Bacterial Expression Systems Based on a Protein A and Protein G Designed for the Production of Immunogens: Applications to Plasmodium falciparum Malaria Antigens

This article describes expression systems based on staphylococcal protein A (SpA) and streptococcal protein G (SpG) which constitute attractive alternatives for the design and production of fusion proteins containing immunogenic structures. A dual expression system that allows the choice between two fusion partners, two synthetic IgG-binding domains (ZZ) of SpA and the serum albumin-binding region BB of SpG, was developed. Genes encoding antigens are expressed in Escherichia coli in parallel as fusions to ZZ and BB and the produced fusion proteins are affinity-purified on human IgG (ZZ fusions) or human serum albumin (BB fusions). The possibility of using ZZ fusions for immunization and the corresponding BB fusions for analysis of the induced immune responses provides a convenient strategy for the generation and analysis of immune responses to selected immunogenic structures. In addition, the cell surface-attaching regions of SpA have been utilized for cell surface display of heterologous antigens on the surface of the Gram-positive bacterium Staphylococcus xylosus. The dual expression system was used to express synthetic gene constructs and genomic gene fragments encoding immunogenic structures from blood-stage antigens of the malaria parasite Plasmodium falciparum. The fusion proteins produced were highly immunogenic in rabbits, mice, and monkeys and induced antibody and T-cell responses to the expressed antigens. Different applications of the SpA- and SpG-based expression systems are described and the immunological properties of the bacterial fusion partners SpA, ZZ, and BB are discussed.     

Potato apyrase reduces granulomatous area and increases presence of multinucleated giant cells in murine schistosomiasis

Granulomas are inflammatory tissue responses directed to a set of antigens. Trapped Schistosoma mansoni eggs promote productive granulomas in the tissues, and they are the main damage caused by schistosomiasis. Some S. mansoni antigenic proteins may have a direct involvement in the resolution of the granulomatous response. The ATP diphosphohydrolases isoforms of this parasite are immunogenic, expressed in all phases of the parasite life cycle and secreted by eggs and adult worms. Potato apyrase is a vegetable protein that cross-reactive with parasite ATP diphosphohydrolases isoforms. In this study, the vegetable protein was purified, before being inoculated in C57BL/6 mice that were later infected with cercariae. Sixty days after infection, adult worms were recovered, antibodies and cytokines were measured, and morphological granuloma alterations evaluated. Immunization of the animals induced significant levels of IgG and IgG1 antibodies and IFN-γ, IL-10 and IL-5 cytokines, but not IL-13, suggesting that potato apyrase is an immunoregulatory protein. Supporting this hypothesis, it was found that liver damage associated with schistosomiasis was mitigated, reducing the size of the areas affected by granuloma to 35% and increasing the presence of multinucleated giant cells in this environment. In conclusion, potato apyrase was found to be effective immunomodulatory antigen for murine schistosomiasis.     

An immunogenic Mr 23,000 integral membrane protein of Schistosoma mansoni worms that closely resembles a human tumor-associated antigen

A Mr 23,000 Ag of the human trematode parasite, Schistosoma mansoni, has been identified by immunoscreening an adult worm cDNA library with antibody affinity purified on the Mr 23,000 to 25,000 integral membrane protein fraction of the parasite. This Ag is immunogenic in infected humans as well as in rabbits exposed to S. mansoni. The protein sequence of the Ag as deduced from cloned DNA sequences is 218 amino acids long and contains four putative transmembrane regions. Of particular significance, the Ag is strikingly similar, with respect to both amino acid sequence (36% identity) and putative domain structure to ME491, a human stage-specific melanoma-associated Ag.     

cDNA expression library screening and identification of two novel antigens: ubiquitin and receptor for activated C kinase (RACK) homologue, of the fish parasite Trypanosoma carassii

Trypanosoma carassii is a kinetoplastid parasite infecting cyprinid fish with a high prevalence in nature. Antibodies have been shown to play a protective role in the immune response against this parasite in common carp, Cyprinus carpio. To identify immunogenic and putative protective T. carassii antigens we constructed a lambdaTriplEx2 expression library of the parasite and screened this with pooled carp immune serum collected 6 weeks post-infection. Screening of the library not only revealed ribosomal proteins but identified ubiquitin and a homologue of the receptor for activated C kinase (RACK) as immunogenic proteins. Equivalents of all these proteins have been identified as immunogenic in expression library screenings of other Trypanosomatida, suggesting an evolutionary conservation of their immunogenicity. The possibility that ubiquitin and/or the homologue of RACK could represent protective antigens and be targets for the design of novel therapies is discussed.     

