Recombinant antigens used as diagnostic tools for lymphatic filariasis.

Lymphatic filariasis (LF) is a parasitic disease caused by the worms Wuchereria bancrofti, Brugia malayi, or Brugia timori. It is a tropical and subtropical illness that affects approximately 67 million people worldwide and that still requires better diagnostic tools to prevent its spread and enhance the effectiveness of control procedures. Traditional parasitological tests and diagnostic methods based on whole protein extracts from different worms are known for problems related to sample time collection, sensitivity, and specificity. More recently, new diagnostic tools based on immunological methods using recombinant antigens have been developed. The current review describes the several recombinant antigens used as tools for lymphatic filariasis diagnosis in antigen and antibody capture assays, highlighting their advantages and limitations as well as the main commercial tests developed based on them. The literature chronology is from 1991 to 2021. First, it describes the historical background related to the identification of relevant antigens and the generation of the recombinant polypeptides used for the LF diagnosis, also detailing features specific to each antigen. The subsequent section then discusses the use of those proteins to develop antigen and antibody capture tests to detect LF. So far, studies focusing on antibody capture assays are based on 13 different antigens with at least six commercially available tests, with five proteins further used for the development of antigen capture tests. Five antigens explored in this paper belong to the SXP/RAL-2 family (BmSXP, Bm14, WbSXP-1, Wb14, WbL), and the others are BmShp-1, Bm33, BmR1, BmVAH, WbVAH, BmALT-1, BmALT-2, and Wb123. It is expected that advances in research with these antigens will allow further development of tests combining both sensitivity and specificity with low costs, assisting the Global Program to Eliminate Lymphatic Filariasis (GPELF).

Screening, construction, and serological identification of the diagnostic antigen molecule EG-06283 for the diagnosis of Echinococcus granulosus.

Several strategies exist to prevent and control echinococcosis, a global parasitic disease. However, most treatments are ineffective and adverse effects are common. Therefore, we aimed to screen protoscolex antigen molecules of Echinococcus granulosus to identify a diagnostic biomarker for hydatid disease. Published E. granulosus transcriptome sequencing data were analyzed to screen for antigen molecules that are highly expressed in protoscoleces but not in oncospheres. The membrane protein EG-06283 (annotated as Frizzled-4) was selected from 16 antigens, and its gene fragment was subjected to codon optimization and synthesis. rEG-06283 expression was induced in the pET-24a/EG-06283/BL21 strain; subsequently, the protein was purified and subcutaneously injected into ICR mice at weeks 0, 2, 4, and 6. Blood sampling occurred periodically to quantify serum immunoglobulin G (IgG) levels via enzyme-linked immunosorbent assays (ELISA). Immunogenicity was determined by western blot assays using sera from normal mice and mice with secondary hydatid infections. The antigen's immune reactivity and diagnostic value were validated using sera of patients with hydatid disease. ELISA results confirmed that the antigen molecule induced specific IgG production in mice, resulting in significantly higher levels than those in the adjuvant and control groups (P < 0.05). The western blot results indicated that the protein was recognized by antibodies in the sera of mice with hydatid infection and the antisera of immunized mice. Quantification of protein levels in the sera of patients with hydatid disease significantly differed from levels in healthy participants (P < 0.05). These results indicate that rEG-06283 is a potential diagnostic antigen for E. granulosus infections.

Schistosoma mansoni venom allergen-like proteins: phylogenetic relationships, stage-specific transcription and tissue localization as predictors of immunological cross-reactivity.

Schistosoma mansoni venom allergen-like proteins (SmVALs) are part of a diverse protein superfamily partitioned into two groups (group 1 and group 2). Phylogenetic analyses of group 1 SmVALs revealed that members could be segregated into subclades (A-D); these subclades share similar gene expression patterns across the parasite lifecycle and immunological cross-reactivity. Furthermore, whole-mount in situ hybridization demonstrated that the phylogenetically, transcriptionally and immunologically-related SmVAL4, 10, 18 and 19 (subclade C) were all localized to the pre-acetabular glands of immature cercariae. Our results suggest that SmVAL group 1 phylogenetic relationships, stage-specific transcriptional profiles and tissue localization are predictive of immunological cross-reactivity.

