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D C Kaslow

Publications and source records attributed to D C Kaslow.

At least 73 records · Page 4Linked to original sources
PubMed1996

A developmental defect in Plasmodium falciparum male gametogenesis.

Asexually replicating populations of Plasmodium parasites, including those from cloned lines, generate both male and female gametes to complete the malaria life cycle through the mosquito. The generation of these sexual forms begins with the induction of gametocytes from haploid asexual stage parasites in the blood of the vertebrate host. The molecular processes that govern the differentiation and development of the sexual forms are largely unknown. Here we describe a defect that affects the development of competent male gametocytes from a mutant clone of P. falciparum (Dd2). Comparison of the Dd2 clone to the predecessor clone from which it was derived (W2'82) shows that the defect is a mutation that arose during the long-term cultivation of asexual stages in vitro. Light and electron microscopic images, and indirect immunofluorescence assays with male-specific anti-alpha-tubulin II antibodies, indicate a global disruption of male development at the gametocyte level with at least a 70-90% reduction in the proportion of mature male gametocytes by the Dd2 clone relative to W2'82. A high prevalence of abnormal gametocyte forms, frequently containing multiple and unusually large vacuoles, is associated with the defect. The reduced production of mature male gametocytes may reflect a problem in processes that commit a gametocyte to male development or a progressive attrition of viable male gametocytes during maturation. The defect is genetically linked to an almost complete absence of male gamete production and of infectivity to mosquitoes. This is the first sex-specific developmental mutation identified and characterized in Plasmodium.

Animals↗
PubMed1996

Clinical immunity to Plasmodium falciparum malaria is associated with serum antibodies to the 19-kDa C-terminal fragment of the merozoite surface antigen, PfMSP-1.

The development of an effective malaria vaccine depends upon identification of antigens that are targets of protective immune responses. An immunoepidemiologic approach has been used to investigate the relationship between antibody responses to a defined region of the major merozoite surface protein of Plasmodium falciparum (PfMSP-1 19) and resistance to clinical malaria in 2 populations of children from West Africa. After allowing for the confounding effects of age, antibodies to PfMSP-1 19 were shown the provide 40% protection against clinical malaria in children in Sierra Leone. In Gambian children, antibodies to one of the epidermal growth factor-like motifs of PfMSP-1 19 were strongly associated with resistance to both clinical malaria and high levels of parasitemia.

Animals↗
PubMed1996

Antibody-mediated inhibition of Aedes aegypti midgut trypsins blocks sporogonic development of Plasmodium gallinaceum.

The peritrophic matrix (PM) that forms around a blood meal is a potential barrier of Plasmodium development in mosquitoes. Previously, we have shown that to traverse the PM, Plasmodium ookinetes secrete a prochitinase and that an inhibitor of chitinase blocks further parasite development. Here we report that it is the mosquito trypsin that activates the Plasmodium prochitinase. Trypsin was identified as the chitinase-activating enzyme by two criteria: (i) trypsin activity and activating activity comigrated on one-dimensional gels, and (ii) activating activity and penetration of the PM by Plasmodium parasites were both hindered by trypsin-specific inhibitors. Subsequently, we examined the effect of antitrypsin antibodies on the parasite life cycle. Antibodies prepared against a recombinant blackfly trypsin effectively and specifically inhibited mosquito trypsin activity. Moreover, when incorporated into an infective blood meal, the antitrypsin antibodies blocked infectivity of Aedes aegypti mosquitoes by Plasmodium gallinaceum. This block of infectivity could be reversed by exogenously provided chitinase, strongly suggesting that the antibodies act by inhibiting prochitinase activation and not on the parasite itself. This work led to the identification of a mosquito antigen, i.e., midgut trypsin, as a novel target for blocking malaria transmission.

Aedes↗
PubMed1996

Dominance of conserved B-cell epitopes of the Plasmodium falciparum merozoite surface protein, MSP1, in blood-stage infections of naive Aotus monkeys.

