Immunoinformatics and Pepscan strategies on the path of a peptide-based serological diagnosis of COVID19.

Several diagnostic tools have been developed for clinical and epidemiological assays. RT-PCR and antigen detection tests are more useful for diagnosis of acute disease, while antibody tests allow the estimation of exposure in the population. Currently, there is an urgent need for the development of diagnostic tests for COVID-19 that can be used for large-scale epidemiological sampling. Through a comprehensive strategy, potential 16 mer antigenic peptides suited for antibody-based SARS-CoV-2 diagnosis were identified. A systematic scan of the three structural proteins (S,N and M) and the non-structural proteins (ORFs) present in the SARS-CoV-2 virus was conducted through the combination of immunoinformatic methods, peptide SPOT synthesis and an immunoassay with cellulose-bound peptides (Pepscan). The Pepscan filter paper sheets with synthetic peptides were tested against pools of sera of COVID-19 patients. Antibody recognition showed a strong signal for peptides corresponding to the S, N and M proteins of SARS-CoV-2 virus, but not for the ORFs proteins. The peptides exhibiting higher signal intensity were found in the C-terminal region of the N protein. Several peptides of this region showed strong recognition with all three immunoglobulins in the pools of sera. The differential reactivity observed between the different immunoglobulin isotypes (IgA, IgM and IgG) within different regions of the S and N proteins, can be advantageous for ensuring accurate diagnosis of all infected patients, with different times of exposure to infection. Few peptides of the M protein showed antibody recognition and no recognition was observed for peptides of the ORFs proteins.

In silico modeling and structural analysis of asparaginyl endopeptidase of schistosoma mansoni (Sm32): Immunological and drug target implications.

Asparaginyl endopeptidase (AE) of Schistosoma mansoni (Sm32), also known as legumain, is a cysteine protease indirectly involved in the digestion of hemoglobin of Schistosoma sp. in the gastrodermis, being a vaccine candidate against this trematode and a potential drug target. This study presents a model for the three-dimensional structure of Sm32 determined by means of homology modeling and a molecular dynamics simulation with explicit solvent refinement. The structure proved to be consistent with other AEs of known crystal structures described in their proenzyme form, revealing a catalytic domain that has a caspase-like overall structure and a C-terminal prodomain that adopts a death-domain-like architecture. We identified amino acid mutations in the ßIV strand, differences in the active site and in the surface electrostatic potentials between Sm32 and its homologous proteins of mouse and human. Additionally, amino acid changes in the activation peptide (AP) of the S. mansoni protein were determined. Our results strongly suggest that Sm32 can be exploited as a potential target for drug design and for the development of biomarkers used in diagnosis and in novel vaccines for the control of parasitic infection, opening the perspective of medicinal chemistry developments.

Use of Synthetic Peptides and Multiple Antigen Blot Assay in the Immunodiagnosis of Hepatitis C Virus Infection.

Acute hepatitis C virus (HCV) infection is usually asymptomatic, therefore, early diagnosis is rare. It may remain undiagnosed in individuals who progress to chronic infection, often until serious liver damage has developed. To incorporate the diagnosis of this viral disease in a multiple-diagnostic assay, we first analyzed by immunoinformatics the HCV subtype 1a polyprotein (specifically Core, E2, NS3, NS5A proteins) to select antigenic peptides to be tested initially by the Pepscan technique. Next, we performed the immunodiagnosis of HCV infection, using the Multiple Antigen Blot Assay (MABA). In 22 patients' sera included in this study, a 20-mer linear peptide belonging to the N-terminus of the worldwide conserved Core protein showed 100% sensitivity and specificity; other sequences showed different levels of antibody recognition. The use of MABA in combination with synthetic peptides as a source of multiple, specific, and nonexpensive antigens for other infectious diseases could represent a rapid, integrated, and inexpensive diagnostic methodology.

