WO2020082145A1 - Polypeptide, cassette d'expression, vecteur d'expression, cellule hôte, trousse pour le triage immunologique du vhc et/ou le diagnostic de l'hépatite c, composition, utilisation d'au moins un polypeptide, et méthodes pour produire un polypeptide, pour le triage immunologique du vhc et le diagnostic de l'hépatite c - Google Patents

Polypeptide, cassette d'expression, vecteur d'expression, cellule hôte, trousse pour le triage immunologique du vhc et/ou le diagnostic de l'hépatite c, composition, utilisation d'au moins un polypeptide, et méthodes pour produire un polypeptide, pour le triage immunologique du vhc et le diagnostic de l'hépatite c Download PDF

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WO2020082145A1
WO2020082145A1 PCT/BR2019/050454 BR2019050454W WO2020082145A1 WO 2020082145 A1 WO2020082145 A1 WO 2020082145A1 BR 2019050454 W BR2019050454 W BR 2019050454W WO 2020082145 A1 WO2020082145 A1 WO 2020082145A1
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polypeptide
hcv
hepatitis
antigen
polypeptides
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PCT/BR2019/050454
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Portuguese (pt)
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Nilson Ivo Tonin ZANCHIN
Marco Aurélio KRIEGER
Lucianna Freitas Oliveira DE LIMA
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Fundação Oswaldo Cruz
Instituto De Biologia Molecular Do Paraná - Ibmp
Instituto De Tecnologia Do Paraná (Tecpar)
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Publication of WO2020082145A1 publication Critical patent/WO2020082145A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/29—Hepatitis virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—DNA viruses
    • C07K14/02—Hepadnaviridae, e.g. hepatitis B virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/18—Togaviridae; Flaviviridae
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis

Definitions

  • POLYPEPTIDE EXPRESSION CASSETTE, EXPRESSION VECTOR, HOSTING CELL, KIT FOR HCV IMMUNOLOGICAL SCREENING AND / OR HEPATITIS C DIAGNOSIS, COMPOSITION, USE OF AT LEAST ONE POLYPEPIDE, AND, METHODS OF METHODOLOGY FOR PRODUCTION AND FOR HEPATITIS C DIAGNOSIS ”
  • the present invention relates to the field of diagnostic medicine and biotechnology. More specifically, the present invention relates to polypeptides for diagnostic application in screening for hepatitis C virus. BACKGROUND OF THE INVENTION
  • Hepatitis C is a serious silent viral disease that can result in long-term health problems.
  • the World Health Organization estimates that approximately 170 million people are chronically infected with the hepatitis C virus.
  • Data from the United States' Center for Disease Control and Prevention revealed that annual hepatitis C-related mortality in 2013 exceeded the total combined deaths 60 other infectious diseases, including HIV infection, pneumococcus and tuberculosis, killing more than any other infectious disease (HEPATITIS C MORTALITY, 2016).
  • the etiologic agent of hepatitis C is a spherical virus of 55-65 nm in diameter, enveloped, whose genome is a 9.3 kilobase molecule with genetic organization of flavi virus (MELLO, 2014).
  • Virus replication occurs via RNA, positive polarity, single-stranded (ssRNA), linear, which contains untranslated regions (RTU's) and which flank an uninterrupted open reading sequence encoding a single 3,011 amino acid polyprotein: NH2-C- El -E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5BCOOH (LIN et al., 1994).
  • This precursor polyprotein is subsequently cleaved by cellular and viral proteases releasing structural (C-El-E2-p7) and non-structural (NS2-NS3-NS4A- NS4B-NS5A-NS5B) proteins, which include elements of controls necessary for translation and viral replication.
  • Non-structural proteins are known to participate in viral replication, while structural proteins are part of the capsid structure and form the envelope glycoproteins. Structural proteins are released from polyprotein after cleavage by cellular proteases, while non-structural proteins are cleaved by viral proteases (by VICENTE et al., 2009; CHATEL-CHAIX, et al., 2011). Each of them assumes different functions in the vima cycle.
  • the nucleocapsid protein regulates cellular processes, contributing to replication and pathogenesis, in addition to being involved in the assembly and packaging of the viral genome (LIN et al., 1993; LIN et al., 1994).
  • This protein has 191 amino acids and is located in the N-terminal region of the viral polyprotein (McLAUCHLAN et al., 2002).
  • the EI and E2 envelope proteins form heterodimers that represent the native form of the HCV envelope. They are highly glycosylated and vulnerable to genetic mutations (ZENG et al., 2012), consequently, they can alter important antigenic properties of the vms promoting the escape of neutralizing antibodies. They also play an important role in the entry of the viruses into the cell (ZENG et al., 2012) and, although the role of the EI region and its association with the cell surface ligands is not yet clear, the role of the E2 protein is better defined, acting directly in connection with cell surface receptors, among them CD81 (PIVER et al., 2010; FREEDMAN et al., 2016).
  • CD81 is expressed in most human cells, including hepatocytes, and the union of this molecule with virus E2 protein induces functional changes and can stimulate the proliferation of non-activated B lymphocytes or the production of inflammatory cytokines (ROSA et al., 2005) .
  • HCV-NS1 The region encoding the p7 protein (HCV-NS1) is an envelope glycoprotein that can be found on the cell membranes of infected individuals.
  • the protein is cleaved in the endoplasmic reticulum in a signal peptide-mediated reaction. This is an essential region for the release of virions, functioning as ion channels (HUANG et al., 2010).
  • the NS2 non-structural region is a 21-23 kDa protein that interacts with other non-structural proteins to participate in the assembly of the viral particle. It is a cysteine with protease activity responsible for the cleavage of the NS2 / NS3 region (POPESCU et al., 2011; KIM et al., 2012).
  • the NS3 protein has catalytic activity of serine protease and helicase which together with the NS4A cofactor, participates in the cleavage of the NS3 / 4A, NS4A / 4B, NS4B / 5A and NS5A / 5B regions during processing (De VICENTE et al ., 2009; FREEDMAN et al., 2016).
  • NS4A is necessary for the phosphorylation of NS5A polymerase.
  • NS5 A plays an important role in viral replication, in the modulation of cell signaling and in the response to interferon (YAMANAKA et al., 2002).
  • the protein B region acts as an RNA-dependent RNA polymerase and plays an important role in the synthesis of the new RNA (PRATT et al., 2005; OLIVEIRA, 2007; KEYVANI et al., 2012).
  • Subtypes 1, 2 and 3 have worldwide distribution, with subtype 1 being the most common. This is the most resistant genotype to treatment and represents 60% of global infection and predominates in North and South America, Northern Europe and Eastern Europe (GUPTA et al., 2014). Genotype 2 represents 9.1% of hepatitis C cases worldwide and is described as one of the genotypes with the best response for treatment (HANAFIAH et al., 2013). Genotype 3 is the second most prevalent in the world, being endemic in Southeast Asia and affecting 30% of the population. Genotype 4 is more prevalent in the Middle East, where it accounts for 90% of hepatitis C cases in Egypt, North Africa and central Africa.
