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.     

Transmission-blocking vaccine of vivax malaria

Malaria remains one of the leading causes of both morbidity and mortality of humans residing in tropical countries. For many malarious regions outside of Africa, development of effective transmission-blocking vaccines will require coverage against both Plasmodium falciparum and Plasmodium vivax. The genes coding for two potential P. vivax transmission-blocking antigens, Pvs25 and Pvs28, have been cloned. Mice vaccinated with yeast-produced recombinant proteins Pvs25 and Pvs28 adsorbed to aluminum hydroxide developed strong antibody responses against the immunogens. The development of oocysts in mosquitoes was completely inhibited when these antisera were ingested with the P. vivax Salvador (Sal) I strain-infected chimpanzee blood. In a large collection of P. vivax field isolates, we found only 5 nucleotide changes that would result in amino acid substitutions in Pvs25. In contrast, the Pvs28 gene had 22 nucleotide changes that would result in conservative amino acid substitutions. How the antigenic polymorphism of Pvs25 and Pvs28 would affect the efficacy of Sal I based vaccine remains to be elucidated. Clinical trials with Pvs25 and the P. falciparum ortholog Pfs25 are in preparation.     

A rapid genotyping method for the vivax malaria transmission-blocking vaccine candidates, Pvs25 and Pvs28

The antigenic diversity observed in many vaccine candidates is one of the difficulties to design effective malaria vaccine. Since it is prerequisite to survey genetic polymorphism of the vaccine candidate antigens for the vaccine development, it is necessary to establish efficient screening method to detect the genetic polymorphism from a large number of samples. Here, we have established efficient polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) method to detect nucleotide diversity of the malaria transmission-blocking vaccine candidates Pvs25 and Pvs28. We can distinguish all 4 haplotypes of Pvs25 by this method. By introducing BsmI-digestion step for Pvs28, we can distinguish 15/16 haplotypes by single electrophoresis. Since this method requires neither sequencing nor radioisotope labeling, it will be easy to transfer the method into a field based high throughput screening of genetic polymorphism.     

Sequence polymorphisms in Pvs48/45 and Pvs47 gametocyte and gamete surface proteins in Plasmodium vivax isolated in Korea

Nucleotide sequence analyses of the Pvs48/45 and Pvs47 genes were conducted in 46 malaria patients from the Republic of Korea (ROK) (n = 40) and returning travellers from India (n = 3) and Indonesia (n = 3). The domain structures, which were based on cysteine residue position and secondary protein structure, were similar between Plasmodium vivax (Pvs48/45 and Pvs47) and Plasmodium falciparum (Pfs48/45 and Pfs47). In comparison to the Sal-1 reference strain (Pvs48/45, PVX_083235 and Pvs47, PVX_083240), Korean isolates revealed seven polymorphisms (E35K, H211N, K250N, D335Y, A376T, I380T and K418R) in Pvs48/45. These isolates could be divided into five haplotypes with the two major types having frequencies of 47.5% and 20%, respectivelfy. In Pvs47, 10 polymorphisms (F22L, F24L, K27E, D31N, V230I, M233I, E240D, I262T, I273M and A373V) were found and they could be divided into four haplotypes with one major type having a frequency of 75%. The Pvs48/45 isolates from India showed a unique amino acid substitution site (K26R). Compared to the Sal-1 and ROK isolates, the Pvs47 isolates from travellers returning from India and Indonesia had amino acid substitutions (S57T and I262K). The current data may contribute to the development of the malaria transmission-blocking vaccine in future clinical trials.     

Identification of two forms of vitellogenin-derived phosvitin and elucidation of their fate and roles during oocyte maturation in the barfin flounder, Verasper moseri