We have shown that conserved B epitopes were immunodominant in animals hyperimmunized with parasite-purified or recombinant merozoite surface protein MSP1 of Plasmodium falciparum. Cross-priming studies also suggested that a conserved T-helper epitope(s) is efficient in inducing the anti-MSP1 antibody response. In this study, we determined whether a similar profile of immune responses was induced during live P. falciparum infections. Naive Aotus monkeys were infected by blood-stage challenge with either one of the two dimorphic MSP1 alleles represented by the FUP and FVO parasites. Sera collected after parasite clearance were analyzed by enzyme-linked immunosorbent assays (ELISAs). Monkeys infected with parasites carrying one allelic form of MSP1 had antibodies that were equally reactive with homologous or heterologous MSP1s. This preferential recognition of conserved epitopes of MSP1 was confirmed by competitive binding ELISAs. Studies with Plasmodium yoelii and P. falciparum show that the C-terminal 19-kDa fragment of MSP1, MSP1(19), is the target of protective immunity. Thus, monkey sera were assayed for recognition with recombinant MSP1(19)s expressing variant and conserved B epitopes. Results of direct and competitive binding ELISAs showed that the anti-MSP1(19) antibodies were also directed primarily against conserved determinants. The similarities between vaccine- or infection-induced antibody responses suggest a possible reciprocal enhancement of the two populations of anti-MSP1 antibodies when a subunit MSP1 vaccine is introduced into populations living in areas where malaria is endemic. This together with previous observations that conserved determinants are important in MSP1-mediated immunity provides an optimistic outlook that a subunit MSP1 vaccine may be effective and practical for field applications in malaria-exposed populations.

Alleles↗
PubMed1996

Natural immune response to the C-terminal 19-kilodalton domain of Plasmodium falciparum merozoite surface protein 1.

We have characterized the natural immune responses to the 19-kDa domain of merozoite surface protein 1 in individuals from an area of western Kenya in which malaria is holoendemic. We used the three known natural variant forms of the yeast-expressed recombinant 19-kDa fragment that are referred to as the E-KNG, Q-KNG, and E-TSR antigens. T-cell proliferative responses in individuals older than 15 years and the profile of immunoglobulin G (IgG) antibody isotypes in individuals from 2 to 74 years old were determined. Positive proliferative responses to the Q-KNG antigen were observed for 54% of the individuals, and 37 and 35% of the individuals responded to the E-KNG and E-TSR constructs, respectively. Considerable heterogeneity in the T-cell proliferative responses to these three variant antigens was observed in different individuals, suggesting that the 19-kDa antigen may contain variant-specific T epitopes. Among responses of the different isotypes of the IgG antibody, IgG1 and IgG3 isotype responses were predominant, and the prevalence and levels of the responses increased with age. We also found that a higher level of IgG1 antibody response correlated with lower parasite density among young age groups, suggesting that IgG1 antibody response may play a role in protection against malaria. However, there was no correlation between the IgG3 antibody level and protection. Furthermore, we observed that although the natural antibodies cross-reacted with all three variant 19-kDa antigens, IgG3 antibodies in 12 plasma samples recognized only the E-KNG and Q-KNG constructs and not the E-TSR antigen. This result suggests that the fine specificity of IgG3 antibodies differentiates among variant-specific natural B-cell determinants in the second epidermal growth factor domain (KNG and TSR) of the antigen.

Adolescent↗
PubMed1996

NYVAC-Pf7: a poxvirus-vectored, multiantigen, multistage vaccine candidate for Plasmodium falciparum malaria.

The highly attenuated NYVAC vaccinia virus strain has been utilized to develop a multiantigen, multistage vaccine candidate for malaria, a disease that remains a serious global health problem and for which no highly effective vaccine exists. Genes encoding seven Plasmodium falciparum antigens derived from the sporozoite (circumsporozoite protein and sporozoite surface protein 2), liver (liver stage antigen 1), blood (merozoite surface protein 1, serine repeat antigen, and apical membrane antigen 1), and sexual (25-kDa sexual-stage antigen) stages of the parasite life cycle were inserted into a single NYVAC genome to generate NYVAC-Pf7. Each of the seven antigens was expressed in NYVAC-Pf7-infected culture cells, and the genotypic and phenotypic stability of the recombinant virus was demonstrated. When inoculated into rhesus monkeys, NYVAC-Pf7 was safe and well tolerated. Antibodies that recognize sporozoites, liver, blood, and sexual stages of P. falciparum were elicited. Specific antibody responses against four of the P.falciparum antigens (circumsporozoite protein, sporozoite surface protein 2, merozoite surface protein 1, and 25-kDa sexual-stage antigen) were characterized. The results demonstrate that NYVAC-Pf7 is an appropriate candidate vaccine for further evaluation in human clinical trials.