The Tao survivorship of schistosomes: implications for schistosomiasis control.

Schistosomiasis, caused by blood flukes of the genus Schistosoma, is a major public health problem which contributes substantially to the economic and financial burdens of many nations in the developing world. An array of survival strategies, such as the unique structure of the tegument which acts as a major host-parasite interface, immune modulation mechanisms, gene regulation, and apoptosis and self-renewal have been adopted by schistosome parasites over the course of long-term evolution with their mammalian definitive hosts. Recent generation of complete schistosome genomes together with numerous biological, immunological, high-throughput "-omics" and gene function studies have revealed the Tao or strategies that schistosomes employ not only to promote long-term survival, but also to ensure effective life cycle transmission. New scenarios for the future control of this important neglected tropical disease will present themselves as our understanding of these Tao increases.

[A comparative analysis of various antigenic proteins found in Haemonchus contortus--a review].

Many innovative researches on the development and introduction of recombinant vaccines against many economically important parasites were carried out in the 20th century. Research continues to hold promise with the development of immunological and molecular approaches for control of these parasites and in this regard it has already been seen that blood-sucking parasites such as Haemonchus contortus and Ostertagia ostertagi are susceptible to control by vaccines containing "novel" or "concealed" antigens. Haemonchus contortus is primarily pathogenic to sheep and its blood-feeding behaviour causes effects ranging from mild anaemia to mortality in young animals. Current means of control which are dependent on repeated treatment with anthelmintics are responsible for the increasing drug resistance of this parasite. Together with the growing concern of residual chemicals in the environment and food chain, this has led to attempts to better understand the biology of the parasite with an aim to develop alternate means of control, including the development of molecular vaccines. More problematic and also important is the formulation and delivery strategy to induce expulsion of this parasite, using vaccines containing recombinant "conventional" antigens. Tremendous progress has been made in the last decade in identifying several antigens from Haemonchus contortus which in their native form stimulate useful levels of protective immunity. Vaccines have been developed against H. contortus using 'novel' gut antigens from the parasite, but variable responsiveness of the host sheep has resulted in varying degrees of protection which are stimulated by these vaccines. Computer models have also been used to simulate vaccine efficacy in worm control and have yielded good results. This review will try to summarise the protective efficacy and also the molecular properties of principal candidate antigens which are expressed by this parasite. The review will try to cover the aspirations, current success, limitations and problems faced by researchers in the control of this economically important parasite.

Schistosome syntenin partially protects vaccinated mice against Schistosoma mansoni infection.

BACKGROUND: Schistosomiasis is a neglected tropical disease caused by several species of trematode of the genus Schistosoma. The disease affects more than 200 million people in the world and causes up to 280,000 deaths per year, besides having high morbidity due to chronic illness that damages internal organs. Current schistosomiasis control strategies are mainly based on chemotherapy, but many researchers believe that the best long-term strategy to control disease is a combination of drug treatment and immunization with an anti-schistosome vaccine. Among the most promising molecules as vaccine candidates are the proteins present in the tegument and digestive tract of the parasite. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we describe for the first time Schistosoma mansoni syntenin (SmSynt) and we evaluate its potential as a recombinant vaccine. We demonstrate by real-time PCR that syntenin is mainly expressed in intravascular life stages (schistosomula and adult worms) of the parasite life cycle and, by confocal microscopy, we localize it in digestive epithelia in adult worms and schistosomula. Administration of siRNAs targeting SmSynt leads to the knock-down of syntenin gene and protein levels, but this has no demonstrable impact on parasite morphology or viability, suggesting that high SmSynt gene expression is not essential for the parasites in vitro. Mice immunization with rSmSynt, formulated with Freund's adjuvant, induces a Th1-type response, as suggested by the production of IFN-γ and TNF-α by rSmSynt-stimulated cultured splenocytes. The protective effect conferred by vaccination with rSmSynt was demonstrated by 30-37% reduction of worm burden, 38-43% reduction in the number, and 35-37% reduction in the area, of liver granulomas. CONCLUSIONS/SIGNIFICANCE: Our report is the first characterization of syntenin in Schistosoma mansoni and our data suggest that this protein is a potential candidate for the development of a multi-antigen vaccine to control schistosomiasis.