  • Genotype 5 has a greater distribution in South Africa and little is known about it, which represents 0.8% of the world population. Genotype 6 corresponds to 5.4% of hepatitis in Southeast Asia and a lower global distribution than genotype 5 (SHEPARD et al., 2005; HANAFIAH et al., 2013; GUPTA et al., 2014; MESSINA et al., 2015).
  • HCV The extensive genetic heterogeneity of HCV has important implications for the immune response, the clinic and the diagnosis of the disease, which poses a challenge to the development of vaccines and the response to therapies.
  • hepatitis C virus There are two main markers of infection for the hepatitis C virus: the anti-HCV serological marker and the presence of the virus RNA in the individual's serum. Serological tests have high sensitivity, but do not distinguish active or resolved infection, and can be developed for strains specific from a region that confer minor sensitivities when used in countries whose circulating genotypes are different (BRAND, 2014). For this reason, it is recommended that the virus be confirmed by supplementary or confirmatory tests for the detection of the viral genome (CLOHERTY et al., 2015) using the polymerase chain reaction (PCR) technique, in order to allow the identification of viral subtypes. However, due to the high cost, this technique is not fully accessible in geographically remote and low-cost areas.
  • PCR polymerase chain reaction
  • the first immunoassays in the EIA / ELIS A format were developed for detection using a single target or by exposing only one epitope. These tests showed high rates of cross-reactivity, low sensitivity and the need for repetitions. In the specific case of the identification of hepatitis C viruses, the diagnosis was based on the non-structural region of the NS4 protein, which, despite having a sensitivity of 40%, allowed the beginning of prevention by transfusion transmission.
  • Microsphere tests are more sensitive than commercial ELISA tests and are capable of analyzing complex mixtures.
  • Luminex Corporation developed a liquid microarray system to analyze fluorescent tests using spheres as solid supports, a system named LUMINEX. This system makes use of microfluidic technology and the binding of target molecules to the microspheres where the reaction takes place.
  • HCV detection assays available in the art despite employing multiepitope antigens, still have many disadvantages, as, for example, the fact that they are developed for a genetic profile of strains circulating in specific regions that may differ from the genotypes that circulate in geographic regions where they are commercialized or not applied in multiplex assays (FONSECA et al., 2011; ARA ⁇ JO et al., 2011; GALDINO et al., 2015 and DIPTI et al., 2006).
  • ELISA assays for screening for hepatitis C show high variability in sensitivity when applied in rich and poor countries, whose performances are considerably lower in poor countries (KHUROO et al., 2015).
  • immunoassays have high rates of false negatives when used to diagnose immunodeficient or hemodialysis patients.
  • serological tests available are not able to differentiate between acute and chronic infection (SHIVKUMAR et al., 2012).
  • the genetic variability for the hepatitis C virus reaches 30-35% (SIMMONDS et al., 1993) and for some epitopes up to 49%, which can generate genotype-specific responses and decrease the sensitivity of an assay .
  • HCV in addition to the execution time, is the presence of a gray zone from which some results are not interpretable (S ALUDES et al., 2014). In the latter case, it is recommended to repeat the serological test in duplicate to confirm the patient's condition. If the repetitions are below the limit of the test, the sample must be considered negative. If the result is greater than or equal to the cutoff point, the sample must be tested by confirmatory tests.
  • the purpose of the present invention is to provide polypeptides from different regions of hepatitis C viruses that solve the main problems of the prior art listed above.
  • the said polypeptides have a high degree of purity and a high capacity for serological discrimination for diagnostic application in screening for the hepatitis C virus, in order to cover 100% of positive patients, considering the variability represented by the different virus genotypes.
  • polypeptides have been developed that can be used alone or together in immunodiagnostic assays with very high discrimination capacity for the screening of the hepatitis C virus.
  • the present invention provides a polypeptide comprising at least two hepatitis C virus (HCV) antigenic regions selected from the group selected from the nucleocapsid region (NC) and NS3, NS4 and NS5 non-structural regions .
  • HCV hepatitis C virus
  • the present invention provides a polynucleotide that encodes the polypeptide.
  • the present invention provides an expression cassette comprising said polynucleotide.
  • the present invention provides an expression vector comprising the nucleic acid and the expression cassette described above.
  • the present invention provides a host cell comprising the nucleic acid, expression vector or expression cassette as defined above.
  • the present invention provides a method for producing a polypeptide comprising: (a) transforming a host cell with the polynucleotide as defined above,
  • the present invention provides a composition comprising a polypeptide as defined above or a combination of two or more polypeptides defined above.
  • the present invention provides a kit for immunological screening for HCV and / or diagnosis of hepatitis C that comprises at least one polypeptide of the present invention.
  • the invention provides for the use of at least one polypeptide, of the composition or kit described above in HCV immunological screening and / or hepatitis C diagnosis.
  • the invention provides a method for immunological screening of HCV, which comprises the steps of:
  • step (c) detecting the antigen / antibody complex formed in step (b), by adding a detection medium, capable of generating a detectable signal in the presence of said antigen / antibody complex.
  • the invention provides a method for diagnosing hepatitis C, which comprises the steps of:
  • step (c) detecting the antigen / antibody complex formed in step (b), by adding a detection medium, capable of generating a detectable signal in the presence of said antigen / antibody complex.
  • Figure 1 shows the quantitative analysis of the fractions obtained after purification of the polypeptides in an ⁇ KTA chromatograph; A) 13% SDS-PAGE gel; B) Western Blotting M: Molecular weight marker.
  • the tables delineate the polypeptides of interest to the present invention.
  • Figure 2 shows an SDS-PAGE gel (13%) revealing in
  • Figure 3 shows the design of the complete AEQ panel composed of 188 sera, of which 23 are reactive for HCV, 26 for HIV 1/2,
  • Figure 4 shows the parameters generated through the analysis of individual antigens performance by EFISA assay.
  • Figure 5 shows the parameters generated through the analysis of antigen performance by EFISA assays in combination.
  • Figure 6 shows the parameters obtained from the performance of Antigen 1, identifying the best coupling condition.
  • Figure 7 shows the parameters obtained from the performance of Antigen 2, identifying the best coupling condition.
  • Figure 8 shows the parameters obtained from the performance of Antigen 3, identifying the best coupling condition.
  • Figure 9 shows the parameters obtained from the performance of Antigen 4, identifying the best coupling condition.
  • Figure 10 shows the parameters obtained from the performance of Antigen 5, identifying the best coupling condition.
  • Figure 11 shows the analysis of the combined tests performed in the multiplex format via LUMINEX system.
