Humoral antibody response and Ig characterization of the specific agglutinins in rabbits during experimental American trypanosomiasis

A comparative follow up study of the specific agglutinins detected by direct agglutination (DA) test and the immune response detected by specific lysis (SL), indirect immunofluorescence (IFA), indirect hemagglutination (IHA) and complement fixation (CF) tests in rabbits inoculated with trypomastigotes of T. cruzi is reported here.The specific antibody response was detected first by DA test. Reductive cleavage of sera with 2-mercaptoethanol produced a drop in the agglutinin titer of the sera during the first 30 days of infection.The next test to become positive was SL and later on the IFA, IHA and CF tests became positive simultaneously.When fractions obtained by column chromatography in Sephadex G-200 were tested serologically it was demonstrated that specific antibodies were detected mainly in fraction I (IgM) of the pooled rabbit sera obtained 15 days after inoculation (acute stage), and in fraction II (IgG) of the pooled sera obtained from rabbits 90 days after inoculation (chronic stage).Antigens prepared with trypsinized and formolized epimastigotes of three T. cruzi strains, belonging to each one of the different immunological groups described, worked similarly in the detection of specific agglutinin antibodies.Trypanosoma cruzi agglutinins were highly specific in their reaction with their homologous T. cruzi antigens as was proved by the low agglutinin titer obtained in sera from infected rabbits when, instead of T. cruzi epimastigotes, promastigotes of L. donovani were used as antigen, and by the incapacity of this parasite to absorb the T. cruzi agglutinins.     

West Nile Complement Fixing antibodies in Nigerian domestic animals and humans

A survey for West Nile Complement Fixing (CF) antibody was carried out in humans and domestic animals in Nigeria. Human sera were obtained from two communities namely Ibadan and Ogbomoso but animal sera were collected from Ibadan and Maiduguri. The overall CF antibody to West Nile virus in the two localities surveyed was 65%. Of 170 persons tested, 53% and 75% were positive in Ibadan and Ogbomoso respectively. Antibody prevalence increased with age in both communities. Tests for antibody against other flaviviruses revealed that monotypic complement fixation reactions were found frequently in young people, but broadly reacting sera were common among the older age groups. Sex distribution of West Nile CF antibody showed that 49/82 (60%) of females and 62/88 (75%) of males had West Nile CF antibody. Tests on animal sera showed that 33% contained CF antibody to West Nile virus. Prevalence of CF antibody in different animal species was 62% in camels, 4% in cattle and 0% in goats.     

Prevalence of antibody to hantaviruses in humans and rodents in the Caribbean region of Colombia determined using Araraquara and Maciel virus antigens

We tested sera from 286 agricultural workers and 322 rodents in the department of Córdoba, northeastern Colombia, for antibodies against two hantaviruses. The sera were analysed by indirect ELISA using the lysate of Vero E6 cells infected with Maciel virus (MACV) or the N protein of Araraquara virus (ARAV) as antigens for the detection of antibodies against hantaviruses. Twenty-four human sera were IgG positive using one or both antigens. We detected anti-MACV IgG antibodies in 10 sera (3.5%) and anti-ARAV antibodies in 21 sera (7.34%). Of the 10 samples that were positive for MACV, seven (70%) were cross-reactive with ARAV; seven of the 21 ARAV-positive samples were cross-reactive with MACV. Using an ARAV IgM ELISA, two of the 24 human sera (8.4%) were positive. We captured 322 rodents, including 210 Cricetidae (181 Zygodontomys brevicauda, 28 Oligoryzomys fulvescens and 1 Oecomys trinitatis), six Heteromys anomalus (Heteromyidae), one Proechimys sp. (Echimyidae) and 105 Muridae (34 Rattus rattus and 71 Mus musculus). All rodent sera were negative for both antigens. The 8.4% detection rate of hantavirus antibodies in humans is much higher than previously found in serosurveys in North America, suggesting that rural agricultural workers in northeastern Colombia are frequently exposed to hantaviruses. Our results also indicate that tests conducted with South American hantavirus antigens could have predictive value and could represent a useful alternative for the diagnosis of hantavirus infection in Colombia.     