Production of full-length cDNA sequences by sequencing and analysis of expressed sequence tags from Schistosoma mansoni

The number of sequences generated by genome projects has increased exponentially, but gene characterization has not followed at the same rate. Sequencing and analysis of full-length cDNAs is an important step in gene characterization that has been used nowadays by several research groups. In this work, we have selected Schistosoma mansoni clones for full-length sequencing, using an algorithm that investigates the presence of the initial methionine in the parasite sequence based on the positions of alignment start between two sequences. BLAST searches to produce such alignments have been performed using parasite expressed sequence tags produced by Minas Gerais Genome Network against sequences from the database Eukaryotic Cluster of Orthologous Groups (KOG). This procedure has allowed the selection of clones representing 398 proteins which have not been deposited as S. mansoni complete CDS in any public database. Dedicated sequencing of 96 of such clones with reads from both 5' and 3' ends has been performed. These reads have been assembled using PHRAP, resulting in the production of 33 full-length sequences that represent novel S. mansoni proteins. These results shall contribute to construct a more complete view of the biology of this important parasite.     

Localization and identification of Schistosoma mansoni/KLH-crossreactive components in infected mice.

KLH (Keyhole limpet hemocyanin) is highly immunogenic, and crossreactive epitopes occur widely in nature. In schistosomiasis, infected hosts generate antibodies reactive with KLH. This is of diagnostic importance but we lack detailed information on the immunogen-carrying molecules and their distribution in the worm. We used anti-KLH antibodies to localize cross-reacting epitopes in the various developmental stages of the parasite in experimental Schistosoma mansoni infection. The staining results show KLH crossreactivity in the life stages of the parasite. By immunoblotting we show that KLH-crossreactive antigenic epitopes in the parasite eggs are carbohydrates, also recognized by antibodies against soluble schistosome egg antigens. The localizations in the larval stages and in adult worms suggest that crossreacting antigenic epitopes are secretory products.     

Mapping of the conserved antigenic domains shared between potato apyrase and parasite ATP diphosphohydrolases: potential application in human parasitic diseases

Evolutionary and closer structural relationships are demonstrated by phylogenetic analysis, peptide prediction and molecular modelling between Solanum tuberosum apyrase, Schistosoma mansoni SmATPase 2 and Leishmania braziliensis NDPase. Specific protein domains are suggested to be potentially involved in the immune response, and also seem to be conserved during host and parasite co-evolution. Significant IgG antibody reactivity was observed in sera from patients with American cutaneous leishmaniasis (ACL) and schistosomiasis using potato apyrase as antigen in ELISA. S. mansoni adult worm or egg, L. braziliensis promastigote (Lb) and Trypanosoma cruzi epimastigote (EPI) have ATP diphosphohydrolases, and antigenic preparations of them were evaluated. In ACL patients, IgG seropositivity was about 43% and 90% for Lb and potato apyrase, respectively, while IgM was lower (40%) or IgG (100%) seropositivity for both soluble egg (SEA) and adult worm (SWAP) antigens was higher than that found for potato apyrase (IgM=10%; IgG=39%). In Chagas disease, IgG seropositivity for EPI and potato apyrase was 97% and 17%, respectively, while the IgM was low (3%) for both antigens. The study of the conserved domains from both parasite proteins and potato apyrase could lead to the development of new drug targets or molecular markers.     

Glycomics analysis of Schistosoma mansoni egg and cercarial secretions

The parasitic helminth Schistosoma mansoni is a major public health concern in many developing countries. Glycoconjugates, and in particular the carbohydrate component of these products, represent the main immunogenic challenge to the host and could therefore represent one of the crucial determinants for successful parasite establishment. Here we report a comparative glycomics analysis of the N- and O-glycans derived from glycoproteins present in S. mansoni egg (egg-secreted protein) and cercarial (0-3-h released protein) secretions by a combination of mass spectrometric techniques. Our results show that S. mansoni secrete glycoproteins with glycosylation patterns that are complex and stage-specific. Cercarial stage secretions were dominated by N-glycans that were core-xylosylated, whereas N-glycans from egg secretions were predominantly core-difucosylated. O-Glycan core structures from cercarial secretions primarily consisted of the core sequence Galbeta1-->3(Galbeta1-->6)GalNAc, whereas egg-secreted O-glycans carried the mucin-type core 1 (Galbeta1-->3GalNAc) and 2 (Galbeta1-->3(GlcNAcbeta1-->6)GalNAc) structures. Additionally we identified a novel O-glycan core in both secretions in which a Gal residue is linked to the protein. Terminal structures of N- and O-glycans contained high levels of fucose and include stage-specific structures. These glycan structures identified in S. mansoni secretions are potentially antigenic motifs and ligands for carbohydrate-binding proteins of the host immune system.     