A new method for visualizing small and multiple phosvitins (Pvs) in oocytes from a marine teleost was developed by a combination of gel filtration, alkaline phosphatase treatment, and SDS-PAGE followed by silver staining. Three distinct Pv polypeptides having molecular masses of 15 kDa, 8 kDa, and 7 kDa were visualized in vitellogenic follicle extract of barfin flounder, Verasper moseri. N-terminal amino acid sequencing identified two different N-termini that fell into the PvA (7 kDa) and PvB (15 kDa and 8 kDa) groups, which were derived from two forms of vitellogenin (Vg), VgA and VgB, respectively. Analysis of time-course change in phosphorus-rich peaks of gel chromatography fractions of follicle extracts from different maturational stages demonstrated a rapid degradation of Pvs during mid-phase of oocyte maturation. Quantitative analysis of free amino acids in maturing follicles revealed an increment of serine content but not of phosphoserine, indicating the occurrence of dephosphorylation concomitant with Pv degradation. Measurement of phosphatase activity in follicles and eggs at different maturational stages demonstrated a significant activation of phosphatase especially under acidic conditions. This suggested that Pv degradation and dephosphorylation are regulated by changes in ooplasm pH during oocyte maturation. Our results also suggested that the Pvs in barfin flounder vitellogenic oocytes bind to much lower amounts of calcium and magnesium than those of masu salmon, Oncorhynchus masou. This indicates that the Pvs in the barfin flounder, a marine teleost spawning its eggs in seawater, do not play a role in the transport and deposition of calcium and magnesium into oocytes.     

The essential mosquito-stage P25 and P28 proteins from Plasmodium form tile-like triangular prisms

P25 and P28 proteins are essential for Plasmodium parasites to infect mosquitoes and are leading candidates for a transmission-blocking malaria vaccine. The Plasmodium vivax P25 is a triangular prism that could tile the parasite surface. The residues forming the triangle are conserved in P25 and P28 from all Plasmodium species. A cocrystal structure shows that a transmission-blocking antibody uses only its heavy chain to bind Pvs25 at a vertex of the triangle.     

Comparison of PvLAP5 and Pvs25 qRT-PCR assays for the detection of Plasmodium vivax gametocytes in field samples preserved at ambient temperature from remote malaria endemic regions of Panama

BackgroundAs the elimination of malaria in Mesoamerica progresses, detection of Plasmodium vivax using light microscopy (LM) becomes more difficult. Highly sensitive molecular tools have been developed to help determine the hidden reservoir of malaria transmission in low transmission settings. In this study we compare the performance of PvLAP5 and Pvs25 qRT-PCR assays to LM for the detection of Plasmodium vivax gametocytes in field samples preserved at ambient temperature from malaria endemic regions of Panama.MethodsFor this purpose, we collected a total of 83 malaria field samples during 2017-2020 preserved in RNAprotect (RNAp) of which 63 (76%) were confirmed P. vivax by LM and selected for further analysis. Additionally, 16 blood samples from local healthy malaria smear negative volunteers, as well as, from 15 malaria naïve lab-bred Aotus monkeys were used as controls. To optimize the assays, we first determined the minimum blood volume sufficient for detection of PvLAP5 and Pv18SrRNA using P. vivax infected Aotus blood that was preserved in RNAp and kept either at ambient temperature for up to 8 days before freezing or was snap-frozen at -80° Celsius at the time of bleeding. We then compared the mean differences in gametocyte detection rates of both qRT-PCR assays to LM and performed a multivariate correlation analysis of study variables. Finally, we determined the sensitivity (Se) and specificity (Sp) of the assays at detecting gametocytes compared to LM.ResultsBlood volume optimization indicated that a blood volume of at least 60 μL was sufficient for detection of PvLAP5 and Pv18SrRNA and no significant differences were found between RNA storage conditions. Both PvLAP5 and Pvs25 qRT-PCR assays showed a 37-39% increase in gametocyte detection rate compared to LM respectively. Strong positive correlations were found between gametocytemia and parasitemia and both PvLAP5 and Pvs25 gametocyte markers. However, no significant differences were detected in the Se and Sp of the Pvs25 and PvLAP5 qRT-PCR assays, even though data from control samples suggested Pvs25 to be more abundant than PvLAP5.ConclusionsThis study shows that the PvLAP5 qRT-PCR assay is as Se and Sp as the gold standard Pvs25 assay and is at least 37% more sensitive than LM at detecting P. vivax gametocytes in field samples preserved in RNAp at ambient temperature from malaria endemic regions of Panama.Author summaryPlasmodium vivax is one of the five species of malaria (P. falciparum, P. malariae, P. ovale and P. knowlesi) that are transmitted to man by the bite of female anopheles mosquitoes. It causes ~14.3 million cases mainly in Southeast Asia, India, the Western Pacific and the Americas annually. In the Americas, malaria remains a major problem in underdeveloped areas and indigenous communities in the Amazon region and eastern Panama, where it is endemic and difficult to eliminate. As malaria elimination progresses, detection of P. vivax by light microscopy (LM) becomes more difficult. Therefore, highly sensitive molecular tools have been developed that use genetic markers for the parasite to help determine the hidden reservoir of malaria transmission. This study compares the performance of two molecular assays based on the genetic markers of mature gametocytes PvLAP5 and Pvs25 with LM. The study shows that the PvLAP5 qRT-PCR assay is as sensitive and specific as the gold standard Pvs25 assay and is at least 37% more sensitive than LM at detecting P. vivax gametocytes. These data suggest that the PvLAP5 qRT-PCR assay can be a useful tool to help determine the hidden reservoir of transmission in endemic foci approaching elimination.     

