Design and development of multiepitope chimeric antigens by bioinformatic and bacterial based recombinant expression methods, with potential application for bovine tuberculosis serodiagnosis.

Bovine tuberculosis (bTB), which is caused by Mycobacterium bovis, is a single health concern, which causes economic losses, is a sanitary barrier and is a zoonotic concern. The golden-pattern intradermic tests have low sensitivity of about 50%. To fix this sensitivity problem, immunoassays could be a powerful tool. However, few studies produced antigens for bTB immunoassays, which needs improvements. Aim of this study was to produce multiepitope chimeric antigens (MCA) to use for bTB diagnosis. To achieve MCA design and development, extensive bibliographic search, antigenic epitope prediction, specificity, hydrophobicity, and 3D structure modeling analyses were performed, as well as cloning, expression and purification. Seven epitopes from four different target proteins (MPB-70, MPB-83, ESAT-6 and GroEL) were combined in five chimeras containing five repetitions of each epitope to enhance antibodies affinity. 3D predicted models revealed that all chimeras have a high percentage of disorder, which could enhance antibody recognition, although taking to protein instability. Each chimera was cloned into pET28a (+) expression plasmids and expressed in six Escherichia coli expression strains. Chimeras 3, 4 and 5 could be solubilized in 8 M urea and purified by ion exchange affinity chromatography. Against bTB positive and negative sera, purified chimera 5 had the best results in indirect dot blot and ELISA, as well as in lateral flow dot blot immunoassay. In conclusion, chimera 5, an MPB-83 containing MCA, could be used for further studies, aimed to develop a serologic or rapid test for bTB diagnosis.

Anti-SARS-CoV-2 and anti-cytokine storm neutralizing antibody therapies against COVID-19: Update, challenges, and perspectives.

Coronavirus disease 2019 (COVID-19) has been declared by the World Health Organization (WHO) as a pandemic since March 2020. This disease is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The only available tools to avoid contamination and transmission of this virus are physical distancing, the use of N95 and surgical masks, and hand hygiene. Vaccines are another essential tool to reduce the impact of the pandemic, though these present challenges in terms of production and logistics, particularly in underdeveloped and developing countries. One of the critical early research findings is the interaction of the spike virus protein with the angiotensin-converting enzyme 2 (ACE2) human receptor. Developing strategies to block this interaction has therefore been identified as a way to treat this infection. Neutralizing antibodies (nAbs) have emerged as a therapeutic approach since the pandemic started. Infected patients may be asymptomatic or present with mild symptoms, and others may evolve to moderate or severe disease, leading to death. An immunological phenomenon known as cytokine storm has been observed in patients with severe disease characterized by a proinflammatory cytokine cascade response that leads to lung injury. Thus, some treatment strategies focus on anti-cytokine storm nAbs. This review summarizes the latest advances in research and clinical trials, challenges, and perspectives on antibody-based treatments (ABT) as therapies against COVID-19.

Correction: Performance of Treponema pallidum recombinant proteins in the serological diagnosis of syphilis.

[This corrects the article DOI: 10.1371/journal.pone.0234043.].

Performance of Treponema pallidum recombinant proteins in the serological diagnosis of syphilis.

Syphilis serodiagnosis is challenging because distinct clinical forms of the infection may influence serological performance and discordant results between tests make clinical decisions difficult. Several recombinant Treponema pallidum-proteins have already been tested for syphilis diagnosis and they are critical to achieve high accuracy in serological testing. Our aim was to assess the varied from performance of T. pallidum-recombinant proteins TmpA, TpN17 and TpN47 for syphilis serodiagnosis. The proteins were evaluated using sera of 338 T. pallidum-negative, 173 T. pallidum-positive individuals and 209 sera from individuals infected with unrelated diseases. The diagnostic potential was validated by analysis of ROC curves. In the liquid microarray analyses, the ROC curve varied from 99.0% for TmpA and TpN17 to 100% for TpN47. The sensitivity score yielded values of up to 90% for TpN17, 100% for TpN47 and 80.0% for TmpA. The lowest and highest specificity values were presented by TpN47 (91.9%) and TmpA antigens (100%), respectively. TpN47 showed the highest accuracy score (95.5%) among all the recombinant proteins assayed. For the ELISA, the ROC curve was 97.2%, 91.8% and 81.6% for TpN17, TmpA and TpN47, respectively. TpN17 and TmpA yielded a sensitivity of 69.9%, while TpN47 obtained a value of 53.8%. Specificity was almost 100% for all three proteins. No cross-reaction was observed for TpN17 in the serum samples from non-bacterial infections. Regarding leptospirosis-positive samples, cross-reactivity score was varied from 8.6 to 34.6%. This is most probably due to conservation of the epitopes in these proteins across bacteria. The use of recombinant proteins in immunoassays for syphilis diagnosis was showed provide greater reliability to results of the treponemal assays. Despite the low sensitivity, the proteins showed high diagnostic capacity due to the AUC values found. However, an improvement in sensitivity could be achieved when antigenic mixtures are evaluated.

