Identification of Zika Virus NS1-Derived Peptides with Potential Applications in Serological Tests.

Zika virus (ZIKV), a mosquito-borne pathogen, is an emerging arbovirus associated with sporadic symptomatic cases of great medical concern, particularly among pregnant women and newborns affected with neurological disorders. Serological diagnosis of ZIKV infection is still an unmet challenge due to the co-circulation of the dengue virus, which shares extensive sequence conservation of structural proteins leading to the generation of cross-reactive antibodies. In this study, we aimed to obtain tools for the development of improved serological tests for the detection of ZIKV infection. Polyclonal sera (pAb) and a monoclonal antibody (mAb 2F2) against a recombinant form of the ZIKV nonstructural protein 1 (NS1) allowed the identification of linear peptide epitopes of the NS1 protein. Based on these findings, six chemically synthesized peptides were tested both in dot blot and ELISA assays using convalescent sera collected from ZIKV-infected patients. Two of these peptides specifically detected the presence of ZIKV antibodies and proved to be candidates for the detection of ZIKV-infected subjects. The availability of these tools opens perspectives for the development of NS1-based serological tests with enhanced sensitivity regarding other flaviviruses.

Identification of zika virus ns1-derived peptides with potential applications in serological tests

Zika virus (ZIKV), a mosquito-borne pathogen, is an emerging arbovirus associated with sporadic symptomatic cases of great medical concern, particularly among pregnant women and newborns affected with neurological disorders. Serological diagnosis of ZIKV infection is still an unmet challenge due to the co-circulation of the dengue virus, which shares extensive sequence conservation of structural proteins leading to the generation of cross-reactive antibodies. In this study, we aimed to obtain tools for the development of improved serological tests for the detection of ZIKV infection. Polyclonal sera (pAb) and a monoclonal antibody (mAb 2F2) against a recombinant form of the ZIKV nonstructural protein 1 (NS1) allowed the identification of linear peptide epitopes of the NS1 protein. Based on these findings, six chemically synthesized peptides were tested both in dot blot and ELISA assays using convalescent sera collected from ZIKV-infected patients. Two of these peptides specifically detected the presence of ZIKV antibodies and proved to be candidates for the detection of ZIKV-infected subjects. The availability of these tools opens perspectives for the development of NS1-based serological tests with enhanced sensitivity regarding other flaviviruses.

Heat-Labile Toxin from Enterotoxigenic Escherichia coli Causes Systemic Impairment in Zebrafish Model.

Heat-labile toxin I (LT-I), produced by strains of enterotoxigenic Escherichia coli (ETEC), causes profuse watery diarrhea in humans. Different in vitro and in vivo models have already elucidated the mechanism of action of this toxin; however, their use does not always allow for more specific studies on how the LT-I toxin acts in systemic tracts and intestinal cell lines. In the present work, zebrafish (Danio rerio) and human intestinal cells (Caco-2) were used as models to study the toxin LT-I. Caco-2 cells were used, in the 62nd passage, at different cell concentrations. LT-I was conjugated to FITC to visualize its transport in cells, as well as microinjected into the caudal vein of zebrafish larvae, in order to investigate its effects on survival, systemic traffic, and morphological formation. The internalization of LT-I was visualized in 3 × 104 Caco-2 cells, being associated with the cell membrane and nucleus. The systemic traffic of LT-I in zebrafish larvae showed its presence in the cardiac cavity, yolk, and regions of the intestine, as demonstrated by cardiac edema (100%), the absence of a swimming bladder (100%), and yolk edema (80%), in addition to growth limitation in the larvae, compared to the control group. There was a reduction in heart rate during the assessment of larval survival kinetics, demonstrating the cardiotoxic effect of LT-I. Thus, in this study, we provide essential new depictions of the features of LT-I.

Hybrid Atypical Enteropathogenic and Extraintestinal Escherichia coli (aEPEC/ExPEC) BA1250 Strain: A Draft Genome.