Protein Microarray Analysis of Antibody Responses to Plasmodium falciparum in Western Kenyan Highland Sites with Differing Transmission Levels

Malaria represents a major public health problem in Africa. In the East African highlands, the high-altitude areas were previously considered too cold to support vector population and parasite transmission, rendering the region particularly prone to epidemic malaria due to the lack of protective immunity of the population. Since the 1980’s, frequent malaria epidemics have been reported and these successive outbreaks may have generated some immunity against Plasmodium falciparum amongst the highland residents. Serological studies reveal indirect evidence of human exposure to the parasite, and can reliably assess prevalence of exposure and transmission intensity in an endemic area. However, the vast majority of serological studies of malaria have been, hereto, limited to a small number of the parasite’s antigens. We surveyed and compared the antibody response profiles of age-stratified sera from residents of two endemic areas in the western Kenyan highlands with differing malaria transmission intensities, during two distinct seasons, against 854 polypeptides of P. falciparum using high-throughput proteomic microarray technology. We identified 107 proteins as serum antibody targets, which were then characterized for their gene ontology biological process and cellular component of the parasite, and showed significant enrichment for categories related to immune evasion, pathogenesis and expression on the host’s cell and parasite’s surface. Additionally, we calculated age-fitted annual seroconversion rates for the immunogenic proteins, and contrasted the age-dependent antibody acquisition for those antigens between the two sampling sites. We observed highly immunogenic antigens that produce stable antibody responses from early age in both sites, as well as less immunogenic proteins that require repeated exposure for stable responses to develop and produce different seroconversion rates between sites. We propose that a combination of highly and less immunogenic proteins could be used in serological surveys to detect differences in malaria transmission levels, distinguishing sites of unstable and stable transmission.     

Isolation and characterization of a protective antigen for adjuvant-free immunization against Schistosoma mansoni

A single, 68,000 m.w. glycoprotein antigen from adult Schistosoma mansoni was purified by immunoaffinity chromatography with the use of a newly developed, protective, anti-schistosome murine monoclonal antibody. Immunization with two doses of 0.5 microgram or 1 microgram of purified antigen, without adjuvants, afforded a mean 28% reduction in parasite recovery in CF1 mice, and 2-% reduction in parasite BALB/c mice. On immunoblotting, the 68,000 m.w. antigen was common to S. mansoni adults and schistosomula, whereas parasite eggs contained only cross-reacting low m.w. antigens of 19,100 and 16,000. Immunization resulted in the development of anti-antigen antibody and enhanced immediate cutaneous hypersensitivity to the 31-3B6 antigen. By contrast, delayed-type hypersensitivity and sensitization to circumoval granuloma formation were not observed in immunized mice. It was concluded that the 68,000 m.w. 31-3B6 antigen represents a candidate vaccine for adjuvant-free immunization against S. mansoni.     

The use of electroblotted antigens of Trichostrongylus colubriformis to induce proliferative responses in sensitized lymphocytes from sheep

Merino sheep were immunized against the intestinal nematode, T. colubriformis, by repeated infections, and proliferative responses of their peripheral blood lymphocytes (PBL) against parasite extracts and excretory-secretory (ES) antigens were monitored over 130 days. Maximal responses occurred 7-14 days after challenge. The ability of soluble proteins and parasite antigens to induce proliferation was compared with that of antigen-bearing particles obtained after antigen was adsorbed onto nitrocellulose. Blank particles increased c.p.m. two- to three-fold above that obtained in medium alone, and to elicit proliferative responses of comparable magnitude between 10 and 100 times more antigen was required when antigen-bearing particles were used instead of soluble extracts or defined proteins. Blood leucocytes as well as T-cell lines established by stimulation with parasite antigens in vitro reacted to moieties of from 5000 to 38,000 mol. wt in ES antigens on nitrocellulose particles. Direct comparisons of T-lymphocyte responses with antibody responses as assessed by immunoblots revealed different profiles of immunogenicity among ES proteins within individual sheep, but the 10,000, 30,000 and 75,000-90,000 mol. wt proteins were immunodominant. These proteins were also those consistently recognized by T-lymphocytes and sera from sheep immunized with ES proteins in adjuvant. Thus, this technique can be applied to identify parasite material which is immunogenic for T-lymphocytes, but the sensitivity of the procedure in sheep is less than reported in human studies.     

