Designed ankyrin repeat proteins: a new approach to mimic complex antigens for diagnostic purposes?

Inhibitory antibodies directed against coagulation factor VIII (FVIII) can be found in patients with acquired and congenital hemophilia A. Such FVIII-inhibiting antibodies are routinely detected by the functional Bethesda Assay. However, this assay has a low sensitivity and shows a high inter-laboratory variability. Another method to detect antibodies recognizing FVIII is ELISA, but this test does not allow the distinction between inhibitory and non-inhibitory antibodies. Therefore, we aimed at replacing the intricate antigen FVIII by Designed Ankyrin Repeat Proteins (DARPins) mimicking the epitopes of FVIII inhibitors. As a model we used the well-described inhibitory human monoclonal anti-FVIII antibody, Bo2C11, for the selection on DARPin libraries. Two DARPins were selected binding to the antigen-binding site of Bo2C11, which mimic thus a functional epitope on FVIII. These DARPins inhibited the binding of the antibody to its antigen and restored FVIII activity as determined in the Bethesda assay. Furthermore, the specific DARPins were able to recognize the target antibody in human plasma and could therefore be used to test for the presence of Bo2C11-like antibodies in a large set of hemophilia A patients. These data suggest, that our approach might be used to isolate epitopes from different sets of anti-FVIII antibodies in order to develop an ELISA-based screening assay allowing the distinction of inhibitory and non-inhibitory anti-FVIII antibodies according to their antibody signatures.

Antigens of the type-three secretion system of Aeromonas salmonicida subsp. salmonicida prevent protective immunity in rainbow trout

Aeromonas salmonicida subsp. salmonicida is the etiologic agent of furunculosis, a frequent and significant disease of fisheries worldwide. The disease is largely controlled by commercial oil adjuvanted vaccines containing bacterins. However, the mechanisms leading to a protective immune response remain poorly understood. The type-three secretion system (T3SS) plays a central role in virulence of A. salmonicida subsp. salmonicida and thus may have an influence on the immune response of the host. The aim of this study was to evaluate the role of the T3SS antigens in mounting a protective immune response against furunculosis. Rainbow trout were intraperitoneally vaccinated in two independent experiments with bacterins prepared from a wild-type A. salmonicida strain and an isogenic strain carrying a deletion in the T3SS (ΔascV). Fish were challenged with the wt strain eight weeks after vaccination. In both trials, the survival rate of trout vaccinated with the ΔascV strain was significantly higher (23-28%) in comparison to the group vaccinated with the wt strain. High-throughput proteomics analysis of whole bacteria showed the ascV deletion in the mutant strain resulted in lower expression of all the components of the T3SS, several of which have a potential immunosuppressive activity. In a third experiment, fish were vaccinated with recombinant AcrV (homologous to the protective antigen LcrV of Yersinia) or S-layer protein VapA (control). AcrV vaccinated fish were not protected against a challenge while fish vaccinated with VapA were partially protected. The presence of T3SS proteins in the vaccine preparations decreased the level of protection against A. salmonicida infection and that AcrV was not a protective antigen. These results challenge the hypothesis that mounting specific antibodies against T3SS proteins should bring better protection to fish and demonstrate that further investigations are needed to better understand the mechanisms underlying effective immune responses against A. salmonicida infection.

The Aeromonas salmonicida subsp. salmonicida exoproteome: global analysis, moonlighting proteins and putative antigens for vaccination against furunculosis