Diarrheagenic Escherichia coli is the major bacterial etiological agent of severe diarrhea and a major concern of public health. These pathogens have acquired genetic characteristics from other pathotypes, leading to unusual and singular genetic combinations, known as hybrid strains and may be more virulent due to a set of virulence factors from more than one pathotype. One of the possible combinations is with extraintestinal E. coli (ExPEC), a leading cause of urinary tract infection, often lethal after entering the bloodstream and atypical enteropathogenic E. coli (aEPEC), responsible for death of thousands of people every year, mainly children under five years old. Here we report the draft genome of a strain originally classified as aEPEC (BA1250) isolated from feces of a child with acute diarrhea. Phylogenetic analysis indicates that BA1250 genome content is genetically closer to E. coli strains that cause extraintestinal infections, other than intestinal infections. A deeper analysis showed that in fact this is a hybrid strain, due to the presence of a set of genes typically characteristic of ExPEC. These genomic findings expand our knowledge about aEPEC heterogeneity allowing further studies concerning E. coli pathogenicity and may be a source for future comparative studies, virulence characteristics, and evolutionary biology.

Análise do sistema toxina-antitoxina HipA/B na indução da persistência bacteriana de cepas de Escherichia coli extraintestinal / The importance of HipA/B Toxin-Antitoxin system in extraintestinal Escherichia Coli persistence

Os sistemas toxina-antitoxinas (TA) compreendem um conjunto de genes que são amplamente difundidos em procariotos. No cromossomo, os sistemas podem estar envolvidos na indução de morte celular em resposta a condições estressantes, indução de persistência, formação de biofilme, colonização de novos nichos, manutenção da mobilidade bacteriana e virulência de bactérias patogênicas. Em E. coli K12, 36 sistemas TA foram descritos, dos quais o do tipo II é o mais abundante e estudado. Dentre as oito toxinas pesquisadas nesse trabalho, o gene da toxina HipA está presente em 76 das 100 cepas de ExPEC estudadas. Apesar da abundância de hipA em ExPEC e em diversos genomas bacterianos, a participação dos sistemas hipA/B na indução da persistência ainda não é clara. Portanto, o sistema hipA/B de duas cepas ExPEC isoladas de infecção sanguínea foi deletado, e estas foram avaliadas quando a indução da persistência bacteriana na presença de antibióticos, formação de biofilme, resistência ao soro e sobrevivência em macrófagos. O sistema TA hipA/B não influenciou no fenótipo de resistência ao soro humano e na sobrevivência intracelular em macrófagos, no entanto, participou da indução da persistência por ciprofloxacino em um isolado (EC182); e da formação de biofilme em superfície de vidro do isolado (EC273)

Toxin-antitoxin (TA) systems comprise a set of genes that are widespread in prokaryotes. On the chromosome, the systems may be involved in the induction of cell death in response to stressful conditions, persistence induction, biofilm formation, colonization of new niches, maintenance of bacterial mobility and virulence. In E. coli K12, 36 TA systems have been described, of which type II is the most abundant. Among the eight toxins searched in this work, hipA is present in 76 bacteria of the 100 ExPEC strains studied. Despite the abundance of hipA in ExPEC and in several bacterial genomes, the participation of hipA/B modules in the persistence is still unclear. Therefore, hipA/B system of two ExPEC strains isolated from blood infection was deleted and consequently evaluated in bacterial persistence induced by antibiotics, serum resistance and macrophage survival. Despite the fact that, the TA hipA/B system did not influence the phenotype of resistance to human serum and intracellular survival in macrophages. Herein, we described that hipA/B was important for persistence induction in one isolate (EC182); and may participate in the biofilm formation on the glass surface in the other studied strain (EC273)

Heat-Labile toxin from enterotoxigenic Escherichia coli causes systemic impairment in Zebrafish model

Heat-labile toxin I (LT-I), produced by strains of enterotoxigenic Escherichia coli (ETEC), causes profuse watery diarrhea in humans. Different in vitro and in vivo models have already elucidated the mechanism of action of this toxin; however, their use does not always allow for more specific studies on how the LT-I toxin acts in systemic tracts and intestinal cell lines. In the present work, zebrafish (Danio rerio) and human intestinal cells (Caco-2) were used as models to study the toxin LT-I. Caco-2 cells were used, in the 62nd passage, at different cell concentrations. LT-I was conjugated to FITC to visualize its transport in cells, as well as microinjected into the caudal vein of zebrafish larvae, in order to investigate its effects on survival, systemic traffic, and morphological formation. The internalization of LT-I was visualized in 3 × 104 Caco-2 cells, being associated with the cell membrane and nucleus. The systemic traffic of LT-I in zebrafish larvae showed its presence in the cardiac cavity, yolk, and regions of the intestine, as demonstrated by cardiac edema (100%), the absence of a swimming bladder (100%), and yolk edema (80%), in addition to growth limitation in the larvae, compared to the control group. There was a reduction in heart rate during the assessment of larval survival kinetics, demonstrating the cardiotoxic effect of LT-I. Thus, in this study, we provide essential new depictions of the features of LT-I.

