Structure of the gene encoding the circumsporozoite protein of Plasmodium yoelii. A rodent model for examining antimalarial sporozoite vaccines

The gene encoding the circumsporozoite protein (CSP) from the rodent malaria parasite, Plasmodium yoelii, has been cloned and the nucleotide sequence has been determined. The gene encodes a protein of 367 amino acids as deduced from the nucleotide sequence. This gene is structurally similar to other Plasmodium spp. CSP genes in that it contains putative hydrophobic signal and anchor sequences at the NH2 and COOH termini, respectively, two small regions (Regions I and II) that are conserved in all CSP genes analyzed to date, and a central region containing the immunodominant repeating peptide sequence. Unlike other CSP genes, however, the immunodominant repeat region of the gene is composed of two distinctly different types of tandem repeats. One repeating unit is six amino acids (Gln-Gly-Pro-Gly-Ala-Pro) in length while the other is only four (Gln-Gln-Pro-Pro) residues long. A synthetic peptide, Gln-Gly-Pro-Gly-Ala-Pro X 3, strongly inhibits the binding of anti-CSP monoclonal antibody to sporozoite antigens while another peptide, Gln-Gln-Pro-Pro X 4, weakly inhibits the binding of this same antibody to sporozoite antigens. This work should allow the construction of a mouse model system to parallel human vaccine trials.     

Two new genotypes of Plasmodium vivax circumsporozoite protein found in the Republic of Korea

The gene encoding Plasmodium vivax circumsporozoite protein (PvCSP) exhibits polymorphism in many geographical isolates. The present study was designed to investigate polymorphism in PvCSP gene of P. vivax isolates in Korea. Thirty isolates, obtained from indigenous cases in Yonchon-gun, Kyonggi-do in 1997, were subjected for sequencing and RFLP analysis of the repeat and post-repeat regions of PvCSP gene and two genotypes (SK-A and SK-B) were identified. The genotype of 19 isolates was SK-A and that of 11 isolates was SK-B. Although the number of 12-base repeats present in SK-A was three while two were found in a Chinese strain CH-5, the repeat sequence of SK-A was identical to that of CH-5 except for one base substitution. Compared with known data there was no identical isolates with SK-B, but the sequence of SK-B was similar to that of a North Korean (NK) isolate. These results indicate that two genotypes of PvCSP coexist in the present epidemic area of Korea and the present parasite may originate from East Asia. RFLP would be useful to classify genotypes of P. vivax population instead of gene sequencing.     

Plasmodium berghei: cloning of the circumsporozoite protein gene

A DNA fragment encoding the carboxy terminal 80% of the Plasmodium berghei circumsporozoite protein was selected from a genomic DNA expression library. Sequencing revealed that the P. berghei circumsporozoite protein was similar in overall structure to circumsporozoite proteins from other malaria species, although the central repeat region was unique in comprising two different blocks of tandem peptide repeats: 11 eight amino acid repeats with predominant sequence DPAPPNAN were followed by 16 two amino repeats, predominantly PQ. The P. berghei circumsporozoite protein exhibited limited, but about equal amino acid homology to circumsporozoite proteins from P. knowlesi, P. vivax, and P. falciparum, indicating that P. berghei is not closely related to any of these other malaria species. Cloning of the P. berghei circumsporozoite protein gene will allow direct testing of sporozoite vaccines in mice.     

