Localization of the ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum in merozoites and ring-infected erythrocytes.

Immunoelectron microscopy with protein A gold has been used to determine the subcellular location of the ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum. RESA was associated with dense vesicles presumed to be micronemes within merozoites. RESA was not detected on the surface of merozoites but was located at the membrane of erythrocytes infected with ring-stage parasites. RESA within merozoites was largely soluble in the nonionic detergent Triton X-100, but was insoluble in this detergent when associated with the erythrocyte membrane.

Building the Human Vaccines Project: strategic management recommendations and summary report of the 15-16 July 2014 business workshop.

The Human Vaccines Project is a bold new initiative, with the goal of solving the principal scientific problem impeding vaccine development for infectious diseases and cancers: the generation of specific, broad, potent and durable immune responses in humans. In the July 2014 workshop, 20 leaders from the public and private sectors came together to give input on strategic business issues for the creation of the Human Vaccines Project. Participants recommended the Project to be established as a nonprofit public-private partnership, structured as a global R&D consortium closely engaged with industrial partners, and located/affiliated with one or more major academic centers conducting vaccine R&D. If successful, participants concluded that the Project could greatly accelerate the development of new and improved vaccines, with the potential to transform disease prevention in the 21st century.

Affinity purification of human antibodies directed against cloned antigens of Plasmodium falciparum.

A technique has been developed for the affinity purification of antibodies recognizing cloned antigens of the malaria parasite Plasmodium falciparum expressed in bacteria. Adsorbents prepared by coupling bacterial lysates to Sepharose were used to isolate monospecific antibodies from human immune sera. Production of an abundant stable fused polypeptide by the bacteria was not a prerequisite for the success of this approach. Also the procedure permits the characterization of antigens which elicit the production of very low levels of antibodies. Affinity-purified human antibodies were used to characterized the corresponding P. falciparum antigens by immunoblotting and a number of antigens identified in this way illustrate some commonly observed features of P. falciparum antigens. Several of these antibody preparations recognized multiple bands in the electrophoretic patterns. Studies on a number of isolates of P. falciparum indicate that many antigens exhibit size polymorphisms. Production of some antigens was shown to be restricted to particular stages of the asexual blood cycle of the parasite while others appear to be specifically processed during the life cycle. Affinity-purified antibodies have also been used to locate antigens within the infected erythrocyte and to delineate subsets of antibodies recognizing different epitopes of a single antigen.

Identification of a molecular marker for the Y chromosome of Brugia malayi.

Random amplification of polymorphic DNA (RAPD) was used to analyse genomic DNA from virgin females and males of Brugia malayi, with a view to identifying sex-specific differences predicted by an XX/XY system of chromosomal sex determination. A product of 2338 bp, amplified with the arbitrary primer 5' GTTGCGATCC 3', was obtained exclusively from males. Primers based on the sequence of this product amplified a DNA fragment of the expected size from each of two independent isolates of B. malayi (from Malaysia and Indonesia) by PCR. No reaction product was obtained from the closely related species Brugia pahangi. In a genetic cross between B. malayi males and B. pahangi females, F1 hybrid microfilariae were PCR-positive, indicating that the locus is paternally-inherited. Southern blotting demonstrated that the target sequence resides in the high molecular weight fraction of genomic DNA, confirming that it is of chromosomal, rather than mitochondrial, origin. Sequencing of the locus revealed significant similarity with members of a family of reverse transcriptase-like genes in Caenorhabditis elegans. In-frame stops indicate that the gene is non-functional, but multiple bands of hybridisation in Southern blots suggest that the RT sequence may be the relic of a transposable element. Multiple repeats of the dinucleotide AT occurred in another region of the sequence. These varied in number between the two isolates of B. malayi in the manner of a microsatellite, surprisingly the first to be described from the B. malayi genome. Because of its association with the Y chromosome, we have given the locus the acronym TOY (Tag On Y). Identification of this chromosome-specific marker confirms the XX/XY heterogametic karyotype in B. malayi and opens the way to elucidation of the role of Y in sex determination.

