Sexual Stages Of Plasmodium Falciparum Research Articles

AGE-DEPENDENT DISTRIBUTION OF PLASMODIUM FALCIPARUM GAMETOCYTES QUANTIFIED BY PFS25 REAL-TIME QT-NASBA IN A CROSS-SECTIONAL STUDY IN BURKINA FASO

Sexual stages of Plasmodium falciparum play a key role in the transmission of malaria. Studies on gametocytes are generally based on microscopic detection, but more sensitive detection methods for P. falciparum gametocytes frequently detect sub-patent gametocytes. We used Pfs25 mRNA quantitative-nucleic acid sequence-based amplification (QT-NASBA) to quantify gametocytes in 412 samples from a cross-sectional study in Burkina Faso, covering all age groups, to determine age-related patterns in gametocyte carriage and gametocyte density. The more sensitive QT-NASBA technique gave estimates of gametocyte prevalence 3.3-fold higher than microscopy (70.1% versus 21.4%, respectively). Prevalence of gametocytes significantly decreased with age. Our data suggest that asexual parasite densities are primarily responsible for the age-related decrease of gametocyte prevalence, possibly because of developing asexual stage immunity. Gametocyte densities decrease also with age, primarily because of decreasing asexual parasite densities; only a small but significant age effect on gametocyte density may be caused by developing sexual stage-specific immunity.

Limited polymorphism in Plasmodium falciparum ookinete surface antigen, von Willebrand factor A domain-related protein from clinical isolates

BackgroundAs malaria becomes increasingly drug resistant and more costly to treat, there is increasing urgency to develop effective vaccines. In comparison to other stages of the malaria lifecycle, sexual stage antigens are under less immune selection pressure and hence are likely to have limited antigenic diversity.MethodsClinical isolates from a wide range of geographical regions were collected. Direct sequencing of PCR products was then used to determine the extent of polymorphisms for the novel Plasmodium falciparum sexual stage antigen von Willebrand Factor A domain-related Protein (PfWARP). These isolates were also used to confirm the extent of diversity of sexual stage antigen Pfs28.ResultsPfWARP was shown to have non-synonymous substitutions at 3 positions and Pfs28 was confirmed to have a single non-synonymous substitution as previously described.ConclusionThis study demonstrates the limited antigenic diversity of two prospective P. falciparum sexual stage antigens, PfWARP and Pfs28. This provides further encouragement for the proceeding with vaccine trials based on these antigens.

Transmission‐reducing immunity is inversely related to age in Plasmodium falciparum gametocyte carriers

Immunity to the sexual stages of Plasmodium falciparum is induced during natural infections and can significantly reduce the transmission of parasites to mosquitoes (transmission reducing activity; TRA) but little is known about how these responses develop with increasing age/exposure to malaria. Routinely TRA is measured in the standard membrane feeding assay (SMFA). Sera were collected from a total of 199 gametocyte carriers (median age 4 years, quartiles 2 and 9 years) near Ifakara, Tanzania; 128 samples were tested in the SMFA and generated TRA data classified as a reduction of > 50% and > 90% of transmission. TRA of > 50% was highest in young children (aged 1-2) with a significant decline with age (chi(2) trend = 5.79, P = 0.016) and in logistic regression was associated with prevalence of antibodies to both Pfs230 and Pfs48/45 (OR 4.03, P = 0.011 and OR 2.43 P = 0.059, respectively). A TRA of > 90% reduction in transmission was not age related but was associated with antibodies to Pfs48/45 (OR 2.36, P = 0.055). Our data confirm that antibodies are an important component of naturally induced TRA. However, whilst a similar but small proportion of individuals at all ages have TRA > 90%, the gradual deterioration of TRA > 50% with age suggests decreased antibody concentration or affinity. This may be due to decreased exposure to gametocytes, probably as a result of increased asexual and/or gametocyte specific immunity.

Parasite infectivity and immunity to Plasmodium falciparum gametocytes in Gambian children.