Performance Assessment of a Trypanosoma cruzi Chimeric Antigen in Multiplex Liquid Microarray Assays.

Diagnosing chronic Chagas disease (CD) requires antibody-antigen detection methods, which are traditionally based on enzymatic assay techniques whose performance depend on the type and quality of antigen used. Previously, 4 recombinant chimeric proteins from the Instituto de Biologia Molecular do Paraná (IBMP-8.1 to 8.4) comprising immuno-dominant regions of diverse Trypanosoma cruzi antigens showed excellent diagnostic performance in enzyme-linked immunosorbent assays. Considering that next-generation platforms offer improved CD diagnostic accuracy with different T. cruzi-specific recombinant antigens, we assessed the performance of these chimeras in liquid microarrays (LMAs). The chimeric proteins were expressed in Escherichia coli and purified by chromatography. Sera from 653 chagasic and 680 healthy individuals were used to assess the performance of these chimeras in detecting specific anti-T. cruzi antibodies. Accuracies ranged from 98.1 to 99.3%, and diagnostic odds ratio values were 3,548 for IBMP-8.3, 4,826 for IBMP-8.1, 7,882 for IBMP-8.2, and 25,000 for IBMP-8.4. A separate sera bank (851 samples) was employed to assess cross-reactivity with other tropical diseases. Leishmania, a pathogen with high similarity to T. cruzi, showed cross-reactivity rates ranging from 0 to 2.17%. Inconclusive results were negligible (0 to 0.71%). Bland-Altman and Deming regression analysis based on 200 randomly selected CD-positive and negative samples demonstrated interchangeability with respect to CD diagnostic performance in both singleplex and multiplex assays. Our results suggested that these chimeras can potentially replace antigens currently used in commercially available assay kits. Moreover, the use of multiplex platforms, such as LMA assays employing 2 or more IBMP antigens, would abrogate the need for 2 different testing techniques when diagnosing CD.

Preliminary multiplex microarray IgG immunoassay for the diagnosis of toxoplasmosis and rubella.

BACKGROUND: During pregnancy, toxoplasmosis and rubella can cause serious damage to the mother and the foetus through vertical transmission. Early diagnosis enables implementation of health measures aimed at preventing vertical transmission and minimising damage caused by these diseases. OBJECTIVE: Here, we report the development of a multiplex assay for simultaneous detection of IgG antibodies produced during toxoplasmosis and rubella infection. METHODS: This assay is based on xMap technology. Initially, by singleplex assays, we evaluated the following antigens: one Toxoplasma gondii lysate; two antigenic extracts of T. gondii (TOX8131 and TOX8122); fragments of T. gondii antigens [SAG-1 (amino acids 45-198), GRA-7 (24-100), GRA-1 (57-149), ROP-4, and MIC-3 (234-306)]; two chimeric antigens composed of fragments of SAG-1, GRA-7, and P35 (CTOX and CTOXH); and fragments of Rubella virus antigens [E-1 (157-176, 213-239, 374-390), E-2 (31-105), and C (1-123)]. FINDINGS: A multiplex assay to simultaneously diagnose toxoplasmosis and rubella was designed with the best-performing antigens in singleplex and multiplex assays, which included CTOXH, T. gondii lysate, TOX8131, E-1, and E-2. The multiplex assay showed 100% sensitivity and specificity for anti-T. gondii IgG detection and 95.6% sensitivity and 100% specificity for anti-R. virus IgG detection. MAIN CONCLUSIONS: We found that, despite the difficulties related to developing multiplex systems, different types of antigens (extracts and recombinant proteins) can be used to develop high-performance diagnostic tests. The assay developed is suitable to screen for prior T. gondii and R. virus infections, because it is a rapid, high-throughput, low-cost alternative to the current standard diagnostic tools, which require multiple individual tests.