Differing Patterns of Selection and Geospatial Genetic Diversity within Two Leading Plasmodium vivax Candidate Vaccine Antigens

Although Plasmodium vivax is a leading cause of malaria around the world, only a handful of vivax antigens are being studied for vaccine development. Here, we investigated genetic signatures of selection and geospatial genetic diversity of two leading vivax vaccine antigens – Plasmodium vivax merozoite surface protein 1 (pvmsp-1) and Plasmodium vivax circumsporozoite protein (pvcsp). Using scalable next-generation sequencing, we deep-sequenced amplicons of the 42 kDa region of pvmsp-1 (n = 44) and the complete gene of pvcsp (n = 47) from Cambodian isolates. These sequences were then compared with global parasite populations obtained from GenBank. Using a combination of statistical and phylogenetic methods to assess for selection and population structure, we found strong evidence of balancing selection in the 42 kDa region of pvmsp-1, which varied significantly over the length of the gene, consistent with immune-mediated selection. In pvcsp, the highly variable central repeat region also showed patterns consistent with immune selection, which were lacking outside the repeat. The patterns of selection seen in both genes differed from their P. falciparum orthologs. In addition, we found that, similar to merozoite antigens from P. falciparum malaria, genetic diversity of pvmsp-1 sequences showed no geographic clustering, while the non-merozoite antigen, pvcsp, showed strong geographic clustering. These findings suggest that while immune selection may act on both vivax vaccine candidate antigens, the geographic distribution of genetic variability differs greatly between these two genes. The selective forces driving this diversification could lead to antigen escape and vaccine failure. Better understanding the geographic distribution of genetic variability in vaccine candidate antigens will be key to designing and implementing efficacious vaccines.     

T cell epitopes of the circumsporozoite protein of Plasmodium vivax. Recognition by lymphocytes of a sporozoite-immunized chimpanzee.

The humoral and cellular antisporozoite immune responses of a laboratory-born chimpanzee were measured following multiple exposures to the bites of Plasmodium vivax-infected mosquitoes. T cell lines and clones derived from the chimpanzee's PBL were used to identify T cell epitopes of the P. vivax circumsporozoite (CS) protein. Two independently obtained cell lines, established by culturing the PBL with either a recombinant P. vivax circumsporozoite (rPvCS) protein or a pool of synthetic peptides spanning the rPvCS sequence, recognized a 20-mer peptide from a nonpolymorphic region of the carboxyl terminus of the CS protein. This peptide overlaps a sequence homologous to region II of the Plasmodium falciparum CS protein. A third T cell line recognized an epitope within the central repeat domain, which has recently been found to be a polymorphic region of the P. vivax CS protein. The CD4+ clones derived from this third T cell line secreted IFN-gamma and IL-2 when stimulated with either the P. vivax repeat peptide (DRAAGQPAG)2 or the rPvCS protein.     

Antigenic analysis of the repeat domain of the circumsporozoite protein of Plasmodium vivax

In the present study we analyzed the fine specificity of mouse monoclonal and human polyclonal antibodies directed against the repeat domain of the circumsporozoite (CS) protein of the human malaria parasite, Plasmodium vivax. Five synthetic peptides, representing monomeric and dimeric repeats of this malarial antigen, were assayed for their capacity to inhibit the binding of these antibodies to a yeast-derived recombinant CS protein. The results revealed the existence of at least two distinct repeated overlapping epitopes in the CS protein of P. vivax. Furthermore, polyclonal sera contain antibodies which recognize additional determinants not represented by the synthetic repeat peptides. Some of these sera contain antibodies recognizing a region flanking the repeat domain (region I). The present findings are in contrast with the antibody response in rodents and humans to the Plasmodium falciparum CS protein, which is directed against a single repeated immunodominant epitope.     

Molecular cloning and characterization of the circumsporozoite protein gene of Plasmodium inui isolated from Formosan macaques (Macaca cyclopis) in Taiwan