Immunity to the sexual stages of Plasmodium falciparum can be induced during natural infections. Characterization of this immunity may facilitate the design of a transmission-blocking vaccine (TBV). This study aimed to assess the prevalence and serological correlates of functional transmission-blocking immunity in Gambian children (aged 1-4 years old) who were P. falciparum gametocyte carriers. Serological assays showed 100% response to fixed, whole parasites but only 42% to live gametes. Responses to the antigens Pfs230 and Pfs48/45 were 54.1% and 37.3%, respectively, in an IgG1 ELISA. 14/55 sera were capable of reducing the infectivity of laboratory isolate NF54 in a standard membrane-feeding assay (SMFA). This activity was strongly correlated with IgG1 responses to Pfs48/45 (r = 0.49, P < 0.001) and to a serological reaction with epitopes of the same molecule (r = 0.38, P = 0.003). A weaker correlation was observed with IgG1 to Pfs230 (r = 0.29, P = 0.03). In direct membrane feeding assays (DMFA) with autologous isolates, sera from 4/29 children showed transmission-blocking activity. There was no correlation with serological assays and the DMFA or between the SMFA and DMFA. This may be caused by variation in sexual stage antigens and/or alternative modes of transmission-blocking immunity, both of which have implications for vaccine implementation.

Identification of an MCM4 homologue expressed specifically in the sexual stage of Plasmodium falciparum

Mini-chromosome maintenance (MCM) proteins play an essential role in DNA replication initiation. We have isolated a novel gene encoding an MCM-like protein from the human malaria parasite Plasmodium falciparum using the vectorette technique. The gene has no introns and comprises an open reading frame encoding 1005 amino acid residues with a predicted Mr of 115 kDa. The encoded protein, termed PfMCM4, contains all conserved sequences in the MCM family and displays the highest homology to the Cdc54 (MCM4) of Saccharomyces cerevisiae. However, PfMCM4 possesses five unique amino acid inserts with sizes ranging from seven to 75 residues. Southern blotting of genomic DNA digests and chromosomal separations showed that the Pfmcm4 gene is present as a single copy per haploid genome and is located on chromosome 13. A 4000-nucleotide transcript of this gene is expressed specifically in the sexual erythrocytic stage, indicating that PfMCM4 may be involved in gametogenesis in which DNA is quickly replicated.

Recombinant Human Antibodies Specific for the Pfs48/45 Protein of the Malaria Parasite Plasmodium falciparum

We report the first construction of two combinatorial human phage display libraries derived from malaria-immune patients. Specific single-chain Fv fragments (scFv) against Pfs48/45, a gamete surface protein of the sexual stages of Plasmodium falciparum, were selected and analyzed extensively. The selected scFv reacted with the surface of extracellular sexual forms of the parasite and showed Pfs48/45 reactivity on immunoblot. The scFv inhibit binding of human malaria sera to native Pfs48/45 from gametocytes. Moreover, the scFv bind to target epitopes of Pfs48/45 exposed in natural infections. Sequence analysis of eight scFv clones specific for epitope III of Pfs48/45 revealed that these clones could be divided into one V(H) family-derived germ-line gene (V(H)1) and two V(L) family segments (V(L)2 and V(K)I).

Molecular Characterization of Mitochondria in Asexual and Sexual Blood Stages of Plasmodium falciparum

Molecular mechanisms that regulate gene expression during development of asexual stage to sexual stage of Plasmodium falciparum in the human erythrocyte are largely unknown. There were apparent variations in ultrastructural characteristics of the mitochondrion between the two developing stages. The asexual stage's mitochondrion had developed less than that of the sexual stage. The respiratory complexes of the mitochondrial electron transport system in the asexual stage were ∼8–10 times less active than those in the sexual stage. Using quantitative polymerase chain reaction to amplify the cytochrome b gene encoding a subunit of mitochondrial cytochrome c reductase, the amount of the cytochrome b gene of the sexual stage was calculated to be ∼3 times higher than that obtained from the asexual stage. Moreover, using quantitative reverse-transcription polymerase chain reaction, a relatively high level of ∼1.3-kb transcript mRNA of the cytochrome b gene was observed in the sexual stage compared to the asexual stage. A known single-copy chromosomal dihydrofolate reductase gene was found to have a similar amount in the two stages. These results suggest that the copy number of the mitochondrial gene, including transcriptional and translational mechanisms, plays a major regulatory role in differential expression during the development of the asexual to sexual stage of P. falciparum in the human cell.