Preliminary multiplex microarray IgG immunoassay for the diagnosis of toxoplasmosis and rubella

BACKGROUND During pregnancy, toxoplasmosis and rubella can cause serious damage to the mother and the foetus through vertical transmission. Early diagnosis enables implementation of health measures aimed at preventing vertical transmission and minimising damage caused by these diseases. OBJECTIVE Here, we report the development of a multiplex assay for simultaneous detection of IgG antibodies produced during toxoplasmosis and rubella infection. METHODS This assay is based on xMap technology. Initially, by singleplex assays, we evaluated the following antigens: one Toxoplasma gondii lysate; two antigenic extracts of T. gondii (TOX8131 and TOX8122); fragments of T. gondii antigens [SAG-1 (amino acids 45-198), GRA-7 (24-100), GRA-1 (57-149), ROP-4, and MIC-3 (234-306)]; two chimeric antigens composed of fragments of SAG-1, GRA-7, and P35 (CTOX and CTOXH); and fragments of Rubella virus antigens [E-1 (157-176, 213-239, 374-390), E-2 (31-105), and C (1-123)]. FINDINGS A multiplex assay to simultaneously diagnose toxoplasmosis and rubella was designed with the best-performing antigens in singleplex and multiplex assays, which included CTOXH, T. gondii lysate, TOX8131, E-1, and E-2. The multiplex assay showed 100% sensitivity and specificity for anti-T. gondii IgG detection and 95.6% sensitivity and 100% specificity for anti-R. virus IgG detection. MAIN CONCLUSIONS We found that, despite the difficulties related to developing multiplex systems, different types of antigens (extracts and recombinant proteins) can be used to develop high-performance diagnostic tests. The assay developed is suitable to screen for prior T. gondii and R. virus infections, because it is a rapid, high-throughput, low-cost alternative to the current standard diagnostic tools, which require multiple individual tests.

Accuracy of chimeric proteins in the serological diagnosis of chronic chagas disease - a Phase II study.

BACKGROUND: The performance of current serologic tests for diagnosing chronic Chagas disease (CD) is highly variable. The search for new diagnostic markers has been a constant challenge for improving accuracy and reducing the number of inconclusive results. METHODOLOGY/PRINCIPAL FINDINGS: Here, four chimeric proteins (IBMP-8.1 to -8.4) comprising immunodominant regions of different Trypanosoma cruzi antigens were tested by enzyme-linked immunosorbent assay. The proteins were used to detect specific anti-T. cruzi antibodies in the sera of 857 chagasic and 689 non-chagasic individuals to evaluate their accuracy for chronic CD diagnosis. The antigens were recombinantly expressed in Escherichia coli and purified by chromatographic methods. The sensitivity and specificity values ranged from 94.3% to 99.3% and 99.4% to 100%, respectively. The diagnostic odds ratio (DOR) values were 6,462 for IBMP-8.1, 3,807 for IBMP-8.2, 32,095 for IBMP-8.3, and 283,714 for IBMP-8.4. These chimeric antigens presented DORs that were higher than the commercial test Pathozyme Chagas. The antigens IBMP-8.3 and -8.4 also showed DORs higher than the Gold ELISA Chagas test. Mixtures with equimolar concentrations were tested in order to improve the diagnosis accuracy of negative samples with high signal and positive samples with low signal. However, no gain in accuracy was observed relative to the individual antigens. A total of 1,079 additional sera were used to test cross-reactivity to unrelated diseases. The cross-reactivity rates ranged from 0.37% to 0.74% even for Leishmania spp., a pathogen showing relatively high genome sequence identity to T. cruzi. Imprecision analyses showed that IBMP chimeras are very stable and the results are highly reproducible. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that the IBMP-8.4 antigen can be safely used in serological tests for T. cruzi screening in blood banks and for chronic CD laboratory diagnosis.