Effect of Porphyromonas gingivalis outer membrane vesicles on gingipain-mediated detachment of cultured oral epithelial cells and immune responses

Porphyromonas gingivalis is a major etiological agent of periodontal diseases and the outer membrane vesicles (OMVs) contain virulence factors such as LPS and gingipains. In this study, we investigated the potential role of the OMVs in host immune response and tissue destruction during P. gingivalis infection. Firstly, we found that sera from periodontitis patients had significantly stronger reactivity against an OMV-producing wild type strain than the isogenic OMV-depleted strain. OMVs were found to be highly antigenic, as absorption of patient sera with OMVs greatly reduced reactivity with whole cells of P. gingivalis. LC-MS/MS analysis of OMVs revealed multiple forms of gingipains and several gingipain-related proteins. Western blots of OMVs using patient sera revealed a conserved immunoreactive antigen profile resembling the profile of OMV antigens that were recognized by gingipain antiserum, suggesting a potential role of OMV-associated gingipains in triggering antibody-mediated immune responses to P. gingivalis infection. When OMVs were added to a monolayer of an oral squamous epithelial cell line, OMVs caused cell detachment, which was inhibited by preincubating OMVs with anti-gingipain antiserum. These data suggest that gingipain-laden OMVs may contribute to tissue destruction in periodontal diseases by serving as a vehicle for the antigens and active proteases.     

Toxoplasma gondii-positive human sera recognise intracellular tachyzoites and bradyzoites with diverse patterns of immunoreactivity

Antibody detection assays have long been the first line test to confirm infection with the zoonotic parasite Toxoplasma gondii. However, challenges exist with serological diagnosis, especially distinguishing between acute, latent and reactivation disease states. The sensitivity and specificity of serological tests might be improved by testing for antibodies against parasite antigens other than those typically found on the parasite surface during the acute stage. To this end, we analysed the reactivity profile of human sera, identified as positive for anti-Toxoplasma gondii IgG in traditional assays, by indirect immunofluorescence reactivity to acute stage intracellular tachyzoites and in vitro-induced latent stage bradyzoites. The majority of anti-Toxoplasma gondii IgG positive sera recognised both intracellularly replicating tachyzoites and in vitro-induced bradyzoites with varying patterns of immune-reactivity. Furthermore, anti-bradyzoite antibodies were not detected in sera that were IgM-positive/IgG-negative. These results demonstrate that anti-Toxoplasma gondii-positive sera may contain antibodies to a variety of antigens in addition to those traditionally used in serological tests, and suggest the need for further investigations into the utility of anti-bradyzoite-specific antibodies to aid in diagnosis of Toxoplasma gondii infection.     

Detection of Toxoplasma gondii Infections using Virus-Like Particles Displaying T. gondii ROP4 Antigen

Toxoplasma gondii ME49 infections are typically diagnosed by serological tests. However, serological diagnosis of RH strain-induced toxoplasmosis remains unknown. In order to develop seradiagnosis of above 2 kinds of infections, we generated recombinant virus-like particles (VLPs) displaying the T. gondii rhoptry protein 4 (ROP4) and evaluated their potential in T. gondii ME49 or RH strain infection diagnostics. Mice were orally infected with either the tachyzoites of T. gondii (RH) or cysts of T. gondii (ME49) at various dosages, and sera were collected at regular intervals. ELISA-based serological tests were performed to assess IgG, IgM, and IgA antibody responses against ROP4 VLP antigen and tissue lysate antigen (TLA). Compared to TLA, IgG, IgM, and IgA levels to ROP4 VLP antigen were significantly higher in the sera of T. gondii RH-infected mice 1 and 2 week post-infection (PI). T. gondii-specific IgG antibody was detected at 1, 2, 4, and 8 week PI in the T. gondii ME49-infected mice with infection dose-dependent manner. These results indicated that the ROP4 VLP antigen was highly sensitive antigens detecting T. gondii RH and ME49 antibodies at an early stage.     

