WO2020082145A1 - Polypeptide, expression cassette, expression vector, host cell, kit for immunological screening for hcv and/or for diagnosing hepatitis c, composition, use of at least one polypeptide and methods for producing a polypeptide, for immunological screening for hcv and for diagnosing hepatitis c - Google Patents

Polypeptide, expression cassette, expression vector, host cell, kit for immunological screening for hcv and/or for diagnosing hepatitis c, composition, use of at least one polypeptide and methods for producing a polypeptide, for immunological screening for hcv and for diagnosing hepatitis 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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French (fr)
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/en

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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

The present invention relates to polypeptides that have immunogenic activity, which can be used alone or together, having high discriminatory capacity for screening the hepatitis C virus (HCV). The polypeptides according to the invention comprise at least two antigenic HCV regions selected from the nucleocapsid region and nonstructural regions NS3, NS4 and NS5. The invention also relates to the nucleic acid, expression cassette, expression vector, host cell, method for producing the polypeptides, composition, use of the polypeptides, kit for immunological screening for HCV and/or for diagnosing hepatitis C, and also to the methods for immunological screening for HCV and for diagnosing hepatitis C.

Description

“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” “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 ”
CAMPO DA INVENÇÃO  FIELD OF THE INVENTION
[001] A presente invenção refere-se ao campo da medicina diagnóstica e da biotecnologia. Mais especificamente, a presente invenção refere-se a polipeptídeos para aplicação diagnóstica na triagem para o vírus da hepatite C. FUNDAMENTOS DA INVENÇÃO  [001] 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
[002] A Hepatite C é uma doença virai silenciosa grave que pode resultar em problemas de saúde a longo prazo. A Organização Mundial da Saúde estima que aproximadamente 170 milhões de pessoas são cronicamente infectadas pelo vírus da hepatite C. Dados do Centro de Controle e Prevenção de Doenças dos Estados Unidos revelaram que a mortalidade anual relacionada à hepatite C em 2013 superou o total de mortes combinadas de outras 60 doenças infecciosas, incluindo infecção pelo HIV, por pneumococos e tuberculose, matando mais que qualquer outra doença infecciosa (HEPATITIS C MORTALITY, 2016).  [002] 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).
[003] Devido à característica silenciosa, 70 a 80% dos infectados desconhecem seu estado sorológico e apenas 20 a 30% podem vir a apresentar sintomas (CHEN e MORGAN, 2006). Por isso, a Organização Mundial da Saúde recomenda que todas as doações de sangue sejam testadas para as hepatites B e C, além de outras doenças infecciosas. O objetivo é garantir a segurança do sangue a ser transfundido e evitar a transmissão virai acidental em pessoas que venham a receber hemoderivados.  [003] Due to the silent characteristic, 70 to 80% of those infected are unaware of their serological status and only 20 to 30% may have symptoms (CHEN and MORGAN, 2006). Therefore, the World Health Organization recommends that all blood donations be tested for hepatitis B and C, in addition to other infectious diseases. The goal is to ensure the safety of the blood to be transfused and to prevent accidental viral transmission in people who will receive blood products.
[004] O agente etiológico da hepatite C é um vírus esférico de 55-65 nm de diâmetro, envelopado, cujo genoma é uma molécula de 9,3 kilobase com organização genética de flavi vírus (MELLO, 2014). A replicação do vírus ocorre via RNA, polaridade positiva, cadeia simples (ssRNA), linear, que contém regiões não traduzidas (UTRs) e que flanqueiam uma sequência de leitura aberta ininterrupta que codifica uma única poliproteína de 3.011 aminoácidos: NH2-C- El-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5BCOOH (LIN et al., 1994). Esta poliproteína precursora é posteriormente clivada por proteases celulares e virais liberando proteínas estruturais (C-El-E2-p7) e não estmturais (NS2-NS3-NS4A- NS4B-NS5A-NS5B), que incluem elementos de controles necessários para a tradução e replicação virai. [004] 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.
[005] Sabe-se que proteínas não estruturais participam da replicação virai, enquanto as estmturais fazem parte da estmtura do capsídeo e formam as glicoproteínas do envelope. As proteínas estmturais são liberadas da poliproteína após clivagem por proteases celulares, enquanto as não-estmturais são clivadas por proteases virais (de VICENTE et al., 2009; CHATEL-CHAIX, et al., 2011). Cada uma delas assume diferentes funções no ciclo do víms.  [005] 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.
[006] A proteína do nucleocapsídeo regula processos celulares, contribuindo para a replicação e patogênese, além de estar envolvida na montagem e empacotamento do genoma virai (LIN et al., 1993; LIN et al., 1994). Esta proteína possui 191 aminoácidos e está localizada na região N-terminal da poliproteína virai (McLAUCHLAN et al., 2002).  [006] 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).
[007] As proteínas do envelope EI e E2 formam heterodímeros que representam a forma nativa do envelope de HCV. São altamente glicosiladas e vulneráveis a mutações genéticas (ZENG et al., 2012), consequentemente, podem alterar importantes propriedades antigênicas do víms promovendo o escape de anticorpos neutralizantes. Também desempenham importante papel na entrada do víms na célula (ZENG et al., 2012) e, embora o papel da região EI e sua associação com os ligantes da superfície da célula ainda não é clara, o papel da proteína E2 está melhor definido, atuando diretamente na ligação a receptores de superfície da célula, dentre eles o CD81 (PIVER et al., 2010; FREEDMAN et al., 2016). O CD81 é expresso na maioria das células humanas, incluindo hepatócitos e a união dessa molécula com a proteína E2 do vírus induz alterações funcionais e pode estimular a proliferação de linfócitos B não ativados ou à produção de citocinas inflamatórias (ROSA et al., 2005). [007] 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) .
A região que codifica para a proteína p7 (HCV-NS1) é uma glicoproteína de envelope que pode ser encontrada nas membranas celulares de indivíduos infectados. A proteína é clivada no retículo endoplasmático em reação mediada por peptídeo sinal. Esta é uma região essencial para a liberação de viriões, funcionando como canais iônicos (HUANG et al., 2010).  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).
[008] A região não estrutural NS2 é uma proteína de 21-23 kDa que interage com outras proteínas não estmturais para participar da montagem da partícula virai. Trata-se de uma cisteína com atividade de protease responsável pela clivagem da região NS2/NS3 (POPESCU et al., 2011 ; KIM et al., 2012). [008] 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).
[009] A proteína NS3 possui atividade catalítica de serina-protease e helicase que juntamente com o cofator NS4A, participa da clivagem das regiões NS3/4A, NS4A/4B, NS4B/5A e NS5A/5B durante o processamento (De VICENTE et al., 2009; FREEDMAN et al., 2016). [009] 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).
[0010] A NS4A é necessária para a fosforilação da polimerase NS5A. [0010] NS4A is necessary for the phosphorylation of NS5A polymerase.
Há pouca informação sobre a função da proteína NS4B. Sugere-se que ela participa da formação de uma membrana onde ocorre a replicação do RNA do HCV (KIM et al., 1996; MASSARIOL et al., 2010). There is little information about the function of the NS4B protein. It is suggested that it participates in the formation of a membrane where HCV RNA replication occurs (KIM et al., 1996; MASSARIOL et al., 2010).
[0011] A NS5 A tem importante papel na replicação virai, na modulação da sinalização celular e na resposta ao interferon (YAMANAKA et al., 2002). A região B da proteína (NS5B) atua como uma RNA polimerase RNA dependente e desempenha uma importante função na síntese do novo RNA (PRATT et al., 2005; OLIVEIRA, 2007; KEYVANI et al., 2012).  [0011] 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 (NS5B) 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).
[0012] A replicação virai rápida e a falta de revisões de erros pela RNA- polimerase virai são as principais razões pelas quais o genoma do HCV sofre frequentes mutações. Isto explica o fato de o vírus da hepatite C possuir oficialmente seis genótipos, identificados por números arábicos (de 1 a 6) que são subdivididos em mais de 50 subtipos (la, lb, 2a, etc.)· A diferença na sequência de nucleotídeos entre os genótipos 1 ao 6 pode alcançar 30 a 35%, ao passo que os subtipos diferem em cerca de 20% (SIMMONDS et al., 1993 (b). [0012] Rapid viral replication and the lack of error revisions by viral RNA polymerase are the main reasons why the HCV genome undergoes frequent mutations. This explains why the hepatitis C virus has officially six genotypes, identified by Arabic numbers (from 1 to 6) that are subdivided into more than 50 subtypes (la, lb, 2a, etc.) · The difference in nucleotide sequence between genotypes 1 to 6 can reach 30 to 35 %, while subtypes differ by about 20% (SIMMONDS et al., 1993 (b).
[0013] Os subtipos 1, 2 e 3 têm distribuição mundial, sendo o subtipo 1 o mais comum. Esse é o genótipo mais resistente ao tratamento e representa 60% da infecção global e predomina na América do Norte e Sul, na Europa do Norte e Europa Oriental (GUPTA et al., 2014). O genótipo 2 representa 9,1% dos casos de hepatite C em todo o mundo e é descrito como um dos genótipos com melhor resposta para o tratamento (HANAFIAH et al., 2013). O genótipo 3 é o segundo mais prevalente no mundo, sendo endémico no sudeste da Asia e acometendo 30% da população. O genótipo 4 tem maior prevalência no Oriente Médio, onde é responsável por 90% dos casos de hepatite C no Egito, no norte da África e na África central. O genótipo 5 possui maior distribuição na África do Sul e pouco se sabe sobre ele, que representa 0,8% da população mundial. O genótipo 6 corresponde a 5,4% das hepatites no sudeste da Asia e uma distribuição mundial inferior ao genótipo 5 (SHEPARD et al., 2005; HANAFIAH et al., 2013; GUPTA et al., 2014; MESSINA et al., 2015). [0013] 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).
[0014] A extensa heterogeneidade genética do HCV tem implicações importantes na resposta imune, na clínica e no diagnóstico da doença, o que toma um desafio o desenvolvimento de vacinas e a resposta às terapias.  [0014] 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.
[0015] Atualmente, o critério mínimo estabelecido pela Organização [0015] Currently, the minimum criteria established by the Organization
Mundial da Saúde para se certificar do diagnóstico do HCV é que ele apresente uma sensibilidade >99% para os testes rápidos e de 100% para ensaios ELISA e que a especificidade seja de >98%. World Health Organization to make sure the HCV diagnosis is that it has a sensitivity> 99% for rapid tests and 100% for ELISA assays and that the specificity is> 98%.