BACKGROUND Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide. Despite the identification of several virulence factors the pathogenesis is still poorly understood. We have used high-throughput proteomics to display the differences between in vitro secretome of A. salmonicida wild-type (wt, hypervirulent, JF5054) and T3SS-deficient (isogenic ΔascV, extremely low-virulent, JF2747) strains in exponential (GP) and stationary (SP) phases of growth. RESULTS Among the different experimental conditions we obtained semi-quantitative values for a total of 2136 A. salmonicida proteins. Proteins of specific A. salmonicida species were proportionally less detected than proteins common to the Aeromonas genus or those shared with other Aeromonas species, suggesting that in vitro growth did not induce the expression of these genes. Four detected proteins which are unidentified in the genome of reference strains of A. salmonicida were homologous to components of the conjugative T4SS of A. hydrophila pRA1 plasmid. Polypeptides of three proteins which are specific to the 01-B526 strain were also discovered. In supernatants (SNs), the number of detected proteins was higher in SP (326 for wt vs 329 for mutant) than in GP (275 for wt vs 263 for mutant). In pellets, the number of identified proteins (a total of 1536) was approximately the same between GP and SP. Numerous highly conserved cytoplasmic proteins were present in A. salmonicida SNs (mainly EF-Tu, EF-G, EF-P, EF-Ts, TypA, AlaS, ribosomal proteins, HtpG, DnaK, peptidyl-prolyl cis-trans isomerases, GAPDH, Enolase, FbaA, TpiA, Pgk, TktA, AckA, AcnB, Mdh, AhpC, Tpx, SodB and PNPase), and several evidences support the theory that their extracellular localization was not the result of cell lysis. According to the Cluster of Orthologous Groups classification, 29% of excreted proteins in A. salmonicida SNs were currently poorly characterized. CONCLUSIONS In this part of our work we elucidated the whole in vitro exoproteome of hypervirulent A. salmonicida subsp. salmonicida and showed the secretion of several highly conserved cytoplasmic proteins with putative moonlighting functions and roles in virulence. All together, our results offer new information about the pathogenesis of furunculosis and point out potential candidates for vaccine development.

Evaluation of an IgE ELISA with Culicoides spp. extracts and recombinant salivary antigens for diagnosis of insect bite hypersensitivity in Warmblood horses

Insect bite hypersensitivity (IBH) in horses represents an immunoglobulin E (IgE)-mediated hypersensitivity to salivary antigens from biting midges (Culicoides spp.). The aim of this study was to evaluate and compare the performances of IgE ELISAs using recombinant Culicoides spp. Obsoletus group salivary gland antigens or crude whole body extracts ('ObsWBE'), C. nubeculosus recombinant proteins (Culn1, 3, 4, 5, 7, 8 and 10) and Obsoletus group recombinant proteins (Culo1 and 2). IgE levels were measured in plasma of 343 Warmblood horses classified as IBH-affected (n=167) and IBH-unaffected (n=176) according to the owners' descriptions. IBH-affected horses were subdivided based on the severity of their clinical signs at sampling and whether or not their IBH history was considered to be classical. The accuracies of the tests increased when clinical signs at sampling were more pronounced or when the IBH history could be considered as classical. A combination of IgE levels against the three best performing Culicoides spp. recombinant proteins (Culn4, Culo1 and Culo2) and ObsWBE resulted in the best performing test. When IBH-affected horses showing a classical history of the disease and severe clinical signs were compared with IBH-unaffected horses, the Youden's index at the optimal cut-off for the three tests in combination was 0.67. This optimal cut-off had a sensitivity of 70%, a specificity of 97% and a total accuracy of 92%. The performance of the IgE ELISA was affected by the severity of IBH clinical signs at sampling and was improved when IgE levels against several recombinant proteins were combined.

Evaluation of the diagnostic value of the ELISA tests developed by using EgHF, Em2 and EmII/3-10 antigens in the serological diagnosis of alveolar echinococcosis