Performance Assessment of Four Chimeric Trypanosoma cruzi Antigens Based on Antigen-Antibody Detection for Diagnosis of Chronic Chagas Disease.

The performance of serologic tests in chronic Chagas disease diagnosis largely depends on the type and quality of the antigen preparations that are used for detection of anti-Trypanosoma cruzi antibodies. Whole-cell T. cruzi extracts or recombinant proteins have shown variation in the performance and cross-reactivity. Synthetic chimeric proteins comprising fragments of repetitive amino acids of several different proteins have been shown to improve assay performances to detect Chagasic infections. Here, we describe the production of four chimeric T. cruzi proteins and the assessment of their performance for diagnostic purposes. Circular Dichroism spectra indicated the absence of well-defined secondary structures, while polydispersity evaluated by Dynamic Light Scattering revealed only minor aggregates in 50 mM carbonate-bicarbonate (pH 9.6), demonstrating that it is an appropriate buffering system for sensitizing microplates. Serum samples from T. cruzi-infected and non-infected individuals were used to assess the performance of these antigens for detecting antibodies against T. cruzi, using both enzyme-linked immunosorbent assay and a liquid bead array platform. Performance parameters (AUC, sensitivity, specificity, accuracy and J index) showed high diagnostic accuracy for all chimeric proteins for detection of specific anti-T. cruzi antibodies and differentiated seropositive individuals from those who were seronegative. Our data suggest that these four chimeric proteins are eligible for phase II studies.

Enhanced target-specific signal detection using an Escherichia coli lysate in multiplex microbead immunoassays with E. coli-derived recombinant antigens.

Diverse techniques have been developed to analyze antibody-mediated responses to infections. However, the most common tests, i.e., enzyme-linked immunosorbent assays, require separate reactions for each antigen and consequently necessitate large sample volumes. Luminex technology allows the detection of multiple antibodies in a single experiment, but nonspecific binding can impair the results. Therefore, we examined the use of Escherichia coli lysates to reduce nonspecific binding and improve the results of liquid microarrays based on Luminex technology. Anti-bacteria antibodies were detected in human serum samples, as evidenced by high median fluorescence intensity (MFI) in assays performed with paramagnetic microspheres coupled with E. coli lysates. Moreover, the addition of an E. coli lysate as a blocker reduced the nonspecific binding of antigens produced by E. coli in a concentration-dependent manner. Tris-HCl reduced MFI values in negative samples, but did not affect MFI for positive samples. For microspheres coupled with different antigens, an E. coli lysate blocker significantly improved the fluorescence signals from positive samples. The addition of Tris-HCl and the E. coli lysate induced antigen-specific differences in MFI. This combination of the E. coli lysate blocker and Tris-HCl yielded a statistically significant improvement in MFI in the assays for Chagas disease and hepatitis C virus samples. However, for the Treponema pallidum p47 antigen improvement in MFI was only observed for the preparation with the E. coli blocker at a concentration of 3%. In conclusion, the addition of an E. coli lysate and Tris-HCl to the microarray assay reduced the nonspecific binding of human anti-bacteria antibodies and, therefore, increased the specific MFI.

Electrospray induced surface activation of polystyrene microbeads for diagnostic applications.

Electrospray is generally regarded as a "soft" technique due to the absence of any observable molecular fragmentation or destruction. This study reports on a novel and easy way to induce surface activation on the surface of polystyrene microbeads through electrospray deposition into a grounded aqueous electrolyte solution bath. This process, nicknamed EISA, which stands for electrospray induced surface activation, proposes that when a highly charged microbead formed by the electrospray process sinks into the aqueous electrolyte solution, it behaves like a highly charged spherical capacitor that discharges in the conductive liquid. The energy released leads to a breakup of the polystyrene surface bonds and water oxidation with oxygen. Further reactions produce a carboxylated surface that was confirmed by X-ray photoelectron spectroscopy (XPS) and protein coupling. An immunoassay based on these modified microbeads was also developed and presented for use in syphilis detection, demonstrating a reliable signal-to-noise ratio between positive and negative results.

Development of a multiplex bead-based assay for detection of hepatitis C virus.