Development and characterization of IgY antibodies against synthetic peptides from Brucella abortus OMP25 and BP26 proteins / Desarrollo y caracterización de anticuerpos IgY contra péptidos sintéticos de las proteínas OMP25 y BP26 de Brucella abortus

Brucellosis is a zoonose produced by bacterial species from the Brucella genus. Its isolation and identification in food using classical microbiological techniques is not practical due to its slow growth rate. Therefore, it is necessary to establish fast and specific methods for the detection of the bacteria in food. The goal of this work was the production and characterization of monospecific polyclonal antibodies in chicken (IgY) against synthetic peptides from Brucella abortus OMP25 and BP26 proteins, suitable for an antigen-capture assay. Conformational as well as antigenic predictions were performed using the ANTHEPROT package. Chemical synthesis was carried out by the multiple manual synthesis using the t-boc strategy. The peptides were used as antigens for the preparation of polyclonal antibodies in chicken. Experimental animals produced specific antibodies against the OMP25 and BP26 peptides constructs determined by ELISA and MABA assays showing correspondence between the predictive study and the immunogenicity obtained in chicken. The IgY proved to be able to recognize B. abortus by MABA assays. The binding activity and specificity of antibodies was determined by Western blot with cell extract from B. abortus. In this study, we demonstrated that OMP25 and BP26 peptides constructs are good candidates for production of specific IgY antipeptide antibodies capable of recognizing proteins from sonicated B. abortus strain S19, indicating the potential usefulness of the IgY antibody for development of immunoassays for detection of Brucella abortus.

La brucelosis es una zoonosis producida por especies del género Brucella. El aislamiento e identificación de la bacteria en alimentos usando las técnicas clásicas de microbiología no es práctico debido a su lenta tasa de crecimiento. Por lo tanto, es necesario establecer métodos rápidos para la detección de la bacteria en alimentos. En el presente trabajo se desarrollaron y caracterizaron anticuerpos policlonales monoespecíficos en gallinas (IgY) contra péptidos sintéticos de las proteínas OMP25 y BP26 de Brucella abortus, que puedan ser utilizados en un ensayo de captura. Para ello, se realizaron estudios conformacionales y de predicción de epítopes en la selección de los péptidos, los cuales se utilizaron como antígenos para la producción de las IgY. Los animales desarrollaron anticuerpos específicos contra los péptidos, mostrando correspondencia entre los estudios predictivos y la inmunogenicidad obtenida. Las IgY reconocieron a B. abortus en un ensayo de MABA y la actividad de unión y especificidad fue determinada por western blot con extracto celular de B. abortus. En este estudio, demostramos que los péptidos de las proteínas OMP25 y BP26 de B. abortus son buenos candidatos para la producción de anticuerpos IgY especificos capaces de reconocer proteínas de extracto de B. abortus cepa S19, indicando el potencial uso de anticuerpos IgY para el desarrollo de inmunoensayos para la detección de Brucella abortus.

Improvements and Variants of the Multiple Antigen Blot Assay-MABA: An Immunoenzymatic Technique for Simultaneous Antigen and Antibody Screening.

This simple, versatile, reliable, reproducible, multipurpose, and inexpensive technique is based on the adhesion of different antigens to a single nitrocellulose strip using, as template, an acrylic device containing 28 parallel channels. The inclusion of channels containing normal human serum improves the quality control of this assay. Antigen-sensitized nitrocellulose strips are cut perpendicularly to the antigen-rows, exposed to immune sera followed by the appropriate conjugate. Positive signals are recorded using chemiluminescent or precipitable colorimetric substrates. This assay allows the simultaneous qualitative demonstration of antigenicity and immunogenicity of antigens obtained as synthetic peptides, recombinant molecules, or crude preparations, with high sensitivity and specificity. Its major value is based on the rapid and simultaneous comparative evaluation of various antigenic preparations allowing the diagnosis of a variety of infectious, allergic, and autoimmune diseases. It can in general be used to detect any type of antibody or circulating antigen. Some improvements and variants of the original technique are included.

A combined proteomic and immunologic approach for the analysis of Schistosoma mansoni cercariae and adult worm protein extracts and the detection of one of the vaccine candidates, Sm28GST, from a Venezuelan parasite isolate.

Understanding the mode of Schistosoma mansoni larval invasion and the mechanism of immune evasion utilized by larvae and adult worms is essential for a rational development of vaccines or drugs to prevent or cure the disease. This parasite has a very complex molecular organization in all parasite stages, and identifying the major parasite proteins would give clues to schistosome metabolism and to the interaction of the parasite with the host immune system. Our goal was the evaluation of the protein parasite repertoire using a proteomic approach, and the characterization of protein extracts from two different parasite stages of a Venezuelan isolate, such as cercariae and adult worms, previously performed by other authors in some other strains. A comparison among authors was made. Besides, we aimed to identify different isoforms of one of the vaccine candidates, the gluthation-S-transferase protein (Sm28GST), by 2D SDS-PAGE and mass spectrometry, and to achieve its immunologic detection using sera from rabbits immunized with synthetic peptides derived from the Sm28GST protein. These techniques allowed the identification of some of the target molecules of the protective immune response that are being evaluated as potential members of a multi-component and multi-stage anti-S. mansoni vaccine and to clarify if the selected peptides induce antibodies that are able to recognize different isoforms of the Sm28GST.