Phase 1 trial of malaria transmission blocking vaccine candidates Pfs25 and Pvs25 formulated with montanide ISA 51

Background

Pfs25 and Pvs25, surface proteins of mosquito stage of the malaria parasites P. falciparum and P. vivax, respectively, are leading candidates for vaccines preventing malaria transmission by mosquitoes. This single blinded, dose escalating, controlled Phase 1 study assessed the safety and immunogenicity of recombinant Pfs25 and Pvs25 formulated with Montanide ISA 51, a water-in-oil emulsion.

Methodology/Principal Findings

The trial was conducted at The Johns Hopkins Center for Immunization Research, Washington DC, USA, between May 16, 2005–April 30, 2007. The trial was designed to enroll 72 healthy male and non-pregnant female volunteers into 1 group to receive adjuvant control and 6 groups to receive escalating doses of the vaccines. Due to unexpected reactogenicity, the vaccination was halted and only 36 volunteers were enrolled into 4 groups: 3 groups of 10 volunteers each were immunized with 5 µg of Pfs25/ISA 51, 5 µg of Pvs25/ISA 51, or 20 µg of Pvs25/ISA 51, respectively. A fourth group of 6 volunteers received adjuvant control (PBS/ISA 51). Frequent local reactogenicity was observed. Systemic adverse events included two cases of erythema nodosum considered to be probably related to the combination of the antigen and the adjuvant. Significant antibody responses were detected in volunteers who completed the lowest scheduled doses of Pfs25/ISA 51. Serum anti-Pfs25 levels correlated with transmission blocking activity.

Conclusion/Significance

It is feasible to induce transmission blocking immunity in humans using the Pfs25/ISA 51 vaccine, but these vaccines are unexpectedly reactogenic for further development. This is the first report that the formulation is associated with systemic adverse events including erythema nodosum.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00295581","term_id":"NCT00295581"}}NCT00295581     

Structure of Fab fragment of malaria transmission blocking antibody 2A8 against P. vivax P25 protein

Understanding the structural basis of recognition between antigen and antibody requires the structural comparison of free and complexed components. Previously, we have reported the crystal structure of the complex between Fab fragment of murine monoclonal antibody 2A8 (Fab2A8) and Plasmodium vivax P25 protein (Pvs25) at 3.2 Å resolution. We report here the crystallization and X-ray structure of native Fab2A8 at 4.0 Å resolution. The 2A8 antibody generated against Pvs25 prevents the formation of P. vivax oocysts in the mosquito, when assayed in membrane feeding experiment.Comparison of native Fab2A8 structure with antigen bound Fab2A8 structure indicates the significant conformational changes in CDR-H1 and CDR-H3 regions of V_H domain and CDR-L3 region of V_L domain of Fab2A8. Upon complex formation, the relative orientation between V_L and V_H domains of Fab2A8 is conserved, while significant differences are observed in elbow angles of heavy and light chains. The combing site residues of complexed Fab2A8 exhibited the reduced temperature factor compared to native Fab2A8, suggesting a loss of conformational entropy upon antigen binding.     

Processing of somatostatin precursors: evidence for enzymatic cleavage by hypothalamic extract

The action of enzymes extracted from rat hypothalamus on the previously characterized high molecular weight forms of hypothalamic somatostatin-like immunoreactivity (4 K SLI and 25 K SLI) has been investigated in vitro in order to further define the role of these molecules as possible precursors for tetradecapeptide somatostatin (SRIF). Studies of the degradation of endogenous SLI and of synthetic SRIF by hypothalamic enzymes showed that the time course of breakdown of endogenous SLI is markedly slower than that of synthetic SRIF due to the relative stability of 25 K SLI as well as the generation of at least two new immunoreactive molecules. Incubation of purified 25 K SLI with SLI-free hypothalamic extract showed after 10 to 30 min newly formed immunoreactive material of an intermediate size between 25 K SLI and 4 K SLI and after 60 min the emergence of material coeluting with SRIF. These data show that the hypothalamus contains the enzymes necessary for degrading endogenous SLI and for processing the 25 K SLI molecule to SRIF providing further evidence that 25 K SLI might be a biosynthetic precursor for SRIF.     

cis,cis-Muconate cyclase from Trichosporon cutaneum.