  • A shows SNR (Signal to Noise Ratio) of the ELISA assay
  • A shows the SNR (Signal to Noise Ratio) of the LUMINEX test
  • the present inventors solved the problem of the state of the art by providing polypeptides constructed from different regions of the hepatitis C virus, of different genotypes, with a high degree of purity and high capacity for serological discrimination.
  • the analyzes revealed that the antigens of the invention, used in immunological screening assays, showed greater performance and better separation capacity between positive and negative sera, in addition to that no cross reaction was detected when exposed to positive samples for other infectious diseases.
  • the present invention provides a polypeptide comprising at least two HCV antigenic regions selected from the group selected from the nucleocapsid region (NC) and non-structural regions NS3, NS4 and NS5.
  • NC nucleocapsid region
  • NS3, NS4 and NS5 non-structural regions
  • HCV antigenic regions are known and have variability in performance, which is widely discussed in the art related to the diagnosis of hepatitis C. Based on the state of the art, mainly with regard to HCV variability, were defined sequences of antigenic regions of the virus to compose drawings of the polypeptides of the present invention.
  • Table 1 identification of the 14 built clones, with the regions included in each and their respective genotypes.
  • clones 4.7, 4.11, 4.12, 4.13 and 4.14 have the lb and 3a genotypes, which are the most prevalent in Brazil, and were constructed using the HCV polyprotein ID # ACH53426.1 and # ACZ60118.1 sequences as reference sequences, respectively.
  • antigens 1 to 5 4.12, 4.13 and 4.14 will also be referred to as antigens 1 to 5, respectively:
  • Antigen 1 SEQ ID NO: 1
  • NC-3a region amino acids 1-119 of SR
  • NS4-3a region amino acids 1691-1731 / 1789-
  • NS5-3a region amino acids 2216-2319 of SR
  • Antigen 2 SEQ ID NO: 3
  • NS4-lb Region amino acids 1691-1731 / 1789-
  • Antigen 3 SEQ ID NO: 5
  • NS3-lb region amino acids 1192-1459 from SR
  • NS5-lb region amino acids 2212-2313 from SR
  • Antigen 4 SEQ ID NO: 7
  • NS3-lb region amino acids 1192-1459 from SR
  • NS4-lb Region amino acids 1691-1731 / 1789-
  • Antigen 5 SEP ID NO: 9
  • NC-lb region amino acids 1-190 of SR
  • NS5-lb region amino acids 2212-2313 from SR
  • polypeptides are at least
  • polypeptides consist of the amino acid sequences of SEQ ID Nos: 1, 3, 5, 7 and 9.
  • polypeptides of the present invention showed reproducibility as to sensitivity and specificity. This suggests that the proteins developed can remain stable for long periods, maintaining their reactive capacity. It is possible that the composition of the stock buffer may have favored its stability, the use of protease inhibitors, the presence of denaturing agent in the buffer or even due to their amino acid sequence. Stable proteins are considered when there is an interest for diagnostic application.
  • amino acid denotes the group a-amino acids that directly or in the form of a precursor can be encoded by a nucleic acid.
  • the individual amino acids are encoded by nucleic acids consisting of three nucleotides, known as codons or base terms. Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is known as "degeneration of the genetic code”.
  • amino acid denotes naturally occurring ⁇ -amino acids, comprising alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.
  • polypeptide can be used interchangeably, and refer to a polymer of amino acids connected by peptide bonds, regardless of the number of amino acid residues that make up this chain.
  • Polypeptides include “variants” or “derivatives” thereof, which refer to a polypeptide that includes variations or modifications, for example, substitution, deletion, addition or chemical modifications in its amino acid sequence in relation to the polypeptide of reference. Examples of chemical modifications are glycosylation, PEGylation, PEG alkylation, alkylation, phosphorylation, acetylation, amidation, etc.
  • the polypeptide can be artificially produced from cloned nucleotide sequences using the recombinant DNA technique or it can be prepared through a known chemical synthesis reaction.
  • polypeptide of the present invention can also be understood as antigen, polyantigen or multiepitope antigen, which consist of a junction of different epitopes that may or may not be interconnected by flexible or rigid linkers, specific to a pathogen or for different pathogens.
  • identity is defined as the degree of equality between DNA or amino acid sequences when compared nucleotide by nucleotide or amino acid by amino acid with a reference sequence.
  • the term "percentage of sequence identity” refers to comparisons between polynucleotides or polypeptides and is determined by two sequences ideally aligned, under certain comparison parameters. This alignment can include spaces (gaps), generating intervals when compared to the reference sequence, which facilitate an adequate comparison of them. In general, the calculation of the percentage of identity considers the number of positions where the same nucleotide or amino acid occurs in the sequences compared to the reference sequence, being carried out through various sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are not limited to, TBLASTN, BLASTP, FASTA, TFASTA, CLUSTALW, FASTDB.
  • polypeptide comprising the antigenic fragments of the present invention can be obtained recombinantly or synthetically.
  • the polypeptides of the present invention are obtained by means of an expression system, which allows obtaining the polypeptides of the present invention.
  • expression system we mean a system comprising nucleotide sequences, which are capable of encoding polypeptides.
  • the present invention provides polynucleotides comprising nucleotide sequences that encode the polypeptides described herein.
  • polynucleotides according to the invention are represented, without limitation, by SEQ ID NOs: 2, 4, 6, 8 and 10 and their degenerations, or sequences with at least 90% identity with SEQ ID NOs : 2, 4, 6, 8 and 10.
  • the polynucleotides of SEQ ID NOs: 2, 4, 6, 8 and 10 encode the polypeptides represented by SEQ ID NO: 1, 3, 5, 7 and 9, respectively.
  • Polynucleotide sequences were constructed to insert a poly-linker at the beginning of the sequences (translated as Met Ala Gly Ser in SEQ ID Nos. 1, 3, 5, 7 and 9), cloning sites (NCO I enzymes, BAM Hl, ECO RI, termination codon and Hind III). The translation of the amino acids corresponding to the codons located downstream of the termination codon (s) are not shown in SEQ ID NOs. 1, 3, 5, 7 and 9.
  • degenerate nucleotide sequence denotes a nucleotide sequence that includes one or more degenerate codons when compared to a reference nucleic acid molecule that encodes a given polypeptide.
  • Degenerate codons contain different nucleotide triplets, but encode the same amino acid residue (eg, GAU and GAC both encode Asp).
  • degenerations are fully supported based on the information provided in the application and common knowledge of the state of the art. For example, the degeneration of the genetic code (that is, different codons being able to encode the same amino acids) is common knowledge in the art and the identity of the amino acid encoded by each codon is well established.
  • preferred codon usage is a term used in the art referring to codons that are most often used in cells of certain species.
  • Preferred codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. The introduction of preferred codon sequences into recombinant DNA can, for example, increase the production of the polypeptide by making translation more efficient in a given cell type. Thus, the polynucleotide sequences of the invention can be optimized for different species.