[0016] São dois os principais marcadores da infecção para o víms da hepatite C: o marcador sorológico anti-HCV e a presença do RNA do víms no soro do indivíduo. Os ensaios sorológicos possuem alta sensibilidade, mas não distinguem infecção ativa ou resolvida, podendo ser desenvolvidos para cepas específicas a partir de uma região que conferem sensibilidades menores quando utilizados em países cujos genótipos circulantes sejam diferentes (BRAND, 2014). Por este motivo, recomenda-se a confirmação da infecção pelo vírus por testes suplementares ou confirmatórios de detecção do genoma virai (CLOHERTY et al., 2015) fazendo uso da técnica da reação em cadeia da polimerase (PCR), de forma a permitir a identificação dos subtipos virais. Contudo, devido ao alto custo, esta técnica não é plenamente acessível em áreas geograficamente remotas e de baixa economia. [0016] 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.
[0017] Os primeiros imunoensaios no formato EIA/ELIS A foram desenvolvidos para a detecção utilizando um único alvo ou pela exposição de apenas um epítopo. Estes ensaios apresentaram altos índices de reatividade cruzada, baixa sensibilidade e a necessidade de repetições. No caso específico da identificação do víms da hepatite C, o diagnóstico tinha por base a região não estrutural da proteína NS4 que, mesmo apresentando sensibilidade de 40%, permitiu o início da prevenção por transmissão transfusional.  [0017] 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.
[0018] Devido às medidas preventivas e com o advento de ensaios mais sensíveis foram introduzidos aos testes para diagnóstico outras regiões antigênicas do vírus, o que permitiu a classificação dos testes ensaios de segunda, terceira e quarta geração (KAMILI et al., 2012; GUPTA et al., 2014), que levaram do aumento da sensibilidade e à diminuição no número de casos de transmissão por via transfusional.  [0018] Due to preventive measures and with the advent of more sensitive assays, other antigenic regions of the virus were introduced to the diagnostic tests, which allowed the classification of the tests of second, third and fourth generation tests (KAMILI et al., 2012; GUPTA et al., 2014), which led to increased sensitivity and a decrease in the number of cases of transfusion transmission.
[0019] Os testes em microesferas apresentam sensibilidade superior aos testes comerciais ELISA e é capaz de analisar misturas complexas. A empresa Luminex Corporation desenvolveu um sistema de microarranjo líquido para analisar ensaios fluorescentes tendo as esferas como suportes sólidos, sistema esse nomeado LUMINEX. Esse sistema faz uso da tecnologia da microfluídica e da ligação de moléculas alvo às microesferas onde acontece a reação.  [0019] 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.
[0020] Os ensaios para detecção de HCV disponíveis na técnica, apesar de empregarem antígenos multiepítopos, ainda apresentam muitas desvantagens, como, por exemplo, o fato de serem desenvolvidos para um perfil genético de cepas circulantes em regiões específicas que podem diferir dos genótipos que circulam em regiões geográficas onde são comercializados ou não serem aplicados em ensaios multiplex (FONSECA et al., 2011 ; ARAÚJO et al., 2011 ; GALDINO et al., 2015 e DIPTI et al., 2006). [0020] The 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; ARAÚJO et al., 2011; GALDINO et al., 2015 and DIPTI et al., 2006).
[0021] Adicionalmente, os ensaios ELISA para triagem da hepatite C apresentam alta variabilidade quanto a sensibilidade quando aplicados em países ricos e pobres, cujos desempenhos são consideravelmente menores em países pobres (KHUROO et al., 2015). Além disso, os imunoensaios apresentam altos índices de falso-negativos quando utilizados para diagnosticar pacientes imunodeficientes ou hemodialisados. Soma-se a isto, o fato de que os testes sorológicos disponíveis não são capazes de diferenciar a infecção aguda da infecção crónica (SHIVKUMAR et al., 2012). Por fim, a variabilidade genética para o vírus da hepatite C chega a apresentar 30-35% (SIMMONDS et al., 1993) e para alguns epítopos até 49%, o que pode gerar respostas genótipo-específicas e diminuir a sensibilidade de um ensaio.  [0021] Additionally, 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). In addition, immunoassays have high rates of false negatives when used to diagnose immunodeficient or hemodialysis patients. Added to this, the fact that the serological tests available are not able to differentiate between acute and chronic infection (SHIVKUMAR et al., 2012). Finally, 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 .
[0022] Outros desafios relacionados aos ensaios sorológicos para [0022] Other challenges related to serological tests for
HCV, além do tempo de execução, é a presença de uma zona cinza a partir da qual alguns resultados não são interpretáveis (S ALUDES et al., 2014). No último caso, recomenda-se a repetição do ensaio sorológico em duplicata para confirmar a condição do paciente. Se as repetições estiverem abaixo do limite do ensaio, a amostra deve ser considerada negativa. Se o resultado for superior ou igual ao ponto de corte, a amostra deve ser testada por testes confirmatórios. 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.
[0023] Apesar da recomendação, os testes confirmatórios para o diagnóstico da hepatite C não são amplamente utilizados em países pobres, por razões financeiras ou relacionadas à sua complexidade ou, ainda, pela falta de laboratórios equipados e pessoal treinado (KAMILI et al., 2012; VITAL SIGNS, 2016). [0023] Despite the recommendation, confirmatory tests for the diagnosis of hepatitis C are not widely used in poor countries, for financial reasons or related to their complexity or, due to the lack of equipped laboratories and trained personnel (KAMILI et al., 2012; VITAL SIGNS, 2016).
[0024] As vantagens da invenção serão evidentes na descrição da invenção fornecida neste documento. [0024] The advantages of the invention will be evident in the description of the provided in this document.
SUMÁRIO DA INVENÇÃO  SUMMARY OF THE INVENTION
[0025] A presente invenção tem por objetivo prover polipeptídeos a partir de diferentes regiões do víms da hepatite C que solucionem os principais problemas do estado da técnica listados anteriormente.  [0025] 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.
[0026] Os referidos polipeptídeos apresentam alto grau de pureza e alta capacidade de discriminação sorológica para aplicação diagnóstica na triagem para o vírus da hepatite C, com a finalidade de cobrir 100% dos pacientes positivos, considerando a variabilidade representada pelos diferentes genótipos do vírus.  [0026] 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.
[0027] Particularmente, foram desenvolvidos polipeptídeos que podem ser utilizados isoladamente ou em conjunto em ensaios imunodiagnósticos com capacidade de discriminação muito alta para a triagem do vírus da hepatite C.  [0027] In particular, 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.
[0028] Em um primeiro aspecto, a presente invenção provê um polipeptídeo compreendendo pelo menos duas regiões antigênicas do vírus da hepatite C (HCV) selecionadas a partir do grupo selecionado de região do nucleocapsídeo (NC) e regiões não estruturais NS3, NS4 e NS5. [0028] In a first aspect, 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 .
[0029] Em um segundo aspecto, a presente invenção provê um polinucleotídeo que codifica o polipeptídeo.  [0029] In a second aspect, the present invention provides a polynucleotide that encodes the polypeptide.
[0030] Em um terceiro aspecto, a presente invenção provê um cassete de expressão compreendendo o referido polinucleotídeo.  [0030] In a third aspect, the present invention provides an expression cassette comprising said polynucleotide.
[0031] Em um quarto aspecto, a presente invenção provê um vetor de expressão que compreende o ácido nucleico o cassete de expressão descritos acima.  [0031] In a fourth aspect, the present invention provides an expression vector comprising the nucleic acid and the expression cassette described above.
[0032] Em um quinto aspecto, a presente invenção provê uma célula hospedeira que compreende o ácido nucleico, vetor de expressão ou cassete de expressão como definidos acima.  [0032] In a fifth aspect, the present invention provides a host cell comprising the nucleic acid, expression vector or expression cassette as defined above.
[0033] Em um sexto aspecto, a presente invenção provê um método para produzir um polipeptídeo que compreende: (a) transformar uma célula hospedeira com o polinucleotídeo como definido acima, [0033] In a sixth aspect, the present invention provides a method for producing a polypeptide comprising: (a) transforming a host cell with the polynucleotide as defined above,
(b) cultivar dita célula para a produção do polipeptídeo; e (b) cultivating said cell for the production of the polypeptide; and
(c) isolar dito polipeptídeo de dita célula ou do meio de cultura circundando dita célula. (c) isolating said polypeptide from said cell or culture medium surrounding said cell.
[0034] Em um sétimo aspecto, a presente invenção provê uma composição que compreende um polipeptídeo como definido acima ou uma combinação de dois ou mais polipeptídeos definidos acima.  [0034] In a seventh aspect, the present invention provides a composition comprising a polypeptide as defined above or a combination of two or more polypeptides defined above.
[0035] Em um oitavo aspecto, a presente invenção provê um kit para triagem imunológica de HCV e/ou diagnóstico de hepatite C que compreende pelo menos um polipeptídeo da presente invenção.  [0035] In an eighth aspect, 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.
[0036] Em um nono aspecto, a invenção provê o uso de pelo menos um polipeptídeo, da composição ou do kit descritos acima na triagem imunológica de HCV e/ou diagnóstico de hepatite C.  [0036] In a ninth aspect, 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.
[0037] Em um décimo aspecto, a invenção provê um método para triagem imunológica do HCV, que compreende as etapas de:  [0037] In a tenth aspect, the invention provides a method for immunological screening of HCV, which comprises the steps of:
(a) prover um ou mais polipeptídeos ou uma composição como definidos acima;  (a) providing one or more polypeptides or a composition as defined above;
(b) contatar um ou mais peptídeos ou composição descritos acima com a amostra biológica a ser testada por um tempo suficiente e sob condições suficientes para formação de complexos anticorpo/ antígeno; e  (b) contacting one or more peptides or composition described above with the biological sample to be tested for a sufficient time and under sufficient conditions to form antibody / antigen complexes; and
(c) detectar o complexo antígeno/anticorpo formado na etapa (b), pela adição de um meio de detecção, capaz de gerar um sinal detectável na presença do referido complexo antígeno/anticorpo.  (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.
