Application of recombinant Cryptosporidium parvum P23 for isolation and prevention.

Cryptosporidium parvum is a coccidian protozoan that causes diarrhea in immunocompromised humans and newborn animals. Billions of oocysts of C. parvum can be released from the infected calves and can contaminate the environment. The severity of the disease depends on the immunological status of the individual. Oocysts of Cryptosporidium are extremely resistant to many environmental stresses, and no effective disinfectant and curative agent against this organism is available. In our study, recombinant C. parvum P23 was prepared for application in the isolation and prevention of cryptosporidiosis. P23 is a glycoprotein that belongs to a family of protein of 23-27 kDa and is believed to be expressed in the different life stages of the parasite. Immunostaining analysis using the indirect fluorescent antibody test showed that P23 could be recognized on the surface of the oocysts. The antibody prepared in rabbit against P23 was bound to Sepharose 4B and used for the isolation of oocysts. The results showed that the prepared column was able to bind specifically only the oocysts. The effect of specific recombinant C. parvum IgY antibody against infection with C. parvum was examined in a mouse model. For this aim, purified egg yolk antibody prepared from immunized hens was used to analyze the protective effect of recombinant P23 specific antibody in immunosuppressed adult mice. The results showed more than 70% reduction in oocyst shedding after challenge with 1 × 10(4) oocysts. These results support previous studies of other investigators regarding the protective effect of P23 as an antigen against C. parvum infection and showed that it could be possible to design a passive immunization strategy against C. parvum based on the anti-P23 yolk antibody in animals and immunosuppressed humans.

High affinity nanobodies against the Trypanosome brucei VSG are potent trypanolytic agents that block endocytosis.

The African trypanosome Trypanosoma brucei, which persists within the bloodstream of the mammalian host, has evolved potent mechanisms for immune evasion. Specifically, antigenic variation of the variant-specific surface glycoprotein (VSG) and a highly active endocytosis and recycling of the surface coat efficiently delay killing mediated by anti-VSG antibodies. Consequently, conventional VSG-specific intact immunoglobulins are non-trypanocidal in the absence of complement. In sharp contrast, monovalent antigen-binding fragments, including 15 kDa nanobodies (Nb) derived from camelid heavy-chain antibodies (HCAbs) recognizing variant-specific VSG epitopes, efficiently lyse trypanosomes both in vitro and in vivo. This Nb-mediated lysis is preceded by very rapid immobilisation of the parasites, massive enlargement of the flagellar pocket and major blockade of endocytosis. This is accompanied by severe metabolic perturbations reflected by reduced intracellular ATP-levels and loss of mitochondrial membrane potential, culminating in cell death. Modification of anti-VSG Nbs through site-directed mutagenesis and by reconstitution into HCAbs, combined with unveiling of trypanolytic activity from intact immunoglobulins by papain proteolysis, demonstrates that the trypanolytic activity of Nbs and Fabs requires low molecular weight, monovalency and high affinity. We propose that the generation of low molecular weight VSG-specific trypanolytic nanobodies that impede endocytosis offers a new opportunity for developing novel trypanosomiasis therapeutics. In addition, these data suggest that the antigen-binding domain of an anti-microbial antibody harbours biological functionality that is latent in the intact immunoglobulin and is revealed only upon release of the antigen-binding fragment.

Recombinant antibody against Trypanosoma cruzi from patients with chronic Chagas heart disease recognizes mammalian nervous system.