Phagocytosis does not play a major role in naturally acquired transmission-blocking immunity to Plasmodium falciparum malaria.

Phagocytosis of Plasmodium falciparum sexual stages in vitro and within the mosquito midgut was assayed in order to assess its role in transmission-blocking immunity to malaria. Both monocytes/macrophages (MM) and polymorphonuclear neutrophils (PMN) phagocytosed malarial gametes in vitro, but levels of phagocytosis were low. Intraerythrocytic gametocytes were not susceptible to phagocytosis. In vitro phagocytosis was positively correlated with levels of antibodies against the gamete surface proteins Pfs230 and Pfs48/45. Immunoglobulin G (IgG) subclass analysis revealed that phagocytosis was correlated with levels of antigamete IgG1. In vivo membrane-feeding experiments were performed in the presence of both pooled and individual malaria immune sera. The phagocytic process proceeded less efficiently in vivo than in vitro, which may be related to the lower ambient temperature (26 degrees C, compared with 37 degrees C). Finally, although we found a correlation between the ability of a serum to promote phagocytosis in vitro and the presence of antibodies against transmission-blocking target antigens, we were unable to demonstrate a role for MM- or PMN-mediated phagocytosis in reduction of infectivity of the malarial parasite to mosquitoes.

Immunization of mice with DNA-based Pfs25 elicits potent malaria transmission-blocking antibodies.

Immunological intervention, in addition to vector control and malaria chemotherapy, will be needed to stop the resurgence of malaria, a disease with a devastating impact on the health of 300 to 500 million people annually. We have pursued a vaccination strategy, based on DNA immunization in mice with genes encoding two antigens present on the sexual stages of Plasmodium falciparum, Pfs25 and Pfg27, to induce biologically important antibodies that can block development of the parasite in the Anopheles mosquito and thus transmission of the disease. DNA encoding Pfs25 when administered by the intramuscular route, either alone or with DNA encoding Pfg27, had the most potent transmission-blocking effects, resulting in up to a 97% decrease in oocyst numbers in mosquito midguts and a 75% decrease in rate of infection. Immunization with DNA encoding a Pfg27-Pfs25 fusion protein was less effective and DNA encoding Pfg27 elicited antibodies in sera that had only modest effects on the infectivity of the parasite. These results show for the first time that DNA vaccination can result in potent transmission-blocking antibodies in mice and suggest that the Pfs25 gene should be included as part of a multicomponent DNA vaccine.

Sexual-stage-specific RNA expression of a new Plasmodium falciparum gene detected by in situ hybridisation

A gene, Pf77, transcribed in the sexual stages of Plasmodium falciparum was isolated from a genomic expression library with a polyclonal antibody raised to gametocyte proteins. The entire coding region was obtained from a series of overlapping genomic and cDNA clones (from gametocyte RNA). A single open reading frame is present with no introns and no tandem repeat sequences. A Pf77 probe hybridised to a single transcript present in RNA prepared from purified gametocytes and could not be detected in RNA prepared from asexual blood stages. In situ hybridisation studies confirmed that the expression of Pf77 mRNA is sexual-stage-specific and in addition, showed that Pf77 mRNA is present only in female gametocytes during the vertebrate stages of the parasite's development.

