about 39×103, 31×103, 33×103 and 30 ×10 3 , respectively .The purified fusion proteins reacted specifically with Plasmodium falciparum-posi-tive serum samples.Moreover, the recombinant protein CSR2a-EGFP could bind to the hepatic cells specif-ically rather than other cells.Conclusion The purified recombinant CSR2a-EGFP protein has a strong binding activity to hepatocytes , indicating its potential value as a targeting molecule for hepatic gene therapy . Key words: Plasmodium falciparum; Circumsporozoite protein; Hepatic cells; Targeting