BACKGROUND: To deeply understand the role of antibodies in the context of Trypanosoma cruzi infection, we decided to characterize A2R1, a parasite antibody selected from single-chain variable fragment (scFv) phage display libraries constructed from B cells of chronic Chagas heart disease patients. METHODS: Immunoblot, ELISA, cytometry, immunofluorescence and immunohistochemical assays were used to characterize A2R1 reactivity. To identify the antibody target, we performed an immunoprecipitation and two-dimensional electrophoresis coupled to mass spectrometry and confirmed A2R1 specific interaction by producing the antigen in different expression systems. Based on these data, we carried out a comparative in silico analysis of the protein target´s orthologues, focusing mainly on post-translational modifications. FINDINGS: A2R1 recognizes a parasite protein of ~50 kDa present in all life cycle stages of T. cruzi, as well as in other members of the kinetoplastid family, showing a defined immunofluorescence labeling pattern consistent with the cytoskeleton. A2R1 binds to tubulin, but this interaction relies on its post-translational modifications. Interestingly, this antibody also targets mammalian tubulin only present in brain, staining in and around cell bodies of the human peripheral and central nervous system. INTERPRETATION: Our findings demonstrate for the first time the existence of a human antibody against T. cruzi tubulin capable of cross-reacting with a human neural protein. This work re-emphasizes the role of molecular mimicry between host and parasitic antigens in the development of pathological manifestations of T. cruzi infection.

Protection against invasive amebiasis by a single monoclonal antibody directed against a lipophosphoglycan antigen localized on the surface of Entamoeba histolytica.

A panel of monoclonal antibodies was raised from mice immunized with a membrane preparation from Entamoeba histolytica, the pathogenic species causing invasive amebiasis in humans. Antibody EH5 gave a polydisperse band in immunoblots from membrane preparations from different E. histolytica strains, and a much weaker signal from two strains of the nonpathogenic species Entamoeba dispar. Although the exact chemical structure of the EH5 antigen is not yet known, the ability of the antigen to be metabolically radiolabeled with [32P]phosphate or [3H]glucose, its sensitivity to digestion by mild acid and phosphatidylinositol-specific phospholipase C, and its specific extraction from E. histolytica trophozoites by a method used to prepare lipophosphoglycans from Leishmania showed that it could be classified as an amebal lipophosphoglycan. Confocal immunofluorescence and immunogold labeling of trophozoites localized the antigen on the outer face of the plasma membrane and on the inner face of internal vesicle membranes. Antibody EH5 strongly agglutinated amebas in a similar way to concanavalin A (Con A), and Con A bound to immunoaffinity-purified EH5 antigen. Therefore, surface lipophosphoglycans may play an important role in the preferential agglutination of pathogenic amebas by Con A. The protective ability of antibody EH5 was tested in a passive immunization experiment in a severe combined immunodeficient (SCID) mouse model. Intrahepatic challenge of animals after administration of an isotype-matched control antibody or without treatment led to the development of a liver abscess in all cases, whereas 11 out of 12 animals immunized with the EH5 antibody developed no liver abscess. Our results demonstrate the importance and, for the first time, the protective capacity of glycan antigens on the surface of the amebas.

Malaria vaccine: the pros and cons.

BACKGROUND: Malaria is an important parasitic disease of humans caused by infection with a parasite of the genus Polasmodium and transmitted by female anopheles. Infection caused by P. falciparum is the most serious of all the other species (P. ovale, P. vivax and P. malariae) especially in terms of morbidity and mortality hence the reason why most of the research has been focussed on this species. The disease affects up to about 40 per cent of the world's population with around 300-500 million people currently infected and mainly in the tropics. It has a high morbidity and mortality especially in resource-poor tropical and subtropical regions with an economic fall of about US$ 12 billion annually in Africa alone. METHOD: relevant literatures were reviewed from medical journals, library search and internet source. Other relevant websites like PATH, Malaria Vaccine Initiative and Global Fund were also visited to source for information. The key words employed were: malaria, vaccine, anopheles mosquito, insecticide treated bed-nets, pyrethroids and Plasmodium. RESULTS: several studies have underscored the need to develop an effective human malaria vaccine for the control and possible eradication of malaria across the globe with the view to reduce the morbidity and mortality associated with the disease, improve on the social and economic losses and also protect those at risk. CONCLUSION: It is very obvious that the need for effective human malaria vaccine is not only to serve those living in malaria endemic regions but also the non-immune travellers especially those travelling to malaria endemic areas; this would offer cost effective means of preventing the disease, reducing the morbidity and mortality associated with it in addition to closing the gap left by other control measures. It is very obvious that there is no single control measure known to be effective in the control of malaria, hence the need for combination of more than one method with the aim of achieving synergy in the total control and possible eradication of the disease. It suffices to say that despite the use of combination of more than one method (e.g., drugs treating patients, breaking the life cycle of the vector mosquito using larvicides, clearing swamps and other mosquito breeding sites), no much progress was made towards achieving this goal, hence the renewed interest especially with regards to vaccine development.