Amino Acid Sequence↗
PubMed1995

Identification of T and B cell epitopes recognized by humans in the C-terminal 42-kDa domain of the Plasmodium falciparum merozoite surface protein (MSP)-1.

The 42-kDa, C-terminal region of the merozoite surface protein-1 (MSP-1) of Plasmodium falciparum is a putative malaria vaccine candidate Ag. Nine synthetic peptides corresponding to predicted T cell sites of MSP-1 in blocks 15 and 16 and eight overlapping peptides representing the conserved block 17 were used to identify naturally immunogenic epitopes. These peptides were tested for their ability to induce proliferation of PBMC from residents in western Kenya, where malaria transmission is holoendemic. Six peptides (PL145, PL146, PL147, PL148, PL149, and PL150) from blocks 15 and 16 induced a positive proliferative response in > 30% of the individuals tested, and three peptides (PL151, PL152, and PL153) induced a proliferative response in < 25% of the donors. Among these peptides, PL146 was from the highly conserved region, PL150 was from a polymorphic region, and all other peptides were from a dimorphic region of blocks 15 and 16. In block 17, only three peptides, PL99, PL100, and PL103, induced proliferation in 30 to 37% of the volunteers. The rest of the peptides induced a proliferative response in approximately 13 to 25% of the donors. The plasma from these donors widely reacted with different allelic forms of 19-kDa recombinant proteins representing block 17 and recognized at least two linear B epitopes, PL104 and PL97. In summary, this study revealed that a majority of immunodominant T and B epitopes are localized in the conserved or dimorphic regions that are nonpolymorphic in the 42-kDa protein of MSP-1. This study suggests that incorporation of T epitopes from the dimorphic blocks 15 and 16 in a vaccine construct may be useful to ensure Ag-specific memory responses.

Amino Acid Sequence↗
PubMed1995

Unique specificity of in vitro inhibition of mosquito midgut trypsin-like activity correlates with in vivo inhibition of malaria parasite infectivity.

Synchrony in the egress of Plasmodium ookinetes from the food bolus and enzymatic digestion of the blood meal in the mosquito midgut suggests that digestive enzymes play a role in the successful transmission of malaria parasites. Previously, we found that parasite-produced chitinase is essential for parasite transmission and can be activated by mosquito midgut protease. To determine the suitability of developing a transmission-blocking vaccine directed against mosquito trypsin-like enzyme(s), Aedes aegypti midgut trypsin-like proteases were characterized biochemically and compared to a mammalian trypsin. Mosquito trypsin is more sensitive to inhibition by aprotinin and less sensitive to egg white trypsin inhibitor than is bovine pancreatic trypsin. Soybean trypsin inhibitor and leupeptin inhibit both enzymes to similar extent. Membrane-feeding assays with aprotinin, leupeptin, and egg white trypsin inhibitor revealed a correlation between in vitro inhibition of mosquito trypsin-like activity and transmission-blocking activity. The results suggest a role for mosquito midgut trypsin(s) in malaria parasite development and indicate that the protease(s) is a potential target for blocking malaria transmission.

Aedes↗
PubMed1995

Plasmodium gallinaceum: mosquito peritrophic matrix and the parasite-vector compatibility.