Chitinase genes expressed by infective larvae of the filarial nematodes, Acanthocheilonema viteae and Onchocerca volvulus.

State-specific products of 220 and 75 kDa were identified by metabolic labelling of infective larvae of the filarial nematode Acanthocheilonema viteae in ticks. Synthesis was temperature sensitive, occurring at 27 degrees C but not at 37 degrees C. These products were secreted 3-6 days after leaving the vector during post-infective development, but subsequent expression was not detected. The smaller protein with a pI of 6.2, was purified by two-dimensional electrophoresis and the N-terminal amino acid sequence was derived. This provisionally identified the protein as a chitinase, which was confirmed biochemically by glycol-chitin substrate gel electrophoresis. The polymerase chain reaction was used to amplify a product from a cDNA library of A. viteae infective larvae. The nucleotide sequence codes for a putative signal peptide of 20 amino acids and a mature protein of 504 residues (Mr 56 kDa), exhibiting 69% identity (81% similarity allowing for conservative substitutions) with the MF1 chitinase described from microfilariae of Brugia malayi. N-linked glycosylation may account for some, or all, of the discrepancy in Mr between the predicted polypeptide and the native parasite product (75 kDa). Primers based on the A. viteae sequence were used to amplify a related sequence from a cDNA library of Onchocerca volvulus infective larvae. The O. volvulus cDNA codes for a 20-amino acid signal peptide followed by 477 residues with an Mr of 54 kDa, and shares 67% identity with the A. viteae chitinase (80% similarity allowing for conservative substitutions) and 69% identity with the B. malayi MF1 molecule. It is proposed that chitinases expressed by infective stages of these filarial nematodes may play a role in ecdysis during post-infective development.

Thioredoxin peroxidase from Onchocerca volvulus: a major hydrogen peroxide detoxifying enzyme in filarial parasites.

Random screening of an Onchocerca volvulus third-stage (L3) cDNA library identified a highly abundant cDNA encoding a newly discovered antioxidant enzyme, thioredoxin peroxidase (TPx), a member of the peroxidoxin superfamily. This TPx cDNA (Ov-tpx-2) encodes a polypeptide of 199 amino acid residues with a calculated molecular weight of 21,890 Da. The Ov-tpx-2 cDNA represents roughly 2.5% of the total cDNAs from the L3 cDNA library. The gene was expressed in Escherichia coli and the protein product was shown to have antioxidant activity. Antiserum raised against Ov-TPX-2 recognized a native protein from extracts of both the L3 and adult-stages with a molecular weight of 22 kD. The localization and stage-specificity of Ov-TPX-2 protein was analyzed by immunocytochemistry and immunoelectron microscopy using monospecific antibodies. Expression was detected in late first-stage larvae during development in the vector and increased in intensity during differentiation to the infective L3-stage. The antigen was also detected in post-infective larvae and adult worms. In larvae, Ov-TPX-2 protein was predominantly localized to the hypodermis and cuticle, with additional sites in the hypodermal chords and multivesicular bodies. In adult worms, the primary sites of expression were the uterine epithelium and intestine, with additional labeling of the body wall and cuticle. Developing embryos and microfilariae in utero were bathed in Ov-TPX-2 protein discharged from epithelial cells. These results suggest that Ov-TPX-2 may protect the parasites from being damaged by host-generated oxidative stress and that Ov-TPX-2 protein provides the H2O2-detoxifying activity predicted but not previously identified in filarial parasites. Its highly upregulated expression in infective larvae may aid in parasite establishment following transmission to the definitive host.

Biochemical and immunochemical characterisation of a 20-kilodalton complex of surface-associated antigens from adult Onchocerca gutturosa filarial nematodes.