The inducible enzyme catalysing the conversion of cis,cis-muconate to (+)-muconolactone was purified 300-fold from the yeast Trichosporon cutaneum, grown on phenol. The enzyme has a sharp pH optimum at pH 6.6. It reacts also with several monohalogen derivatives and with one monomethyl derivative of cis,cis-muconate, but not with cis,trans- or trans,trans-muconate or 3-carboxy-cis,cis-muconate. In contrast with the corresponding enzymes in bacteria, the yeast enzyme does not require added divalent metal ions for activity and is not inhibited by EDTA. The purified enzyme can be resolved into two peaks by isoelectric focusing. The two forms have pI 4.58 (cis,cis-muconate cyclase I) and pI 4.74 (cis, cis-muconate cyclase II), respectively. Each of these is homogenous on polyacrylamide-gel electrophoresis in the absence or presence of sodium dodecyl sulphate. The two enzyme forms have the same molecular weight (50000) as determined by gel filtration and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. They have the same Km value (25 microM) for cis,cis-muconate. They differ with respect to their content of free thiol groups. cis, cis-Muconate cyclase I contains one thiol group, essential for activity, but relatively stable upon storage. cis, cis-Muconate cyclase II contains two thiol groups that are readily oxidized during storage with concomitant loss of activity.     

Purification and some physico-chemical properties of myocardial adenylate deaminase

A procedure for isolation of adenylate deaminase from duck heart muscle has been developed. The method includes extraction of enzyme, chromatography on cellulose phosphate, fractionation by ammonium sulfate, chromatography on Sephadex G-25 and ion-exchange chromatography on DEAE-cellulose. The enzyme was purified approximately 4000-fold with a yield of 25%. Electrophoresis in polyacrylamide gel revealed that the enzyme contains no proteins other than adenylate deaminase. The enzyme has a UV absorption spectrum typical for proteins which contain no nucleic acid impurities. Using sievorptive chromatography, it was shown that the myocardial extract contains two adenylate deaminase forms, which are tetramers with mol. weights of 190 000 and 240 000. The molecular weights of the subunits are 47 000 and 63 000, respectively. In the oligomeric form the enzyme is only detected at high enzyme concentrations and in the presence of large amounts of substrate.     

Molecular biology of gelsolin, a calcium-regulated actin filament severing protein

Gelsolin is a Ca2+-binding protein of mammalian leukocytes, platelets and other cells which has multiple and closely regulated powerful effects on actin. In the presence of micromolar Ca2+, gelsolin severs actin filaments, causing profound changes in the consistency of actin polymer networks. A variant of gelsolin containing a 25-amino acid extension at the NH2-terminus is present in plasma where it may be involved in the clearance of actin filaments released during tissue damage. Gelsolin has two sites which bind actin cooperatively. These sites have been localized using proteolytic cleavage and monoclonal antibody mapping techniques. The NH2-terminal half of the molecule contains a Ca2+-insensitive actin severing domain while the COOH-terminal half contains a Ca2+-sensitive actin binding domain which does not sever filaments. These data suggest that the NH2-terminal severing domain in intact gelsolin is influenced by the Ca2+-regulated COOH-terminal half of the molecule. The primary structure of gelsolin, deduced from human plasma gelsolin cDNA clones, supports the existence of actin binding domains and suggests that these may have arisen from a gene duplication event, and diverged subsequently to adopt their respective unique functions. The plasma and cytoplasmic forms of gelsolin are encoded by a single gene, and preliminary results indicate that separate mRNAs code for the two forms. Further application of molecular biological techniques will allow exploration into the structural basis for the multifunctionality of gelsolin, as well as the molecular basis for the genesis of the cytoplasmic and secreted forms of gelsolin.     