We characterized the complete nucleic and amino acid sequences of the Plasmodium inui circumsporozoite protein (Pincsp) gene and analyzed nucleotide diversity across the entire Pincsp gene by using 7 field isolates and strains Taiwan I and II obtained from Formosan macaques (Macaca cyclopis) in Taiwan. The length of the circumsporozoite protein ( CSP ) gene ranged from 1077 to 1125 bp. Size polymorphisms were due to variations in the number of tandem repeat units. The non-repetitive (NR) region exhibited high homology (99.1 ～ 100 and 98.7 ～ 100% at the nucleotide and amino acid levels, respectively) and was conserved among the variants (nucleotide diversities, π, of the 5'NR and 3'NR regions were 0.00364 and 0.00392, respectively). In the central repetitive (CR) region, we decomposed the sequences into 2 kinds of repeating amino acid motifs, i.e., a repeat unit R1, PA(P/A)(P/A)A(E)GG (n = 11-13), and a following repeat unit R2: P(A/G)(A/P/G)(P/Q)AQ(N/K) (n = 9-10). Analyzing these repeat sequences showed evidence of 3 genetic mechanisms for generating variations in the repeats of the Pincsp gene, i.e., point mutation, insertion, and recombination. These findings suggest that polymorphisms in the Pincsp gene are essentially limited to the CR region, which showed much greater variability in terms of length, number of repeats, and sequence.     

Antibody response of naturally infected individuals to recombinant Plasmodium vivax apical membrane antigen-1

In the present study, we evaluate the naturally acquired antibody response to the Plasmodium vivax apical membrane antigen 1 (PvAMA-1), a leading vaccine candidate against malaria. The gene encoding the PvAMA-1 ectodomain region (amino acids 43-487) was cloned by PCR using genomic DNA from a Brazilian individual with patent P. vivax infection. The predicted amino acid sequence displayed a high degree of identity (97.3%) with a previously published sequence from the P. vivax Salvador strain. A recombinant protein representing the PvAMA-1 ectodomain was expressed in Escherichia coli and refolded. By ELISA, this recombinant protein reacted with 85 and 48.5% of the IgG or IgM antibodies, respectively, from Brazilian individuals with patent P. vivax malaria. IgG1 was the predominant subclass of IgG. The frequency of response increased according to the number of malaria episodes, reaching 100% in individuals in their fourth malaria episode. The high degree of recognition of PvAMA-1 by human antibodies was confirmed using a second recombinant protein expressed in Pichia pastoris (PV66/AMA-1). The observation that recognition of the bacterial recombinant PvAMA-1 was only slightly lower than that of the highly immunogenic 19kDa C-terminal domain of the P. vivax Merozoite Surface Protein-1 was also important. DNA sequencing of the PvAMA-1 variable domain from 20 Brazilian isolates confirmed the limited polymorphism of PvAMA-1 suggested by serological analysis. In conclusion, we provide evidence that PvAMA-1 is highly immunogenic during natural infection in humans and displays limited polymorphism in Brazil. Based on these observations, we conclude that PvAMA-1 merits further immunological studies as a vaccine candidate against P. vivax malaria.     

Plasmodium falciparum: in vitro characterization and human infectivity of a cloned line.

The culture-adapted NF54 isolate of Plasmodium falciparum was subjected in vitro to three sequential limiting dilution titrations and the resulting clone was given the designation CVD1. DNA sequence analysis of the gene encoding the circumsporozoite (CS) protein revealed differences between CVD1 and the published NF54 CS gene. CVD1 had 1191 bp, 397 amino acids, and 42 repeat units while NF54 had 1218 bp, 405 amino acids, and 44 repeat units. The CVD1 clone was more sensitive to chloroquine than was the parental line, in vitro. Anopheles stephensi mosquitoes were infected equally by the cloned and uncloned parasites. Volunteers were readily infected by NF54 and CVD1 following infectious mosquito bites. The availability of a well-characterized, chloroquine-sensitive clone which safety infects humans should facilitate performance of experimental challenge studies to assess vaccine efficacy.     