Targeted glycoproteomic analysis reveals that kappa-5 is a major, uniquely glycosylated component of Schistosoma mansoni egg antigens

Glycans present on glycoproteins from the eggs of the parasite Schistosoma mansoni are mediators of various immune responses of the human host, including T-cell modulation and granuloma formation, and they are the target of glycan-specific antibodies. Here we have analyzed the glycosylation of kappa-5, a major glycoprotein antigen from S. mansoni eggs using a targeted approach of lectin purification followed by mass spectrometry of glycopeptides as well as released glycans. We demonstrate that kappa-5 has four fully occupied N-glycosylation sites carrying unique triantennary glycans composed of a difucosylated and xylosylated core region, and immunogenic GalNAcβ1-4GlcNAc (LDN) termini. Furthermore, we show that the kappa-5 specific IgE antibodies in sera of S. mansoni-infected individuals are directed against the core region of the kappa-5 glycans. Whereas two previously analyzed immunomodulatory egg glycoproteins, IPSE/alpha-1 and omega-1, both express diantennary N-glycans with a difucosylated core and one or two Galβ1-4(Fucα1-3)GlcNAc (Lewis X) antennae, the kappa-5 glycosylation appears unique among the major soluble egg antigens of S. mansoni. The distinct structural and antigenic properties of kappa-5 glycans suggest a specific role for kappa-5 in schistosome egg immunogenicity.     

Diagnostic Mr 31,000 Schistosoma mansoni proteins: requirement of infection, but not immunization, and use of the "miniblot" technique for the production of monoclonal antibodies

Antibodies directed against diagnostic Mr 31,000 polypeptide(s) of adult Schistosoma mansoni were already formed in mice during prepatency. In contrast, repeated immunization of mice with homogenates of adult schistosomes failed to elicit antibodies detectable in immunoblots in the Mr 31,000 region. Therefore, spleen cells of infected mice were used to produce hybridoma lines. The "miniblot technique" was developed in order to detect in hybridoma supernatants antibodies against schistosome Mr 31,000 components. Electrophoretically separated total S. mansoni proteins were transferred onto nitrocellulose, and the position of the Mr 31,000 components was determined with polyclonal antisera and immunoblotting. Pieces of about 3 square mm of nitrocellulose bearing the diagnostic proteins were incubated with about 100 microliter of hybridoma supernatant in microtitre plates and subsequently probed with peroxidase-conjugated antibody to mouse IgG. This screening technique identified hybridomas secreting antibody to the relevant S. mansoni antigens. It is applicable to other defined parasite antigens, which are, however, not available in biochemically purified form. The monoclonal antibodies produced against the proteins with diagnostic potential reacted with antigens localized in the schistosome gut.     

Mass spectrometric analysis of the Schistosoma mansoni tegumental sub-proteome

Schistosoma mansoni is a parasitic worm that lives in the blood vessels of its host. We mapped the S. mansoni tegumental outer-surface structure proteome by 1D SDS-PAGE and LC-MS/MS and an EST-database from the ongoing genome-sequencing project. We identified 740 proteins of which 43 were tegument-specific. Many of these proteins show no homology to any nonschistosomal protein, demonstrating that the schistosomal outer-surface comprises specific and unique proteins, likely to be critical for parasite survival.     

T and B cell epitope mapping of SM23, an integral membrane protein of Schistosoma mansoni.

SM23 is an integral membrane protein of the blood-vessel dwelling parasitic worm Schistosoma mansoni. This protein has been detected with antibodies in all stages of the parasite found in the human host, notably the lung stage, and therefore is of interest as a vaccine candidate. In addition SM23 has been shown to be a member of a proposed new superfamily of membrane proteins whose structures do not conform to the previously known classifications. To date there are 13 members including ME491 (CD63, Pltgp40), CD9 (p23), TAPA-1, CD37, CD53, MRC OX-44, CO-029, MRP-1, L6, the gene product of TI-1, the target of mAb AD-1, SM23, and SJ23 (the Schistosoma japonicum homologue). Most of these molecules except for those in the two blood vessel-dwelling parasites are found in membranes of hemopoietic and/or malignant cells and all have unknown function. In this study we used recombinantly expressed full-length and partial molecules as well as synthesized peptides to map T cell and B cell epitopes of SM23. The two predicted external hydrophilic domains were found to be highly immunogenic and contained several B cell epitopes. There were at least four T cell epitopes in the large hydrophilic domain. One segment of 23 amino acids contained both a T cell and B cell epitope as well as the putative glycosylation site. This particular segment was recognized by immune sera and cells of every mouse strain tested. The elucidation of these epitopes demonstrates the immunogenic nature of this molecule and raises questions as to the role of SM23 in the host/parasite relationship.     