[Preliminary evaluation on the diagnostic performance of five native antigens from Echinococcus granulosus].

OBJECTIVE: To evaluate the diagnostic potential of 5 native antigens obtained from Echinococcus granulosus. METHODS: The cyst fluid and protoscoleces were collected from infected livers of slaughtered sheep near Urumqi. The protoscolex somatic soluble antigen (EgPS), crude hydatid cyst fluid antigen (EgHF), partially purified hydatid fluid antigen (Burstein's antigen, EgBu) and the native antigen B (EgAgB) were prepared. Echinococcus granulosus adult worms were collected from experimentally infected dogs, and then used to prepare adult somatic soluble antigen (EgAS). Serum samples from patients with cystic echinococcosis (369 cases), alveolar echinococcosis (14 cases), cysticercosis (20 cases), schistosomiasis (50 cases), paragonimiasis westermani (10 cases), visceral leishmaniasis (5 cases), and healthy individuals (366 cases) were examined for the antibodies against 5 native antigens with indirect ELISA. A ROC analysis was performed to determine a cut-off value for each native antigen. RESULTS: EgPS presented the highest AUC(BOC) (0.958 +/- 0.067), sensitivity (88.7%), positive predictive value (0.96), positive likelihood ratio (16.61), diagnostic efficiency (91.7%), and odds ratio (138.475). The specificity of EgBu (96.1%) was the highest, followed by EgAgB (95.7%), EgPS (94.7%), EgAS (89.0%), and EgHF (80.4%). The Youden index was ranked in order of EgPS>EgBu>EgHF>EgAS>EgAgB. The antibody reactive intensity of EgBu-ELISA was highest (4.055 +/- 1.365) and the EgAS-ELISA was lowest (2.947 +/- 1.276), and there was no significant difference between them (P > 0.05). The five native antigens presented high cross-reactivity with sera from alveolar echinococcosis (9/14-12/14) and from cysticercosis (8/20-16/20). CONCLUSION: There are significant differences among the five native antigen-ELISA. The EgPS antigen has the best diagnostic performance , which is followed by EgBu and EgAgB.

Identification of novel antigens within the Schistosoma japonicum tetraspanin family based on molecular characterization.

Tetraspanins (TSPs) are proteins found on the surface of the parasite Schistosoma mansoni that have been regarded as potential protective antigens. However, divergent evolution may occur among the species of S. mansoni and Schistosoma japonicum under different environmental pressure. Thus, it was essential to characterize the S. japonicum TSP family members before selecting potential candidate TSP antigens. In this study, we used bioinformatics and experimental validation to investigate 29 TSP members from S. japonicum, including all known genes, Sj23, TE736, Sj25, and Sj-TSP-2. Five TSP members were found to be variable, and two others (Sj-tsp genes) were alternatively spliced. The phylogenetic analysis showed that schistosome TSPs were highly divergent from those of other phyla. Quantitative RT-PCR revealed that the Sj-tsp genes were differentially transcribed in the developmental stages of cercariae, schistosomula, adult worms, and eggs. Six Sj-tsp genes were significantly up-regulated during the transformation from cercariae to schistosomula. Four Sj-tsp genes, including Sj-tsp-1, Sj-tsp-8, Sj-tsp-14, and Sj-tsp-26 were confirmed as potential protective antigens based on the molecular characterization. RNAi was preformed against the conserved Sj-tsp genes which were highly expressed in schistosomula to explore gene functions. These data will promote the identification of candidate antigens within the TSP family for developing novel vaccines against S. japonicum infections.