  • nucleic acids of the present invention had the codons optimized.
  • the nucleic acids of the present invention had the codons optimized for replacement of the rare codons, distribution of GC content and removal of repetitive sequences for the transcription and stability and translation of the mRNA, to obtain high levels expression in prokaryotic system.
  • nucleic acids of the invention are optimized for use in codon for Escherichia coli.
  • nucleotide sequences are optimized for replacing rare codons.
  • the present invention provides an expression cassette comprising the nucleic acid according to the invention. Said cassette is placed under conditions that lead to the expression of the polypeptides of the present invention.
  • the expression cassette can also comprise sequences necessary for its expression, such as promoters, enhancer and terminator sequences compatible with the expression system. Furthermore, the expression cassette may comprise suitable spacer sequences, linker sequences and restriction sites. In addition, the cassette may further comprise a coding sequence for histidine tail.
  • the present invention provides an expression vector comprising a nucleic acid or an expression cassette of according to the invention.
  • This expression vector can be used to transform a host cell and allow expression of the nucleic acid according to the invention in said cell.
  • the expression vector comprises regulatory elements that allow the expression of the nucleic acid and elements that allow its selection in the host cell according to the invention.
  • the methods for selecting these elements depending on the host cell in which the expression is desired, are well known to the person skilled in the art and widely described in the literature.
  • Vectors can be constructed by classical molecular biology techniques, well known to those skilled in the art.
  • Non-limiting examples of expression vectors suitable for expression in host cells are plasmids and viral or bacterial vectors.
  • the present invention provides a host cell transiently / transfected in a transient or stable manner with the nucleic acid, cassette or vector of the invention.
  • the nucleic acid, cassette or vector can be contained in the cell in the form of an episome or in a chromosomal form.
  • the host cell can be a cell of bacteria, yeast, filamentous fungi, protozoa, insects, animal and plant cells.
  • the host cell is a bacterial cell, preferably an Escherichia coli cell.
  • the present invention provides a method for producing the polypeptide according to the invention, comprising inserting a nucleic acid, a cassette or an expression vector according to the invention into an in vivo expression system and the collection of the polypeptide produced by said system.
  • a method for producing the polypeptide according to the invention comprising inserting a nucleic acid, a cassette or an expression vector according to the invention into an in vivo expression system and the collection of the polypeptide produced by said system.
  • Numerous in vivo expression systems comprising the use of suitable host cells, are commercially available and the use of these systems is well known to the person skilled in the art.
  • Particularly suitable expression systems include microorganisms, such as bacteria transformed with bacteriophage, plasmid or cosmid recombinant DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (eg, baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV [cauliflower mosaic virus ⁇ ; tobacco mosaic virus, TMV [tobacco mosaic virus]) or with bacterial expression vectors (for example, Ti or pBR322 plasmids); or animal cell systems. It is also possible to employ cell-free translation systems to produce the polypeptides of the invention.
  • microorganisms such as bacteria transformed with bacteriophage, plasmid or cosmid recombinant DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (eg, baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV [ca
  • nucleic acid, expression cassette or the vector encoding a recombinant or synthetic protein of the present invention into host cells can be carried out using methods described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986) and Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1989).
  • the transformed or transfected host cell described above is then cultured in a suitable nutrient medium under conditions conducive to the expression of the recombinant proteins of the invention.
  • the medium used to grow the cells can be any conventional medium suitable for developing the host cells, such as minimal or complex medium containing appropriate supplements. Suitable media are available from commercial suppliers or can be prepared according to published recipes (for example, in the catalogs of the American Type Culture Collection).
  • the proteins of the invention produced by the cells can then be recovered from the cell or the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the aqueous protein components of the supernatant or filtered through a salt, for example, ammonium sulphate, purification by a variety of chromatographic procedures, for example ion exchange chromatography, exclusion chromatography, interaction chromatography hydrophobic, gel filtration chromatography, affinity chromatography or similar, depending on the type of polypeptide in question.
  • a salt for example, ammonium sulphate
  • the obtained recombinant polypeptide is then purified and characterized biochemically, using, for example, methods common to the field of biochemistry, such as HPLC, SDS-PAGE, Western Blotting, isoelectric focusing with pH gradient, circular dichroism.
  • methods common to the field of biochemistry such as HPLC, SDS-PAGE, Western Blotting, isoelectric focusing with pH gradient, circular dichroism.
  • Polypeptides can be expressed "fused" to a tag.
  • tag or the English term “tog” refers to coding sequences incorporated near the multiple cloning site of an expression vector, enabling its translation concurrently and adjacent to the sequence of the cloned recombinant polypeptide. Thus, the tag is expressed fused to the recombinant polypeptide.
  • Such tags are well known in the art and include compounds and peptides such as polyhistidine, polyarginine, FLAG, glutathione-S-transferase, maltose-binding protein (MBP), cellulose-binding domain (CBD), Beta-Gal , OMNI, thioredoxin, NusA, mistin, chitin-binding domain, cutinase, fluorescent compounds (such as GFP, YFP, FITC, rhodamine, lanthanides), enzymes (such as peroxidase, luciferase, alkaline phosphatase), chemiluminescent compounds, biotinyl groups, recognized epitopes by antibodies like leucine zipper, c-myc, metal-binding domains and binding sites for secondary antibodies.
  • compounds and peptides such as polyhistidine, polyarginine, FLAG, glutathione-S-transferase, maltose-binding protein (MB
  • Polypeptides can also be obtained synthetically using methods known in the art.
  • Direct synthesis of the polypeptides of the invention can be carried out using solid phase synthesis, solution synthesis or other conventional means, generally using a-aminogroup, a-carboxyl and / or functional groups of the amino acid side chains.
  • solid phase synthesis a suitably protected amino acid residue is attached through its carboxyl group to an insoluble polymeric support, such as a cross-linked polystyrene or polyamide resin.
  • Solid-phase synthesis methods include both BOC and FMOC methods, which use tert-butyloxycarbonyl, and 9-fluorenylmethyloxycarbonyl as a-amino protecting groups, respectively, both well known to those skilled in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, NY; Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, New York, 1995).
  • the following protecting groups can be examples used for the synthesis of the polypeptides of the invention: 9-fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), carbobenzyloxy (Cbz), 2-chloro-3-indenylmethoxycarbonyl (Climoc), benz (f) indene-3-yl-methoxycarbonyl (Bimoc), 1,1-dioxobenzo [b] thiophene-2-yl-methoxycarbonyl (Bsmoc), 2,2,2-trichloroethoxycarbonyl (Troe), 2- (trimethylsilyl) ethoxycarbonyl (Teoc), homobenzyloxycarbonyl (hZ), 1,1-dimethyl-2,2,2-trichloroetoxycarbonyl (TCBoc), 1-methyl-1 (4-biphenyl) ethoxycarbonyl (Bpoc),
  • the polypeptides can be separated and purified by a known purification method.