Surface radioiodination of adult Onchocerca parasites reveals a restricted range of proteins associated with the cuticle. We present data to show that prominent among these is a complex of low-molecular-weight proteins which can be released in soluble form by homogenisation of surface-labelled Onchocerca gutturosa in phosphate-buffered saline (PBS). One of this groups of proteins, designated gp20, has a molecular mass of 20,000, is glycosylated with two N-linked carbohydrate side chains, and has a basic pI. Other PBS-soluble, 125I-labelled proteins of similar size appear not to be glycosylated. A distinct group of molecules are released only in the presence of reducing agents, and are likely to be cuticular collagens. The low-molecular-weight components are antigenic and cross-reactive with Onchocerca volvulus infection sera. Cross-reactions are also observed in immunoprecipitation experiments using sera from Brugia-immunised animals and infected humans. Comparative two-dimensional analyses of these immunoprecipitates reveal at least two Onchocerca specific components. As an alternative to radiolabelling and PBS homogenisation, incubation of worms in medium containing the reducing agent 2-mercaptoethanol resulted in a similar set of molecules being released into the medium. Since surface antigens of O. gutturosa from bovines and O. volvulus from humans appear similar in size and are antigenically cross-reactive, the more readily available parasite is being used to study further the properties of these molecules and to provide reagents for raising antisera reactive to the equivalent O. volvulus antigens.

Developmentally regulated expression and secretion of a polymorphic antigen by Onchocerca infective-stage larvae.

In order to analyse the developmental biology of Onchocerca spp. with a view to identifying molecules with specialised functions, we have devised a novel method for labelling proteins synthesised by larvae during growth in the vectors. Pulse labelling of Onchocerca lienalis by micro-injections of [35S]methionine into blackflies have revealed a major acidic protein of 23 kDa which is developmentally expressed almost exclusively by infective, third-stage larvae. The protein appears to be antigenically conserved between O. lienalis and Onchocerca volvulus, but exhibits size polymorphisms both among species and among individual organisms. It continues to be elaborated after terminal differentiation of the parasite in flies, but not by post-infective larvae entering the phase of development in the vertebrate host. A shift in temperature from 26 degrees C to 37 degrees C triggers secretion of the 23-kDa molecule as a discrete event 24-72 h after transmission. The labelling technique has been successfully employed with filarial species that develop in mosquitoes, and in principle should be widely applicable to the study of endoparasite gene expression within arthropods.

Characterization of the heat-shock protein 60 chaperonin from Onchocerca volvulus.

Chaperonin 60 (cpn60) belongs to the group of ubiquitous molecular chaperones that comprise the heat shock proteins, nucleoplasmins and chaperonins. Antibodies to recombinant CPN60 from humans was used to screen a cDNA library of Onchocerca volvulus and antigen-positive clones were selected. Sequencing of the DNA inserts confirmed their identity as cpn60 transcripts. These are distinct from a cpn60 sequence recorded previously from O. volvulus (GenBank accession number Y09416) that appears to be of endobacterial origin, rather than derived from the parasite itself. The full-length sequence of the cDNA (designated Ov-cpn60) codes for a protein of 64.3kDa (598 amino acid residues) and shares significant identity with homologous gene products from Caenorhabditis elegans (72%), humans (69%), yeast (53%) and Escherichia coli (50%). The endobacterial and parasite sequences are 41% conserved. Ov-CPN60 migrates with an apparent molecular mass of 65kDa on SDS-PAGE and is present in all life-cycle stages, as determined by immunoblotting with rabbit antibodies raised against the recombinant protein. Immunogold electron microscopy identified the protein within mitochondria, as expected, but also in extra-mitochondrial sites, including inclusion bodies of the glandular oesophagus (in infective larvae), the uterine wall, cytosol of developing spermatids, and the hypodermis and cuticle. Endobacteria were also labelled, indicating cross-reactivity between CPN60 from the parasite and its intracellular symbiont. In human infections, serum antibodies to Ov-CPN60 were present in only 11% of cases from Ecuador, but in 81-89% of subjects in three separate foci from West Africa. There was no relationship between antibody levels and age, sex, or infection intensity, and no consistent association between the serological response and immune status. An evaluation of antibody specificities in individual sera revealed a mixture of parasite-specific and host crossreactive anti-CPN60 antibodies, the ratio of which varied amongst geographic areas. It is concluded that antibody responses to Ov-CPN60 are unlikely to contribute either to host protection or pathology in onchocerciasis.