[0038] Em um décimo primeiro aspecto, a invenção provê um método para diagnóstico de hepatite C, que compreende as etapas de:  [0038] In an eleventh aspect, the invention provides a method for diagnosing hepatitis C, which comprises the steps of:
(b) prover um ou mais polipeptídeos ou uma composição como definidos acima;  (b) providing one or more polypeptides or a composition as defined above;
(b) contatar um ou mais peptídeos ou composição descritos acima com a amostra biológica a ser testada por um tempo suficiente e sob condições suficientes para formação de complexos anticorpo/ antígeno; e (b) contacting one or more peptides or composition described above with the biological sample to be tested for a sufficient time and under sufficient conditions to form antibody / antigen complexes; and
(c) detectar o complexo antígeno/anticorpo formado na etapa (b), pela adição de um meio de detecção, capaz de gerar um sinal detectável na presença do referido complexo antígeno/anticorpo.  (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.
BREVE DESCRIÇÃO DAS FIGURAS  BRIEF DESCRIPTION OF THE FIGURES
[0039] A Figura 1 mostra a análise quantitativa das frações obtidas após a purificação dos polipeptídeos em cromatógrafo ÀKTA; A) Gel SDS-PAGE 13 %; B) Western Blotting M: Marcador de massa molecular. Os quadros delimitam os polipeptídeos de interesse para a presente invenção.  [0039] Figure 1 shows the quantitative analysis of the fractions obtained after purification of the polypeptides in an ÀKTA chromatograph; A) 13% SDS-PAGE gel; B) Western Blotting M: Molecular weight marker. The tables delineate the polypeptides of interest to the present invention.
[0040] A Figura 2 mostra um gel de SDS-PAGE (13%) revelando em [0040] Figure 2 shows an SDS-PAGE gel (13%) revealing in
A) o estado dos polipeptídeos logo a purificação e em B) o estado dos polipeptídeos após a sequência de descongelamentos em banho de gelo, após um ano. Os quadros delimitam os polipeptídeos de interesse para a presente invenção. A) the state of the polypeptides after purification and in B) the state of the polypeptides after the thaw sequence in an ice bath, after one year. The tables delineate the polypeptides of interest to the present invention.
[0041] A Figura 3 mostra o desenho do painel completo AEQ composto por 188 soros, dos quais 23 são reativos para HCV, 26 para HIV 1/2, [0041] 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,
30 HTLV 1/2, 31 HBV, 30 Sífilis, 28 Chagas e 21 soros são negativos para todos os patógenos que compõe o painel. 30 HTLV 1/2, 31 HBV, 30 Syphilis, 28 Chagas and 21 serums are negative for all pathogens that make up the panel.
[0042] A Figura 4 mostra os parâmetros gerados através das análises de desempenho individual dos antígenos por ensaio EFISA.  [0042] Figure 4 shows the parameters generated through the analysis of individual antigens performance by EFISA assay.
[0043] A Figura 5 mostra os parâmetros gerados através das análises de desempenho dos antígenos por ensaios EFISA em combinação.  [0043] Figure 5 shows the parameters generated through the analysis of antigen performance by EFISA assays in combination.
[0044] A Figura 6 mostra os parâmetros obtidos a partir do desempenho do Antígeno 1 , identificando a melhor condição de acoplamento.  [0044] Figure 6 shows the parameters obtained from the performance of Antigen 1, identifying the best coupling condition.
[0045] A Figura 7 mostra os parâmetros obtidos a partir do desempenho do Antígeno 2, identificando a melhor condição de acoplamento. [0045] Figure 7 shows the parameters obtained from the performance of Antigen 2, identifying the best coupling condition.
[0046] A Figura 8 mostra os parâmetros obtidos a partir do desempenho do Antígeno 3, identificando a melhor condição de acoplamento. [0047] A Figura 9 mostra os parâmetros obtidos a partir do desempenho do Antígeno 4, identificando a melhor condição de acoplamento. [0046] Figure 8 shows the parameters obtained from the performance of Antigen 3, identifying the best coupling condition. [0047] Figure 9 shows the parameters obtained from the performance of Antigen 4, identifying the best coupling condition.
[0048] A Figura 10 mostra os parâmetros obtidos a partir do desempenho do Antígeno 5, identificando a melhor condição de acoplamento. [0048] Figure 10 shows the parameters obtained from the performance of Antigen 5, identifying the best coupling condition.
[0049] A Figura 11 mostra a análise dos ensaios em combinação realizados no formato multiplex via sistema LUMINEX. [0049] Figure 11 shows the analysis of the combined tests performed in the multiplex format via LUMINEX system.
[0050] A Figura 12 mostra parâmetros gráficos para avaliação da capacidade individual de separação dos antígenos por ensaios no formato ELISA, em que A mostra SNR ( Signal to Noise Ratio) do ensaio ELISA e B mostra a média dos sinais por ensaio ELISA, na qual os sinais“Pos” = a média dos soros positivos e“Neg” = a média dos soros negativos.  [0050] Figure 12 shows graphical parameters for assessing the individual ability to separate antigens by ELISA assays, in which A shows SNR (Signal to Noise Ratio) of the ELISA assay and B shows the average of the signals per ELISA assay, in which the signs “Pos” = the average of the positive sera and “Neg” = the average of the negative sera.
[0051] A Figura 13 mostra os parâmetros gráficos para avaliação da capacidade individual de separação dos antígenos por ensaios no formato LUMINEX singlepex, em que A mostra o SNR ( Signal to Noise Ratio) do ensaio LUMINEX e B mostra média dos sinais obtidos, na qual os“Sinais Pos” = a média dos soros positivos e“Sinais Neg” = a média dos soros negativos.  [0051] Figure 13 shows the graphical parameters for assessing the individual capacity of antigen separation by tests in the LUMINEX singlepex format, in which A shows the SNR (Signal to Noise Ratio) of the LUMINEX test and B shows the average of the obtained signals, in which the “Pos signals” = the average of the positive sera and “Neg signals” = the average of the negative sera.
[0052] A Figura 14 mostra parâmetros gráficos para avaliação da capacidade de separação de combinações dos antígenos por ensaios no formato ELISA combinado, em que A mostra SNR do ensaio ELISA e B mostra média dos sinais das combinações de antígenos, na qual os sinais“Sinais +” = a média dos soros positivos e“Sinais -” = a média dos soros negativos. [0052] Figure 14 shows graphical parameters for evaluating the ability to separate combinations of antigens by assays in the combined ELISA format, in which A shows the SNR of the ELISA assay and B shows the mean of the signals from the antigen combinations, in which the signs “ Signals + ”= the mean of positive sera and“ Signals - ”= the mean of negative sera.
[0053] A Figura 15 mostra parâmetros gráficos para avaliação da capacidade de separação de combinações dos antígenos por ensaios no formato LUMINEX multiplex , em que A mostra SNR ( Signal to Noise Ratio) das combinações no ensaio LUMINEX e B mostra média dos sinais obtidos para as combinações no LUMINEX, na qual os sinais“Positivos” = a média dos soros positivos e“Negativos” = a média dos soros negativos.  [0053] Figure 15 shows graphic parameters for evaluating the ability to separate combinations of antigens by assays in the LUMINEX multiplex format, in which A shows SNR (Signal to Noise Ratio) of the combinations in the LUMINEX and B assay shows the average of the signals obtained for the combinations in LUMINEX, in which the signs "Positive" = the mean of positive sera and "Negative" = the mean of negative sera.
DESCRIÇÃO DETALHADA DA INVENÇÃO  DETAILED DESCRIPTION OF THE INVENTION
[0054] A não ser que sejam definidos de maneira diferente, todos os termos técnicos e científicos aqui utilizados têm o mesmo significado entendido por um técnico no assunto ao qual a invenção pertence. A terminologia utilizada na descrição da invenção tem finalidade de descrever concretizações particulares somente, e não tem a intenção de limitar o escopo dos ensinamentos. A não ser que seja indicado de forma diferente, todos os números expressando quantidades, porcentagens e proporções, e outros valores numéricos usados no relatório descritivo e nas reivindicações, devem ser entendidos como sendo modificados, em todos os casos, pelo termo“cerca de”. Assim, a não ser que seja indicado o contrário, os parâmetros numéricos mostrados no relatório descritivo e nas reivindicações são aproximações que podem variar, dependendo das propriedades a serem obtidas. [0054] Unless they are defined differently, all technical and scientific terms used here have the same meaning understood by a technician in the subject to which the invention belongs. The terminology used in describing the invention is intended to describe particular embodiments only, and is not intended to limit the scope of the teachings. Unless otherwise indicated, all numbers expressing quantities, percentages and proportions, and other numerical values used in the specification and in the claims, should be understood as being modified, in all cases, by the term "about" . Thus, unless otherwise indicated, the numerical parameters shown in the specification and in the claims are approximations that may vary, depending on the properties to be obtained.
[0055] A prática da presente invenção irá empregar, a menos que indicado de forma diferente, métodos convencionais de química, bioquímica, técnicas de DNA recombinante e imunologia, dentro do conhecimento da técnica. Tais técnicas são explicadas completamente na literatura. Veja, por exemplo, Fundamental Virology, 2a Edição, vols. I & II (B.N. Fields e D. M. Knipe, eds.); Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir e C.C. Blackwell eds., Blackwell Scientific Publications); T.E. Creighton, Proteins: Structures e Molecular Properties (W.H. Freeman e Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., edição atual); Sambrook, e cols., Molecular Cloning: A Laboratory Manual (2a Edição, 1989); Methods In Enzymology (S. Colowick e N. Kaplan eds., Academic Press, Inc.). [0055] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, recombinant DNA techniques and immunology, within the skill of the art. Such techniques are fully explained in the literature. See, eg, Fundamental Virology, 2nd Edition, vols. I & II (BN Fields and DM Knipe, eds.); Handbook of Experimental Immunology, Vols. I-IV (DM Weir and CC Blackwell eds., Blackwell Scientific Publications); TE Creighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); AL Lehninger, Biochemistry (Worth Publishers, Inc., current edition); Sambrook et al, Molecular Cloning:. A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.).
[0056] Os presentes inventores solucionaram o problema do estado da técnica provendo polipeptídeos construídos a partir de diferentes regiões do vírus da hepatite C, de diferentes genótipos, com alto grau de pureza e alta capacidade de discriminação sorológica. As análises revelaram que os antígenos da invenção, usados em ensaios de triagem imunológica, apresentaram maior desempenho e melhor capacidade de separação entre os soros positivos e negativos, além de não ter sido detectada qualquer reação cruzada quando expostos a amostras positivas para outras doenças infecciosas. [0056] 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.