Characterization of Entamoeba histolytica intermediate subunit lectin-specific human monoclonal antibodies generated in transgenic mice expressing human immunoglobulin loci.

Four fully human monoclonal antibodies (MAbs) to Entamoeba histolytica intermediate subunit lectin (Igl) were prepared in XenoMouse mice, which are transgenic mice expressing human immunoglobulin loci. Examination of the reactivities of these MAbs to recombinant Igl1 and Igl2 of E. histolytica showed that XEhI-20 {immunoglobulin G2(kappa) [IgG2(kappa)]} and XEhI-28 [IgG2(kappa)] were specific to Igl1, XEhI-B5 [IgG2(kappa)] was specific to Igl2, and XEhI-H2 [IgM(kappa)] was reactive with both Igls. Gene analyses revealed that the V(H) and V(L) germ lines were VH3-48 and L2 for XEhI-20, VH3-21 and L2 for XEhI-28, VH3-33 and B3 for XEhI-B5, and VH4-4 and A19 for XEhI-H2, respectively. Flow cytometry analyses showed that the epitopes recognized by all of these MAbs were located on the surfaces of living trophozoites. Confocal microscopy demonstrated that most Igl1 and Igl2 proteins were colocalized on the surface and in the cytoplasm, but different localization patterns in intracellular vacuoles were also present. The preincubation of trophozoites with XEhI-20, XEhI-B5, and XEhI-H2 caused significant inhibition of the adherence of trophozoites to Chinese hamster ovary cells, whereas preincubation with XEhI-28 did not do so. XEhI-20, XEhI-B5, and XEhI-H2 were injected intraperitoneally into hamsters 24 h prior to intrahepatic challenge with E. histolytica trophozoites. One week later, the mean abscess size in groups injected with one of the three MAbs was significantly smaller than that in controls injected with polyclonal IgG or IgM isolated from healthy humans. These results demonstrate that human MAbs to Igls may be applicable for immunoprophylaxis of amebiasis.

The importance of human FcgammaRI in mediating protection to malaria.

The success of passive immunization suggests that antibody-based therapies will be effective at controlling malaria. We describe the development of fully human antibodies specific for Plasmodium falciparum by antibody repertoire cloning from phage display libraries generated from immune Gambian adults. Although these novel reagents bind with strong affinity to malaria parasites, it remains unclear if in vitro assays are predictive of functional immunity in humans, due to the lack of suitable animal models permissive for P. falciparum. A potentially useful solution described herein allows the antimalarial efficacy of human antibodies to be determined using rodent malaria parasites transgenic for P. falciparum antigens in mice also transgenic for human Fc-receptors. These human IgG1s cured animals of an otherwise lethal malaria infection, and protection was crucially dependent on human FcgammaRI. This important finding documents the capacity of FcgammaRI to mediate potent antimalaria immunity and supports the development of FcgammaRI-directed therapy for human malaria.

Passive transfer of Plasmodium falciparum MSP-2 pseudopeptide-induced antibodies efficiently controlled parasitemia in Plasmodium berghei-infected mice.