The structure of human thrombospondin, an adhesive glycoprotein with multiple calcium-binding sites and homologies with several different proteins

Thrombospondin is one of a class of adhesive glycoproteins that mediate cell-to-cell and cell-to-matrix interactions. We have used two monoclonal antibodies to isolate cDNA clones of thrombospondin from a human endothelial cell cDNA library and have determined the complete nucleotide sequence of the coding region. Three regions of known amino acid sequence of human platelet thrombospondin confirm that the clones are authentic. Three types of repeating amino acid sequence are present in thrombospondin. The first is 57 amino acids long and shows homology with circumsporozoite protein from Plasmodium falciparum. The second is 50-60 amino acids long and shows homology with epidermal growth factor precursor. The third occurs as a continuous eightfold repeat of a 38-residue sequence; structural homology with parvalbumin and calmodulin indicates that these repeats constitute the multiple calcium-binding sites of thrombospondin. The amino acid sequence arg-gly-asp-ala is included in the last type 3 repeat. This sequence is probably the site for the association of thrombospondin with cells. In addition, localized homologies with procollagen, fibronectin, and von Willebrand factor are present in one region of the thrombospondin molecule.     

Development of a polymorphic strain of Plasmodium vivax in monkeys.

A strain of Plasmodium vivax from Thailand with a polymorphic repeat unit of the circumsporozoite protein was established in Saimiri sciureus boliviensis and 3 species of Aotus monkeys. All 11 attempts to transmit infection via sporozoite inoculation, 4 times to splenectomized S. sciureus boliviensis, 2 times to splenectomized Aotus nancymai, and 5 times to intact Saimiri monkeys, were successful. Anopheles freeborni, Anopheles stephensi, Anopheles dirus, and Anopheles gambiae mosquitoes were infected by feeding on parasitemic blood from a chimpanzee and an Aotus azarae boliviensis monkey. Our results indicate that this strain may be useful in antisporozoite vaccine trials.     

Sequence variation in putative functional domains of the circumsporozoite protein of Plasmodium falciparum. Implications for vaccine development

Sequences of the circumsporozoite protein gene from five isolates of the human malaria parasite Plasmodium falciparum are compared, and the extent of sequence variability within putative functional domains is assessed in terms relating to vaccine efficacy. Nucleotide substitutions were observed outside of the immunodominant domain. Of the substitutions observed outside of the repeat domain, none were silent. The substitutions correlated with biologically functional regions, such as a helper T cell epitope (Th2R) and a region (N1) which may be important in liver invasion. Contrary to previous impressions, the small numbers of amino acid changes in these areas of the protein seem potentially very significant. The immunodominant repeat region displays several characteristics that implicate a rapid evolutionary mechanism, most probably involving recombination. The data supporting this are 1) variable numbers of repeats, 2) a shifting pattern of substitutions among the isolates, and 3) codon bias. The region thus has the potential for very rapid change should an effective anti-repeat vaccine come into use. We conclude that strain variability is significant, that the potential for large scale variation in the repeats is great, and that regions that may be critical for an effective vaccine are polymorphic. Their potential impact on malaria vaccine development must be addressed.     

Immunogenicity of the repetitive and nonrepetitive peptide regions of the divergent CS protein of Plasmodium knowlesi

The circumsporozoite (CS) protein of the Nuri strain of the simian malarial parasite Plasmodium knowlesi was expressed as a fusion protein in E. coli. This fusion protein cross-reacted with the polyclonal monkey sera raised against irradiated sporozoites of another strain (H strain) of P. knowlesi. The antibody against the repeat units of the H strain CS protein was affinity purified from the polyclonal sera by using synthetic repeat peptides. The affinity-purified antibody did not cross-react with the Nuri CS fusion protein. The immunogenicity of different regions of the CS protein was additionally studied by using several synthetic peptides. All but the most COOH-terminal peptide showed cross-reactivity with the polyclonal sera. Because the repeat regions of the CS protein of the two strains are diverse, whereas the non-repetitive regions are immunogenic and conserved, the latter may be better suited for a potential vaccine.     

Infectivity of two strains of Plasmodium vivax to Anopheles albitarsis mosquitoes from Colombia

Anopheles albitarsis obtained from Villavicencio, Colombia, were colonized in the laboratory using force-mating techniques. Laboratory reared mosquitoes were allowed to feed on Aotus monkeys infected with the Salvador II or the Rio Meta strains of Plasmodium vivax from El Salvador and Colombia, respectively. In comparison with other species, the An. albitarsis were less susceptible than Anopheles freeborni, Anopheles culicifacies and strains of Anopheles albimanus from El Salvador, Panama and Colombia and more susceptible than a strain of An. albimanus from Haiti.     