The primary antibody response of malaria patients to Plasmodium falciparum sexual stage antigens which are potential transmission blocking vaccine candidates

Thirty serum samples collected from adult patients attending the Hospital for Tropical Diseases, London, with P. falciparum malaria, were studied. Sera were screened by indirect immunofluorescence for anti-gametocyte antibodies. Twelve of the serum samples taken from 14 patients with primary infections were found to have both IgM and IgG antibodies to gametocyte antigens and total Ig titres comparable with those of patients who had had previous malaria attacks. Sera of individuals from hyperendemic areas have been found to immunoprecipitate the 230 and 48/45 kD gametocyte surface antigens which are known targets of transmission blocking antibodies. To investigate the epitope specificity of the serum samples from our adult patients, competitive ELISAs with 3 monoclonal antibodies (MAbs) that block transmission and recognize different epitopes on the 48/45 Kd antigen, were carried out. Specific antibodies for these epitopes were found in 60% of the sera while nearly a third were able to inhibit the binding of at least two MAbs.

Immunogenicity of Plasmodium falciparum sexual stage antigens: Implications for the design of a transmission blocking vaccine

Immunogenicity of sexual stage antigens and boosting of transmission blocking antibodies following a natural infection are two critical factors in the design of an effective, subunit vaccine to block the transmission of malaria from man to mosquito. Immunogenicity and boosting are both T cell-dependent. Antigens, such as the 230-kDa, the 48/45-kDa, and the 40/10-kDa, expressed early in the extracellular forms of the sexual stage of Plasmodium falciparum, have limited immunogenicity in humans and in mice. In contrast, Pfs25, expressed predominantly in zygotes and ookinetes, has widespread immunogenicity in mice. Pfs25 expressed by a recombinant vaccinia virus (vSIDK) is also widely immunogenic in mice, and induces transmission blocking antibodies following multiple inoculations with vSIDK. The implications of these immunogenicity data are discussed relative to the design of an effective transmission blocking vaccine.

Plasmodium falciparum sexual stage antigens: Immunogenicity and cell-mediated responses

Antibody and cell-mediated immune responses to the transmission-blocking target antigens of Plasmodium falciparum, Pfs 48/45, were determined in infected non-immune patients and in immune individuals from an endemic area. Characterization of the B cell epitopes with monoclonal antibodies showed that there were five regions identifiable but there could be interactions between them causing either competitive or enhancing effects. Sera from infected non-immune patients contained antibodies that would compete with one or more of the mAbs to the different epitopes. Immune responsiveness to purified Pfs 48/45 in P. falciparum-immune adults measured as lymphoproliferation, production of interferon-γ, or as Pfs 48/45-specific antibody was very limited. This did not appear to be due to MHC class II restriction, to diversity in structure of the parasite antigens or to a failure of immunological memory. The antibody-response data were more consistent with down-regulation of immunity as a result of prolonged exposure to infection.

Minimal variation in a vaccine candidate from the sexual stage of Plasmodium falciparum

Minimal variation in a vaccine candidate from the sexual stage of Plasmodium falciparum

A microprocessor-based control unit for Plasmodium falciparum culture apparatus.

We describe a microprocessor-based control unit for apparatus which effects changes of culture medium on Plasmodium falciparum parasites maintained in vitro. The unit has required minimal attention during the two years it has been in operation and it has markedly facilitated the production of mature sexual stages of Plasmodium falciparum on a routine basis.

Characterization of Plasmodium falciparum sexual stage antigens and their biosynthesis in synchronised gametocyte cultures

Synchronised gametocyte cultures were used to study the biosynthesis of the sexual stage target antigens ( M r 230 000, 48 000 and 25 000) for anti gamete/zygote antibodies. These antigens were shown to be synthesized during gametocyte development from day 2–3 onwards until gametogenesis occurred. After gametogenesis a 25 kDa protein was predominantly synthesized, whereas synthesis of the other target proteins was hardly detectable. The 48, 45, and 25 kDa proteins appeared to be glycosylated, in addition the 25 kDa was also acylated in that it bound [ 3H]palmitic acid covalently. The iso-electric point (p I) of these proteins was assessed as being 6.0 ± 0.1 (for both 48 and 45 kDa) and 5.6 ± 0.1 (for 25 kDa).