  • An example of such purification methods may include a combination of solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like.
  • the method for producing the polypeptide of the present invention comprises the steps of:
  • the polynucleotide from step (a) encodes the polypeptide comprising at least 90% identity with any of SEQ ID NOs: 1, 3, 5, 7 and 9.
  • the polynucleotide from step (a) comprises the nucleic acid sequence of any of SEQ ID NO: 2, 4, 6, 8 and 10 and their degenerations.
  • the transformation of the host cell with the polynucleotide of the present invention is carried out by means of a vector of expression.
  • the vector is pET28a and the transformed host cell is E. coli.
  • step (b) The conditions for cultivating the host cell referred to in step (b) are known to a person skilled in the art.
  • cultivation is done in LB medium in the presence of an antibiotic, under agitation.
  • the antibiotic is kanamycin.
  • Cultivation can be carried out at different temperatures for different periods of time.
  • cultivation can be done for about 4 to about 16 hours, at a temperature of about 16 ° C to about 37 ° C.
  • cultivation is carried out at 37 ° C for 16 hours, under agitation.
  • step (b) The production of the polypeptide referred to in step (b) can be carried out with any technique known in the prior art.
  • the expression of the polynucleotides of the invention is induced by adding IPTG to the culture medium, after obtaining adequate optical density.
  • the inclusion bodies are solubilized before the isolation of the polypeptide from step (c).
  • Solubilization can be carried out with any chaotropic agent known in the art, with non-limiting examples being urea and guanidine.
  • solubilization is carried out with buffer containing guanidine.
  • step (c) The isolation of the polypeptide referred to in step (c) can be carried out with any technique known in the art.
  • purification is done by chromatography techniques.
  • purification is performed by affinity chromatography.
  • Non-limiting examples include the nickel resin affinity method, ion exchange, other affinity or adsorption methods, ion pair, reverse phase and molecular exclusion.
  • these contaminating proteins can compete with the antigens of interest for binding sites in the solid phases of the immunoassays (microspheres), which can result in the loss of performance of the assay.
  • High levels of purity are also important for the stability of antigens, since among E. coli contaminants there are many proteolytic enzymes that cause the degradation of expressed antigens. The results presented for the tests reveal the purity characteristic for the purified antigens.
  • the invention provides a composition, comprising one, two, three, four or five of the polypeptides of the invention.
  • the composition is used as a reagent for methods for immunological screening of HCV and diagnosis of hepatitis C.
  • stabilized compositions of the antigens of the present invention are provided.
  • the stabilized compositions result in the absence of protein aggregates, which guarantees non-interference in the antigen-antibody interaction, related to the aggregation, in the immunoassays of the present invention.
  • storage buffer formulations are provided for stabilizing the antigens of the invention.
  • the components of the buffers used in the present invention are known in the art.
  • the storage buffer comprises urea.
  • the storage buffer comprises 4M urea.
  • Luminex Corporation recommends testing different concentrations of the proteins of interest in different buffers when you want to optimize a reaction through the microfluidic platform and observe its functionality.
  • the protein storage buffer containing 4M urea which was the buffer that proved to be most suitable for coupling, as it prevents precipitation or the formation of aggregates between the coupled microspheres.
  • developing HCV proteins for use in immunodiagnosis that remain stable in a buffer containing up to 4M urea was also an attractive result for the present invention, since urea is considered possible to interfere in diagnostic tests when used in high concentrations, he observed It is understood that low concentrations of the agent, during coupling, may not significantly interfere with the results.
  • Luminex Corporation notifies that urea can potentially interfere in the coupling of proteins to microspheres, from the results obtained in this invention, better performances, better areas under the curve (AUC, from English Area Under Curve), less deviations for negative sera and even greater separation capacity (SNR, from English Signal to Noise Ratio) for proteins 1, 2 and 3, from the tests whose antigens were coupled to the microspheres in the presence of time containing 4M urea. Therefore, coupling in the presence of urea was essential to maintain the presentation of the epitopes of interest.
  • AUC area under Curve
  • SNR separation capacity
  • HCV hepatitis C virus
  • non-aggregating concentrations of antigen were also determined.
  • concentrations of the antigens of the present invention in the composition vary, generally from 0.007 to 0.015 mg / ml, preferably from 0.02 to 1? mg / ml.
  • the invention provides a kit for immunological screening for HCV and / or diagnosis of hepatitis C comprising one, two, three, four or five of the polypeptides of the present invention.
  • the kit also includes instructions for use and a reaction control.
  • the reaction control is a positive reaction control.
  • the kit may also comprise a means of detecting the antigen / antibody complex, which may comprise a signal generator, capable of generating a detectable signal.
  • the detection means can be those known in the art.
  • a non-limiting example of the detection means may be a conjugate comprised of an antibody coupled to a signal generating compound, capable of generating a detectable signal.
  • kits are developed for use in immunoassays.
  • immunoassays are ELISA or LUMINEX.
  • the invention provides for the use of one or more of the polypeptides or the composition or kit as described herein for HCV immunological screening and / or for diagnosis of hepatitis C.
  • the invention also provides methods for immunological screening of HCV and for the diagnosis of hepatitis C that involve the steps of:
  • step (c) detecting the antigen / antibody complex formed in step (b), by adding a detection medium, capable of generating a detectable signal in the presence of said antigen / antibody complex.
  • the indirect immunoassay model is performed. Even more preferably, the immunoassay is an ELISA assay or LUMINEX assay.
  • coupling concentrations of 7.5 to 25 pg / mL are applied. In a specific embodiment, coupling concentrations of 7.5 to 15 pg / mL are applied.
  • immunoassays can be performed in singleplex or multiplex format.
  • the antigens were evaluated for the best performance from the AUC (Area Below the Curve) of the ROC curve (English, Receiver Operating Characteristic), the greatest sensitivity and specificity, the greatest separation capacity, as well as the smallest standard deviation for negative samples.
  • the combination of two or more of the polypeptides of the present invention can be employed.
  • the combination of the five polypeptides described herein is employed.
  • the background signals or backgrounds were considered low.
  • the bottom signal indicates the efficiency or not of the washing steps or blocking.
  • This signal may also be related to the purity of the antigens used in the presence of heterologous components that can cause an increase in the background signal, such as for example, contaminating proteins from E. coli. Since the tests performed showed low background signals, this allows us to conclude that obtaining the antigens from a single chromatographic step in this invention, was sufficient to obtain proteins with a high degree of purity, confirmed the efficiency of the washing steps during the tests and the additional blockade performed with the E. coli extract.
  • the combination made up of the five antigens demonstrated the best performance, presenting a discriminatory capacity equivalent to 36 times the separation capacity obtained by the commercial chimeric antigen used as the test control, (ProsPec), the smallest deviation between negative samples and the greatest separation between MFI and IR values between negative and positive samples.