The role of Pvs28 in sporozoite development in Anopheles sinensis and its longevity in BALB/c mice

To develop a vivax malaria vaccine for blocking malarial transmission, the ookinete surface protein Pvs28 was cloned from Korean malaria patients using polymerase chain reaction. The Pvs28 gene consists of 726 bp and encodes 241 amino acids. It was subcloned into the expression vector pQE30 and expressed in Escherichia coli. The expressed recombinant protein, rPvs28, has a molecular weight of about 28 kDa in SDS–PAGE analysis. A monoclonal antibody against rPvs28 was produced using BALB/c mice. It inhibited sporozoite development in Anopheles sinensis mosquitoes (n = 81) which is one of the malaria vectors in Korea, with relatively high antibody titer against rPv28 persisting for more than 6 months. These results indicate that rPvs28 induces an immune response in mice that effectively blocks sporozoite development in mosquitoes. Therefore it could be a vaccine candidate for preventing vivax malaria in Korea.     

Chemical identification of lipid components in the membranous form of rat liver alkaline phosphatase

Membranous and soluble forms of rat liver alkaline phosphatase were selectively prepared by extracting microsomes with n-butanol at pH 8.5 and 5.5, respectively, and purified in homogeneous forms by the method previously established (Miki et al. (1986) Eur. J. Biochem. 160, 41-48). When subjected to polyacrylamide gel electrophoresis, the two forms migrated to the same position in the presence of sodium dodecyl sulfate, while the membranous form remained at the top of gels in the absence of the detergent. Treatment of the membranous form with phosphatidylinositol-specific phospholipase C resulted in its conversion to a soluble form with the same electrophoretic mobility even in the absence of the detergent as that of the soluble form extracted at pH 5.5. Automated Edman degradation analysis showed that the two forms have the same N-terminal amino acid sequence up to the 30th residue determined. Chemical analyses of hydrolysates of the two forms by gas-liquid chromatography demonstrated that the membranous form contains palmitic acid, stearic acid, and inositol, while the soluble form contains inositol but is devoid of the fatty acids. Taken together, these results suggest that rat liver alkaline phosphatase is covalently attached to phosphatidylinositol acylated with palmitic acid and stearic acid, which functions as the membrane-anchoring domain of the enzyme molecule.     

Synapsoarchitectonics of the septum of the cat brain

In the medial and lateral septal nuclei, 4 types of axonal terminals are distinguished. Type I contains spherical vesicles and forms asymmetric synapses on small and middle stems and spines of the dendrites; type I terminals comprise 63% in the medial nucleus of the total number of axons, and in the lateral one--52%. Type II contains polymorphic vesicles and forms symmetrical synapses on the soma and large dendrites. In the medial nucleus they comprise 6%, and in the lateral one--3%. Type III contains either clear spherical (IIIa), or polymorphic (IIIb) vesicles, as well as 1-2 vesicles with a dense core. They form axodendritic, axospine and axosomatic synapses. In the medial nucleus they comprise 25% and 3%, respectively, in the lateral one--40% and 2%. Type IV contains a great number of vesicles with a dense core. These terminals in both septal nuclei comprise 3% and do not participate in formation of active contacts.     

Mammalian FKBP-25 and its associated proteins

Soluble proteins from porcine brain were divided into two packs: (1) proteins which pass freely through CM52-cellulose, and (2) proteins retained on CM52. Each of these two packs of proteins was fractionated on preparative flat-bed isoelectrofocusing gel in the range of pH 2-12. Native FKBP-25 and its truncated forms were found among other proteins retained on CM52-cellulose. Immunoblotting with anti-FKBP-25 showed two bands in the range 27-30 kDa, one due to unmodified FKBP-25 and other due to FKBP-25 mixed with high-mobility group II protein (HMG-II). Selective immunostaining with anti-FKBP-25 antibodies of proteins which were not retained on CM52-cellulose showed several bands within the range of pI 7-5 and mass of 23 +/- 2 kDa. These fractions of proteins were next resolved on two-dimensional gels and immunostained with anti-FKBP-25 antibodies. Six proteins in the pI range 7-5 were detected. Edman degradation of alpha-chymotrypsin digests of the major spot suggests that it contains the GTP-binding protein Rab5 co-migrating with guanylyl kinase, whereas MALDI-TOF showed that a residual content of FKBP-25 may be also associated with these two proteins. A residual quantity of FKBP-25 was also associated with the phosphatidylethanolamine-binding protein which is abundant in the brain.