Contributions to the study of Trichinella spiralis TSL-1 antigens in host immunity.

The observation on different hosts infected with Trichinella spiralis that recognized similar muscle larvae (ML) antigens and the fact that different monoclonal antibodies (mAb) had a similar reactivity to ML components prompted a proposal to define a useful classification system for these antigens. For this purpose, an international workshop provided a platform for the classification of T. spiralis antigens. ML antigens were classified in eight groups -- Trichinella spiralis larvae groups, TSL-1 to TSL-8. TSL-1 antigens are highly immunogenic and a number of important studies have been performed to analyse the role of these antigens in the host-parasite interplay. In this context, we have focused on the analysis of the role of TSL-1 antigens in the induction of innate immune responses with particular emphasis on the activation of mast cells (MC) by an IgE-independent pathway. These studies provided evidence on the role of mediator release from TSL-1-activated MC in the development of Type 2 immune responses. The protective role of TSL-1 in T. spiralis-infected mice has been described. In addition, it has been demonstrated that the use of TSL-1 antigens allows for a more sensitive and specific diagnosis of human and animal trichinellosis.

Trichinella antigens: a review.

This paper presents a review of the Trichinella antigens within the context of species variation. As with other parasites, Trichinella antigens can be classified according to their localisation as surface, excretory/secretory (ES) and somatic components. Surface antigens are mainly constituents of the outer cuticle although secretions from inner parts of the body wall as well as from the oesophagus can temporarily accumulate in the surface. ES antigens come mainly from the excretory granules of the stichosome, whereas somatic constitutive antigens come from the internal parts of the worms. ES products are considered very important from an immunological point of view as they are easily targeted by the immune system, whereas parasite death is required for exposure of somatic products. Some of the antigenic components have been characterised chemically. Phosphorylcholine is an important hapten that modulates the immune responses in Trichinella infections. Glycoproteins are the major components of surface and excretory/secretory products. A 43-kDa glycoprotein has been regarded as a good candidate for diagnosis and vaccination purposes. Recently some glycans have received special attention either as relevant epitopes or as parasite evasion strategies.

A novel C-type lectin secreted by a tissue-dwelling parasitic nematode.

Many parasitic nematodes live for surprisingly long periods in the tissues of their hosts, implying sophisticated mechanisms for evading the host immune system. The nematode Toxocara canis survives for years in mammalian tissues, and when cultivated in vitro, secretes antigens such as TES-32. From the peptide sequence, we cloned TES-32 cDNA, which encodes a 219 amino-acid protein that has a domain characteristic of host calcium-dependent (C-type) lectins, a family of proteins associated with immune defence. Homology modelling predicted that TES-32 bears remarkable structural similarity to mammalian immune-system lectins. Native TES-32 acted as a functional lectin in affinity chromatography. Unusually, it bound both mannose- and galactose-type monosaccharides, a pattern precluded in mammalian lectins by a constraining loop adjacent to the carbohydrate-binding site. In TES-32, this loop appeared to be less obtrusive, permitting a broader range of ligand binding. The similarity of TES-32 to host immune cell receptors suggests a hitherto unsuspected strategy for parasite immune evasion.

Activities of the EM10 protein from Echinococcus multilocularis in cultured mammalian cells demonstrate functional relationships to ERM family members.

