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.

Anti-DENV-NS1 monoclonal antibody for the differential histopathological diagnosis of hemorrhagic fever caused by dengue.

Dengue is the most prevalent arboviral disease in humans in tropical and subtropical regions, especially in urban areas, and can cause major epidemics. Although a self-limiting illness, it may sometimes have serious hemorrhagic manifestations, and the outcome of dengue hemorrhagic fever has similar clinical manifestations as in other infections, which could result in death. Therefore, autopsy procedures are required under certain circumstances such as in hemorrhagic fevers, sometimes to confirm or to clarify the diagnosis that may have epidemiological consequences. Normally, the Immunohistochemistry Laboratory of the Pathology Center of Adolfo Lutz Institute receives autopsy samples from different hospitals in Sao Paulo State to confirm a previous diagnosis, especially hemorrhagic fever of infectious etiology. For this diagnosis, we have been using a mouse polyclonal antibody to dengue virus that often does not provide a clear conclusion, because of background staining or no relevant immunostaining, which hampers the histopathological analysis. Accordingly, in the present study, anti-DENV-NS1 monoclonal antibody (4H2) was tested to determine its accuracy in immunohistochemical analysis. Twenty-four autopsy cases of hemorrhagic febrile syndrome showing histopathological alterations compatible with dengue disease were studied: twenty cases were confirmed by RT-PCR for DENV-2 and in four by RT-PCR for yellow fever virus. Samples from autopsied cases of deaths caused by other infectious diseases (two meningitis C and two severe acute respiratory syndrome caused by influenza A H1N1) were included as negative control cases. Positive immunostaining for DENV-NS1 was detected in 16/20 (80%) liver samples and 11/15 (73%) spleen samples from autopsied hemorrhagic dengue patients, whereas the polyclonal antibody detected DENV antigens in 12/20 (60%) liver and in 6/15 (40%) spleen samples from the same cases. Positive results were not obtained with liver biopsy samples from yellow fever or Neisseria meningitides and Flu-A cases. 4H2 mAb recognizes the native protein of the four DENV serotypes in infected cells and did not cross-react with native ZIKV- or CHKV-infected cells by immunohistochemical assay, so it is a useful tool for differential histopathological conclusion of acute febrile hemorrhagic deaths.

Recombinant PilS: cloning, expression and biochemical characterization of a Pil-Fimbriae subunit

Pil-fimbriae is a type IV pili member, which is a remarkably versatile component with a wide variety of functions, including motility, attachment to different surfaces, electrical conductance, DNA acquisition, and secretion of a broad range of structurally distinct protein substrates. Despite the previous functional characterization of Pil, more studies are required to understand the regulation of Pil expression and production, since the exact mechanisms involved in these steps are still unknown. Therefore it is extremely important to have a protein with the correct secondary and tertiary structure that will enable an accurate characterization and a specific antisera generation. For this reason, the aim of this work was to generate potential tools for further investigations to comprehend the mechanisms involved in Pil regulation and its role in pathogenic E. coli infections with the obtaining of a precise native-like recombinant PilS and the corresponding antisera. The pilS gene was successfully cloned into an expression vector, and recombinant PilS (rPilS) was efficiently solubilized and purified by metal affinity chromatography. Protein characterization analyses indicated that rPilS presented native-like secondary and tertiary structures after the refolding process. The generated anti-rPilS sera efficiently recognized recombinant and native proteins from atypical enteropathogenic E. coli strains.

Paracoccidioides HSP90 Can Be Found in the Cell Surface and Is a Target for Antibodies with Therapeutic Potential.

Paracoccidioidomycosis (PCM) is one of the most frequent systemic mycoses in Latin America. It affects mainly male rural workers in impoverished regions, and the therapy can last up to two years or use drugs that are very toxic. Given the need for novel safe and effective approaches to treat PCM, we have been developing monoclonal antibodies (mAbs) that could be used not only to block specific fungal targets, but also modulate the host's antifungal immunity. In this work we show the generation of and promising results with an mAb against Heat Shock Protein (HSP)90, a molecular chaperone that is an important virulence factor in fungi. Using recombinant Paracoccidioides lutzii (Pb01) and P. brasiliensis (Pb18) HSP90 proteins produced in E. coli, we immunized mice and generated polyclonal antibodies and an IgG1 hybridoma mAb. The proteins were very immunogenic and both the polyclonal serum and mAb were used in immunofluorescence experiments, which showed binding of antibodies to the yeast cell surface. The mAb successfully opsonized P. lutzii and P. brasiliensis cells in co-incubations with J774.16 macrophage-like cells. Our results suggest that this mAb could serve as the basis for new immunotherapy regimens for PCM.

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.