[0057] Em um primeiro aspecto, a presente invenção provê um polipeptídeo compreendendo pelo menos duas regiões antigênicas do HCV selecionadas a partir do grupo selecionado de região do nucleocapsídeo (NC) e regiões não estruturais NS3, NS4 e NS5.  [0057] In a first aspect, 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.
[0058] As referidas regiões antigênicas do HCV são conhecidas e apresentam variabilidade no desempenho, o que é amplamente discutido na arte relacionada ao diagnóstico da hepatite C. Com base no estado da técnica, principalmente no que se refere à variabilidade do HCV, foram definidas sequências de regiões antigênica do vírus para os compor desenhos dos polipeptídeos da presente invenção.  [0058] The referred 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.
[0059] Atentando-se para a baixa identidade entre epítopos e considerando a variabilidade entre os genótipos existentes, os inventores desenvolveram polipeptídeos específicos com a possibilidade de utilizá-los individualmente ou em conjunto, a fim de obter maior representatividade, tendo em vista que a variabilidade apresentada pode levar ao não reconhecimento de anticorpos genótipo-específico.  [0059] Paying attention to the low identity between epitopes and considering the variability between the existing genotypes, the inventors developed specific polypeptides with the possibility of using them individually or together, in order to obtain greater representativeness, considering that the presented variability may lead to the non-recognition of genotype-specific antibodies.
[0060] Dessa forma, os inventores desenvolveram polipeptídeos multiepítopo obtidos pela junção de regiões estruturais e não estruturais do HCV.  [0060] In this way, the inventors developed multiepitope polypeptides obtained by joining structural and non-structural regions of HCV.
[0061] Inicialmente, foram desenvolvidos 14 clones (nomeadas por códigos numéricos de 4.1 a 4.14, conforme identificado nas figuras), os quais continham diferentes combinações entre a região imunogênica da proteína estrutural do nucleocapsídeo e as regiões não estmturais NS3, NS4 e NS5 para os genótipos 1 ao 6. A Tabela 1 abaixo identifica as construções correspondentes para cada um dos 14 clones. [0061] Initially, 14 clones were developed (named by numeric codes from 4.1 to 4.14, as identified in the figures), which contained different combinations between the immunogenic region of the structural protein of the nucleocapsid and the non-structural regions NS3, NS4 and NS5 for genotypes 1 to 6. Table 1 below identifies the corresponding constructs for each of the 14 clones.
Tabela 1 : identificação dos 14 clones construídos, com as regiões incluídas em cada um e seus respectivos genótipos.Table 1: identification of the 14 built clones, with the regions included in each and their respective genotypes.
Figure imgf000014_0001
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000015_0001
[0062] Após testes de expressão e purificação, os clones 4.2, 4.3, 4.4,[0062] After expression and purification tests, clones 4.2, 4.3, 4.4,
4.6 e 4.10 foram descartados, tendo em vista os resultados negativos obtidos com os mesmos (resultados não mostrados). 4.6 and 4.10 were discarded, in view of the negative results obtained with them (results not shown).
[0063] Os nove clones expressos e purificados com sucesso (clones 4.1, [0063] The nine clones successfully expressed and purified (clones 4.1,
4.5, 4.7, 4.8, 4.9, 4,11, 4.12, 4.13 e 4.14) foram testados em imunoensaios, evidenciando que todos os antígenos purificados possuem eficiência compatível com as características desejadas para a aplicação diagnóstica na triagem da hepatite C. 4.5, 4.7, 4.8, 4.9, 4.11, 4.12, 4.13 and 4.14) were tested in immunoassays, showing that all purified antigens have efficiency compatible with the desired characteristics for the diagnostic application in the screening of hepatitis C.
[0064] Dentre os clones desenvolvidos pelos inventores, o uso combinado de cinco deles apresentou melhor resultado em ensaios de combinação, quais sejam, os clones 4.7, 4.11, 4.12, 4.13 e 4.14. Esses clones apresentam os genótipos lb e 3a, os quais são os mais prevalentes no Brasil, e foram construídos tendo como sequências de referência as sequências da poliproteína de HCV ID #ACH53426.1 e #ACZ60118.1, respectivamente. [0065] No contexto da presente invenção, os polipeptídeos 4.7, 4.11,[0064] Among the clones developed by the inventors, the combined use of five of them showed better results in combination tests, namely, clones 4.7, 4.11, 4.12, 4.13 and 4.14. These clones 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. [0065] In the context of the present invention, polypeptides 4.7, 4.11,
4.12, 4.13 e 4.14 serão também referidos como antígenos 1 a 5, respectivamente: 4.12, 4.13 and 4.14 will also be referred to as antigens 1 to 5, respectively:
Antígeno 1 : SEQ ID NO: 1  Antigen 1: SEQ ID NO: 1
Sequência de Referência (SR): ID #ACZ60118.1  Reference String (SR): ID # ACZ60118.1
Região NC-3a: aminoácidos 1-119 da SR  NC-3a region: amino acids 1-119 of SR
Região NS4-3a: aminoácidos 1691-1731/1789- NS4-3a region: amino acids 1691-1731 / 1789-
1867/1922-1940 da SR SR 1867 / 1922-1940
Região NS5-3a: aminoácidos 2216-2319 da SR  NS5-3a region: amino acids 2216-2319 of SR
Antígeno 2: SEQ ID NO: 3  Antigen 2: SEQ ID NO: 3
Sequência de Referência (SR): ID #ACH53426.1 Região NC-lb: aminoácidos 2-119 da SR  Reference Sequence (SR): ID # ACH53426.1 NC-lb Region: amino acids 2-119 of the SR
Região NS4-lb: aminoácidos 1691-1731/1789- NS4-lb Region: amino acids 1691-1731 / 1789-
1867/1922-1940 da SR SR 1867 / 1922-1940
Antígeno 3: SEQ ID NO: 5  Antigen 3: SEQ ID NO: 5
Sequência de Referência (SR): ID #ACH53426.1  Reference String (SR): ID # ACH53426.1
Região NS3-lb: aminoácidos 1192-1459 da SR  NS3-lb region: amino acids 1192-1459 from SR
Região NS5-lb: aminoácidos 2212-2313 da SR  NS5-lb region: amino acids 2212-2313 from SR
Antígeno 4: SEQ ID NO: 7  Antigen 4: SEQ ID NO: 7
Sequência de Referência (SR): ID #ACH53426.1  Reference String (SR): ID # ACH53426.1
Região NS3-lb: aminoácidos 1192-1459 da SR  NS3-lb region: amino acids 1192-1459 from SR
Região NS4-lb: aminoácidos 1691-1731/1789- NS4-lb Region: amino acids 1691-1731 / 1789-
1867/1922-1940 da SR SR 1867 / 1922-1940
Antígeno 5: SEP ID NO: 9  Antigen 5: SEP ID NO: 9
Sequência de Referência (SR): ID #ACH53426.1  Reference String (SR): ID # ACH53426.1
Região NC-lb: aminoácidos 1-190 da SR  NC-lb region: amino acids 1-190 of SR
Região NS5-lb: aminoácidos 2212-2313 da SR  NS5-lb region: amino acids 2212-2313 from SR
[0066] Em um aspecto preferencial, os polipeptídeos são pelo menos[0066] In a preferred aspect, polypeptides are at least
90%, pelo menos 91%, pelo menos 92%, pelo menos 93%, pelo menos 94%, pelo menos 95%, pelo menos 96%, pelo menos 97%, pelo menos 98% ou pelo menos 99% idênticos às sequências de aminoácidos das SEQ ID NOs: 1, 3, 5, 7 e 9, em que um ou mais aminoácidos são opcionalmente substituídos, adicionados ou deletados em relação à sequência das SEQ ID NOs: 1, 3, 5, 7 e 9. 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least at least 99% identical to the amino acid sequences of SEQ ID NOs: 1, 3, 5, 7 and 9, where one or more amino acids are optionally substituted, added or deleted in relation to the sequence of SEQ ID NOs: 1, 3, 5 , 7 and 9.
[0067] Em um aspecto ainda mais preferencial, os polipeptídeos consistem nas sequências de aminoácidos das SEQ ID NOs: 1, 3, 5, 7 e 9.  [0067] In an even more preferred aspect, polypeptides consist of the amino acid sequences of SEQ ID NOs: 1, 3, 5, 7 and 9.
[0068] Os polipeptídeos da presente invenção apresentaram reprodutibilidade quanto à sensibilidade e especificidade. Isto sugere que as proteínas desenvolvidas podem se manter estáveis por longos períodos, mantendo sua capacidade reativa. E possível que a composição do tampão de estoque pode ter favorecido sua estabilidade, o uso de inibidores de proteases, a presença de agente desnaturante no tampão ou, até mesmo, devido à sequência de aminoácidos das mesmas. Proteínas estáveis são consideradas quando existe interesse de aplicação diagnóstica.  [0068] The 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.
[0069] Conforme empregado ao longo do presente pedido, o termo [0069] As used throughout this application, the term
“aminoácido” denota o grupo a-aminoácidos que diretamente ou na forma de um precursor pode ser codificado por um ácido nucleico. Os aminoácidos individuais são codificados por ácidos nucleicos consistindo de três nucleotídeos, conhecidos como códons ou temo de bases. Cada aminoácido é codificado por pelo menos um códon. O fato do mesmo aminoácido ser codificado por diferentes códons é conhecido como“degeneração do código genético”. O termo“aminoácido”, como usado no presente pedido, denota os a-aminoácidos que ocorrem naturalmente, compreendendo alanina, arginina, asparagina, ácido aspártico, cisteína, glutamina, ácido glutâmico, glicina, histidina, isoleucina, leucina, lisina, metionina, fenilalanina, prolina, serina, treonina, triptofano, tirosina e valina. "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". The term "amino acid", as used in this application, denotes naturally occurring α-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.