Alveolar echinococcosis (AE), caused by larva stage of Echinococcus multilocularis, is one of the lethal parasitic diseases of man and a major public health problem in many countries in the northern hemisphere. When the living conditions and habits in Turkey were considered in terms of relation with the life cycle of the parasite, it was suggested that AE has been much more common than reported mainly from the Eastern Anatolia region of Turkey. Since in vitro serologic diagnosis tests with high specificity for AE have not been used in our country, most of the cases with liver lesions were misdiagnosed by radiological investigations as malignancies. The aim of this study was to evaluate the diagnostic value of the in-house ELISA methods developed by using three different antigens (EgHF, Em2, EmII/3-10) in the serological diagnosis of AE. The study samples included a total of 100 sera provided by Bern University Parasitology Institute where samples were obtained from patients with helminthiasis and all were confirmed by clinical, parasitological and/or histopathological means. Ten samples from each of the cases infected by E.multilocularis, E.granulosus, Taenia solium, Wuchereria bancrofti, Strongyloides stercolaris, Ascaris lumbricoides, Toxocara canis, Trichinella spiralis, Fasciola hepatica and Schistosoma haematobium were studied. In the study, EgHF (E.granulosus hydatid fluid) antigens were prepared in our laboratory from the liver cyst fluids of sheeps with cystic echinococcosis, however Em2 (E.multilocularis metacestode-purified laminated layer) and EmII/3-10 (E.multilocularis recombinant protoscolex tegument) antigens were provided by Bern University Parasitology Institute. Flat bottom ELISA plates were covered with EgHF, Em2 and EmII/3-10 antigens in the concentrations of 2.5 µg, 1 µg and 0.18 µg per well, respectively, and all sera were tested by EgHF-ELISA, Em2-ELISA and EmII/3-10-ELISA methods. For each tests, the samples which were reactive above the cut-off value (mean OD of negative controls+2 SD) were accepted as positive. The sensitivity of the ELISA tests performed with EgHF, Em2 and Em2II/3-10 antigens were estimated as 100%, 90% and 90%, respectively, whereas the specificity were 63%, 91% and 91%, respectively. When Em2-ELISA and EmII/3-10-ELISA tests were evaluated together, the specificity increased to 96%. Our data indicated that the highest sensitivity (100% with EgHF-ELISA) and specificity (96% with Em2-ELISA + EmII/3-10-ELISA) for the serodiagnosis of AE can be achieved by the combined use of the ELISA tests with three different antigens. It was concluded that the early and accurate diagnosis of AE in our country which is endemic for that disease, could be supported by the use of highly specific serological tests such as Em2-ELISA ve EmII/3-10-ELISA contributing radiological data.

Demonstration of species-specific and cross-reactive components of Taenia solium metacestode antigens.

Analysis of human serum reactivities to antigenic components of soluble Taenia solium metacestode proteins showed the predominant presence of determinants shared by T. solium, Echinococcus multilocularis and E. granulosus. Two polypeptides were demonstrated by SDS-PAGE and Western blot or enzyme-linked immunoelectrotransfer blot (EITB) assay to bind serum and CSF antibodies only from T. solium cysticercosis patients. These species-specific antigenic polypeptides focused between pH 4.6 and 3.9 after resolution by isoelectric focusing followed by EITB. The high species-specificity demonstrated by the present techniques offers the opportunity to confirm serologically an infection by T. solium metacestode.

An immunoassay for the detection of circulating antigens in human echinococcosis.

An immunoassay (double-antibody-sandwich-ELISA) was developed to detect circulating antigens (CAg) in patients with cystic (Echinococcus granulosus) echinococcosis. Echinococcus antigens derived from heterologous intermediate hosts were used to immunize rabbits and to purify the rabbit-IgG-fraction obtained by affinity-chromatography, thus avoiding major interference with host components. The purified rabbit anti-hydatid IgG was immunosorbed with bovine and human sera. One part of the resulting IgG served as coating agent in a double antibody sandwich-ELISA; the other part, coupled to alkaline phosphatase, as detecting conjugate. The specificity of the antibody reaction was demonstrated by immunoelectrophoresis. Sera of 21 patients with cystic echinococcosis were examined with this test system. In seven of the patients' sera CAg were detected in concentrations ranging between 310 ng and 680 ng protein per ml serum. Comparing pre- and postoperative serum samples obtained from nine patients operated on for cystic echinococcosis, four sera were found to be CAg-positive before and three after operation.

A collaborative study on larval excretory/secretory antigens of Toxocara canis for the immunodiagnosis of human toxocariasis with ELISA.