Expression of Trypanosoma brucei gambiense Antigens in Leishmania tarentolae. Potential for Use in Rapid Serodiagnostic Tests (RDTs)

The development of rapid serodiagnostic tests for sleeping sickness and other diseases caused by kinetoplastids relies on the affordable production of parasite-specific recombinant antigens. Here, we describe the production of recombinant antigens from Trypanosoma brucei gambiense (T.b. gambiense) in the related species Leishmania tarentolae (L. tarentolae), and compare their diagnostic sensitivity and specificity to native antigens currently used in diagnostic kits against a panel of human sera. A number of T.b. gambiense protein antigen candidates were chosen for recombinant expression in L. tarentolae based on current diagnostics in field use and recent findings on immunodiagnostic antigens found by proteomic profiling. In particular, the extracellular domains of invariant surface glycoprotein 65 (ISG65), variant surface glycoproteins VSG LiTat 1.3 and VSG LiTat 1.5 were fused with C-terminal histidine tags and expressed as soluble proteins in the medium of cultured, recombinant L. tarentolae. Using affinity chromatography, on average 10 mg/L of recombinant protein was purified from cultures and subsequently tested against a panel of sera from sleeping sickness patients from controls, i.e. persons without sleeping sickness living in HAT endemic countries. The evaluation on sera from 172 T.b. gambiense human African trypanosomiasis (HAT) patients and from 119 controls showed very high diagnostic potential of the two recombinant VSG and the rISG65 fragments with areas under the curve between 0.97 and 0.98 compared to 0.98 and 0.99 with native VSG LiTat 1.3 and VSG LiTat 1.5 (statistically not different). Evaluation on sera from 78 T.b. rhodesiense HAT patients and from 100 controls showed an acceptable diagnostic potential of rISG65 with an area under the curve of 0.83. These results indicate that a combination of these recombinant antigens has the potential to be used in next generation rapid serodiagnostic tests. In addition, the L. tarentolae expression system enables simple, cheap and efficient production of recombinant kinetoplatid proteins for use in diagnostic, vaccine and drug discovery research that does not rely on animal use to generate materials.     

Failure to prove arenavirus infection among the small mammals from an endemic area of Korean hemorrhagic nephrosonephritis.

In the light of recent knowledge on a complex of diseases caused by a new group of viruses, arenaviruses, virological studies largely directed toward small field mammals were undertaken during 1973-1974 aiming at etiological clarification of Korean hemorrhagic nephrosonephritis (KHNN). Specimens were collected in an endemic area of KHNN located north to northeast of Seoul. Virus isolation tests with 299 urine specimens and 131 mite pools recovered from small mammals and 14 acute stage sera from typical cases yielded negative results. Complement-fixation (CF) tests failed to detect antibodies against the antigens of Congo, lymphocytic choriomeningitis (LCM), Tacaribe, and Pichinde viruses among 366 small mammal sera. In addition, CF tests of 59 of the above sera against Apoi and Lassa virus antigens were negative. The results do not support the likelihood of an arenavirus being transmitted among Korean small field mammals, the overwhelming majority of which were Apodemus agrarius. A hypothesis that KHNN is caused by a virus of small field mammal origin was not proved within the technical limit of relatively unsophisticated methods employed herein.     

Immunodominant antigens of Leishmania chagasi associated with protection against human visceral leishmaniasis

Background

Protection and recovery from visceral leishmaniasis (VL) have been associated with cell-mediated immune (CMI) responses, whereas no protective role has been attributed to humoral responses against specific parasitic antigens. In this report, we compared carefully selected groups of individuals with distinct responses to Leishmania chagasi to explore antigen-recognizing IgG present in resistant individuals.