The ezrin-radixin-moesin (ERM) homolog EM10 is expressed by the larval stage of the parasite E. multilocularis and shows 46.9% overall identity in the primary structure with human ezrin. To determine whether EM10 has similar activities to ERM proteins, we investigated properties of the protein expressed in mammalian cells. In particular, we transiently expressed haemagglutinin-tagged (HA-tagged) versions of the full-length EM10 as well as the amino- and the carboxy-terminal halves of EM10 in HtTA-1 cells. In addition we stably transfected NIH-3T3 cells with untagged full-length EM10. The data demonstrate that EM10 polypeptides behave like their corresponding portions of radixin when transiently expressed in mammalian cells. The full-length and amino-terminal EM10 polypeptides were localized to cortical structures. Cells expressing the carboxy-terminal polypeptide of EM10 showed long actin-filled protrusions. Cells expressing full-length EM10 showed a reduction in endogenous moesin-staining at cortical structures. In stably transfected NIH-3T3 cells EM10 was not crisply localized but rather was diffuse throughout the cytoplasm. These cells showed a conspicuous loss of stress-fibers, a phenotype that was not seen in analogous experiments with ERM proteins. The results demonstrate both similarities and differences between the functional properties of EM10 and ERM proteins expressed in vertebrate cells.

[Experimental studies on dynamic changes of circulating antigens of Schistosoma japonicum in infected rabbits].

AIM: To observe the dynamic changes and disappearance of three kinds of schistosome circulating antigens(CAgs) in infected rabbits before and after praziquantel treatment. METHODS: Dot-ELISA assays were developed by using 3 McAbs to determine the dynamic changes of the gut-associated antigen (GAA), membrane-associated antigen (MAA) and soluble egg antigen (SEA) respectively in sera of rabbits infected with S. japonicum. RESULTS: By the 4th week after infection 39% of the rabbits infected with S. japonicum were GAA positive and only 8.6% were MAA or SEA positive. All the infected rabbits becOme positive with GAA, MAA and SEA by the 8th week after infection. The titers of the GAA, MAA and SEA in sera of the infected rabbits reached their peaks by the 10th week after infection. Titers from rabbits of the untreated group rabbits remained high level until the 27th week after infection. SEA titers were found negative in 10% of the infected rabbits in the treatment group by the 6th week after treatment. The titers of GAA of, MAA' and SEA were found negative in 20%, 80% and 70% of the rabbits in the treatment group, respectively the 8th after treatment. GAA, MAA and SEA in sera except one of the treated rabbits were all negative by the 14th week after treatment. CONCLUSION: The determination of GAA is useful for the early diagnosis of schistosomiasis, while the determination of, MAA and SEA might be used for the efficacy evaluation.

Evaluation of IgG4 response in ascariasis by ELISA for serodiagnosis.

The excretory/secretory (ES) antigen(s) of Ascaris lumbricoides was fractionated into 10 fractions by gel chromatography on a Suparose 12 column in FPLC. Of these, the third fraction (Al III), showing binding activity with both IgE and IgG antibodies of A. lumbricoides infected patients' sera, was further resolved into 2 fractions (Al IIIa and Al IIIb) on passage through a Mono Q column. Al IIIb was found to be the most potent antigen due to its high binding affinity with IgE and IgG antibodies of Ascaris infected patients as evidenced by ELISA inhibition. Although a two to five-fold increase of serum IgE level was observed in all helminthic parasite infected patients studied compared to control subjects, Al IIIb specific IgE was detected in sera of all Ascaris infected and only 40% of hookworm infected patients. When Al IIIb was tested by ELISA with sera of control subjects, Ascaris, hookworm, Strongyloides and Trichuris infected patients, strong binding was observed with the IgE and also IgG of all the Ascaris infected patients; however, it cross-reacted with IgG in 50% of hookworm, 28.6% of Trichuris trichura and 22.2% of Strongyloides infected patients' sera; but with IgE only in 40% of hookworm infected patients' sera. Further study showed specific detection of IgG4 in all the serum samples of 65 Ascaris infected patients when Al IIIb antigen was allowed to react with different subclasses of IgG by ELISA, giving a sensitivity of 100%. Reactivities of Al IIIb with IgG1 and IgG3 were only 47.6 and 11.8% respectively and there was no reactivity with IgG2 subclass. No IgG4 reactivity against Al IIIb was observed in the sera of hookworm, Trichuris or Strongyloides infected patients and was similar to that observed with control subjects showing the 100% specificity of the test system. This study may therefore be regarded as a novel technique for serodiagnosis of ascariasis by measuring Ascaris specific IgG4.