Two batches of excretory/secretory (E/S) antigens from second stage larvae of Toxocara canis maintained in vitro were prepared independently in two different laboratories (Zürich and Basel) and analysed in order to obtain information for future efforts to standardize the enzyme-linked immunosorbent assay (ELISA) used for the serodiagnosis of human toxocariasis. SDS-PAGE and "Western-blotting" revealed at least 10 different antigenic components common to the two antigen preparations. However, distinct qualitative and quantitative differences among the two E/S-antigens were observed, since one antigen had a more complex composition than the other. Despite these differences, an accordance of serodiagnosis was obtained in 80% of 25 sera from patients with suspected Toxocara infection tested independently in two different ELISA systems (Basel and Zürich) with the corresponding E/S-antigens. The specificity was 93% as determined (BS-antigen, BS-ELISA) by testing 46 out of 3396 sera from patients with parasitologically proven extra-intestinal helminthic infections. Cross-reactions occurred mainly with sera from patients infected with filariae (5 from 13 cases) exhibiting very high extinction values in their homologous ELISA-system. The reproducibility (intra- and inter-test variations) of two ELISA systems using the corresponding E/S-antigens varied from 5-15%. The results demonstrate that T. canis E/S-antigens may well be applicable for standardization of the ELISA used for the serodiagnosis of human toxocariasis.

Antibodies against major histocompatibility complex class II antigens directly inhibit the growth of T cells infected with Theileria parva without affecting their state of activation

We have analyzed the effect of antibodies (Abs) directed against major histocompatibility complex (MHC) class II Abs on the proliferation of Theileria parva-infected (Tpi) T cells. Anti-MHC class II Abs exert a direct effect on Tpi T cells causing an acute block in their proliferation. The inhibition does not involve apoptosis and is also entirely reversible. The rapid arrest of DNA synthesis caused by anti-MHC class II Abs is not due to interference with the state of activation of the T cells since the transcriptional activator NF-kappa B remains activated in arrested cells. In addition, interleukin 2 (IL-2), IL-2R, and c-myc gene expression are also unaffected. By analyzing the cell-cycle phase distribution of inhibited cells, it could be shown that cells in all phases of the cell cycle are inhibited. The signal transduction pathway that results in inhibition was shown to be independent of protein kinase C and extracellular Ca2+. Tyrosine kinase inhibitors, however, partly reduced the level of inhibition and, conversely, phosphatase inhibitors enhanced it. The possible relevance of this phenomenon in other systems is discussed.

Serological diagnosis of echinococcosis: the diagnostic potential of native antigens

PURPOSE: Human alveolar (AE) and cystic echinococcosis (CE) caused by the metacestode stages of Echinococcus multilocularis and E. granulosus, respectively, lack pathognomonic clinical signs. Diagnosis therefore relies on the results of imaging and serological studies. The primary goal of this study was to evaluate the efficacy of several easy-to-produce crude or partially purified E. granulosus and E. multilocularis metacestode-derived antigens as tools for the serological diagnosis and differential diagnosis of patients suspicious for AE or CE. METHODS: The sera of 51 treatment-naïve AE and 32 CE patients, 98 Swiss blood donors and 38 patients who were initially suspicious for echinococcosis but suffering from various other liver diseases (e.g., liver neoplasia, etc.) were analysed. RESULTS: According to the results of enzyme-linked immunosorbent assays (ELISA), metacestode-derived antigens of E. granulosus had sensitivities varying from 81 to 97% and >99.9% for the diagnosis of CE and AE, respectively. Antigens derived from E. multilocularis metacestodes had sensitivities ranging from 84 to 91% and >99.9% for the diagnosis of CE and AE, respectively. Specificities ranged from 92 to >99.9%. Post-test probabilities for the differential diagnosis of AE from liver neoplasias, CE from cystic liver lesions, and screening for AE in Switzerland were around 95, 86 and 2.2%, respectively. Cross-reactions with antibodies in sera of patients with other parasitic affections (fasciolosis, schistosomosis, amebosis, cysticercosis, and filarioses) did occur at variable frequencies, but could be eliminated through the use of confirmatory testing. CONCLUSIONS: Different metacestode-derived antigens of E. granulosus and E. multilocularis are valuable, widely accessible, and cost-efficient tools for the serological diagnosis of echinococcosis. However, confirmatory testing is necessary, due to the lack of species specificity and the occurrence of cross-reactions to other helminthic diseases.