Hepatitis C virus (HCV) infection is a major burden to public health worldwide, affecting approximately 3% of the human population. Although HCV detection is currently based on reliable tests, the field of medical diagnostics has a growing need for inexpensive, accurate, and quick high-throughput assays. By using the recombinant HCV antigens NS3, NS4, NS5, and Combined, we describe a new bead-based multiplex test capable of detecting HCV infection in human serum samples. The first analysis, made in a singleplex format, showed that each antigen coupled to an individual bead set presented high-level responses for anti-HCV-positive reference serum pools and lower-level responses for the HCV-negative pools. Our next approach was to determine the sensitivity and specificity of each antigen by testing 93 HCV-positive and 93 HCV-negative sera. When assayed in the singleplex format, the NS3, NS4, and NS5 antigens presented lower sensitivity values (50.5%, 51.6%, and 55.9%, respectively) than did the Combined antigen, which presented a sensitivity of 93.5%. All antigens presented 100% specificity. These antigens were then multiplexed in a 4-plex assay, which resulted in increased sensitivity and specificity values, performing with 100% sensitivity and 100% specificity. The positive and negative predictive values for the 4-plex assay were 100%. Although preliminary, this 4-plex assay showed robust results that, aligned with its small-sample-volume requirements and also its cost- and time-effectiveness, make it a reasonable alternative to tests currently used for HCV screening of potentially infected individuals.

A novel hepatitis C virus genotyping method based on liquid microarray.

The strategy used to treat HCV infection depends on the genotype involved. An accurate and reliable genotyping method is therefore of paramount importance. We describe here, for the first time, the use of a liquid microarray for HCV genotyping. This liquid microarray is based on the 5'UTR - the most highly conserved region of HCV - and the variable region NS5B sequence. The simultaneous genotyping of two regions can be used to confirm findings and should detect inter-genotypic recombination. Plasma samples from 78 patients infected with viruses with genotypes and subtypes determined in the Versant™ HCV Genotype Assay LiPA (version I; Siemens Medical Solutions, Diagnostics Division, Fernwald, Germany) were tested with our new liquid microarray method. This method successfully determined the genotypes of 74 of the 78 samples previously genotyped in the Versant™ HCV Genotype Assay LiPA (74/78, 95%). The concordance between the two methods was 100% for genotype determination (74/74). At the subtype level, all 3a and 2b samples gave identical results with both methods (17/17 and 7/7, respectively). Two 2c samples were correctly identified by microarray, but could only be determined to the genotype level with the Versant™ HCV assay. Genotype "1" subtypes (1a and 1b) were correctly identified by the Versant™ HCV assay and the microarray in 68% and 40% of cases, respectively. No genotype discordance was found for any sample. HCV was successfully genotyped with both methods, and this is of prime importance for treatment planning. Liquid microarray assays may therefore be added to the list of methods suitable for HCV genotyping. It provides comparable results and may readily be adapted for the detection of other viruses frequently co-infecting HCV patients. Liquid array technology is thus a reliable and promising platform for HCV genotyping.

Viability study of a multiplex diagnostic platform for Chagas disease.

A new multiplex assay platform was evaluated to detect Trypanosoma cruzi infection using the recombinant antigens CRA, FRA, CRAFRA fusion and parasite lysate. The antigens presented different sensitivity and specificity in a singleplex test when compared to a serial dilution of two pools comprising 10 positive serum samples and one pool of 10 negative samples. The recombinant protein CRA presented lower sensitivity (55%) in contrast to the 100% specificity and sensitivity of FRA, CRAFRA and T. cruzi lysate. These antigens also showed good results in a duplex test and the duplex test with CRAFRA/T. cruzi lysate showed better performance with 100% specificity and sensitivity, as well as a lower cut-off value in comparison to the other duplex test, FRA/T. cruzi lysate. Hence, when the antigens were used in duplex format, both tests showed decreased cut-off values and no interference between different bead sets, resulting in increasing sensitivity and specificity. The results of these multiplex tests show that they could be an alternative to singleplex detection for Chagas disease, and also indicate the necessity of using multiplex diagnostic tools to increase the sensitivity and specificity for diagnostic tests. Emerging data from the T. cruzi genome and from its ORFeome project will also allow the identification of new antigens for this disease detection application.