  • the trial also showed no cross reaction after tests performed with HIV, HTLV, HBV, Syphilis and Chagas positive sera.
  • the optimal condition of the protein set was achieved by making use of low concentrations of proteins for coupling.
  • concentrations used in the present invention are considerably lower than the concentrations of couplings used for commercial antigens such as, for example, the commercial protein used as an assay control.
  • the discriminatory capacity of the tests through the LUMINEX platform was superior to the ELISA in all aspects AUC (95% CI), capacity of separation between the samples and probability of positivity through IR and SNR.
  • the LUMINEX system has many advantages, among which the reaction kinetics on the caboxylated surface of the polystyrene magnetic microspheres is superior to the reaction kinetics of other methods. Among the advantages that the LUMINEX assay has over ELISA, the ability to multiplex is considered in the technique as the most attractive.
  • Serological markers used in blood banks must be highly reliable, present good reproducibility and maximum sensitivity and specificity.
  • Another important factor for the implementation of new markers in this type of laboratory is that the cut-off for the tests used must be lower, in order to increase the sensitivity. This strategy aims to ensure the safety of the recipient, as it aims to avoid transfusion of blood bags from infected donors that have low titers for the markers.
  • the set of proteins in combination 4 was the set that had the lowest cutoff point among all the tests performed, cut-off equal to 50, with a higher probability of positivity than the other combinations or the individual protein evaluations.
  • the present invention revealed antigens that promoted high levels of serological discrimination, obtaining assays with 100% specificity for a total of 165 negative sera tested for HCV and without the presence of cross reaction, which counts positively for their diagnostic application.
  • EXAMPLE 1 Cloning of the coding sequences into an expression vector
  • nucleotide sequences of interest were synthesized and underwent an optimization process for the replacement of rare codons, distribution of GC content and removal of repetitive sequences for the transcription and stability and translation of the mRNA aiming at obtaining high levels of expression in prokaryotic system. They were also designed to receive two combinations of Bam HI / Hind III and Nco I / Eco RI restriction sites at their ends aiming at subcloning in the expression vector pET28a in order to allow the positioning of hexa-histidine in the N-terminal or C region -terminal.
  • vector transfer subcloning was performed.
  • 5pg of the vector pUC57, containing the synthetic polynucleotide was linearized with the enzymes Bam Hl and Hind III at 37 ° C, as recommended by the manufacturer, considering the final reaction volume of 50 pL. After visualization and excision of the bands on the 0.8% agarose gel, the fragments were purified using the QIAquick Gel Extraction® kit (Qiagen).
  • the vector pET28a was linearized by digestion with the enzymes Bam Hl and Hind III and had the ends dephosphorylated by Alkaline Phosphatase (FastAp - ThermoFischer) at 37 ° C, according to the manufacturer's protocol and the connection of the inserts containing the synthetic genes was carried out using the enzyme Ligase T4 (Thermo Scientific), at 16 ° C, for 16h. An aliquot of the content of the binding reaction was used for transformation into E. coli DH5a (Promega) by thermal shock and confirmed by analysis of the fragments on the SDS-PAGE gel.
  • the control of the ligation reaction consisted of adding only the dephosphorylated pET28a and the enzyme T4 DNA ligase without the presence of the insert in a tube.
  • the hexa-histidine tag was positioned in the N-terminal region.
  • the coding sequences for antigens 2 to 5 were obtained from PCR amplification of the NS4 and NS5 regions. For this, forward (reverse) and reverse (reverse) initiators (SEQ ID Nos: 11 to 16) were used.
  • the coding sequence was obtained by isolating the NS4 region of plasmid pUC57 (clone 4.5 or 4.11) and the amplified and digested product was linked to plasmid pET28a + NS3 (clone 4.1) using the EcoR I and Hind III enzymes. Primers of SEQ ID Nos: 15 and 16 were used to isolate the NS4 region.
  • the amplification reaction was performed with 1U of Taq Platinum High Fidelity (Invitrogen) and with 20 to 40ng of plasmid template DNA. The reaction was carried out paying attention to the dissociation temperature (Tm) of the primers. Once the sizes of the amplified fragments were confirmed by 0.8% agarose gel, they were purified using the Qiagen PCR product purification kit. Then, the ends of the sequences were digested with the enzymes Bam Hl and Hind III to generate cohesive ends and obtain the binding in the linearized pET28A vector in the presence of T4 Ligase, at 16 ° C for 16h.
  • Tm dissociation temperature
  • Polypeptides demonstrated high levels of expression in the E. coli BL21 Star (DE3) strain grown in LB (Luria Bertani - 10g Tryptone, 5g Yeast Extract and 5g NaCl / L).
  • the cells were washed with 100mL 1X PBS and again centrifuged. The supernatant was discarded and the biomass was weighed. The biomass weight was recorded in order to evaluate the expression yield for each protein after the purification step.
  • the material could be stored at -20 ° C until the moment of use.
  • Polypeptides were expressed as inclusion bodies, whose analyzes by SDS-PAGE 13% gel reveal that the positions of the bands, after electrophoresis, correspond to the expected sizes for the constructions ( Figure 01).
  • the buffers were evaluated according to the pi of each protein, as well as the need to add reducing agents, detergents or glycerol in an attempt to maintain their stability or solubilization.
  • the lysis of cells for small volumes of culture were performed using Promega FastBreak Cell Lysis Reagent according to the manufacturer's description, followed by two sonication cycles using the Cole Parmer processor ultrasonic equipment at 40% amplitude, 30 seconds on and one minute off or adding 200pg / mL of lysozyme to the tubes containing the cells and incubating them for up to 30 minutes in buffer containing 50mM sodium phosphate pH 7.4, 300mM NaCl, 5% glycerol, 0.05% Tween 20 and 7mM b-mercaptoethanol (b-ME).
  • protease inhibitors such as PMSF lmM and Roche's protease inhibitor cocktail.
  • the cell wall rupture was performed by sonication using the Cole Parmer processor ultrasonic equipment for six cycles of lysis at 40% amplitude, 30 seconds of pulse and 60 seconds of interval at 4 ° C.
  • the extract was clarified by centrifugation (20 minutes, 4 ° C, at 20,000 g).
  • the biomass lysis from cultures for volumes above 1L was performed in microfluidizer equipment (Microfluidics Corporation, USA) using a pressure of approximately 80 Psi in an ice bath.
  • the cells were resuspended in Lysis Buffer (50 mM sodium phosphate pH 7.4, 300 mM NaCl, 5% glycerol, 0.05% Tween 20, 7 mM b-mercaptoethanol (b-ME) and protease inhibitors) using 3- 4mL of buffer for each 1g of cells.