Transmission of malaria parasites occurs by relatively few species of mosquitoes. One proposed mechanism of refractoriness is an inability of certain Plasmodium spp. to cross the peritrophic matrix (PM) in the midgut of an incompatible mosquito. We have tested this hypothesis by studying sporogonic development of Plasmodium gallinaceum in susceptible (Aedes aegypti and Anopheles gambiae G3) and refractory (Anopheles stephensi) mosquito species in the presence and absence of the PM. In the presence of the PM the number of oocytes that developed in A. gambiae G3 was about 20% of that in A. aegypti, whereas no oocysts developed in A. stephensi. To disrupt PM formation we added, to an infectious bloodmeal, either exogenous fungal chitinase or polyoxin D, the latter being a potent inhibitor of chitin synthase. The absence of the PM did not increase the susceptibility of A. aegypti and A. gambiae nor did it make A. stephensi susceptible to P. gallinaceum infection. The data indicate that the PM is not the primary determinant of P. gallinaceum compatibility in these mosquitoes and suggest that determinant(s) of refractoriness occurs after the parasite crosses the mosquito PM.

Aedes↗
PubMed1995

A genetic locus on Plasmodium falciparum chromosome 12 linked to a defect in mosquito-infectivity and male gametogenesis.

Infection of mosquitoes by Plasmodium spp. requires sexual differentiation of the malarial parasite in the vertebrate host and mating of the heterogametes in the vector midgut. A Plasmodium falciparum clone, Dd2, differentiates into normal-appearing gametocytes, yet poorly infects mosquitoes. The Dd2 clone, however, effectively cross-fertilized HB3, a Central American P. falciparum clone, and yielded several independent recombinant progeny. We have examined 11 HB3 x Dd2 progeny for their ability to infect mosquitoes and to differentiate into male gametes. Our analyses indicate that the poor mosquito-infectivity of the Dd2 clone results from a defect in male gametogenesis. This defect was inherited as a single locus in the independent recombinant progeny of HB3 x Dd2. Comparison with a restriction fragment length polymorphism map of the HB3 x Dd2 cross indicates that the defective phenotype of Dd2 maps to a locus on P. falciparum chromosome 12. This genetic locus may contain determinants that play a crucial role in male gametogenesis by P. falciparum.

Animals↗
PubMed1995

Recombinant Pfs230, a Plasmodium falciparum gametocyte protein, induces antisera that reduce the infectivity of Plasmodium falciparum to mosquitoes.

Six regions of malaria transmission-blocking target antigen, Pfs230, encoding 80% of the 363-kDa protein, were expressed as recombinant proteins in E. coli as fusions with maltose-binding protein (MBP). Antisera generated against amylose-purified recombinant Pfs230/MBP fusion proteins (r230/MBP.A-r230/MBP.F) all recognized the 360-kDa form of parasite-produced Pfs230 by immunoblot. However, only antisera against the four carboxy regions (C-F) of Pfs230 and not the two amino regions (A and B) recognized the 310-kDa form of Pfs230, the form expressed on the surface of gametes. The data suggest that the 310-kDa form of Pfs230 arises from the cleavage of 50 kDa from the amino terminus of the 360-kDa form. Furthermore, antisera against r230/MBP.C bound to the surface of intact gametes and significantly reduced (by 71.2-89.8% (rank sum analysis, P < 0.01)) the infectivity of P. falciparum parasites to mosquitoes. This is the first report of a recombinant form of a P. falciparum gametocyte protein capable of inducing antisera that reduce malaria parasite infectivity to mosquitoes.

ATP-Binding Cassette Transporters↗
PubMed1995

Human immune recognition of recombinant proteins representing discrete domains of the Plasmodium falciparum gamete surface protein, Pfs230.

The 230 kD gametocyte/gamete-specific surface protein of Plasmodium falciparum, Pfs230, is a target of antibodies which inhibit the development of the parasite inside the mosquito vector. A transmission blocking vaccine based on Pfs230 may be a powerful tool for malaria control. As a first step, Pfs230 has been expressed in E. coli as a series of recombinant proteins, fused to maltose binding protein. We have used the fusion proteins to assess cellular and humoral immune responses to Pfs230 in malaria-immune adult Gambian blood donors; responses to the fusion proteins have been compared with responses to native Pfs230. The tetrapeptide repeat region of the molecule appears to be immunodominant for both antibody-producing cells and peripheral blood T cells. We postulate that this may represent a mechanism for immune evasion since the N-terminal repeat region of the molecule is cleaved from the mature protein and shed into the plasma. Responses to fusion proteins representing the seven-cysteine motifs were correlated within individual donors, suggesting that cross-reactive epitopes occur within the motifs. Antibody responses to recombinant proteins were poorly correlated with responses to native Pfs230 suggesting that dominant epitopes of the native protein are not adequately represented in the recombinant proteins. Although prokaryotic expression products may be suitable for induction of cellular immune responses to Pfs230, alternative expression systems may be needed for creation of appropriate B cell epitopes.