Methodology and Principal Findings

VL patients with negative delayed-type hypersensitivity (DTH) were classified into the susceptible group. Individuals who had recovered from VL and converted to a DTH+ response, as well as asymptomatic infected individuals (DTH+), were categorized into the resistant group. Sera from these groups were used to detect antigens from L. chagasi by conventional and 2D Western blot assays. Despite an overall reduction in the reactivity of several proteins after DTH conversion, a specific group of proteins (approximately 110–130 kDa) consistently reacted with sera from DTH converters. Other antigens that specifically reacted with sera from DTH+ individuals were isolated and tandem mass spectrometry followed by database query with the protein search engine MASCO were used to identify antigens. The serological properties of recombinant version of the selected antigens were tested by ELISA. Sera from asymptomatic infected people (DTH+) reacted more strongly with a mixture of selected recombinant antigens than with total soluble Leishmania antigen (SLA), with less cross-reactivity against Chagas disease patients'' sera.

Significance

Our results are the first evidence of leishmania proteins that are specifically recognized by sera from individuals who are putatively resistant to VL. In addition, these data highlight the possibility of using specific proteins in serological tests for the identification of asymptomatic infected individuals.     

Evaluation of the sensitivity of selected serological tests and activity of Coxiella burnetii antigens in the diagnosis of Q fever]

Sensitivity of three serological tests: indirect immunofluorescence assay (If), complement fixation test (CF), and microagglutination test (MA) was evaluated. Sera (118 samples) of humans suspected of C. burnetii infection were tested. Phase II antibodies were detected in 68.6% of sera and phase I antibodies--in 38.2% of sera. Among seropositive to phase II antigen--93.8% of sera reacted in IF, 62.9% in MA, and 32.1% in CF; among seropositive to phase I antigens--100% of samples reacted in IF, 2.6% in MA and 2.6% in CF. Calculated sensitivity of above tests was as followed: IF-93.8%, MA-67.1%, CF-34.2%. Some human sera (6.1%) reacted with hen egg antigens in CF. Reactivity of diagnostic antigens prepared from reference Henzerling strain and four others isolated in Poland with rabbit immune sera and sera of individuals suspected of C. burnetii infection in IF was compared. Generally, the immune sera reacted in highest titres with homologous antigens derived from homologous strains. Human sera showed differentiated activity to particular antigens. The titres of phase I antibodies fluctuated from 0 to 16 depending on the antigen applied. Because of that fact diagnostic antigens should be prepared from the mixture of reference strains and isolates from a region under study.     

Recombinant Antigens Expressed in Pichia pastoris for the Diagnosis of Sleeping Sickness Caused by Trypanosoma brucei gambiense

Background

Screening tests for gambiense sleeping sickness, such as the CATT/T. b. gambiense and a recently developed lateral flow tests, are hitherto based on native variant surface glycoproteins (VSGs), namely LiTat 1.3 and LiTat 1.5, purified from highly virulent trypanosome strains grown in rodents.

Methodology/Principal Findings

We have expressed SUMO (small ubiquitin-like modifier) fusion proteins of the immunogenic N-terminal part of these antigens in the yeast Pichia pastoris. The secreted recombinant proteins were affinity purified with yields up to 10 mg per liter cell culture.

Conclusions/Significance

The diagnostic potential of each separate antigen and a mixture of both antigens was confirmed in ELISA on sera from 88 HAT patients and 74 endemic non-HAT controls. Replacement of native antigens in the screening tests for sleeping sickness by recombinant proteins will eliminate both the infection risk for the laboratory staff during antigen production and the need for laboratory animals. Upscaling production of recombinant antigens, e.g. in biofermentors, is straightforward thus leading to improved standardisation of antigen production and reduced production costs, which on their turn will increase the availability and affordability of the diagnostic tests needed for the elimination of gambiense HAT.     

