Knowlesi Circumsporozoite Protein Research Articles

Cloning, expression and purification of Plasmodium knowlesi circumsporozoite protein and immunoblot analysis with P. knowlesi strain A1H1 protein extract.

Circumsporozoite protein (CSP) is a sporozoite major surface protein of Plasmodium species. The protein showed promising protection level as a vaccine candidate against Plasmodium falciparum infection. There is a lack of studies on P. knowlesi CSP (PkCSP) as a vaccine candidate due to the high polymorphic characteristic of central repeat region. Recent studies showed the protein has a relatively conserved region at the C-terminal, which consists of T- and B-cell epitopes. This could be the target region for vaccine development against the pre-erythrocytic stage of the parasite. In this study, recombinant PkCSP was expressed using Escherichia coli system. Recombinant PkCSP was immunized in animal models and the antiserum was evaluated using immunoblot analysis. Results showed that PkCSP can be successfully expressed using the bacterial system. Endpoint titre of the antiserum were ranged up to 1:819200. Immunoblot analysis showed the antiserum recognized recombinant PkCSP but not total protein extract from P. knowlesi erythrocytic stage. In conclusion, PkCSP could elicit strong immune response in animal models. However, serum antibodies could not recognize protein from the parasite's erythrocytic stage extract indicating it is not expressed at the erythrocytic stage. Therefore, PkCSP remains as a potential pre-erythrocytic vaccine candidate against P. knowlesi infection.

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.

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.

Anopheles dirus co-infection with human and monkey malaria parasites in Vietnam

The feasibility of identifying parasite DNA and specific mRNAs from wild-caught Anopheles dirus mosquitoes was assessed using dried mosquito salivary glands preserved on filter paper. We were able to detect Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium knowlesi DNA by conventional PCR and, furthermore, detected P. falciparum gametocyte-specific genes, pfg377 and pfs16 mRNA, P. knowlesi circumsporozoite protein (CSP) and sporozoite surface protein 2 (SSP2) mRNA by reverse transcription-PCR. Using this technique, we were able to confirm the presence of P. vivax, P. falciparum and P. knowlesi in one particular wild-caught mosquito. These results indicate that P. knowlesi may be transmitted by the primary human malaria vector in forested areas in Vietnam. This study also shows that the preservation of mosquito salivary glands on filter paper, and the down-stream extraction of parasite DNA and RNA from those, offers a powerful resource for molecular epidemiological studies on malaria.

Molecular Mechanism of Host Specificity in Plasmodium falciparum Infection: ROLE OF CIRCUMSPOROZOITE PROTEIN

Plasmodium falciparum sporozoites invade liver cells in humans and set the stage for malaria infection. Circumsporozoite protein (CSP), a predominant surface antigen on sporozoite surface, has been associated with the binding and invasion of liver cells by the sporozoites. Although CSP across the Plasmodium genus has homology and conserved structural organization, infection of a non-natural host by a species is rare. We investigated the role of CSP in providing the host specificity in P. falciparum infection. CSP from P. falciparum, P. gallinaceum, P. knowlesi, and P. yoelii species representing human, avian, simian, and rodent malaria species were recombinantly expressed, and the proteins were purified to homogeneity. The recombinant proteins were evaluated for their capacity to bind to human liver cell line HepG2 and to prevent P. falciparum sporozoites from invading these cells. The proteins showed significant differences in the binding and sporozoite invasion inhibition activity. Differences among proteins directly correlate with changes in the binding affinity to the sporozoite receptor on liver cells. P. knowlesi CSP (PkCSP) and P. yoelii CSP (PyCSP) had 4,790- and 17,800-fold lower affinity for heparin in comparison to P. falciparum CSP (PfCSP). We suggest that a difference in the binding affinity for the liver cell receptor is a mechanism involved in maintaining the host specificity by the malaria parasite.

Protection of rhesus macaques against lethal Plasmodium knowlesi malaria by a heterologous DNA priming and poxvirus boosting immunization regimen.