ATP-Binding Cassette Transporters↗
PubMed1995

Serum antibodies from malaria-exposed people recognize conserved epitopes formed by the two epidermal growth factor motifs of MSP1(19), the carboxy-terminal fragment of the major merozoite surface protein of Plasmodium falciparum.

The major merozoite surface protein of Plasmodium falciparum (PfMSP1) is a candidate antigen for a malaria vaccine. A 19-kDa C-terminal processing product of PfMSP1 (PfMSP1(19)) is composed of two domains sharing a cysteine-rich motif with epidermal growth factor (EGF) and is the target of monoclonal antibodies which block erythrocyte invasion in vitro. We have evaluated human antibody responses to PfMSP1(19) by using recombinant proteins representing the EGF motifs encoded by the two main alleles of the MSP1 gene. We find that both EGF motifs are antigenic but that only 10 to 20% of malaria-exposed individuals have serum antibodies that recognized either of the motifs. When both EGF motifs were expressed together as a single protein, they were recognized by more than 40% of sera from malaria-exposed individuals. Major epitopes recognized by human antibodies are dependent upon the correct tertiary structure of the protein and are cross-reactive between the different allelic sequences of PfMSP1(19). This suggests that antibodies induced by vaccination with one or the other allelic forms of the protein could recognize all strains of P. falciparum. Immunoglobulin G (IgG) subclass-specific enzyme immunoassays indicate that PfMSP1(19) antibodies are predominantly of the IgG1 subclass.

Adolescent↗
PubMed1995

Immunogenicity and in vivo efficacy of recombinant Plasmodium falciparum merozoite surface protein-1 in Aotus monkeys.

BACKGROUND: The carboxy-terminus of the merozoite surface protein-1 (MSP1) of Plasmodium falciparum has been implicated as a target of protective immunity. MATERIALS AND METHODS: Two recombinant proteins from the carboxy-terminus of MSP1, the 42 kD fused to GST (bMSP1(42)) and the 19 kD (yMSP1(19)), were expressed in Escherichia coli and secreted from Saccharomyces cerevisiae, respectively. To determine if vaccination with these recombinant proteins induces protective immunity, we conducted a randomized, blinded vaccine trial in two species of Aotus monkeys, A. nancymai and A. vociferans. After three injections using Freund's adjuvant, the monkeys were challenged with the virulent Vietnam Oak Knoll (FVO) strain of P. falciparum. RESULTS: All three control monkeys required treatment by Day 19. Two of three monkeys vaccinated with bMSP1(42) required treatment by Day 17, whereas the third monkey controlled parasitemia for 28 days before requiring treatment. In contrast, both of the A. nancymai vaccinated with yMSP1(19) self-resolved an otherwise lethal infection. One of the two yMSP1(19)-vaccinated A. vociferans had a prolonged prepatent period of > 28 days before requiring treatment. No evidence of mutations were evident in the parasites recovered after the prolonged prepatent period. Sera from the two A. nancymai that self-cured had no detectable effect on in vitro invasion. CONCLUSIONS: Vaccination of A. nancymai with yMSP1(19) induced protective immune responses. The course of recrudescing parasitemias in protected monkeys suggested that immunity is not mediated by antibodies that block invasion. Our data indicate that vaccine trials with the highly adapted FVO strain of P. falciparum can be tested in A. nancymai and that MSP1(19) is a promising anti-blood-stage vaccine for human trials.

Amino Acid Sequence↗
PubMed1994

Immunogenicity of the C-terminal 19-kDa fragment of the Plasmodium falciparum merozoite surface protein 1 (MSP1), YMSP1(19) expressed in S. cerevisiae.