Taenia solium: a two-dimensional Western blotting method combined with the use of an EST-library for the identification of immunogenic proteins recognized by sera from neurocysticercosis patients

Commercial antigens used to diagnose human neurocysticercosis (NCC) are obtained from either a soluble parasite extract or a parasite-derived glycoprotein fraction. The aim of the present study was to identify antigenic proteins as potential diagnostic candidates in this context. Soluble immunogenic proteins from Taenia solium cysticerci were identified by two-dimensional electrophoresis Western blotting using human sera from Nicaragua confirmed to be positive for NCC by computer tomography. Six antigenic proteins were identified and sequenced by liquid chromatography–mass spectrometry. Among these immunogenic proteins, a novel sequence was found and named Tsol-p27. To determine the antigenicity of Tsol-p27, the previously reported antigen TsolHSP36 and the new Tsol-p27 were expressed as recombinant proteins and evaluated serologically. Immunoblotting demonstrated that Tsol-p27 was recognized by sera from 13 NCC-positive humans, whereas TsolHSP36 was identified by only two of those 13 positive sera. None of the antigens were recognized by negative control sera. Despite the limited number of serum samples evaluated in this study, the results indicate that Tsol-p27 might be a suitable candidate for diagnosis of human NCC.     

IPSE/alpha-1, a major secretory glycoprotein antigen from schistosome eggs, expresses the Lewis X motif on core-difucosylated N-glycans

Schistosomes are parasitic flatworms that infect millions of people in (sub)tropical areas around the world. Glycoconjugates of schistosomes play a critical role in the interaction of the different developmental stages of the parasite with the host. In particular, glycosylated components of the eggs produced by the adult worm pairs living in the bloodstream are strongly immunogenic. We have investigated the glycosylation of interleukin-4-inducing factor from schistosome eggs (IPSE/alpha-1), a major secretory egg antigen from Schistosoma mansoni that triggers interleukin-4 production in human basophils, by MS analysis of tryptic glycopeptides. Nanoscale LC-MS(/MS) and MALDI-TOF(/TOF)-MS studies combined with enzymatic degradations showed that monomeric IPSE/alpha-1 contains two N-glycosylation sites, which are each occupied for a large proportion with core-difucosylated diantennary glycans that carry one or more Lewis X motifs. Lewis X has been reported as a major immunogenic glycan element of schistosomes. This is the first report both on the expression of Lewis X on a specific schistosome egg protein and on a protein-specific glycosylation analysis of schistosome eggs.     

Schistosoma mansoni: structural and biochemical characterization of two distinct Venus Kinase Receptors

Venus Kinase Receptors (VKRs) are atypical transmembrane proteins composed of an extracellular Venus FlyTrap module linked through a single helix to a tyrosine kinase domain similar to that of insulin receptors. This structure was first described in Schistosoma mansoni, then in a selected range of invertebrates, including many insects. The preferential expression of VKRs in larvae and gonads suggested their role in development and reproduction. While a single vkr gene was consistently found in all genomes, we identified two distinct vkr genes in S. mansoni. Our data indicated that Smvkr1 and Smvkr2 are very similar in structure and likely originated from gene duplication. Both genes are expressed in all the parasite stages and encode homologous proteins with a conserved VKR structure. Recombinant SmVKR1 and SmVKR2 exhibit tyrosine kinase activities dependent on the binding of distinct small ligand molecules. SmVKR1 and SmVKR2 could represent paralogs with different functions in the parasite.     

Immunoglobulins to surface-associated biofilm immunogens provide a novel means of visualization of methicillin-resistant Staphylococcus aureus biofilms

Antigens from the methicillin-resistant Staphylococcus aureus (MRSA) cell wall have been shown to be immunogenic in vivo and upregulated during biofilm growth. In this study, we created purified, recombinant forms of selected antigens and biofilm-upregulated, cell wall-associated proteins. These proteins were shown to cause a robust polyclonal immunoglobulin G (IgG) response when used to immunize rabbits. Antibodies against these recombinant proteins bound to the native forms of each protein as harvested from in vitro grown biofilms of MRSA, as determined both via Western blot analysis and immunofluorescence confocal microscopy. These IgGs could be utilized as imaging tools that localize to areas of specific protein production within a biofilm. This work illustrates that immunogenic, cell wall-associated, biofilm-upregulated proteins are promising for in vitro visualization of biofilm growth, architecture, and space-function relationships.