We have developed monoclonal antibodies directed against the pseudopeptide psi-130, derived from the highly conserved malarial antigen Plasmodium falciparum merozoite surface protein 2 (MSP-2), for obtaining novel molecular tools with potential applications in the control of malaria. Following isotype switching, these antibodies were tested for their ability to suppress blood-stage parasitemia through passive immunization in malaria-infected mice. Some proved totally effective in suppressing a lethal blood-stage challenge infection and others reduced malarial parasitemia. Protection against P. berghei malaria following Ig passive immunization can be associated with specific immunoglobulins induced by a site-directed designed MSP-2 reduced amide pseudopeptide.

Interaction of malaria parasite-inhibitory antibodies with the merozoite surface protein MSP1(19) by computational docking.

Merozoite surface protein 1 (MSP1) of the malaria parasite Plasmodium falciparum is an important vaccine candidate antigen. Antibodies specific for the C-terminal maturation product, MSP1(19), have been shown to inhibit erythrocyte invasion and parasite growth. Specific monoclonal antibodies react with conformational epitopes contained within the two EGF-like domains that constitute the antigen MSP1(19). To gain greater insight into the inhibitory process, the authors selected two strongly inhibitory antibodies (designated 12.8 and 12.10) and modeled their structures by homology. Computational docking was used to generate antigen-antibody complexes and a selection filter based on NMR data was applied to obtain plausible models. Molecular Dynamics simulations of the selected complexes were performed to evaluate the role of specific side chains in the binding. Favorable complexes were obtained that complement the NMR data in defining specific binding sites. These models can provide valuable guidelines for future experimental work that is devoted to the understanding of the action mechanism of invasion-inhibitory antibodies.

Identification of Novel Pre-Erythrocytic Malaria Antigen Candidates for Combination Vaccines with Circumsporozoite Protein.

Malaria vaccine development has been hampered by the limited availability of antigens identified through conventional discovery approaches, and improvements are needed to enhance the efficacy of the leading vaccine candidate RTS,S that targets the circumsporozoite protein (CSP) of the infective sporozoite. Here we report a transcriptome-based approach to identify novel pre-erythrocytic vaccine antigens that could potentially be used in combination with CSP. We hypothesized that stage-specific upregulated genes would enrich for protective vaccine targets, and used tiling microarray to identify P. falciparum genes transcribed at higher levels during liver stage versus sporozoite or blood stages of development. We prepared DNA vaccines for 21 genes using the predicted orthologues in P. yoelii and P. berghei and tested their efficacy using different delivery methods against pre-erythrocytic malaria in rodent models. In our primary screen using P. yoelii in BALB/c mice, we found that 16 antigens significantly reduced liver stage parasite burden. In our confirmatory screen using P. berghei in C57Bl/6 mice, we confirmed 6 antigens that were protective in both models. Two antigens, when combined with CSP, provided significantly greater protection than CSP alone in both models. Based on the observations reported here, transcriptional patterns of Plasmodium genes can be useful in identifying novel pre-erythrocytic antigens that induce protective immunity alone or in combination with CSP.

Current Mathematical Models for Analyzing Anti-Malarial Antibody Data with an Eye to Malaria Elimination and Eradication.

The last decade has witnessed a steady reduction of the malaria burden worldwide. With various countries targeting disease elimination in the near future, the popular parasite infection or entomological inoculation rates are becoming less and less informative of the underlying malaria burden due to a reduced number of infected individuals or mosquitoes at the time of sampling. To overcome such problem, alternative measures based on antibodies against specific malaria antigens have gained recent interest in malaria epidemiology due to the possibility of estimating past disease exposure in absence of infected individuals. This paper aims then to review current mathematical models and corresponding statistical approaches used in antibody data analysis. The application of these models is illustrated with three data sets from Equatorial Guinea, Brazilian Amazonia region, and western Kenyan highlands. A brief discussion is also carried out on the future challenges of using these models in the context of malaria elimination.

Measurement of Antibody-Mediated Reduction of Plasmodium yoelii Liver Burden by Bioluminescent Imaging.