Whole-genome sequencing of a Plasmodium vivax isolate from the China-Myanmar border area

Currently, there is a trend of an increasing number of Plasmodium vivaxmalaria cases in China that are imported across its Southeast Asia border, especially in the China-Myanmar border area (CMB). To date, little is known about the genetic diversity of P. vivax in this region. In this paper, we report the first genome sequencing of a P. vivaxisolate (CMB-1) from a vivax malaria patient in CMB. The sequencing data were aligned onto 96.43% of the P. vivax Salvador I reference strain (Sal I) genome with 7.84-fold coverage as well as onto 98.32% of 14 Sal I chromosomes. Using the de novo assembly approach, we generated 8,541 scaffolds and assembled a total of 27.1 Mb of sequence into CMB-1 scaffolds. Furthermore, we identified all 295 known virgenes, which is the largest subtelomeric multigene family in malaria parasites. These results provide an important foundation for further research onP. vivax population genetics.     

Cellular immune response of humans to the circumsporozoite protein of Plasmodium vivax.

The cellular immune response to the circumsporozoite (CS) protein of Plasmodium vivax of individuals from malaria-endemic areas of Brazil was studied. We examined the in vitro proliferative response of the peripheral blood mononuclear cells (PBMC) of 22 individuals when stimulated with a CS recombinant protein (rPvCS-2) and two other synthetic peptides based on the sequence of the P. vivax CS protein. Seven of the individuals from malaria-endemic area displayed an antigen-specific in vitro proliferative response to the recombinant protein PvCS-2 and one out of 6, proliferative response to the peptide 308-320. In contrast, none of the individuals displayed a proliferative response when stimulated with the D/A peptide which represent some of the repeated units present in this CS protein. Our study, therefore, provides evidence for the presence, within the major surface antigen of P. vivax sporozoites, of epitopes capable to induce proliferation of human PBMC.     

Infection of Peruvian Aotus nancymai monkeys with different strains of Plasmodium falciparum, P. vivax, and P. malariae

Aotus nancymai (karyotype I) monkeys from Peru were studied for their susceptibility to infection with Plasmodium falciparum, P. vivax, and P. malariae. Three strains of P. falciparum (Santa Lucia from El Salvador, Indochina I/CDC from Thailand, and Uganda Palo Alto) were inoculated into 38 monkeys. The results indicated that this species of Aotus monkey is highly susceptible to infection. The Uganda Palo Alto and the Santa Lucia strain parasites appear to be the most useful for immunologic and chemotherapeutic studies. Five strains of P. vivax (Chesson, ONG, Vietnam Palo Alto, Salvador I, and Honduran I/CDC) were inoculated into 28 monkeys. The Vietnam Palo Alto strain produced the highest level parasitemias ranging from 23,800 to 157,000/mm3. Mosquito infections were obtained with the ONG, Chesson, and Salvador I strains. Two out of 6 attempts to transmit P. vivax via sporozoite inoculation to splenectomized monkeys were successful with prepatent periods of 39 and 57 days. Five monkeys were infected with the Uganda I/CDC strain of P. malariae. Maximum parasitemias ranged from 10 to 5,390/mm3.     