Hybrid atypical enteropathogenic and extraintestinal Escherichia coli (aEPEC/ExPEC) BA1250 strain: a draft genome

Diarrheagenic Escherichia coli is the major bacterial etiological agent of severe diarrhea and a major concern of public health. These pathogens have acquired genetic characteristics from other pathotypes, leading to unusual and singular genetic combinations, known as hybrid strains and may be more virulent due to a set of virulence factors from more than one pathotype. One of the possible combinations is with extraintestinal E. coli (ExPEC), a leading cause of urinary tract infection, often lethal after entering the bloodstream and atypical enteropathogenic E. coli (aEPEC), responsible for death of thousands of people every year, mainly children under five years old. Here we report the draft genome of a strain originally classified as aEPEC (BA1250) isolated from feces of a child with acute diarrhea. Phylogenetic analysis indicates that BA1250 genome content is genetically closer to E. coli strains that cause extraintestinal infections, other than intestinal infections. A deeper analysis showed that in fact this is a hybrid strain, due to the presence of a set of genes typically characteristic of ExPEC. These genomic findings expand our knowledge about aEPEC heterogeneity allowing further studies concerning E. coli pathogenicity and may be a source for future comparative studies, virulence characteristics, and evolutionary biology.

Immunological tests for diarrhoea caused by diarrhoeagenic Escherichia coli targeting their main virulence factors

Gene detection does not assure the expression of the corresponding virulence factor. Therefore, the antibody-based assays comprise the largest group of rapid methods that can be employed for the detection of virulence factor production/secretion. For these antibody-based assays, polyclonal, monoclonal, or recombinant antibodies can be employed. Thus, this chapter presents protocols for antibodies that are generated as well as the strategies of standardized and developed immunoassays for diarrhoeagenic Escherichia coli diagnosis targeting their main virulence factors.

Development and Validation of Shiga Toxin-Producing Escherichia coli Immunodiagnostic Assay.

Shiga toxin (Stx)-producing Escherichia coli (STEC) and its subgroup enterohemorrhagic E. coli are important pathogens involved in diarrhea, which may be complicated by hemorrhagic colitis and hemolytic uremic syndrome, the leading cause of acute renal failure in children. Early diagnosis is essential for clinical management, as an antibiotic treatment in STEC infections is not recommended. Previously obtained antibodies against Stx1 and Stx2 toxins were employed to evaluate the sensitivity and specificity of the latex Agglutination test (LAT), lateral flow assay (LFA), and capture ELISA (cEIA) for STEC detection. The LAT (mAb Stx1 plus mAb stx2) showed 99% sensitivity and 97% specificity. Individually, Stx1 antibodies showed 95.5% and 94% sensitivity and a specificity of 97% and 99% in the cEIA and LFA assay, respectively. Stx2 antibodies showed a sensitivity of 92% in both assays and a specificity of 100% and 98% in the cEIA and LFA assay, respectively. These results allow us to conclude that we have robust tools for the diagnosis of STEC infections.

Development and validation of shiga toxin-producing escherichia coli immunodiagnostic assay

Shiga toxin (Stx)–producing Escherichia coli (STEC) and its subgroup enterohemorrhagic E. coli are important pathogens involved in diarrhea, which may be complicated by hemorrhagic colitis and hemolytic uremic syndrome, the leading cause of acute renal failure in children. Early diagnosis is essential for clinical management, as an antibiotic treatment in STEC infections is not recommended. Previously obtained antibodies against Stx1 and Stx2 toxins were employed to evaluate the sensitivity and specificity of the latex Agglutination test (LAT), lateral flow assay (LFA), and capture ELISA (cEIA) for STEC detection. The LAT (mAb Stx1 plus mAb stx2) showed 99% sensitivity and 97% specificity. Individually, Stx1 antibodies showed 95.5% and 94% sensitivity and a specificity of 97% and 99% in the cEIA and LFA assay, respectively. Stx2 antibodies showed a sensitivity of 92% in both assays and a specificity of 100% and 98% in the cEIA and LFA assay, respectively. These results allow us to conclude that we have robust tools for the diagnosis of STEC infections.