  • Lysis Buffer 50 mM sodium phosphate pH 7.4, 300 mM NaCl, 5% glycerol, 0.05% Tween 20, 7 mM b-mercaptoethanol (b-ME) and protease inhibitors
  • the material was centrifuged at 20,000g for 30 minutes and, again, taken to the ultrasonic equipment with a buffer composed of 50mM sodium phosphate pH 7.4, 300mM NaCl, 7mM b-ME in order to remove traces of the Triton X-100 present in the anterior buffer.
  • the inclusion bodies were recovered by centrifugation and stored at -20 ° C until the moment of use.
  • solubilization buffer composed of 8M urea, 7mM b-ME, 150mM NaCl, 1-20mM Dithiothreitol (DTT), lmM PMSF or 6M Guanidine, 7mM b-ME , 150mM NaCl, 1-20mM Dithiothreitol (DTT) and 1mM PMSF in the proportion of 3 to 7mL of buffer per gram of material that was incubated at room temperature for up to 16h using an orbital shaker (20 revolutions per minute).
  • Protein solubilization was obtained in buffer containing 6M Guanidine, which allowed reversing the protein affinity to the column and obtaining the purified samples (see Figure 6.4, gel C or D) and elutions were obtained in buffer containing 4M of urea and 500 mM imidazole.
  • the tube containing the precipitated protein was kept at room temperature for a few minutes for complete ethanol evaporation. Protein resolubilization was performed after adding 6pL of distilled water and 4pL of running buffer (IX) to the tubes that were heated to 95 ° C and whose fractions were analyzed by SDS-PAGE gel or Western Blotting.
  • IX running buffer
  • Step 1 Buffer A, 10 VC
  • Step 2 0 to 10% buffer B, 10 VC,
  • Table 2 Composition of the buffers used for the standardization of protein purification. All buffers received protease inhibitors such as 1 mM PMSF (Phenylmethanesulfonyl fluoride), protease inhibitor cocktail (Roche) and 7 mM b-mercaptoethanol (b-ME).
  • protease inhibitors such as 1 mM PMSF (Phenylmethanesulfonyl fluoride), protease inhibitor cocktail (Roche) and 7 mM b-mercaptoethanol (b-ME).
  • the antigens were diluted to concentrations below 5mM DTT (due to incompatibility with the affinity chromatographic technique) and filtered.
  • Proteins were not purified in the presence of buffer containing 8M urea.
  • the bond between the solubilized proteins and the nickel-containing resin was obtained only in the presence of a buffer containing 6M guanidine.
  • the replacement of the denaturing agent was essential for the efficiency of binding proteins to nickel resin, which allowed their purification. The reason why this occurred may be related to the lack of exposure of the hexa-histidine tag.
  • the yields obtained with the expression and purification systems of the present invention corresponded to obtaining 10 mg / L for Antigen 1, 8.4mg / mL for Antigen 2, 24mg / L for Antigen 3, 18mg / L for Antigen 4 and 9.6 mg / mL for Antigen 5 that presented purity above> 95%, as shows Figure 1.
  • Table 3 Parameters obtained from the in silico analysis of the amino acid sequences for the polypeptides.
  • the storage buffer was defined after experiments with the objective of preventing losses due to precipitation, maintaining protein stability and removing traces of urea, guanidine or imidazole and decreasing the concentration of salt, since these agents can act as interferents for the immunoassay techniques to be employed.
  • the storage buffer for the antigens after purification, was composed of 4M urea, 10mM sodium phosphate (pH 7.5), 20mM NaCl, 7mM B-mercaptoethanol, 1mM PMSF.
  • Protein stability and integrity analyzes were performed after thawing, during the period of 12 months, of which, periodically, tubes containing 200uL of protein in concentrations ranging from 1.5 to 3 mg / mL were kept at - 20 ° C in stock buffer.
  • the indirect test model consisting of the coupling the antigens to the microspheres or in the coating of antigens to the wells of the polystyrene microplates in order to interact with specific antibodies present in the serum of infected individuals.
  • the assays went through optimization steps until complete standardization with the evaluation of the best protein concentration to be used in the coupling or the ideal mass of antigens for the coating of the microsphere surfaces. Also evaluated were the type of block and concentration, the working dilution of the serum and the secondary antibody, in addition to the estimated reaction time.
  • the immunodiagnostic assays both by the microarray platform and by ELISA were performed using the proteins from the purification by the ⁇ KTA equipment in front of the complete panel of External Quality Analysis (AEQ), whose main characteristic is high reactivity to disease markers.
  • AEQ External Quality Analysis
  • the AEQ panel is characterized by FIOCRUZ, INCQS and consists of 192 sera, of which 23 are reactive for HCV, 26 for HIV 1/2, 30 HTLV 1/2, 31 HBV, 30 Syphilis, 28 Chagas and 21 serums are negative for all pathogens that make up the panel (Figure 3).
  • the supernatant was discarded and the pellet recovered in 10 ml of PBS buffer.
  • the biomass was lysed using the Sonicador Ultrasonic Homogenizer® at 40% amplitude for three cycles of 20 seconds on and 1 minute q / in an ice bath.
  • the lysed extract was centrifuged at 10,000 rpm for 20 minutes and the supernatant was filtered through a 0.45 pm Millipore membrane, followed by a new 0.22 pm membrane filtration.
  • the pellet was discarded and 0.1% sodium azide solution was added to the extract for preservation. After quantification, the material was aliquoted in microtubes that were stored at -20 ° C.
  • the working solution was used at a concentration of 10 mg / mL diluted in PBS buffer pH 7.2 and kept at 4 ° C.
  • the tests were performed with the addition of 50pL of the carbonate / bicarbonate buffer pH 9.6 containing 20ng of the protein of interest for adhesion.
  • the plate was sealed with sealant film and stored at 4 ° C for 16 hours.
  • the excess coating solution was removed by washing with 150pL of PBS-T (3X) and 100pL of a PBS-T blocking solution containing 2% casein was added to the wells, the plate was incubated for 1 hour at 37 ° C, under slight agitation (200rpm).
  • Table 4 Parameters generated through analysis of individual antigens performance by simple ELISA assay.
  • Table 6 Parameters generated through the analysis of antigen performance by ELISA assays in combination.
  • Proteins were coupled to microspheres in the presence of buffers: (1) 1M urea in PBS pH 7.4, (2) Storage buffer containing 4M urea, 20 mM Phosphate pH 7.5, 50mM NaCl , 7mM B-mercaptoethanol, (3) PBS pH 7.4 and (4) 100mM Sodium Carbonate, pH 9.0. These tests were performed in front of the complete AEQ panel containing the 188 sera.
  • Antigen 1 they were coupled at a concentration of 15pg / mL.
  • the coupling in the presence of a buffer containing 4M urea did not show precipitation, nor was there any decrease in the count of the number of microspheres.