Viability study of a multiplex diagnostic platform for Chagas disease

A new multiplex assay platform was evaluated to detect Trypanosoma cruzi infection using the recombinant antigensCRA, FRA, CRAFRA fusion and parasite lysate. The antigens presented different sensitivity and specificity in a singleplex test when compared to a serial dilution of two pools comprising 10 positive serum samples and one pool of 10 negative samples. The recombinant protein CRA presented lower sensitivity (55%) in contrast to the 100% specificity and sensitivity of FRA, CRAFRA and T. cruzi lysate. These antigens also showed good results in a duplex test and the duplex test with CRAFRA/T. cruzi lysate showed better performance with 100% specificity and sensitivity, as well as a lower cut-off value in comparison to the other duplex test, FRA/T. cruzi lysate. Hence, when the antigens were used in duplex format, both tests showed decreased cut-off values and no interference between different bead sets, resulting in increasing sensitivity and specificity. The results of these multiplex tests show that they could be an alternative to singleplex detection for Chagas disease, and also indicate the necessity of using multiplex diagnostic tools to increase the sensitivity and specificity for diagnostic tests. Emerging data from the T. cruzi genome and from its ORFeome project will also allow the identification of new antigens for this disease detection application.

Caracterização genômica da bactéria endossimbiótica do tripanosomatídeo Crithidia deanei e de suas DNA topoisomerases / Genomic characterization of the endosymbiotic bacteria of the trypanosome Crithidia deanei and its DNA topoisomerases

O principal objeto de estudo do presente trabalho são as DNA topoisomerases do tipo II do endossimbionte,devido ao seu papel essencial tanto nos processos replicação e transcrição,como também na recombinação e segregação dos cromossomos bacterianos.Existem duas topoisomerases do tipo II em bactérias:a DNA girase e DNA topoisomerase IV. A DNA girase introduz superenovelamento negativo no DNA circular,enquanto a topo IV tem como sua principal função a decatenação do DNA durante a segregação dos cromossomos.Para esses estudos foi gerada uma biblioteca genômica do endossimbionte em pUC18 e seqüenciados 7.500 clones inicialmente,abrangendo uma cobertura de 1,2 MB. As seqüências obtidas foram analisadas através do algoritmo BLAST.A análise mostrou que o genoma do endossimbionte apresenta uma grande homologia com os genomas de bactérias do gênero Bordetella,mas diferem no conteúdo A+T,que no genoma do endossimbionte é de aproximadamente 70por cento,enquanto que no gênero Bordetella é de apenas 32por cento.Esta estratégia permitiu encontrar as seqüências dos genes que codificam as subunidades A(GyrA)e B(GyrB)da DNA girase. Identificamos também o gene que codifica a topoisomerase III(topo III),mas não fomos capazes de identificar os genes que codificam as enzimas topo IV e I(topoisomerase I),que formam juntamente com a DNA girase e topo III o repertório de topoisomerases encontrado em outras bactérias.Baseados nas seqüências dos genes gyrA e gyrB foram desenhados iniciadores para amplificação dos alvos através de PCR.Os produtos das amplificações foram clonados para expressão em E.coli.As proteínas recombinantes foram purificadas para a produção de antissoros policlonais.Ensaio de western blot...preditas a partir da seqüência dos genes gyrA e gyrB.Análise por imunofluorescência mostrou que os antissoros contra as subunidades A e B da DNA girase reconhecem antígenos específicos do endossimbionte,inoculados por todo o citoplasma da bactéria.Os genes gyrA e gyrB também...

TcZFP1: a CCCH zinc finger protein of Trypanosoma cruzi that binds poly-C oligoribonucleotides in vitro.

We have identified two zinc finger proteins of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease in humans. These proteins, named tcZFP1 and tcZFP2, share the unusual zinc finger motif (CCCH) found in a diverse range of RNA-binding proteins involved in various aspects of the control of cell homeostasis and differentiation. We report here the functional expression of a recombinant tcZFP1, and the relative affinity and stability of the specific complexes formed between the protein and synthetic oligoribonucleotides containing C-rich sequences.