Development and validation of shiga toxin-producing escherichia coli immunodiagnostic assay

Shiga toxin (Stx)–producing Escherichia coli (STEC) and its subgroup enterohemorrhagic E. coli are important pathogens involved in diarrhea, which may be complicated by hemorrhagic colitis and hemolytic uremic syndrome, the leading cause of acute renal failure in children. Early diagnosis is essential for clinical management, as an antibiotic treatment in STEC infections is not recommended. Previously obtained antibodies against Stx1 and Stx2 toxins were employed to evaluate the sensitivity and specificity of the latex Agglutination test (LAT), lateral flow assay (LFA), and capture ELISA (cEIA) for STEC detection. The LAT (mAb Stx1 plus mAb stx2) showed 99% sensitivity and 97% specificity. Individually, Stx1 antibodies showed 95.5% and 94% sensitivity and a specificity of 97% and 99% in the cEIA and LFA assay, respectively. Stx2 antibodies showed a sensitivity of 92% in both assays and a specificity of 100% and 98% in the cEIA and LFA assay, respectively. These results allow us to conclude that we have robust tools for the diagnosis of STEC infections.

Structural Changes in Stx1 Engineering Monoclonal Antibody Improves Its Functionality as Diagnostic Tool for a Rapid Latex Agglutination Test.

Stx1 toxin is one of the AB5 toxins of Shiga toxin-producing Escherichia coli (STEC) responsible for foodborne intoxication during outbreaks. The single-chain variable fragment (scFv) is the most common recombinant antibody format; it consists of both variable chains connected by a peptide linker with conserved specificity and affinity for antigen. The drawbacks of scFv production in bacteria are the heterologous expression, conformation and stability of the molecule, which could change the affinity for the antigen. In this work, we obtained a stable and functional scFv-Stx1 in bacteria, starting from IgG produced by hybridoma cells. After structural modifications, i.e., change in protein orientation, vector and linker, its solubility for expression in bacteria was increased as well as the affinity for its antigen, demonstrated by a scFv dissociation constant (KD) of 2.26 × 10-7 M. Also, it was able to recognize purified Stx1 and cross-reacted with Stx2 toxin by ELISA (Enzyme-Linked Immunosorbent Assay), and detected 88% of Stx1-producing strains using a rapid latex agglutination test. Thus, the scFv fragment obtained in the present work is a bacteria-produced tool for use in a rapid diagnosis test, providing an alternative for STEC diagnosis.

Large-scale evaluation of a rapid diagnostic test for diarrhea caused by enterotoxigenic Escherichia coli targeting the heat-labile toxin.

We standardized an immunochromatographic test (IC) for heat-labile toxin I (LT-I) detection using LT-I antibodies and a specific platform containing the apparatus for application, assembly and cutting. IC detected as little as 62.5ng/mL of purified LT-I toxin and presented 91% sensitivity, 99.5% specificity and 96.0% accuracy, thereby proving to be an excellent point-of-care test for the diagnosis of enterotoxigenic E. coli infection in low-income countries.

Large-scale evaluation of a rapid diagnostic test for diarrhea caused by enterotoxigenic Escherichia coli targeting the heat-labile toxin

We standardized an immunochromatographic test (IC) for heat-labile toxin I (LT-I) detection using LT-I antibodies and a specific platform containing the apparatus for application, assembly and cutting. IC detected as little as 62.5 ng/mL of purified LT-I toxin and presented 91% sensitivity, 99.5% specificity and 96.0% accuracy, thereby proving to be an excellent point-of-care test for the diagnosis of enterotoxigenic E. coli infection in low-income countries.