  • the parameters obtained from the analysis of the ROC curve for the microspheres coupled in the buffer containing 4M urea demonstrated a superior performance when compared to the results obtained with the couplings in buffers containing 1M urea, PBS or sodium carbonate. Tests carried out with concentrations below 15pg / mL did not favor the performance of the tests for these proteins and there was a loss of sensitivity.
  • the coupling was performed by diluting the antigens to the final volume of 100pL.
  • the antigens were diluted and kept refrigerated until use.
  • the microspheres were homogenized in the matrix flask with the aid of a vortex mixer, followed by an ultrasound bath in Cole-Parmer (Vernon Hills-IL, USA).
  • the matrix flask containing the solution with the microspheres (12,500,000 microspheres / mL) was then shaken in the vortex and in the ultrasound bath for 30 seconds for up to three times. Then, the 80m1 volume containing 1,000,000 microspheres were transferred to a 96-well, flat-bottomed, low-adhesion microplate well.
  • microspheres were washed twice in the Hydroflex plate washer (TECAN, Durham - NC) using the MAGSOAK program.
  • 80pL of Activation Buffer 0.1M NaH 2 P0 4 , pH 6.2
  • the wells were homogenized with the aid of a pipette and the plate was incubated at 25 ° C for 20 minutes, under agitation (250rpm), protected from light. Given the incubation period, the solution was automatically aspirated by selecting the ASP program from the washer, followed by the addition of 100pL of the desired Coupling Buffer in order to balance the environment where the microspheres meet to receive the previously diluted antigen.
  • the plates were sealed with a film, covered in aluminum foil and incubated at 37 ° C for 2 hours, under agitation (250rpm).
  • the MAGSOAK 2 program was activated for the last wash using 100pL of PBS / TBN Blocking Buffer (PBS Buffer pH 7.2, 1% bovine albumin, Tween 20 0.02%, NaN 3 0.01%).
  • PBS Buffer pH 7.2, 1% bovine albumin, Tween 20 0.02%, NaN 3 0.01% The microspheres were transferred to a low-binding Eppendorf tube (USA Scientific) and kept in Blocking Buffer for a minimum of 24 h at 4 ° C as recommended by Luminex Corporation.
  • each well would receive 50pL of a solution containing 2,500 microspheres coupled to the antigen of interest.
  • the matrix flask was vortexed and ultrasound bathed and the working volume was transferred to a 15 or 20mL Falcon tube containing PBS / TBN buffer and 2% E. coli extract solution (10mg / mL).
  • the secondary goat anti-human IgG antibody conjugated to phycoerythrin (R-PE) (Moss Inc., Pasadena-CA, USA) was diluted 1: 1000 in blocking buffer PBS / TBN and 50pL of this dilution were added to the wells.
  • the plate was again incubated for 20 minutes at 37 ° C under protection from light and shaking (250rpm) followed by a washing sequence where, at the end, the washer discarded 100 pL of a buffer called SheathFluid in each well.
  • SheathFluid is a specific buffer developed and marketed by Luminex Corporation for use in reading microspheres by the xMAP 200 platform, acquiring 100 independent events per well.
  • the plate was taken to read the reaction by identifying the microsphere codes and detecting the presence of the secondary antibody.
  • the analysis of the results was performed considering the signs of the Median Intensity of Fluorescence (MFI) and the code of the microsphere coupled in the Luminex 200® equipment to evaluate 100 events per well.
  • MFI Median Intensity of Fluorescence
  • Figures 6 to 10 show the individual performance of Antigens 1 to 5, respectively, presented in the form of a graph through the standardization obtained by the reactivity index (IR). The dotted line represents the cutoff point and the shaded area represents the gray zone (cut off ⁇ 10%). The analysis of the results was performed considering the median signs of immunofluorescence (MFI) and a 95% confidence interval.
  • MFI median signs of immunofluorescence
  • combination 4 was the one that clearly showed the greatest discriminatory capacity among sera, with the greatest distance between MFIs for negative and positive sera.
  • the IR was up to 140 times higher than the IR obtained for negative sera. This assay also demonstrated the smallest standard deviation for negative sera.
  • Figure 15 shows the bar graph where combination 4 presents the greatest separation capacity among the average of the signals for positive and negative samples, whose probability of positivity for this combination was 1,053.7 times. By far, this was the best performance achieved in the present invention. In addition, separation capacities for the other combinations in the multiplex assay were high when compared to the performance of the control antigen.
  • E. Y-box-binding protein 1 interacts with hepatitis C viras NS3 / 4A and influences the equilibrium between viral RNA replication and infectious particle production. Journal of Virology, vol. 85, p. 11022-37, 2011.
  • DIPTI C. A., JAIN, S. K., NAVIN, K.
  • HEPATITIS C MORTALITY. Hepatitis C Kills More Americans than Any Other Infectious Disease. CDC 24/7. May 4, 2016.
  • HCV hepatitis C viras
  • Inhibitors of HCV NS5B polymerase synthesis and stracture-activity relationships of N-1-heteroalkyl-4-hydroxyquinolon-3-ylbenzothiadiazines. Bioorganic and amp; Med Chemistry Letters, v. 15, p. 1577-82, 2005.
  • VITAL SIGNS evaluation of hepatitis C viras infection testing and reporting. MMWR, U.S. sites, CDC. V. 62 (18), 2013.
  • HCV core protein regulates its ability for p53 activation and p21 suppression.

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Abstract

La présente invention concerne des polypeptides à activité immunogène, pouvant être utilisés de manière isolée ou ensemble, et présentant une haute capacité de discrimination pour le triage du virus de l'hépatite C (VHC). Les polypeptides selon l'invention comprennent au moins deux régions antigéniques du VHC sélectionnées parmi une région du nucléocapside et des régions non structurales NS3, NS4 et NS5. L'invention concerne également un acide nucléique, un cassette d'expression, un vecteur d'expression, une cellule hôte, une méthode de production des polypeptides, une composition, l'utilisation des polypeptides, une trousse pour le triage immunologique du VHC et/ou le diagnostic de l'hépatite C, ainsi que des méthodes pour le triage immunologique du VHC et le diagnostic de l'hépatite C.
PCT/BR2019/050454 2018-10-22 2019-10-22 Polypeptide, cassette d'expression, vecteur d'expression, cellule hôte, trousse pour le triage immunologique du vhc et/ou le diagnostic de l'hépatite c, composition, utilisation d'au moins un polypeptide, et méthodes pour produire un polypeptide, pour le triage immunologique du vhc et le diagnostic de l'hépatite c WO2020082145A1 (fr)

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BR102018071672-7A BR102018071672A2 (pt) 2018-10-22 2018-10-22 Polipeptídeo, cassete de expressão, vetor de expressão, célula hospedeira, kit para triagem imunológica de hcv e/ou diagnóstico de hepatite c, composição, uso de pelo menos um polipeptídeo, e, métodos para produzir um polipeptídeo, para triagem imunológica de hcv e para o diagnóstico de hepatite c

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