[0070] Os termos“peptídeo”,“polipeptídeo” ou“proteína” podem ser utilizados intercambiavelmente, e fazem referência a um polímero de aminoácidos conectado por ligações peptídicas, independentemente do número de resíduos de aminoácido que constituem esta cadeia. Os polipeptídeos, como aqui usados, incluem“variantes” ou“derivados” dos mesmos, que se referem a um polipeptídeo que inclui variações ou modificações, por exemplo, substituição, deleção, adição ou modificações químicas em sua sequência de aminoácido em relação ao polipeptídeo de referência. Exemplos de modificações químicas são glicosilação, PEGlação, PEG alquilação, alquilação, fosforilação, acetilação, amidação, etc. O polipeptídeo pode ser produzido artificialmente a partir de sequências nucleotídicas clonadas através da técnica de DNA recombinante ou pode ser preparado através de uma reação de síntese química conhecida. [0070] The terms "peptide", "polypeptide" or "protein" 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, as used herein, 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.
[0071] Mais especificamente, o termo polipeptídeo da presente invenção pode também ser entendido como antígeno, poliantígeno ou antígeno multiepítopo, que consistem em uma junção de epítopos diferentes que podem ou não estar interligados por ligantes ( linkers ) flexíveis ou rígidos, específicos para um único patógeno ou para patógenos diferentes.  [0071] More specifically, the term 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.
[0072] O termo“identidade” é definido como o grau de igualdade entre sequências de DNA ou de aminoácidos quando comparados nucleotídeo por nucleotídeo ou aminoácido por aminoácido com uma sequência de referência.  [0072] The term "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.
[0073] O termo“porcentagem de identidade de sequências” refere-se a comparações entre polinucleotídeos ou polipeptídeos e é determinado por duas sequências idealmente alinhadas, sob determinados parâmetros de comparação. Este alinhamento pode compreender espaços ( gaps ), gerando intervalos quando comparadas à sequência de referência, que facilitam uma comparação adequada das mesmas. De maneira geral, o cálculo da porcentagem de identidade considera o número de posições onde o mesmo nucleotídeo ou aminoácido ocorre nas sequências comparadas à sequência referência, sendo realizado através de diversos algoritmos de comparação de sequências e programas conhecidos no estado da arte. Tais algoritmos e programas incluem, mas não são limitados a, TBLASTN, BLASTP, FASTA, TFASTA, CLUSTALW, FASTDB. [0073] 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.
[0074] O polipeptídeo compreendendo os fragmentos antigênicos da presente invenção podem ser obtidos de forma recombinante ou sintética. Em uma modalidade particular, os polipeptídeos da presente invenção são obtidos por meio de um sistema de expressão, o qual permite a obtenção dos polipeptídeos da presente invenção.  [0074] The polypeptide comprising the antigenic fragments of the present invention can be obtained recombinantly or synthetically. In a particular embodiment, the polypeptides of the present invention are obtained by means of an expression system, which allows obtaining the polypeptides of the present invention.
[0075] Por sistema de expressão, entende-se um sistema compreendendo as sequências nucleotídicas, as quais são capazes de codificar polipeptídeos.  [0075] By expression system, we mean a system comprising nucleotide sequences, which are capable of encoding polypeptides.
[0076] Em um segundo aspecto, a presente invenção provê polinucleotídeos compreendendo sequências nucleotídicas que codificam os polipeptídeos aqui descritos.  [0076] In a second aspect, the present invention provides polynucleotides comprising nucleotide sequences that encode the polypeptides described herein.
[0077] Os polinucleotídeos de acordo com a invenção são representados, de forma não limitativa, pelas SEQ ID NOs: 2, 4, 6, 8 e 10 e suas degenerações, ou sequências com pelo menos 90% de identidade com as SEQ ID NOs: 2, 4, 6, 8 e 10. Os polinucleotídeos das SEQ ID NOs: 2, 4, 6, 8 e 10 codificam os polipeptídeos representados pelas SEQ ID NO: 1, 3, 5, 7 e 9, respectivamente.  [0077] The 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.
[0078] As sequências polinucleotídicas foram construídas para inserção de um poly-linker no início das sequências (traduzido como Met Ala Gly Ser nas SEQ ID Nos. 1, 3, 5, 7 e 9), sítios de clonagem (enzimas NCO I, BAM Hl, ECO RI, códon de terminação e Hind III). A tradução dos aminoácidos correspondentes aos códons localizados à jusante do(s) códon(s) de terminação não estão mostrados nas SEQ ID NOs. 1, 3, 5, 7 e 9.  [0078] 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.
[0079] O termo“sequência de nucleotídeos degenerada” denota uma sequência de nucleotídeos que inclui um ou mais códons degenerados quando comparada com uma molécula de ácido nucleico de referência que codifica um dado polipeptídeo. Códons degenerados contêm diferentes tripletes de nucleotídeos, mas codificam o mesmo resíduo de aminoácido (p.ex., GAU e GAC ambos codificam Asp). [0079] The term "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).
[0080] Um técnico no assunto reconheceria que as degenerações são integralmente suportadas com base nas informações fornecidas no pedido e no conhecimento comum do estado da técnica. Por exemplo, a degeneração do código genético (isto é, diferentes códons podendo codificar os mesmos aminoácidos) é um conhecimento comum na técnica e a identidade do aminoácido codificado por cada códon é bem estabelecida.  [0080] A person skilled in the art would recognize that 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.
[0081] Com base nas informações bem conhecidas e estabelecidas no estado da técnica, o técnico no assunto é capaz de identificar as substituições de nucleotídeos que não alteram a sequência de aminoácidos resultante. Assim, quando em posse tanto da sequência de nucleotídeos de um gene quanto da sequência de aminoácidos da proteína codificada, o técnico no assunto identificará facilmente as degenerações que codificam a mesma proteína, com a mesma sequência de aminoácidos.  [0081] Based on information well known and established in the state of the art, the person skilled in the art is able to identify nucleotide substitutions that do not alter the resulting amino acid sequence. Thus, when in possession of both the nucleotide sequence of a gene and the amino acid sequence of the encoded protein, the person skilled in the art will easily identify the degenerations that encode the same protein, with the same amino acid sequence.
[0082] O uso dos códons preferidos pode ser adaptado de acordo com a célula hospedeira na qual o ácido nucleico deve ser transcrito. Estas etapas podem ser realizadas de acordo com métodos bem conhecidos do versado na técnica e dos quais alguns são descritos no manual de referência Sambrook et al. (Sambrook et al, 2001).  [0082] The use of the preferred codons can be adapted according to the host cell in which the nucleic acid is to be transcribed. These steps can be performed according to methods well known to the person skilled in the art and some of which are described in the reference manual Sambrook et al. (Sambrook et al, 2001).
[0083] Neste sentido, diferentes espécies podem exibir um“códon usage” preferencial. Vide Grantham et al., Nuc. Acids Res. 8: 1893 (1980), Haas et al. Curr. Biol. 6:315 (1996), Wain-Hobson et al., Gene 13:355 (1981), Grosjean and Fiers, Gene 18: 199 (1982), Holm, Nuc. Acids Res. 14:3075 (1986), Ikemura, J. Mol. Biol. 158:573 (1982), Sharp and Matassi, Curr. Opin. Genet. Dev. 4:851 (1994), Kane, Curr. Opin. Biotechnol. 6:494 (1995), and Makrides, Microbiol. Rev. 60:512 (1996). Como usado aqui, o termo“ códon usage preferencial”, ou“códons preferenciais” é um termo usado na arte referindo-se a códons que são mais frequentemente utilizados em células de certas espécies. Códons preferenciais para uma espécie em particular podem ser introduzidos nos polinucleotídeos da presente invenção por uma variedade de métodos conhecidos na arte. A introdução de sequências de códons preferenciais em um DNA recombinante pode, por exemplo, aumentar a produção do polipeptídeo ao tomar a tradução mais eficiente em um determinado tipo de célula. Assim, as sequências de polinucleotídeo da invenção podem ser otimizadas para diferentes espécies. [0083] In this sense, different species may exhibit a preferential “codon usage”. See Grantham et al., Nuc. Acids Res. 8: 1893 (1980), Haas et al. Curr. Biol. 6: 315 (1996), Wain-Hobson et al., Gene 13: 355 (1981), Grosjean and Fiers, Gene 18: 199 (1982), Holm, Nuc. Acids Res. 14: 3075 (1986), Ikemura, J. Mol. Biol. 158: 573 (1982), Sharp and Matassi, Curr. Opin. Genet. Dev. 4: 851 (1994), Kane, Curr. Opin. Biotechnol. 6: 494 (1995), and Makrides, Microbiol. Rev. 60: 512 (1996). As used here, the term "preferred codon usage", or "preferred codons" 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.
[0084] Em um aspecto preferencial, os ácidos nucleicos da presente invenção tiveram os códons otimizados.  [0084] In a preferred aspect, the nucleic acids of the present invention had the codons optimized.
[0085] Em um aspecto ainda mais preferencial, os ácidos nucleicos da presente invenção tiveram os códons otimizados para substituição dos códons raros, distribuição de conteúdo GC e remoção de sequências repetitivas visando a transcrição e estabilidade e tradução do mRNA, para obtenção de altos níveis de expressão em sistema procarioto.  [0085] In an even more preferred aspect, 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.
[0086] Em um aspecto particular, os ácidos nucleicos da invenção são otimizados para uso de códon para Escherichia coli. Em uma modalidade preferida, as sequências de nucleotídeos são otimizadas para substituição de códons raros.  [0086] In a particular aspect, the nucleic acids of the invention are optimized for use in codon for Escherichia coli. In a preferred embodiment, the nucleotide sequences are optimized for replacing rare codons.
[0087] Em um terceiro aspecto, a presente invenção provê um cassete de expressão compreendendo o ácido nucleico de acordo com a invenção. O referido cassete é colocado em condições que conduzam à expressão dos polipeptídeos da presente invenção.  [0087] In a third aspect, 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.
[0088] O cassete de expressão pode compreender, ainda, sequências necessárias à sua expressão, tais como, promotores, sequências intensificadoras e terminadoras compatíveis com o sistema de expressão. Ademais, o cassete de expressão pode compreender sequências espaçadoras, sequências ligadoras e sítios de restrição adequados. Além disso, o cassete pode compreender, ainda, uma sequência codificante para cauda de histidina.  [0088] 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.
[0089] Em um quarto aspecto, a presente invenção provê um vetor de expressão compreendendo um ácido nucleico ou um cassete de expressão de acordo com a invenção. Este vetor de expressão pode ser utilizado para transformar uma célula hospedeira e permitir a expressão do ácido nucleico de acordo com a invenção na referida célula. [0089] In a fourth aspect, 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.