The immunogenicity of the C-terminal 19-kDa fragment of Plasmodium falciparum MSP1 expressed in yeast as a nonfusion product, YMSP1(19), was studied. Immunization with YMSP1(19) in rabbits induced high titers of Abs specific for native conformational epitopes on parasite MSP1. In mice, immunogenicity was dependent on the mouse strain and the adjuvant formulation. This suggests that different adjuvants may alter the immunogenicity of MSP1(19) in a genetically diverse population. Although YMSP1(19) induced anti-MSP1 Abs, they did not inhibit in vitro parasite growth. This contrasts with the strong inhibitory activities of Abs produced against a recombinant MSP1(42) (BVp42), which contains the entire MSP1(19) coding sequence. Further analyses showed that YMSP1(19) was the target of the inhibitory, anti-BVp42 Abs because YMSP1(19) could completely block binding of anti-BVp42 Abs to parasite MSP1 or BVp42. Moreover, depletion of YMSP1(19)-specific Abs completely abolished the parasite inhibitory activities of anti-BVp42 sera. Anti-YMSP1(19) sera did not block the inhibitory activities of anti-BVp42 sera, suggesting that inhibitory epitopes were not in close structural proximity with noninhibitory epitopes. The finding that YMSP1(19) possessed inhibitory epitopes but induced anti-MSP1 Abs that were not inhibitory suggests that although the T-epitope(s) produced by immunization with YMSP1(19) could provide help for Ab production, it did not induce an effective inhibitory Ab response. We hypothesize that the nature/specificity of T helper epitopes on MSP1 may be crucial in efficient induction of biologically relevant and/or protective Abs.

Adjuvants, Immunologic↗
PubMed1994

A novel glucose-6-phosphate dehydrogenase in Plasmodium falciparum: cDNA and primary protein structure.

The structure of the parasite-encoded G6PD (PfG6PD) may provide clues about the relative protection against malaria in humans with glucose-6-phosphate dehydrogenase (G6PD) deficiency. We have cloned Pfg6pd cDNA encoding a predicted 856 amino acid residues polypeptide with a calculated molecular mass of > 94 kDa. The predicted amino acid sequence is highly homologous to G6PD from other organisms. Pfg6pd maps as a single or low copy number gene to chromosome 14. The unusually large N-terminus and the distance between the NADP-binding site and G6PD-binding site is novel for the parasite G6PD. The differences between parasite and human G6PD proteins could potentially be exploited for designing new chemotherapeutic agents.

Amino Acid Sequence↗
PubMed1994

Crystallization, sequence and preliminary crystallographic data for transmission-blocking anti-malaria Fab 4B7 with cyclic peptides from the Pfs25 protein of P. falciparum.

X-ray quality crystals of an Fab fragment from a transmission-blocking monoclonal antibody 4B7 (MAb 4B7) against a sexual stage protein Pfs25 of Plasmodium falciparum were grown as uncomplexed and peptide-complexed forms. Initially, the intact immunoglobulin was crystallized because cleavage with pepsin or papain did not produce a homogeneous product. Further proteolytic trials with elastase produced a suitable Fab fragment from which crystals have been obtained, both for the free Fab and in complex with cyclic peptides and in the presence of linear peptides. While linear peptides bind to MAb 4B7, cyclic peptides modeled on a predicted beta-hairpin loop of the third EGF-like domain of Pfs25 bind better and readily co-crystallize with the Fab. The genes for the variable domain of the Fab have been cloned, sequenced and the primary amino-acid sequence for the complete Fab deduced. This work explores the use of glycerol as an additive and the modification of the peptide sequence outside the epitope for improving in the crystallization. Data sets have been collected from crystals of several Fab-peptide complexes and from uncomplexed Fab to resolutions ranging from 2.4 to 3.3 A. The packing arrangements of several crystal forms have been determined by molecular replacement, and refinement of their three-dimensional structures is in progress. The three-dimensional structure of this Fab complexed with the various peptides will aid in an understanding of the mode by which this antibody recognizes and prevents transmission of the malaria parasite.

Journal Article↗