We tested a cytokine-enhanced, multiantigen, DNA priming and poxvirus boosting vaccine regimen for prevention of malaria in the Plasmodium knowlesi-rhesus macaque model system. Animals were primed with a mixture of DNA plasmids encoding two preerythrocytic-stage proteins and two erythrocytic-stage proteins from P. knowlesi and combinations of the cytokines granulocyte-macrophage colony-stimulating factor, interleukin-4, and tumor necrosis factor alpha and were boosted with a mixture of four recombinant, attenuated vaccinia virus strains encoding the four P. knowlesi antigens. Two weeks after boosting, the geometric mean immunofluorescence titers in the immunized groups against sporozoites and infected erythrocytes ranged from 160 to 8,096 and from 1,810 to 5,120, respectively. The geometric mean anti-P. knowlesi circumsporozoite protein (PkCSP) titers ranged from 1,761 to 24,242. Peripheral blood mononuclear cells (PBMC) from the immunized monkeys produced gamma interferon (IFN-gamma) in response to incubation with pooled peptides from the PkCSP at frequencies of 10 to 571 spot-forming cells/10(6) PBMC. Following challenge with 100 infectious P. knowlesi sporozoites, 2 of 11 immunized monkeys were sterilely protected, and 7 of the 9 infected monkeys resolved their parasitemias spontaneously. In contrast, all four controls became infected and required treatment for overwhelming parasitemia. Early protection was strongly associated with IFN-gamma responses against a pool of peptides from the preerythrocytic-stage antigen, PkCSP. These findings demonstrate that a multistage, multiantigen, DNA priming and poxvirus boosting vaccine regimen can protect nonhuman primates from an otherwise lethal malaria sporozoite challenge.

ELISPOT assay for detection of peptide specific Interferon-γ secreting cells in rhesus macaques

A reliable procedure to measure antigen specific T cell responses in rhesus macaques is required to determine the efficacy of vaccines and immunotherapies. The currently available T cell assays are poorly quantifiable or technically difficult to perform. Classical 51Cr-release cytotoxic T cell (CTL) assays are cumbersome and difficult to quantitate reproducibly. Detection of specific T-cell using MHC-peptide tetrameric complexes is highly sensitive, but requires knowledge of MHC type and prior identification of T cell epitopes. We therefore developed a rhesus interferon-γ (IFN-γ) ELISPOT assay capable of detecting IFN-γ secretion in response to stimulation with pooled 20-mer peptides. Peripheral blood mononuclear cells (PBMCs) from rhesus monkeys immunized with a DNA vaccine and recombinant canary pox encoding the Plasmodium knowlesi circumsporozoite protein (PkCSP) were incubated with pools of peptides from PkCSP. Positive responses to peptide pools and individual peptides ranging from 100 to 450 spot forming cells (SFC)/10 6 PBMC were detected in four of four immunized monkeys and in zero of two control monkeys. In two monkeys studied in detail, the IFN-γ response was focussed on a single 20-mer peptide, QGDGANAGQPQAQGDGANAG, and was dependent on CD4 +, but not CD8 +, T cells. Background responses in control monkeys and preimmunization PBMCs ranged from 10 to 50 SFC/10 6 PBMC. The average within assay and between assay coefficients of variation (CV) for this peptide ELISPOT were 21.9 and 24.7%, respectively. This peptide IFN-γ assay will be a useful tool for evaluation of T cell responses in rhesus macaques.

Production of anti-sporozoite antibodies in absence of response to carrier by coupling an MDP derivative to a malaria peptide-tetanus toxoid conjugate

A synthetic peptide (pep) representing a portion of the Plasmodium knowlesi circumsporozoite protein attached to a tetanus toxoid (TT) carrier, has been shown to be immunogenic when delivered in saline with derivatives of the synthetic adjuvant, muramyl dipeptide (MDP). The present study was designed to determine if the degree of substitution of pep and of MDP derivatives on the tetanus toxoid (TT) carrier, as well as the choice of MDP derivative used play a role in determining anti-pep and anti-TT antibody levels. One of the MDP derivatives used in the conjugates was ε-amino-caproic Murabutide, since Murabutide which is currently in clinical trials cannot be conjugated. The results show that low doses of this derivative coupled with pep on TT can be used to stimulate high levels of circulating anti-pep antibodies without augmenting the anti-carrier response. In addition, anti-pep antibodies elicited in response to one of the conjugates were biologically active since they produced shedding of the circumsporozoite coat of live parasites.

Identification and chemical synthesis of a tandemly repeated immunogenic region of Plasmodium knowlesi circumsporozoite protein.

Complementary DNA clones that code for the immunogenic region of the Plasmodium knowlesi circumsporozoite protein were shown to contain a tandemly repeating 36-base pair unit. A synthetic dodecapeptide corresponding to the predicted reading frame of the repeating nucleotide unit behaved, in an immunoradiometric assay, identically with the native P. knowlesi circumsporozoite protein. The repeating 36-base pair unit occurred 12 times within the gene and accounts for at least one-third of the amino acid sequence of the surface antigen protein.