[0090] Com vantagem, o vetor de expressão compreende elementos reguladores que permitem a expressão do ácido nucleico e elementos que permitem a sua seleção na célula hospedeira de acordo com a invenção. Os métodos para selecionar estes elementos em função da célula hospedeira na qual a expressão é desejada, são bem conhecidos do versado na técnica e amplamente descritos na literatura.  [0090] Advantageously, 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.
[0091] Os vetores podem ser construídos por técnicas clássicas de biologia molecular, bem conhecidas do versado na técnica. Exemplos não limitantes de vetores de expressão adequados para expressão em células hospedeiras são plasmídeos e vetores virais ou bacterianos.  [0091] 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.
[0092] Em um quinto aspecto, a presente invenção provê uma célula hospedeira transformada/transfectada de maneira transitória ou estável com o ácido nucleico, o cassete ou o vetor da invenção. O ácido nucleico, o cassete ou o vetor pode estar contido na célula sob a forma de epissoma ou sob forma cromossômica.  [0092] In a fifth aspect, 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.
[0093] A célula hospedeira pode ser uma célula de bactéria, levedura, fungos filamentosos, protozoários, insetos, células animais e vegetais.  [0093] The host cell can be a cell of bacteria, yeast, filamentous fungi, protozoa, insects, animal and plant cells.
[0094] Em um aspecto específico, a célula hospedeira é uma célula bacteriana, preferivelmente uma célula de Escherichia coli.  [0094] In a specific aspect, the host cell is a bacterial cell, preferably an Escherichia coli cell.
[0095] Em um sexto aspecto, a presente invenção provê método para produzir o polipeptídeo de acordo com a invenção, compreendendo a inserção de um ácido nucleico, um cassete ou um vetor de expressão de acordo com a invenção em um sistema de expressão in vivo e a coleta do polipeptídeo produzido pelo referido sistema. Numerosos sistemas de expressão in vivo , compreendendo o uso de células hospedeiras adequadas, estão disponíveis no comércio e a utilização destes sistemas é bem conhecida do versado na técnica. [0096] Sistemas de expressão particularmente adequados incluem microrganismos, como bactérias transformadas com vetores de expressão de DNA recombinante de bacteriófago, plasmídeo ou cosmídeo; levedura transformada com vetores de expressão de levedura; sistemas de células de insetos infectadas com vetores de expressão de vírus (por exemplo, baculovírus); sistemas de células de plantas transformados com vetores de expressão de vírus (por exemplo, vírus do mosaico da couve-flor, CaMV [cauliflower mosaic virus\ ; vírus do mosaico do tabaco, TMV [tobacco mosaic virus ]) ou com vetores de expressão bacterianos (por exemplo, plasmídeos Ti ou pBR322); ou sistemas de células animais. Também é possível empregar sistemas de tradução isentos de células para produzir os polipeptídeos da invenção. [0095] In a sixth aspect, 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. 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. [0096] 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.
[0097] A introdução do ácido nucleico, cassete de expressão ou o vetor que codifica uma proteína recombinante ou sintética da presente invenção em células hospedeiras pode ser realizada por meio de métodos descritos em muitos manuais de laboratório padrão, como Davis et al., Basic Methods in Molecular Biology (1986) e Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY (1989).  [0097] The introduction of the 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).
[0098] A célula hospedeira transformada ou transfectada descrita acima é depois cultivada em um meio nutriente adequado sob condições conducentes que permitam a expressão das proteínas recombinantes da invenção. O meio usado para cultivar as células pode ser qualquer meio convencional adequado para desenvolver as células hospedeiras, tal como meio mínimo ou complexo contendo suplementos apropriados. Os meios adequados estão disponíveis a partir de fornecedores comerciais ou podem ser preparados de acordo com receitas publicadas (por exemplo, nos catálogos da American Type Culture Collection). As proteínas da invenção produzidos pelas células podem ser depois recuperados da célula ou do meio de cultura por procedimentos convencionais incluindo separar as células hospedeiras do meio pela centrifugação ou filtração, precipitando os componentes aquosos de proteína do sobrenadante ou filtrado por meio de um sal, por exemplo, sulfato de amónio, purificação por uma variedade de procedimentos cromatográficos, por exemplo cromatografia por troca iônica, cromatografia por exclusão, cromatografia de interação hidrofóbica, cromatografia por filtração em gel, cromatografia por afinidade ou similares, dependente do tipo de polipeptídeo em questão. 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.
[0099] O polipeptídeo recombinante obtido é, então, purificado e caracterizado bioquimicamente, utilizando-se, por exemplo, métodos comuns ao campo da bioquímica, como HPLC, SDS-PAGE, Western Blotting, focalização isoelétrica com gradiente de pH, dicroísmo circular. Por meio desses métodos, é possível determinar características como, por exemplo, o rendimento da expressão do polipeptídeo recombinante; a determinação das características das estruturas secundárias, além de outras características cuja determinação é importante para o desenvolvimento de uma composição para uso como reagente para diagnóstico da Hepatite C.  [0099] 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. Through these methods, it is possible to determine characteristics such as, for example, the expression yield of the recombinant polypeptide; the determination of the characteristics of the secondary structures, in addition to other characteristics whose determination is important for the development of a composition for use as a reagent for the diagnosis of Hepatitis C.
[00100] Os polipeptídeos podem ser expressos “fusionados” à uma etiqueta. O termo“etiqueta” ou o termo em inglês“tog” refere-se a sequências codificadoras incorporadas próximas ao sítio múltiplo de clonagem de um vetor de expressão, possibilitando a sua tradução concomitante e adjacente à sequência do polipeptídeo recombinante clonada. Assim, a etiqueta é expressa fusionada ao polipeptídeo recombinante. Tais etiquetas são bem conhecidas no estado da técnica e incluem compostos e peptídeos como poli-histidina, poli- arginina, FLAG, glutationa-S-transferase, proteína ligante a maltose (MBP), domínio ligante a celulose (CBD), Beta-Gal, OMNI, tioredoxina, NusA, mistina, domínio ligante a quitina, cutinase, compostos fluorescentes (como GFP, YFP, FITC, rodamina, lantanídeos), enzimas (como peroxidase, luciferase, fosfatase alcalina), compostos quimioluminescentes, grupos biotinila, epítopos reconhecidos por anticorpos como zíper de leucina, c-myc, domínios ligantes a metais e sítios de ligação para anticorpos secundários. [00100] Polypeptides can be expressed "fused" to a tag. The term "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.
[00101] Os polipeptídeos também podem ser obtidos sinteticamente usando métodos conhecidos na arte. Síntese direta dos polipeptídeos da invenção pode ser realizada usando síntese em fase sólida, síntese em solução ou outros meios convencionais, utilizando geralmente grupos de proteção do a- aminogrupo, da a-carboxila e/ou dos grupos funcionais das cadeias laterais dos aminoácidos. Por exemplo, na síntese em fase sólida, um resíduo de aminoácido adequadamente protegido é ligado através do seu grupo carboxila a um suporte polimérico insolúvel, tais como uma resina reticulada de poliestireno ou poliamida. Métodos de síntese em fase sólida incluem tanto métodos BOC e FMOC, que utilizam tert-butiloxicarbonil, e 9- fluorenilmetiloxicarbonila como grupos protetores a-amino, respectivamente, ambos bem conhecidos pelos técnicos no assunto (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.; Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, New York, 1995). [00101] 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. For example, in 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).
[00102] Os seguintes grupos protetores podem ser exemplos utilizados para a síntese dos polipeptídeos da invenção: 9-fluorenilmetiloxicarboil (Fmoc), tert-butiloxicarbonil (Boc), carbobenziloxi (Cbz), 2-cloro-3- indenilmetoxicarbonil (Climoc), benz(f)indeno-3-il-metóxicarbonil (Bimoc), l,l-dioxobenzo[b]tiofeno-2-il-metoxicarbonil (Bsmoc), 2,2,2- tricloroetóxicarbonil (Troe), 2-(trimetilsilil)etoxicarbonil (Teoc), homobenziloxicarbonil (hZ), l,l-dimetil-2,2,2-tricloroetóxicarbonil (TCBoc), 1-metil-l (4-bifenil)etoxicarbonil (Bpoc), l-(3,5-di-t-butilfenil)-l- metiletoxicarbonil (t-Bumeoc), 2-(2’- or 4’-piridil)etoxicarbonil (Pyoc), viniloxicarbonil (Voc),l-isopropilaliloxicarbonil (Ipaoc), 3-(piridil)alil- oxicarbonil (Paloc), p-metoxibenziloxicarbonil (Moz), p-nitrocarbamato (PNZ), 4-azidobenzyloxycarbonyl (AZBZ), Benzil (Bn) MeO, BnO, Metoximetil (Mom), metiltiometil (MTM), fenildimetilsililmetoximetil (SMOM), t-butildimetilsilil (TBDMS), benziloximetil (BOM), p- metoxibenziloximetil (PMBM), nitrobenziloximetil (NBOM), p-anisiloxymetil (p-AOM), pBu0CH20-, 4-penteniloximetil (POM), 2-metoxietóximetil (MEM), 2-(trimetilsilil)etoximetil (SEM), mentoximetil (MM), tetrahidropiranil (THP), -OCOCOph, Acetil, C1CH2C02-, -C02CH2CC13, 2- (Trimetilsilil)etil (TMSE), 2(p-toluenosulfonil)etil (Tse). (Greene T.W. Wuts P.G.M., Protective groups in organic synthesis, 3rd ed., John Wiley & Sons, INC, Nova York, EUA, 1999). [00102] 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), l- (3,5-di- t-butylphenyl) -1-methylethoxycarbonyl (t-Bumeoc), 2- (2'- or 4'-pyridyl) ethoxycarbonyl (Pyoc), vinyloxycarbonyl (Voc), l-isopropylalyloxycarbonyl (Ipaoc), 3- (pyridyl) allyl- oxycarbonyl (Paloc), p-methoxybenzyloxycarbonyl (Moz), p-nitrocarbamate (PNZ), 4-azidobenzyloxycarbonyl (AZBZ), Benzyl (Bn) MeO, BnO, Methoxymethyl (Mom), methylthiomethyl (MTM), phenyldimethylsilmetoxy (SMOM), t-butyldimethylsilyl (TBDMS), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), nitrobenzyloxymethyl (NBOM), p-anisiloxymethyl (p-AOM), pBu0CH20-, 4-pentenyloxymethyl (2-pentenyloxymethyl) (MEM), 2- (trimethylsilyl) ethoxymethyl (SEM), menthoxymethyl (MM), tetrahydropyranyl (THP), -OCOCOph, Acetyl, C1CH2C02-, -C02CH2CC13, 2- (Trimethylsilyl) ethyl (TMSE), 2 (p-toluenesulfonyl ) ethyl (Tse). (Greene TW Wuts PGM, Protective groups in organic synthesis, 3rd ed., John Wiley & Sons, INC, New York, USA, 1999).