Paracoccidioides HSP90 can be found in the cell surface and Is a target for antibodies with therapeutic potential

Paracoccidioidomycosis (PCM) is one of the most frequent systemic mycoses in Latin America. It affects mainly male rural workers in impoverished regions, and the therapy can last up to two years or use drugs that are very toxic. Given the need for novel safe and effective approaches to treat PCM, we have been developing monoclonal antibodies (mAbs) that could be used not only to block specific fungal targets, but also modulate the host’s antifungal immunity. In this work we show the generation of and promising results with an mAb against Heat Shock Protein (HSP)90, a molecular chaperone that is an important virulence factor in fungi. Using recombinant Paracoccidioides lutzii (Pb01) and P. brasiliensis (Pb18) HSP90 proteins produced in E. coli, we immunized mice and generated polyclonal antibodies and an IgG1 hybridoma mAb. The proteins were very immunogenic and both the polyclonal serum and mAb were used in immunofluorescence experiments, which showed binding of antibodies to the yeast cell surface. The mAb successfully opsonized P. lutzii and P. brasiliensis cells in co-incubations with J774.16 macrophage-like cells. Our results suggest that this mAb could serve as the basis for new immunotherapy regimens for PCM

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.

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.

Immunogenic Domains and Secondary Structure of Escherichia coli Recombinant Secreted Protein Escherichia coli-Secreted Protein B.

Several pathogenic bacteria are able to induce the attaching and effacing (A/E) lesion. The A/E lesion is caused by effector proteins, such as Escherichia coli-secreted protein B (EspB), responsible together with Escherichia coli-secreted protein D for forming a pore structure on the host cell, which allows the translocation of effector proteins. Different variants of this protein can be found in E. coli strains, and during natural infection or when this protein is injected, this leads to variant-specific production of antibodies, which may not be able to recognize other variants of this bacterial protein. Herein, we describe the production of a hybrid recombinant EspB toxin that comprises all known variants of this protein. This recombinant protein could be useful as an antigen for the production of antibodies with broad-range detection of EspB-bearing bacteria, or as an antigen that could be used in vaccine formulation to generate antibodies against different EspB variants, thereby increasing immunization potential. In addition, the recombinant protein allowed us to analyze its secondary structure, to propose the immunogenic regions of EspB variants, and also to characterize anti-EspB antibodies. Our results suggest that this hybrid protein or a protein composed of the conserved immunogenic regions could be used for a variety of clinical applications.

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.

Immunogenic domains and secondary structure of escherichia coli recombinant secreted protein escherichia coli-secreted protein B

Several pathogenic bacteria are able to induce the attaching and effacing (A/E) lesion. The A/E lesion is caused by effector proteins, such as Escherichia coli-secreted protein B (EspB), responsible together with Escherichia coli-secreted protein D for forming a pore structure on the host cell, which allows the translocation of effector proteins. Different variants of this protein can be found in E. coli strains, and during natural infection or when this protein is injected, this leads to variant-specific production of antibodies, which may not be able to recognize other variants of this bacterial protein. Herein, we describe the production of a hybrid recombinant EspB toxin that comprises all known variants of this protein. This recombinant protein could be useful as an antigen for the production of antibodies with broad-range detection of EspB-bearing bacteria, or as an antigen that could be used in vaccine formulation to generate antibodies against different EspB variants, thereby increasing immunization potential. In addition, the recombinant protein allowed us to analyze its secondary structure, to propose the immunogenic regions of EspB variants, and also to characterize anti-EspB antibodies. Our results suggest that this hybrid protein or a protein composed of the conserved immunogenic regions could be used for a variety of clinical applications.

Development of a prototype immunochromatographic test for rapid diagnosis of respiratory adenovirus infection

Human adenoviruses comprise an important group of etiologie agents that are responsible for various diseases in adults and children, such as respiratory, ocular, gastroenteric, and urinary infections. In immunocompromised and organ-transplanted individuals, these agents can cause generalized infections. Rapid diagnostic methods for detecting these infectious agents are not widely available.& para;& para;The aim of this work was to produce monoclonal and polyclonal anti-adenovirus antibodies to be used in a rapid diagnostic test for respiratory infections.& para;& para;Adenovirus hexons were satisfactorily purified by ultracentrifugation and chromatography. After virus purification, anti-hexon monoclonal antibodies were produced and characterized, following classical methods. Antibodies were specific for adenoviruses 2,3,5, and 41. The proposed immunochromatographic test was standardized using colloidal gold.& para;& para;The standardization of the rapid test was sufficient to detect adenovirus antigens (in nasopharyngeal lavage samples) with sensitivity of 100% and specificity of 85% when compared to direct immunofluorescence.& para;& para;The immunochromatographic assay prototype was sufficiently sensitive to detect B (3), C (2 and 5), and F (41) adenovirus samples. Although based on preliminary data, the test demonstrated the same performance as direct immunofluorescence, but with the advantage of being a point-of-care test. Further studies are still needed to confirm its effectiveness in clinical practice.