OvB20, an Onchocerca volvulus-cloned antigen selected by differential immunoscreening with vaccination serum in a cattle model of onchocerciasis.

A cDNA of Onchocerca volvulus has been isolated by differential immunoscreening of an adult worm expression library using sera raised in cattle against the related species, O. lienalis. It was selected because of its recognition by antibodies from cattle immunized with irradiated third-stage (L3) larvae and not by antibodies from animals infected with non-irradiated larvae. The original 311-bp clone was used to isolate a 1478-bp cDNA. Designated OvB20, this codes for 460 amino acid residues, hybridizes with a approximately 1.6 kBp transcript and appears to be transcribed from a filarial-specific, single copy gene. It is expressed in developing stages from embryo to L4 larva, but not in the adult. The product of OvB20 appears to undergo co- or post-translational processing: in vitro transcription and translation give rise to a polypeptide consistent with the deduced amino acid sequence (approximately 52 kDa), whilst products of 52 and 65 kDa are detected in larvae by immunoblotting and following in vitro translations to which exogenous microsomes have been added. A 42-kDa protein was also detected in all in vitro translations. No homologous genes were found in the computer databases, although there are regions of weak sequence similarity with C-reactive proteins. The functional role of OvB20 may warrant further attention, as it has recently been shown that the recombinant protein confers host protection against a related rodent filaria following active immunization (Taylor, M.J., Abdel-Wahab, N., Wu, Y., Jenkins, R.E. and Bianco, A.E. (1995) Onchocerca volvulus larval antigen, OvB20 induces partial protection in a rodent model of onchocerciasis. Infect. Immun. 63, 4417-4422).

Variable antigen associated with the surface of erythrocytes infected with mature stages of Plasmodium falciparum.

Immune human sera were used to select a cDNA clone expressing an asexual blood-stage antigen of Plasmodium falciparum. Antibodies affinity-purified on extracts from this clone were used to characterize the antigen by immunoblotting and immunofluorescence. The antigen is present in mature-stage parasites as a high molecular weight protein of about 250 kDa and is apparently processed to smaller fragments in the merozoite. It varies in molecular weight and antibody reactivity in different isolates, and has been localized at the erythrocyte membrane by immunoelectronmicroscopy. Part of the protein is composed of exactly repeated hexapeptide units that constitute the strain-specific determinant. This molecule has similar characteristics to the strain-specific molecule believed to be responsible for cytoadherence.

A cDNA clone expressing a rhoptry protein of Plasmodium falciparum.

Antibodies from immune humans were used to select a cDNA clone expressing an asexual blood stage antigen of Plasmodium falciparum. The expressed fused polypeptide was used as an affinity reagent to purify human antibodies specific for the corresponding parasite antigen. Western blotting and immunoelectronmicroscopy demonstrated that the antigen was a 105 kDa protein located in the rhoptries of merozoites. The cDNA encodes the carboxy terminus of the rhoptry antigen, a sequence rich both in charged and hydroxy amino acids.

Putative glycophorin-binding protein is secreted from schizonts of Plasmodium falciparum.

A cDNA clone expressing an antigen of Plasmodium falciparum, selected by screening an expression library cloned in Escherichia coli, encodes a portion of the protein identified as a glycophorin-binding protein [Kochan et al. (1986) Cell 44, 689-696]. Human antibodies affinity-purified on extracts from this clone were used to characterize the antigen by immunoblotting. This protein was present in all isolates tested, restricted to mature trophozoites and schizonts. It was abundant in culture supernatants at the time of merozoite release but present in minor amounts if at all in merozoites. The pattern of antigen distribution over schizont-infected cells observed by immunoelectron microscopy differed from that of the precursor of the major merozoite surface antigens in that most of the antigen appeared to be located over the erythrocyte cytoplasm without any obvious association with organelles. It thus appears unlikely that this antigen is present on the merozoite surface prior to schizont rupture.

The effect of gamma-radiation and heat shock on protein synthesis and antioxidant enzymes in the gastrointestinal parasite, Heligmosomoides polygyrus.