Antibodies against the infectious sporozoite stage of malaria have been shown to be effective in preventing infection of the liver and in mitigating the ensuing blood stage. However, only a handful of antibody targets have been vetted and shown to be successful in mediating in vivo protection. Even more limited are the means with which to measure how effectively antibodies can reduce the number of parasites establishing infection in the liver. Traditionally, only qPCR of infected mouse livers could accurately measure liver parasite burden. However, this procedure requires sacrifice of the animal and precludes monitoring of the ensuing blood stage infection. Herein we describe a method of accurately assessing antibody-mediated reduction of parasite liver burden by combining passive or active immunization of mice and mosquito bite challenge with luciferase-expressing transgenic P. yoelii parasites. This method is rapid, reliable and allows for observation of blood stage disease in the same animal. This model will prove integral in screening the efficacy of novel antibody targets as the search for a more effective malaria vaccine continues.

The stage-specific in vitro efficacy of a malaria antigen cocktail provides valuable insights into the development of effective multi-stage vaccines.

Multicomponent vaccines targeting different stages of Plasmodium falciparum represent a promising, holistic concept towards better malaria vaccines. Additionally, an effective vaccine candidate should demonstrate cross-strain specificity because many antigens are polymorphic, which can reduce vaccine efficacy. A cocktail of recombinant fusion proteins (VAMAX-Mix) featuring three diversity-covering variants of the blood-stage antigen PfAMA1, each combined with the conserved sexual-stage antigen Pfs25 and one of the pre-erythrocytic-stage antigens PfCSP_TSR or PfCelTOS, or the additional blood-stage antigen PfMSP1_19, was produced in Pichia pastoris and used to immunize rabbits. The immune sera and purified IgG were used to perform various assays determining antigen specific titers and in vitro efficacy against different parasite stages and strains. In functional in vitro assays we observed robust inhibition of blood-stage (up to 90%), and sexual-stage parasites (up to 100%) and biased inhibition of pre-erythrocytic parasites (0-40%). Cross-strain blood-stage efficacy was observed in erythrocyte invasion assays using four different P. falciparum strains. The quantification of antigen-specific IgGs allowed the determination of specific IC50 values. The significant difference in antigen-specific IC50 requirements, the direct correlation between antigen-specific IgG and the relative quantitative representation of antigens within the cocktail, provide valuable implementations for future multi-stage, multi-component vaccine designs.

Parasitologic and clinical human response to immunoglobulin administration in falciparum malaria.

The protective effect of African IgG antibodies against Plasmodium falciparum malaria was investigated by passive transfer in Thai patients. Sera from 333 African adults were collected in the Cote d'Ivoire and subjected to extensive screening. One hundred fifty-three samples were discarded for safety reasons, and IgG was extracted from those remaining under conditions allowing their use by the intravenous (iv) route. Eight Thai patients with P. falciparum parasitemia were treated by iv inoculation of the IgG: six with a 100 mg/kg dose given over three days, one with a single 20 mg/kg dose, and one with a single 200 mg/kg dose. To ensure a safety margin of at least 48 hours, subjects were chosen among patients having a recrudescent parasitemia following quinine treatment failure at the RI level. At that stage, symptoms were mild or absent and parasitemia was low but increasing (range 4, 200-9,000/microliters). The IgG pool exerted a profound, stage-specific, but non-sterilizing effect on each of the strains tested, and proved to be safe. Asexual parasitemia decreased by a mean 728-fold (range 46-1,086), while gametocytes were unaffected. Clearance of parasites and symptoms was as fast or faster than with drugs, and was consistent in the eight patients treated, suggesting that target antigens were equally expressed in geographically remote isolates. In peripheral blood smears, no mature forms were seen at any time during the followup, which does not support the hypothesis that reversal of cytoadherence occurred. After the disappearance of the transferred antibodies, recrudescent parasites from three patients were found to be susceptible to the same extent (mean decrease of 1,310-fold) to the same IgG preparation, indicating that selection of parasites able to escape the effect of antibodies had not occurred. No adverse side-effects were detected during the followup, which lasted one year.