Evaluation of Neospora caninum serodiagnostic antigens for bovine neosporosis

Abortion and reproductive failure caused by Neospora caninum infection has a dramatic negative economic impact on the cattle industry. To date, no definitive serodiagnostic tool for assessing N. caninum abortion has been reported. In this study, we evaluated the diagnostic performance of numerous N. caninum antigens in relation to abortion in cattle. Five recombinant proteins with potential as diagnostic antigens (NcGRA6, NcGRA7, NcGRA14, NcCyP, and NcSAG1) were compared by indirect enzyme-linked immunosorbent assay (iELISA) using sera from mice and cattle experimentally infected with N. caninum. The best-performing three antigens (NcSAG1, NcGRA7, and NcGRA6) were evaluated by IgG-iELISAs to assess their utility in diagnosing Neospora abortion using sera from confirmed N. caninum-aborted dams based on immunohistochemical assays (IHC). Additionally, all samples were tested using a commercial N. caninum antibody competitive ELISA (cELISA). The iELISAs against both NcSAG1 and NcGRA7 could efficiently distinguish IHC positive and negative samples compared with iELISAs against NcGRA6 and the cELISA. Furthermore, antibody levels against NcSAG1 and NcGRA7 were significantly higher in aborting cows comparing with infected but non-aborted dams in a herd experiencing a Neospora abortion outbreak. Tracking the dynamics of antibody levels during pregnancy revealed a marked increase in NcSAG1- and NcGRA7-specific antibodies at the last trimester of pregnancy. In contrast, no marked differences in antibody levels against either antigen were noted in neurologically symptomatic calves compared with non-symptomatic infected calves. Our data suggests NcSAG1 and NcGRA7 as indicators for Neospora abortion.     

Immunization against Taenia taeniaeformis in mice: studies on the characterization of antigens from oncospheres

Mature eggs of Taenia taeniaeformis hatched readily in the presence of sodium hypochlorite and no loss in infectivity of oncospheres for mice was observed after hatching. Crude and sodium deoxycholate-solubilized antigens (termed TtO-DOC) prepared from such oncospheres stimulated high levels of protection against T. taeniaeformis infection in immunized mice similar to those described previously for oncospheres prepared by other methods. Mice immunized with TtO-DOC antigens that had been exposed to potassium metaperiodate remained significantly protected against infection. Exposure of TtO-DOC antigens to pronase and thermolysin, or to trypsin, significantly reduced the ability of these antigens to protect mice against infection. These data suggest that the antigens which immunize mice against infection include protein components. ¹²⁵I-labelled TtO-DOC antigens were immunoprecipitated with sera from mice infected with T. taeniaeformis and the immunoprecipitates analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunoprecipitation with sera from C3H/He mice infected for 28 days revealed a single major labelled protein antigen having a relative molecular mass (M_r) of 31,000. Sera from 5-month infected C3H/He mice immunoprecipitated at least thirteen labelled antigens, including one at M_r 31,000. Attempts to use SDS-PAGE separated proteins to immunize mice showed that oncosphere antigens exposed to the reducing conditions prior to SDS-PAGE lost their ability to protect mice against infection. It was concluded that SDS-PAGE was an unsatisfactory technique for the isolation of a host protective fraction of TtO-DOC antigens. TtO-DOC proteins were resolved by PAGE performed in the presence of sodium deoxycholate (DOC-PAGE) and mice were vaccinated with cut-outs from the gel. A fraction of the DOC-polyacrylamide gel was found to be effective in immunizing mice against infection. Thus, although the characteristics of the protein antigens in this DOC-PAGE fraction have yet to be determined, an important fractionation technique has been identified. It was shown that partial removal of DOC from oncosphere antigen preparations solubilized in 1% DOC was required for the antigen to stimulate protective immunity. These findings will facilitate further antigen characterization studies towards the development of a defined-antigen vaccine in murine cysticercosis. This is particularly so as attempts to raise anti-oncospheral monoclonal antibodies capable of passively transferring protection to mice by using crude antigen preparations to immunize donor mice have not been successful.     