Protein synthesis and antioxidant enzyme activities were investigated in gamma-irradiated (300 Gy) and heat shocked (42 degrees C) larval stages of the gastrointestinal parasite, Heligmosomoides polygyrus bakeri (H. polygyrus). No qualitative or quantitative differences were observed in the incorporation of (35S)-methionine into somatic proteins of unirradiated or irradiated exsheathed third-stage (L3) larvae at either 37 degrees C or 42 degrees C. The rate of protein synthesis doubled in L3 stages maintained at 42 degrees C compared with 37 degrees C, irrespective of whether the larvae had been irradiated or not. The composition of excretory/secretory (ES) proteins varied between unirradiated and irradiated exsheathed L3 larvae maintained under identical conditions. Prominent heat-inducible proteins of 26 and 17 kDa were synthesised and excreted at 42 degrees C by both unirradiated and irradiated L3 stages. No major differences in protein synthesis could be detected between unirradiated and irradiated fourth-stage (L4) larvae. Temperature elevation significantly reduced protein synthesis in L4 stages, most notably in unirradiated parasites. Heat-inducible proteins were not detected in response to either irradiation or temperature elevation in L4 larvae. Immune sera recognised a similar spectrum of antigens in both unirradiated and irradiated L4 somatic and ES preparations and reacted with antigens from irradiated L4 parasites with less intensity than with antigens from unirradiated L4 larvae. Catalase was the only antioxidant enzyme examined with activity that changed significantly in irradiated parasites, being reduced to approximately 36% of normal levels in irradiated L4 stages. No significant difference existed between irradiated and unirradiated parasites in the levels of activity of superoxide dismutase and glutathione reductase.

The effects of gamma radiation on the development of Heligmosomoides polygyrus bakeri in mice.

The development of a Heligmosomoides polygyrus bakeri (H. polygyrus) primary infection in its definitive host was severely effected by a wide range of gamma radiation doses (10-400 Gy). Male worms were more susceptible to gamma radiation than female worms. A dose of 400 Gy prevented the development of L3 larvae to mature female worms and 200 Gy abrogated the maturation of males. At 300 Gy, a dose known to stimulate high levels of protective immunity, male worms were unable to moult to the L4 stage and females failed to develop into morphologically normal adults. An experiment to select for a radiation resistant parasite line provided data on the cumulative effects of gamma rays on successive parasite generations. Parasite fitness data demonstrated that worm development, at the level of embryogenesis, was far more sensitive to radiation damage than either post embryonic development or adult worm fecundity. The parasite line died out on the 14th generation of selection after receiving an accumulated dose of 420 Gy. It is concluded that gamma radiation profoundly alters the developmental biology of H. polygyrus in a dose-dependent manner, with maximal sensitivity exhibited during embryogenesis.

Ocular immunopathologic findings of experimental onchocerciasis.

Ocular immunopathologic responses of inbred guinea pigs infected with Onchocerca microfilariae from domesticated animals were studied as a laboratory model of human ocular onchocerciasis. A single intracorneal infection of normal guinea pigs with microfilariae produced only minimal ocular lesions. In contrast, intracorneal infection of guinea pigs previously immunized by systemic infection with microfilariae produced intense corneal and uveal inflammation. Transfer of splenic lymphocytes from immunized donors to syngeneic normal recipients substituted effectively for the active immunization. Cell recipients produced marked corneal inflammatory reactions when challenged by a single intracorneal infection. Fresh and cryopreserved microfilariae produced identical reactions. The corneal inflammatory infiltrates were composed primarily of eosinophils, neutrophils, and plasma cells and resembled human onchocercal keratitis. Diethylcarbamazine citrate administration after a challenge intracorneal infection increased the severity of the corneal inflammatory response in immunized animals.

Stage-specific and species cross-reactive antibody responses in experimental Onchocerca infections of cattle.