ELISA detection of vivax malaria with recombinant multiple stage-specific antigens and its application to survey of residents in endemic areas

An ELISA was developed for the diagnosis of vivax malaria using multiple stage-specific recombinant antigens of Plasmodium vivax. The DNA from the whole blood of a malaria patient was used as template to amplify the coding regions for the antigenic domains of circumsporozoite protein (CSP-1), merozoite surface protein (MSP-1), apical merozoite antigen (AMA-1), serine repeat antigen (SERA), and exported antigen (EXP-1). Each amplified DNA fragment was inserted into pQE30 plasmid to induce the expression of His-tagged protein in Escherichia coli (M15 strain) by IPTG. His-tagged proteins were purified by Ni-NTA metal-affinity chromatography and used as antigens for ELISA with patient sera that were confirmed previously by blood smear examinations. When applied to patient sera, 122 (80.3%) out of 152 vivax malaria cases reacted to at least one antigen, while no reactions were observed with 128 uninfected serum samples. We applied this ELISA to the screening of 3,262 civilian residents in endemic regions near the DMZ, which resulted in 236 positively detected (7.2%) cases. This method can be applied to serological diagnosis and mass screening in endemic regions, or can be used as a safety test for transfusion blood in endemic areas.     

Sequence polymorphism in two novel Plasmodium vivax ookinete surface proteins, Pvs25 and Pvs28, that are malaria transmission-blocking vaccine candidates.

BACKGROUND: For many malarious regions outside of Africa, development of effective transmission-blocking vaccines will require coverage against both Plasmodium falciparum and P. vivax. Work on P. vivax transmission-blocking vaccines has been hampered by the inability to clone the vaccine candidate genes from this parasite. Materials and METHODS: To search for genes encoding the ookinete surface proteins from P. vivax, the DNA sequences of the eight known proteins in the P25 subfamily (Pfs25, Pgs25, Pys25, Pbs25) and in the P21/28 subfamily (Pfs28, Pgs28, Pys21, Pbs21) of zygote/ookinete surface proteins were aligned. Regions of highest identity were used to design degenerate PCR oligonucleotides. Genomic DNA from the Sal I strain of P. vivax and genomic and splinkerette DNA libraries were used as PCR templates. To characterize the polymorphisms of Pvs25 and Pvs28, these two genes were PCR amplified and the DNA sequences were determined from genomic DNA extracted from patients infected with P. vivax. RESULTS: Analysis of the deduced amino acid sequence of Pvs28 revealed a secretory signal sequence, four epidermal growth factor (EGF)-like domains, six copies of the heptad amino acid repeat (GSGGE/D), and a short hydrophobic region. Because the fourth EGF-like domain has four rather than six cysteines, the gene designated Pvs28 is the presumed homologue of P21/28 subfamily members. Analysis of the deduced amino acid sequence of Pvs25 revealed a similar structure to that of Pvs28. The presence of six rather than four cysteines in the fourth EGF-like domain suggested that Pvs25 is the homologue of P25 subfamily members. Several regions of genetic polymorphisms in Pvs25 and Pvs28 were identified in field isolates of P. vivax. CONCLUSIONS: The genes encoding two ookinete surface proteins, Pvs28 and Pvs25, from P. vivax have been isolated and sequenced. Comparison of the primary structures of Pvs25, Pvs28, Pfs25, and Pfs28 suggest that there are regions of genetic polymorphism in the P25 and P21/28 subfamilies.     

Structure of a malaria parasite antigenic determinant displayed on filamentous bacteriophage determined by NMR spectroscopy: implications for the structure of continuous peptide epitopes of proteins

The NANP repeating sequence of the circumsporozoite protein of Plasmodium falciparum was displayed on the surface of fd filamentous bacteriophage as a 12-residue insert (NANP)(3) in the N-terminal region of the major coat protein (pVIII). The structure of the epitope determined by multidimensional solution NMR spectroscopy of the modified pVIII protein in lipid micelles was shown to be a twofold repeat of an extended and non-hydrogen-bonded loop based on the sequence NPNA, demonstrating that the repeating sequence is NPNA, not NANP. Further, high resolution solid-state NMR spectra of intact hybrid virions containing the modified pVIII proteins demonstrate that the peptides displayed on the surface of the virion adopt a single, stable conformation; this is consistent with their pronounced immunogenicity as well as their ability to mimic the antigenicity of their native parent proteins.