Workshop on a detailed characterization of Trichinella spiralis antigens: a platform for future studies on antigens and antibodies to this parasite.

In order to characterize immunodominant components of T. spiralis a workshop was organized. In this the reactivity of monoclonal and polyclonal antibodies, provided by different research groups, towards total extracts from adult, new born larvae and muscle larvae as well as to excretory/secretory components of muscle larvae were tested by ELISA, Western blot and immunoprecipitation assays. As a result of this workshop T. spiralis ML antigens have been classified into eight groups (TSL-1-TSL-8) according to their recognition by monoclonal and polyclonal antibodies. Among them, TSL-1 antigens have been the most extensively characterized both biochemically and immunologically. These antigens are stage specific, originate in the muscle stichosome and are abundant in both E/S and on the larval cuticular surface. The TSL-1 antigens share an immunodominant carbohydrate epitope (tyvelose), which is unique for Trichinella and is not associated with phosphorylcholine. The data collected in this workshop has allowed both the unification of the nomenclature for T. spiralis antigens and their biochemical characterization. It also has provided a platform for further studies on the characterization of other T. spiralis antigens and indeed for other Trichinella species.

Fasciola hepatica: characterization of somatic and excretory-secretory antigens of adult flukes recognized by infected sheep.

The immune response of sheep to somatic components and excretory/secretory products of adult Fasciola hepatica, was studied during an experimental infection. Antibodies against adult fluke somatic and excretory/secretory antigens were detected by thin layer immunoassay from the second week post infection. Similarly, the results of Western blot analysis showed specific recognition of several components as early as two weeks after infection. However, an increase in the number and intensity of bands with time of infection was observed in the patterns of antigenic recognition. Most notorious was the strong reactivity of all infected sheep sera towards components of 20-23 kDa in the somatic preparation and components of 23-27 kDa in the excretory/secretory products of adult F. hepatica, specially noticeable after the sixth week post-infection. Since these polypeptides were recognized by all infected animals, they could play an important role in the diagnosis of sheep fascioliasis.

[Comparative study on antigens of Cysticercus cellulosae by ELIB and ELISA].

Urea-soluble antigens (Ag-u) and water-soluble antigens (Ag-w) of Cysticercus cellulosae were studied by using ELIB and ELISA, respectively. The results showed that using Ag-u and Ag-w, 9 and 14 bands of polypeptides could be recognized by sera from cysticercosis patients; 4 and 10 bands could be recognized by sera from hydatidosis patients; whereas 4 and 8 bands could be recognized by sera from clornochiasis patients. Using ELISA at serum dilutions of 1:400, the positive rate of sera against Ag-u and Ag-w was 96.6% and 86.6%, respectively in 60 cysticercosis patients; 16.6% and 46.6% respectively in 30 hydatidosis patients and negative in 24 clornorchiasis patients and 50 normal healthy persons, indicating that both Ag-u and Ag-w share common antigen components causing cross reaction with the sera from hydatidosis and clonorchiasis patients. However, such components were fewer in Ag-u than in Ag-w. In view of the fact that Ag-u is superior to Ag-w in both sensitivity and specificity, Ag-u might be better used for the diagnosis of cysticercosis.

Biotechnology in the development of vaccines for animal parasites.

Attempts to develop vaccines for protozoan and helminth parasites of livestock have been generally unproductive. Difficulties have been encountered in identifying antigens which induce protective immune responses and in obtaining sufficient quantities of antigens for vaccine trials. Use of monoclonal antibody and genetic engineering technologies provides the necessary tools to overcome these problems. Application of these technologies in animal parasitology should provide for significant breakthroughs in vaccine development.