Cattle experimentally infected with Onchocerca lienalis were examined by enzyme-linked immunosorbent assay and immunoblotting to determine the degree of stage- and species-specificity in the immune response to infection. Levels of serum antibodies to antigens derived from third-stage larvae increased little after the first three weeks of infection, and the range of antibody specificities remained limited following the appearance of microfilariae (mf) in the skin. In contrast, antibodies to antigens from adult worms of either sex exhibited a vigorous response, characterized by a series of peaks arising 15-30, 79, and > 266 days after infection that were coincident with the timings of larval molts and the onset of a patent infection. Antibody specificities to the adult worms included many directed to molecules that were shared with other life-cycle stages, but some were stage-specific and others were confined to one sex. A response cross-reactive with antigens from mf was initiated during the prepatent period, but antibody levels increased steeply after the infection became patent. This was followed by a major expansion of antibody specificities to products exclusively directed to mf, most notably in the range of 12-18 kilodaltons. Sera from O. lienalis-infected cattle cross-reacted extensively with antigens derived from O. volvulus adult worms and the profiles of antibody levels over time were indistinguishable from those obtained with O. lienalis extracts. The dominant response was of IgG1, although limited IgG2 and IgM reactivities were found, while no Onchocerca-specific IgA was detected. These results demonstrate that parasite development has a profound influence on the level and repertoire of antibodies produced during Onchocerca infections, and that extensive cross-reactivity exists between O. lienalis and O. volvulus, lending support to the role of cattle models in the study of human onchocerciasis.

Patterns of antigen expression in asexual blood stages and gametocytes of Plasmodium falciparum.

The stage specificity and localization of 12 Plasmodium falciparum antigens were determined by immunofluorescence using acetone-fixed parasites reacted with monospecific antibodies against cloned antigens. Antibodies were prepared by immunization of rabbits with recombinant proteins or by affinity purification of human plasma against cloned antigen adsorbents. Most of the antigens occurred predominantly in mature asexual parasites, two were abundant in ring stages and three were absent in rings. Four of the 12 antigens were detected in asexual stages but not in gametocytes. Grouping of antigens by localization within blood stages was difficult because of the complexity of fluorescence patterns observed. With some antibodies, fluorescence was apparently distributed evenly over the parasites, but in other cases label was concentrated within discrete compartments or organelles. Extraparasitic intraerythrocytic fluorescence was also observed.

Prospective evaluation of enzyme-linked immunosorbent assay and immunoblot methods for the diagnosis of endemic Strongyloides stercoralis infection.

Recently described enzyme-linked immunosorbent assay (ELISA) and immunoblot methods for the detection of serum IgG against Strongyloides stercoralis larval antigens were prospectively evaluated for the diagnosis of endemic strongyloidiasis. A modification of the ELISA involved preincubation of sera with Onchocerca gutturosa phosphate-buffered saline-soluble extract to remove cross-reactivity with other helminths. The sensitivity of the ELISA increased from 80% to 85% following preincubation. Similarly, there was an increase in specificity from 94% to 97%. The IgG recognition of 41-, 31-, and 28-kD filariform larval components showed sensitivities of 100%, 85%, and 65%, respectively. Both the ELISA following incubation of sera with O. gutturosa extract and serum IgG reactivity to a 41-kD larval component using immunoblotting are sensitive and specific techniques for diagnosing endemic strongyloidiasis.

Separation of viable and non-viable Onchocerca microfilariae using an ion exchanger.

A technique has been developed for separating viable and non-viable Onchocerca lienalis microfilariae (mff) by passing a suspension of the parasites through a column of DEAE cellulose. A proportion (between 42% and 87%) of normally motile parasites, whether unfrozen or cryopreserved, passed through the cellulose columns and retained their viability. Non-motile and sluggish mff were retained by the cellulose. The proportion of viable cryopreserved mff was greater after passage through the column as assessed by motility, migration in mice and development in the insect vector. Numbers of cryopreserved mff eluted through the columns peak quickly at a relatively high level after about 20 ml of eluate had been passed through, whereas normal unfrozen parasites rose and fell in numbers more slowly. By using sodium chloride gradients and buffers of differing conductivity it was concluded that the separation of the mff is probably due to net charge changes as well as motility. It is believed that this technique offers considerable promise as a tool to provide populations of mff of more uniform viability following freezing and thawing with existing cryopreservation techniques.