A combined proteomic and immunologic approach for the analysis of Schistosoma mansoni cercariae and adult worm protein extracts and the detection of one of the vaccine candidates, Sm28GST, from a Venezuelan parasite isolate / Aproximación al análisis proteómico e inmunológico de extractos proteicos de cercaria y verme adulto de Schistosoma mansoni y detección de uno de los candidatos a vacuna, Sm28GST, de un aislado venezolano

Understanding the mode of Schistosoma mansoni larval invasion and the mechanism of immune evasion utilized by larvae and adult worms is essential for a rational development of vaccines or drugs to prevent or cure the disease. This parasite has a very complex molecular organization in all parasite stages, and identifying the major parasite proteins would give clues to schistosome metabolism and to the interaction of the parasite with the host immune system. Our goal was the evaluation of the protein parasite repertoire using a proteomic approach, and the characterization of protein extracts from two different parasite stages of a Venezuelan isolate, such as cercariae and adult worms, previously performed by other authors in some other strains. A comparison among authors was made. Besides, we aimed to identify different isoforms of one of the vaccine candidates, the gluthation-S-transferase protein (Sm28GST), by 2D SDS-PAGE and mass spectrometry, and to achieve its immunologic detection using sera from rabbits immunized with synthetic peptides derived from the Sm28GST protein. These techniques allowed the identification of some of the target molecules of the protective immune response that are being evaluated as potential members of a multi-component and multi-stage anti-S. mansoni vaccine and to clarify if the selected peptides induce antibodies that are able to recognize different isoforms of the Sm28GST.

Es esencial comprender la forma como las larvas de Schistosoma mansoni invaden y los mecanismos de evasión inmune utilizados por larvas y adultos, para el desarrollo racional de vacunas o drogas para prevenir o curar la esquistosomiasis. Este parásito tiene una organización molecular muy compleja en todos sus estadíos, por lo que la identificación de las proteínas más importantes es clave para investigar el metabolismo del esquistosoma y la interacción del parásito con el sistema inmune del hospedero. El objetivo de este trabajo fue evaluar el repertorio proteico del parásito utilizando una aproximación proteómica y la caracterización de extractos proteicos de dos estadios parasitarios diferentes de un aislado venezolano, como la cercaria y el verme adulto, previamente realizado por otros autores en otras aislados. Se realizó una comparación entre autores. Además, se identificaron diferentes isoformas de uno de los candidatos a vacuna, la glutation S transferasa (Sm28GST) por 2D SDS-PAGE y espectrometría de masas y se logró su detección inmunológica, usando sueros de conejos inmunizados con péptidos sintéticos derivados de la proteína Sm28GST. Estas técnicas permitieron identificar algunas de las moléculas blanco de la respuesta inmune protectora que están siendo evaluados como miembros potenciales de una vacuna multi-estadio y multi-componente y aclarar si los péptidos seleccionados indujeron anticuerpos capaces de reconocer diferentes isoformas de la Sm28GST.

Immunogenicity of synthetic peptides derived from Plasmodium falciparum proteins.

To obtain antibodies suitable to be used in an antigen-capture assay, we have identified, synthesized, and evaluated a series of peptides from different Plasmodium falciparum excretory-secretory proteins: glutamate-rich protein (GLURP); histidine-rich protein 2; histidine-rich protein 3; Falciparum interspersed repeat antigen and, serine-rich antigen homologous. Conformational as well as antigenic predictions were performed using the ANTHEPROT package. Chemical synthesis was carried out by the multiple manual synthesis using the t-boc strategy. The peptides were used as antigens for the preparation of polyclonal antibodies in rabbits. Out of the 14 peptide constructs, eight by ELISA and, six by MABA elicited antibodies that showed correspondence between the predictive study and the immunogenicity obtained in rabbits. All antipeptide (GLURP, HRP2, and FIRA) antisera were found to bind to the corresponding synthetic sequence in an ELISA assay. The binding activity and specificity of antibodies were determined by Western blot with supernatant culture from P. falciparum. Anti-GLURP (IMT-94 and IMT-200) antisera bound to five molecules present in supernatant with molecular weight of 73, 82, 116, 124, and 128 kDa. Anti-HRP2 (IMT-192) antisera recognized a band of 58 kDa. In both cases, the specific molecules were inhibited by preincubation with the homologous peptide. Anti-HRP3, anti-FIRA neither anti-SERPH antisera showed reactivity. Anti-peptides HRP2 antibodies recognized the recombinant protein present in Parasight-F test. The same way, synthetic peptides from HRPII molecule were recognized by monoclonal antibody present in the Parasight-F assay. Our results confirm the potential value of synthetic peptides when inducing monospecific polyclonal antibodies for the development of diagnostic tests based on the capture of antigens.