[00103] Após a reação química, os polipeptídeos podem ser separados e purificados por um método de purificação conhecido. Um exemplo de tais métodos de purificação pode incluir uma combinação de extração por solvente, destilação, cromatografia por coluna, cromatografia líquida, recristalização e similares.  [00103] After the chemical reaction, 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.
[00104] Em uma concretização preferida, o método para produzir o polipeptídeo da presente invenção compreende as etapas de:  [00104] In a preferred embodiment, the method for producing the polypeptide of the present invention comprises the steps of:
(a) transformar uma célula hospedeira um polinucleotídeo da presente invenção para obter uma célula hospedeira transformada;  (a) transforming a host cell into a polynucleotide of the present invention to obtain a transformed host cell;
(b) cultivar a célula hospedeira transformada para a produção do polipeptídeo;  (b) cultivating the transformed host cell for the production of the polypeptide;
(c) isolar o polipeptídeo da presente invenção da célula ou do meio de cultura circundando dita célula.  (c) isolating the polypeptide of the present invention from the cell or culture medium surrounding said cell.
[00105] Em uma modalidade, o polinucleotídeo da etapa (a) codifica o polipeptídeo compreendendo pelo menos 90% de identidade com qualquer uma das SEQ ID NOs: 1, 3, 5, 7 e 9.  [00105] In one embodiment, 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.
[00106] Em outra modalidade, o polinucleotídeo da etapa (a) compreende a sequência de ácidos nucleicos de qualquer uma das SEQ ID NO: 2, 4, 6, 8 e 10 e suas degenerações.  [00106] In another embodiment, 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.
[00107] Em uma modalidade, a transformação da célula hospedeira com o polinucleotídeo da presente invenção é realizada por meio de um vetor de expressão. Em uma modalidade específica, o vetor é pET28a e a célula hospedeira transformada é E. coli. [00107] In one embodiment, the transformation of the host cell with the polynucleotide of the present invention is carried out by means of a vector of expression. In a specific embodiment, the vector is pET28a and the transformed host cell is E. coli.
[00108] As condições de cultivo da célula hospedeira referido na etapa (b) são de conhecimento de um técnico no assunto. Em uma concretização, o cultivo é feito em meio LB na presença de um antibiótico, sob agitação. Em uma modalidade específica, o antibiótico é canamicina. O cultivo pode ser realizado a diferentes temperaturas durante diferentes períodos de tempo. Em uma modalidade específica, o cultivo pode ser feito por cerca de 4 a cerca de 16 horas, a uma temperatura de cerca de 16°C a cerca de 37°C. Em uma concretização preferida, o cultivo é feito a 37°C por 16 horas, sob agitação.  [00108] The conditions for cultivating the host cell referred to in step (b) are known to a person skilled in the art. In one embodiment, cultivation is done in LB medium in the presence of an antibiotic, under agitation. In a specific embodiment, the antibiotic is kanamycin. Cultivation can be carried out at different temperatures for different periods of time. In a specific embodiment, cultivation can be done for about 4 to about 16 hours, at a temperature of about 16 ° C to about 37 ° C. In a preferred embodiment, cultivation is carried out at 37 ° C for 16 hours, under agitation.
[00109] A produção do polipeptídeo referida na etapa (b) pode ser realizada com qualquer técnica conhecida no estado da técnica. Em uma concretização, a indução da expressão dos polinucleotídeo da invenção é feita por meio de adição de IPTG ao meio de cultura, após obtenção de densidade óptica adequada. [00109] The production of the polypeptide referred to in step (b) can be carried out with any technique known in the prior art. In one embodiment, the expression of the polynucleotides of the invention is induced by adding IPTG to the culture medium, after obtaining adequate optical density.
[00110] Em uma etapa opcional, é realizada a solubilização de corpos de inclusão antes do isolamento do polipeptídeo da etapa (c). A solubilização pode ser realizada com qualquer agente caotrópico conhecido no estado da técnica, sendo exemplos não limitativos a ureia e a guanidina. Em uma concretização, a solubilização é realizada com tampão contendo guanidina.  [00110] In an optional step, 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. In one embodiment, solubilization is carried out with buffer containing guanidine.
[00111] O isolamento do polipeptídeo referido na etapa (c) pode ser realizado com qualquer técnica conhecida no estado da técnica. Em uma concretização, a purificação é feita por técnicas de cromatografia. Em uma modalidade específica, a purificação é realizada por cromatografia de afinidade. Exemplos não limitativos incluem o método de afinidade à resina de níquel, troca iônica, outros métodos por afinidade ou adsorção, par iônico, fase reversa e exclusão molecular. [00111] The isolation of the polypeptide referred to in step (c) can be carried out with any technique known in the art. In one embodiment, purification is done by chromatography techniques. In a specific embodiment, 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.
[00112] Os sistemas de expressão e purificação desenvolvidos nesta invenção permitiram a obtenção abundante dos antígenos citados por meio de apenas uma etapa cromatográfica (cromatografia por afinidade). Esta característica foi especialmente relevante no que se refere à qualidade dos imunoensaios, pois anticorpos anti-E. coli estão presentes no soro humano, todavia, podem interagir com proteínas de E. coli que, porventura, tenham sido purificadas juntamente, com os antígenos desenvolvidos no trabalho, gerando resultados falso-positivos. [00112] The expression and purification systems developed in this invention allowed the abundant obtaining of the mentioned antigens by means of only one chromatographic step (affinity chromatography). This characteristic was especially relevant with regard to the quality of the immunoassays, as anti-E antibodies. coli are present in human serum, however, they can interact with E. coli proteins that may have been purified together with the antigens developed in the work, generating false positive results.
[00113] Além disso, essas proteínas contaminantes podem competir com os antígenos de interesse pelos sítios de ligação nas fases sólidas dos imunoensaios (microesferas), o que pode acarretar a perda de desempenho do ensaio. Altos níveis de pureza são também importantes para a estabilidade dos antígenos, pois entre os contaminantes de E. coli existem muitas enzimas proteolíticas que causam a degradação dos antígenos expressos. Os resultados apresentados para os ensaios revelam a característica de pureza para os antígenos purificados.  [00113] In addition, 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.
[00114] Ao comparar os rendimentos obtidos na presente invenção com os rendimentos de referências do estado da técnica para a obtenção de proteínas para o vírus da hepatite C, pode-se afirmar que os rendimentos obtidos para purificação das proteínas desenvolvidas podem ser considerados altos. O estado da técnica aponta para rendimentos inferiores para a maior parte das proteínas desenvolvidas para o diagnóstico da hepatite C. Deste modo, pode-se afirmar que os polipeptídeos desenvolvidas nesta invenção, expressas em Escherichia coli, apresentaram rendimentos entre 8 a 25 mg por litro de cultura que se apresentaram com 95% de pureza utilizando apenas um passo cromatográfico, o que é vantajoso e considerado satisfatório tendo como objetivo uma produção em larga escala para aplicação diagnóstica.  [00114] When comparing the yields obtained in the present invention with the reference yields of the prior art for obtaining proteins for the hepatitis C virus, it can be said that the yields obtained for purification of the developed proteins can be considered high. The state of the art points to lower yields for most proteins developed for the diagnosis of hepatitis C. Thus, it can be said that the polypeptides developed in this invention, expressed in Escherichia coli, showed yields between 8 to 25 mg per liter of culture that presented themselves with 95% purity using only one chromatographic step, which is advantageous and considered satisfactory with the objective of large-scale production for diagnostic application.
[00115] Em um sétimo aspecto, a invenção provê uma composição, compreendendo um, dois, três, quatro ou cinco dos polipeptídeos da invenção. [00116] Em um aspecto particular, a composição é utilizada como reagente para métodos para triagem imunológica de HCV e diagnóstico de hepatite C. [00115] In a seventh aspect, the invention provides a composition, comprising one, two, three, four or five of the polypeptides of the invention. [00116] In a particular aspect, the composition is used as a reagent for methods for immunological screening of HCV and diagnosis of hepatitis C.
[00117] Adicionalmente, são providas composições estabilizadas dos antígenos da presente invenção. As composições estabilizadas resultam na ausência de agregados proteicos, o que garante a não interferência na interação antígeno-anticorpo, relacionada à agregação, nos imunoensaios da presente invenção.  [00117] Additionally, 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.
[00118] Em um aspecto particular da presente invenção, são providas formulações de tampões de armazenamento para estabilização dos antígenos da invenção. Os componentes dos tampões utilizados na presente invenção são conhecidos na arte. Em uma modalidade específica, o tampão de armazenamento compreende ureia. Em uma concretização preferida, o tampão de armazenamento compreende ureia 4M.  [00118] In a particular aspect 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. In a specific embodiment, the storage buffer comprises urea. In a preferred embodiment, the storage buffer comprises 4M urea.
[00119] A Luminex Corporation recomenda testar diferentes concentrações das proteínas de interesse em diferentes tampões quando se deseja otimizar uma reação através da plataforma de microfluídica e observar a sua funcionalidade. Dentre eles, o tampão de armazenamento das proteínas contendo 4M de ureia, que foi o tampão que se revelou mais adequado para o acoplamento, por evitar a precipitação ou a formação de agregados entre as microesferas acopladas. Assim, desenvolver proteínas para o HCV para uso em imunodiagnóstico que se mantenham estáveis em tampão contendo até 4M de ureia também foi um resultado atrativo para a presente invenção, pois sendo a ureia considerada possível interferente em testes para diagnósticos quando utilizada em altas concentrações, observou-se que baixas concentrações do agente, durante o acoplamento, podem não interferir significativamente nos resultados. Neste contexto, embora a Luminex Corporation notifique que a ureia pode potencialmente interferir no acoplamento das proteínas às microesferas, a partir dos resultados obtidos nesta invenção, foi alcançado os melhores desempenhos, melhores áreas abaixo da curva (AUC, do inglês Area Under Curve), menores desvios para os soros negativos e ainda a maior capacidade de separação (SNR, do inglês Signal to Noise Ratio) para as proteínas 1, 2 e 3, a partir dos ensaios cujos antígenos foram acoplados às microesferas na presença de tempão contendo 4M ureia. Portanto, o acoplamento na presença de ureia foi essencial para manter a apresentação dos epítopos de interesse. [00119] 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. Among them, 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. Thus, 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. In this context, although 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.