Bioassay of three strains of Bacillus sphaericus on field-collected mosquito larvae.

Bacillus sphaericus strains 1593, 1404, and SSII-1 were assayed for infectivity against field-collected larvae of Psorophora columbiae, Culex nigripalpus, and Aedes taeniorhynchus in southwest Florida. Results indicate that all three strains are highly active against the Psorophora and Culex species. A. taeniorhynchus is also susceptible but requires higher dosages to achieve lethal responses. Tests were also conducted on the rate of infection and the differences in susceptibility of different instars to B. sphaericus. These tests indicate that nearly 75% of the mortality that occurs in the course of exposure to B. sphaericus occurs within 48 hr post-incubation with the bacteria. Furthermore, our tests indicate P. columbiae larvae decrease in susceptibility to the Bacillus with increase in larval age (instar). This investigation shows B. sphaericus to be a feasible biological control agent that warrants further study.     

Comparison of the Complement-Fixation Test and the Microscopic-Agglutination Test (Agglutination-Lysis) for the Detection of Leptospiral Serogroup Antibodies

The MA and CF tests using alcohol extracted antigens have been compared with sera from infected rabbits and calves. The MA test was highly serotype specific with serum from both animal species. The CF test was broadly reactive with serum from rabbits infected with various serotypes. However, when bovine serum was used cross reactivity was reduced and it was necessary to use pooled antigens to detect heterologous serotypes.     

Evaluation of purified H and M antigens of histoplasmin as reagents in the complement fixation test.

Complement-fixation (CF) tests were performed with purified H and M antigens, histoplasmin, and Histoplasma capsulatum whole cell yeast phase antigen using sera of 126 patients with proven or suspected histoplasmosis. Specific titers for either H or for M antibody were obtained with the individual purified antigens; the highest titers were comparable to those obtained with histoplasmin. However, in sera containing only anti-M antibody, the titers obtained with the purified M antigen were 2 to 16 times those obtained with the histoplasmin or yeast phase antigens. The CF test for either H or M antibody was 4 to 32 times as reactive as the agar-gel microimmunodiffusion test; in general precipitin lines were obtained with either H or M antigens from sera with CF titers greater than or equal to 8. With sera containing H antibody, there was an excellent correlation between the CF titers obtained with purified M antigen and histoplasmin. The correlations of CF titers with H antigen and either histoplasmin or yeast phase antigen were very low.     

Stage-specific antigens of Physarum polycephalum

Antisera were prepared in isogenic F₁ hybrid rats against three amoebal strains and against two genetically related plasmodial strains of Physarum polycephalum. Differences in specificities between the antisera were studied using immunofluorescence tests and Ouchterlony double diffusion tests. There were no strain-specific differences between any of the three anti-amoebal sera, nor were any strain-specific differences found between the two anti-plasmodial sera. However, both ubiquitous and stage-specific antigens were detected.     

The specificity and significance of the inhibition of Fc receptor binding by anti H-2 sera

The possibility that Ia antigens are unique among H-2 antigens in their relationship to the Fc receptor was investigated in an EA rosette assay. Antibody specific for antigens in various regions of theH-2 complex was incubated with mouse cells, and the ability of the cells to form rosettes with antibody-coated chicken erythrocytes was tested. Antibody raised against the H-2 antigens of Ia-negative tumor cells was highly effective in inhibiting rosette formation. A variety of antisera againstK-, I-, andD-region antigens tested in recombinant mice inhibited EA rosette formation, suggesting that antigens in each of these regions could be detected in rosette inhibition. The F(ab′)₂ fragments of all antisera tested also produced specific EA rosette inhibition. Finally, antibody against Ia antigens failed to inhibit bone marrow RFCs, although antibody against H-2K and H-2D antigens did inhibit. Although H-2 serology is in a state of rapid change at present, it must be concluded that in this assay, antibody against antigens in theK andD regions as well as theI region can inhibit EA rosette formation. Inhibition of these rosettes by anti H-2 sera is therefore not due to a special association of Ia antigens with Fc receptors.     