Immunogenicity of Sm32 synthetic peptides derived from the Schistosoma mansoni adult worm.

The previously called "hemoglobinase" Sm32 molecule of the adult worm of Schistosoma mansoni was chemically synthesized in 22 polymeric peptides based on the t-boc strategy. Their immunogenicity was evaluated in rabbits to which a mixture of five to six peptides of 20 amino acids long were given in three doses with Freund's adjuvant. Seventeen peptides were found to be immunogenic, and sera from immunized rabbits corresponding to the molecule from the first 335 amino acids, recognized the 32 kDa native protein from the adult worm antigen by western blot. Of those, the relevant peptides responsible of the recognition of the original molecule corresponded to amino acids 101-120, 121-140 and 244-268, based on inhibition competitive assays. Because Sm32 is one of the excretory and secretory molecules released with the vomitus of the adult worm, it is one of the target antigens for detection in plasma of infected individuals. The production of these polyclonal monospecific antibodies against the synthetic peptides could be of value in the immunodiagnosis of this parasitosis.

Immunological similarity between Schistosoma and bovine cathepsin D.

IgG antibodies from sera of rabbits immunized with a mixture of three synthetic peptides of highly conserved surface-exposed sequences between Schistosoma japonicum and S. mansoni cathepsin D, and a rabbit anti-bovine cathepsin D serum strongly recognized a 45 kDa molecule on immunoblots of adult S. mansoni worm saline extracts (AWSE). This recognition was abolished by immunoadsorption with two of the three selected peptides. The anti-peptide antibodies fixed onto Protein A-Sepharose specifically immunoprecipitated a S. mansoni AWSE component that was able to degrade bovine hemoglobin at pH 3.8. This reaction was inhibited by 7 microM pepstatin A, a classical aspartyl protease inhibitor, suggesting that the parasite cathepsin D was immunoprecipitated. The anti-peptide antibodies also recognized on a dot-blot assay a purified, commercially obtained bovine cathepsin D preparation but not the purified human counterpart. On the other hand, the anti-bovine cathepsin D serum recognized the two above-mentioned schistosome peptides. In addition, S. mansoni-infected patient sera recognized on immunoblots the bovine but not the human cathepsin D. These results, together with a comparative analysis of the selected peptide sequence regions between the schistosome and the two mammal enzymes, allowed us to pinpoint to one amino acid the cross-reactivity between parasite and bovine cathepsin D and the lack of it with human cathepsin D. This difference might be of relevance for immunodiagnosis.

Péptidos sintéticos para el diagnóstico y vigilancia epidemiológica en esquistosomosis mansoni / Synthetics peptides for the diagnose and vigilance epidemiologic in schitosomiasis mansoni

El diagnóstico de laboratorio de la esquistosomosis es uno de los problemas por resolver, especialmente en áreas de bajas cargas parasitarias, como Venezuela, además de ser también una de las limitantes la evaluación de las nuevas vacunas contra esta parasitosis. Por esta razón, nos planteamos desarrollar métodos de inmunodiagnóstico basados en la detección de anticuerpos y de antígenos circulantes utilizando la estrategia de la síntesis química, para elaborar péptidos derivados de la captesina B (Sm31) y la asparraginil endopeptidasa (Sm32). Tres de los péptidos de la Sm31 fueron reconocidos al menos por el 49 por ciento de los pacientes y de ellos el IMT-180, lo fue por el 86 por ciento, exhibiendo además una alta especificidad (100 por cierto). Dos de los péptidos de la Sm31 y 3 de la Sm32 indujeron en conejos, el reconocimiento de las respectivas moléculas, inclusive en cortes histológicos. Resultados preliminares de inmunoensayos de captura anti-Sm32 han revelado una baja sensibilidad