Treatment of murine cryptosporidiosis with anticryptosporidial immune rat bile.

Persistent cryptosporidiosis was established in nu/nu BALB/c mice by oral inoculation with Cryptosporidium parvum oocysts. The model was used to determine the impact of anticryptosporidial immune rat bile on the resolution of the disease. Presence of C. parvum-specific IgA in the immune rat bile was determined by enzyme-linked immunosorbent assay. Infection of mice was verified by stool analysis for oocytes and by hematoxylin and eosin-stained intestinal sections from control mice (infected but untreated). Efficacy of treatment was determined in control and treated mice by analysis of identical, hematoxylin and eosin-stained sections of the small intestine and cecum. Semi-quantitative comparisons were made by determining the percent of crypts infected with Cryptosporidium organisms. The scores of treated mice were significantly lower then controls. Microscopic analysis of intestinal sections showed less villus atrophy, crypt hyperplasia, and fewer organisms per crypt in the immune bile-treated mice than in controls. These results support a role for humoral immunity in the eradication of cryptosporidiosis.

Antibodies to ring-infected erythrocyte surface antigen (Pf155/RESA) protect against P. falciparum parasitemia in highly exposed multigravidas women in Malawi.

To determine whether antibodies to defined B-cell epitopes of Plasmodium falciparum antigens were related to protection against parasitemic attacks in highly exposed pregnant women, two samples of 235 with no initial P. falciparum parasitemia (NP) and 89 multigravidas who presented initial P. falciparum parasitemia (IP) were enrolled in an antimalarial prophylaxis trial in the Mangochi District in Malawi. Sera were collected under effective prophylaxis and tested for antibody measurement using FAST-ELISA. Mean antibody titers to synthetic peptides reproducing the 3 major B-cell epitopes of the ring-infected erythrocyte surface antigen (Pf155/RESA), as (EENV)4, (EENVEHDA)4 and (DDEHVEEPTVA)3, were higher in the NP than in the IP multigravidas, and this remained consistent within the season of malaria transmission (all p < 0.05). All antibodies to Pf155/RESA were positively intercorrelated within each group. Mean antibody titers to peptide (PNAN)5 reproducing the major B-cell epitope of the circumsporozoite protein (CS protein) were similar between NP and IP multigravidas in both dry and rainy season. Antibodies to Pf155/RESA epitopes may contribute to immune protection against blood-stage parasite multiplication in these highly malaria-exposed pregnant women.

Prophylactic and therapeutic use of an anti-brevetoxin (PbTx-2) antibody in conscious rats.

A polyclonal antiserum was investigated for prophylactic and therapeutic use in the treatment of brevetoxin intoxication. Conscious, tethered male rats were pre-treated with 1 ml of anti-brevetoxin IgG (PbAb) or control IgG by a 10 min infusion, then given brevetoxin (25 micrograms/kg) by a 1 hr infusion. Rats pre-treated with control IgG demonstrated signs of brevetoxin intoxication; these signs were absent in rats pre-treated with PbAb. In therapy studies, rats were infused for 1 hr with 100 micrograms/kg brevetoxin, followed immediately by 2 ml of either PbAb or control IgG. During toxin infusion, both groups showed signs of brevetoxin intoxication. Rats treated with control antibody died within 6 hr. In rats treated with PbAb, respiratory rates began to return toward baseline almost immediately, and fewer neurological signs developed. After 24 hr, nearly all neurological signs had disappeared and both core and peripheral temperatures had returned to normal. There was a time differential between two groups of signs, suggesting high and low accessibility compartments for the antibody. These compartments probably represent central and peripheral nervous system. All animals treated with PbAb survived at least 8 days. These results suggest that PbAb has both therapeutic and prophylactic potential in the treatment of brevetoxin intoxication. Further, because of the differential in efficacy in reversing central and peripheral nervous system signs of brevetoxin intoxication, it provides useful new information on the mechanism of action of this toxin.