Recognition of cytoplasmic yeast antigens of Cryptococcus neoformans var. neoformans and Cryptococcus neoformans var. gattii by immune human sera

The humoral immune response of patients infected with Cryptococcus neoformans var. neoformans and C. neoformans var. gattii to cytoplasmic (non-capsular) antigens from the two varieties of Cryptococcus has been investigated. Cytoplasmic antigens from C. neoformans (one clinical isolate and one acapsular mutant of var. neoformans and two clinical isolates from var. gattii) were subject to isoelectric focusing, SDS-PAGE and Western blotting; patients sera was then used in the immunoenzyme development of the Western blots. The humoral response from the 20 patients (all HIV+) infected with var. neoformans against the var. neoformans antigens was predominantly IgG based, with a large number of bands recognised; the most commonly recognised bands were at 26, 52, 74, 100, 115 and 144 kDa. The IgM response was less pronounced and the IgA response was practically non-existent. The humoral response of the sera from the 15 patients (all but one HIV-) infected with var. gattii against var. gattii antigens was also predominantly IgG based with bands at 37, 55, 65, 74, 94 and 115 kDa being most commonly recognised. Periodate treatment of cytoplasmic antigens reduced the intensity of antigen recognition, though it did not absolutely destroy reactivity to any individual antigen. Comparison of immunodevelopment of cytoplasmic antigens from both varieties grown at 25°C and 37°C revealed that culture temperature made no differences in the number of bands recognised although there were differences in the intensity of recognition. This is the first report on the pattern of serological recognition of the non-capsular antigens from the two varieties of Cryptococcus and it identifies a number of major antigenic components.     

Serodiagnosis of Echinococcus spp. Infection: Explorative Selection of Diagnostic Antigens by Peptide Microarray

Background

Production of native antigens for serodiagnosis of helminthic infections is laborious and hampered by batch-to-batch variation. For serodiagnosis of echinococcosis, especially cystic disease, most screening tests rely on crude or purified Echinococcus granulosus hydatid cyst fluid. To resolve limitations associated with native antigens in serological tests, the use of standardized and highly pure antigens produced by chemical synthesis offers considerable advantages, provided appropriate diagnostic sensitivity and specificity is achieved.

Methodology/Principal Findings

Making use of the growing collection of genomic and proteomic data, we applied a set of bioinformatic selection criteria to a collection of protein sequences including conceptually translated nucleotide sequence data of two related tapeworms, Echinococcus multilocularis and Echinococcus granulosus. Our approach targeted alpha-helical coiled-coils and intrinsically unstructured regions of parasite proteins potentially exposed to the host immune system. From 6 proteins of E. multilocularis and 5 proteins of E. granulosus, 45 peptides between 24 and 30 amino acids in length were designed. These peptides were chemically synthesized, spotted on microarrays and screened for reactivity with sera from infected humans. Peptides reacting above the cut-off were validated in enzyme-linked immunosorbent assays (ELISA). Peptides identified failed to differentiate between E. multilocularis and E. granulosus infection. The peptide performing best reached 57% sensitivity and 94% specificity. This candidate derived from Echinococcus multilocularis antigen B8/1 and showed strong reactivity to sera from patients infected either with E. multilocularis or E. granulosus.

Conclusions/Significance

This study provides proof of principle for the discovery of diagnostically relevant peptides by bioinformatic selection complemented with screening on a high-throughput microarray platform. Our data showed that a single peptide cannot provide sufficient diagnostic sensitivity whereas pooling several peptide antigens improved sensitivity; thus combinations of several peptides may lead the way to new diagnostic tests that replace, or at least complement conventional immunodiagnosis of echinococcosis. Our strategy could prove useful for diagnostic developments in other pathogens.