Structural changes in Stx1 engineering monoclonal antibody improves its functionality as diagnostic tool for a rapid latex agglutination test

Stx1 toxin is one of the AB(5) toxins of Shiga toxin-producing Escherichia coli (STEC) responsible for foodborne intoxication during outbreaks. The single-chain variable fragment (scFv) is the most common recombinant antibody format; it consists of both variable chains connected by a peptide linker with conserved specificity and affinity for antigen. The drawbacks of scFv production in bacteria are the heterologous expression, conformation and stability of the molecule, which could change the affinity for the antigen. In this work, we obtained a stable and functional scFv-Stx1 in bacteria, starting from IgG produced by hybridoma cells. After structural modifications, i.e., change in protein orientation, vector and linker, its solubility for expression in bacteria was increased as well as the affinity for its antigen, demonstrated by a scFv dissociation constant (K-D) of 2.26 x 10(-7) M. Also, it was able to recognize purified Stx1 and cross-reacted with Stx2 toxin by ELISA (Enzyme-Linked Immunosorbent Assay), and detected 88% of Stx1-producing strains using a rapid latex agglutination test. Thus, the scFv fragment obtained in the present work is a bacteria-produced tool for use in a rapid diagnosis test, providing an alternative for STEC diagnosis.

Large-scale evaluation of a rapid diagnostic test for diarrhea caused by enterotoxigenic Escherichia coli targeting the heat-labile toxin

We standardized an immunochromatographic test (IC) for heat-labile toxin I (LT-I) detection using LT-I antibodies and a specific platform containing the apparatus for application, assembly and cutting. IC detected as little as 62.5 ng/mL of purified LT-I toxin and presented 91% sensitivity, 99.5% specificity and 96.0% accuracy, thereby proving to be an excellent point-of-care test for the diagnosis of enterotoxigenic E. coli infection in low-income countries.

Structural changes in Stx1 engineering monoclonal antibody improves its functionality as diagnostic tool for a rapid latex agglutination test

Stx1 toxin is one of the AB(5) toxins of Shiga toxin-producing Escherichia coli (STEC) responsible for foodborne intoxication during outbreaks. The single-chain variable fragment (scFv) is the most common recombinant antibody format; it consists of both variable chains connected by a peptide linker with conserved specificity and affinity for antigen. The drawbacks of scFv production in bacteria are the heterologous expression, conformation and stability of the molecule, which could change the affinity for the antigen. In this work, we obtained a stable and functional scFv-Stx1 in bacteria, starting from IgG produced by hybridoma cells. After structural modifications, i.e., change in protein orientation, vector and linker, its solubility for expression in bacteria was increased as well as the affinity for its antigen, demonstrated by a scFv dissociation constant (K-D) of 2.26 x 10(-7) M. Also, it was able to recognize purified Stx1 and cross-reacted with Stx2 toxin by ELISA (Enzyme-Linked Immunosorbent Assay), and detected 88% of Stx1-producing strains using a rapid latex agglutination test. Thus, the scFv fragment obtained in the present work is a bacteria-produced tool for use in a rapid diagnosis test, providing an alternative for STEC diagnosis.

Epitope Sequences in Dengue Virus NS1 Protein Identified by Monoclonal Antibodies.

Dengue nonstructural protein 1 (NS1) is a multi-functional glycoprotein with essential functions both in viral replication and modulation of host innate immune responses. NS1 has been established as a good surrogate marker for infection. In the present study, we generated four anti-NS1 monoclonal antibodies against recombinant NS1 protein from dengue virus serotype 2 (DENV2), which were used to map three NS1 epitopes. The sequence 193AVHADMGYWIESALNDT209 was recognized by monoclonal antibodies 2H5 and 4H1BC, which also cross-reacted with Zika virus (ZIKV) protein. On the other hand, the sequence 25VHTWTEQYKFQPES38 was recognized by mAb 4F6 that did not cross react with ZIKV. Lastly, a previously unidentified DENV2 NS1-specific epitope, represented by the sequence 127ELHNQTFLIDGPETAEC143, is described in the present study after reaction with mAb 4H2, which also did not cross react with ZIKV. The selection and characterization of the epitope, specificity of anti-NS1 mAbs, may contribute to the development of diagnostic tools able to differentiate DENV and ZIKV infections.