[00120] Um grande número de proteínas comerciais disponíveis no mercado para aplicação no diagnóstico laboratorial para o vírus da hepatite C estão disponíveis para venda em tampões de armazenamento contendo ureia com concentrações que variam de 6 a 8M.  [00120] A large number of commercial proteins available on the market for application in laboratory diagnosis for the hepatitis C virus are available for sale in storage buffers containing urea with concentrations ranging from 6 to 8M.
[00121] Os atuais ensaios licenciados comercialmente para o vírus da hepatite C (VHC) utilizam proteínas recombinantes contendo epítopos lineares. Há provas, no entanto, que os epítopos conformacionais para HCV seriam mais imunorreativos. Por outro lado, é extremamente difícil a obtenção de proteínas recombinantes para HCV na forma solúvel quando a expressão se dá em E. coli.  [00121] Current commercially licensed assays for hepatitis C virus (HCV) use recombinant proteins containing linear epitopes. There is evidence, however, that HCV conformational epitopes would be more immunoreactive. On the other hand, it is extremely difficult to obtain recombinant proteins for HCV in soluble form when the expression occurs in E. coli.
[00122] A única informação de conhecimento dos inventores da presente invenção sobre ensaios de acoplamento que levam ureia são ensaios multiplex para avaliarem respostas de anticorpos contra doenças parasitárias de importância para a saúde pública no Camboja, para a pesquisa de anticorpos anti-imunoglobulina G contra antígenos recombinantes de Plasmodium falciparum e P. vivax, Wuchereria bancrofti, Toxoplasma gondii, Taenia solium e Strongyloides stercoralis, cujos acoplados às microesferas foram realizados na presença de até 2M de ureia. Ainda não há relatos, na literatura científica para ensaios realizados na plataforma LUMINEX utilizando 4M de ureia para o acoplamento. [00122] The only information known to the inventors of the present invention about coupling assays that carry urea are multiplex assays to evaluate antibody responses against parasitic diseases of public health importance in Cambodia, for the search for anti-immunoglobulin G antibodies against recombinant antigens from Plasmodium falciparum and P. vivax, Wuchereria bancrofti, Toxoplasma gondii, Taenia solium and Strongyloides stercoralis, whose coupled to the microspheres were performed in the presence of up to 2M urea. There are still no reports in the scientific literature for tests performed on the LUMINEX platform using 4M urea for coupling.
[00123] Em modalidades específicas da invenção foram também determinadas as concentrações não agregantes de antígeno. Deste modo, as concentrações dos antígenos da presente invenção na composição variam, de um modo geral, de 0,007 a 0,015 mg/mL, preferencialmente, de 0,02 a 1? mg/mL. [00123] In specific embodiments of the invention, non-aggregating concentrations of antigen were also determined. Thus, the 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.
[00124] Em um oitavo aspecto, a invenção provê um kit para triagem imunológica de HCV e/ou diagnóstico de hepatite C que compreende um, dois, três, quatro ou cinco dos polipeptídeos da presente invenção.  [00124] In an eighth aspect, 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.
[00125] Opcionalmente, o kit compreende ainda instruções de uso e um controle de reação.  [00125] Optionally, the kit also includes instructions for use and a reaction control.
[00126] Em uma modalidade, o controle de reação é um controle de reação positivo.  [00126] In one embodiment, the reaction control is a positive reaction control.
[001] Adicionalmente, o kit pode ainda compreender um meio de detecção do complexo antígeno/anticorpo, o qual pode compreender um gerador de sinal, capaz de gerar um sinal detectável.  [001] Additionally, 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.
[002] Os meios de detecção podem ser aqueles conhecidos na técnica. [002] The detection means can be those known in the art.
Um exemplo não limitativo do meio de detecção pode ser um conjugado compreendido por um anticorpo acoplado a um composto gerador de sinal, capaz de gerar um sinal detectável. 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.
[00127] Em uma modalidade, os kits são desenvolvidos para uso em imunoensaios. Em uma modalidade mais específica, os imunoensaios são do tipo ELISA ou LUMINEX.  [00127] In one embodiment, the kits are developed for use in immunoassays. In a more specific modality, immunoassays are ELISA or LUMINEX.
[00128] Em um nono aspecto, a invenção provê o uso de um ou mais dos polipeptídeos ou da composição ou do kit como aqui descritos para triagem imunológica de HCV e/ou para diagnóstico de hepatite C.  [00128] In a ninth aspect, 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.
[00129] A invenção ainda provê métodos para triagem imunológica de HCV e para o diagnóstico de hepatite C que envolvem as etapas de:  [00129] The invention also provides methods for immunological screening of HCV and for the diagnosis of hepatitis C that involve the steps of:
(a) prover um ou mais polipeptídeos como definidos em qualquer uma das reivindicações 1 a 4 ou uma composição como definida na reivindicação 11 ou 12;  (a) providing one or more polypeptides as defined in any one of claims 1 to 4 or a composition as defined in claim 11 or 12;
(b) contatar os ditos um ou mais peptídeos ou a dita composição com a amostra biológica a ser testada por um tempo suficiente e sob condições suficientes para formação de complexos anticorpo/antígeno; e(b) contacting said one or more peptides or said composition with the biological sample to be tested for a sufficient time and under conditions sufficient to form antibody / antigen complexes; and
(c) detectar o complexo antígeno/anticorpo formado na etapa (b), pela adição de um meio de detecção, capaz de gerar um sinal detectável na presença do referido complexo antígeno/anticorpo. (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.
[00130] Em uma concretização preferencial, é realizado o modelo de imunoensaio indireto. Ainda mais preferencialmente, o imunoensaio é ensaio ELISA ou ensaio LUMINEX.  [00130] In a preferred embodiment, the indirect immunoassay model is performed. Even more preferably, the immunoassay is an ELISA assay or LUMINEX assay.
[00131] Em uma modalidade, para os ensaios ELISA, são aplicados de 2,5 a 1.000 ng de antígeno por poço. Em uma modalidade específica, são aplicados de 20 a 50 ng de antígenos por poço.  [00131] In one mode, for ELISA assays, 2.5 to 1,000 ng of antigen are applied per well. In a specific modality, 20 to 50 ng of antigens are applied per well.
[00132] Em uma modalidade, para ensaios LUMINEX, são aplicadas concentrações de acoplamento de 7,5 a 25 pg/mL. Em uma modalidade específica, são aplicadas concentrações de acoplamento de 7,5 a 15 pg/mL.  [00132] In one embodiment, for LUMINEX assays, 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.
[00133] No contexto da presente invenção, os imunoensaios podem ser realizados em formato singleplex ou multiplex. [00133] In the context of the present invention, immunoassays can be performed in singleplex or multiplex format.
[00134] Os antígenos foram avaliados quanto aos melhores desempenhos a partir da AUC (Area Abaixo da Curva) da curva ROC (do inglês, Receiver Operating Characteristic ), da maior sensibilidade e especificidade, maior capacidade de separação, bem como o menor desvio padrão para as amostras negativas.  [00134] 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.
[00135] Todas as proteínas desenvolvidas apresentaram ótimos desempenhos e capacidade de separação entre os soros negativos e positivos e todas elas foram avaliadas individualmente e em conjuntos. Todos os antígenos confirmaram antigenicidade demonstrando sensibilidade de até 100%.  [00135] All proteins developed showed excellent performance and separation capacity between negative and positive sera and all of them were evaluated individually and in groups. All antigens confirmed antigenicity, showing sensitivity of up to 100%.
[00136] Nos ensaios realizados no formato ELISA para a análise individual de desempenho para os antígenos, a melhor sensibilidade e especificidade (100%) foi alcançada para o antígeno 3. Dos desempenhos apresentados, o índice de reatividade para os soros positivos foi, em geral, acima de 12 e para os soros negativos, abaixo de 0,8. [00137] Nos ensaios LUMINEX formato singleplex, foram obtidos 100% de sensibilidade e especificidade para os antígenos 1, 3, 4 e 5 (para o antígeno 2, foi de 95,65%). A maior capacidade de separação entre os soros positivos e negativos foi obtida para o antígeno 4. [00136] In the tests performed in the ELISA format for individual performance analysis for antigens, the best sensitivity and specificity (100%) was achieved for antigen 3. Of the performances presented, the reactivity index for positive sera was, in general, above 12 and for negative sera, below 0.8. [00137] In the LUMINEX singleplex assays, 100% sensitivity and specificity were obtained for antigens 1, 3, 4 and 5 (for antigen 2, it was 95.65%). The greatest capacity for separation between positive and negative sera was obtained for antigen 4.
[00138] No formato multiplex, pode ser empregada a combinação de dois ou mais dos polipeptídeos da presente invenção. Preferencialmente, é empregada a combinação dos cinco polipeptídeos aqui descritos.  [00138] In the multiplex format, the combination of two or more of the polypeptides of the present invention can be employed. Preferably, the combination of the five polypeptides described herein is employed.
[00139] A combinação dos cinco polipeptídeos em ensaio ELISA combinado apresentou sensibilidade e especificidade de 100%, com desempenhos superiores aos obtidos quando os antígenos foram avaliados individualmente no mesmo ensaio. A combinação dos cinco antígenos demonstrou o melhor desempenho e capacidade discriminatória.  [00139] The combination of the five polypeptides in the combined ELISA assay showed sensitivity and specificity of 100%, with performances superior to those obtained when the antigens were evaluated individually in the same assay. The combination of the five antigens demonstrated the best performance and discriminatory capacity.
[00140] Os resultados obtidos concordam com os dados do ensaio LUMINEX em formato multiplex, dentre os quais as maiores AUCs foram obtidas pela combinação dos cinco polipeptídeos.  [00140] The results obtained agree with the data from the LUMINEX assay in multiplex format, among which the largest AUCs were obtained by combining the five polypeptides.
[00141] Foi observado que a combinação dos antígenos 2 e 3 (combinação 1), 2, 3, 4 e 5 (combinação 2) e 1, 2, 3, 4 e 5 (combinação 3) apresentaram 100% de sensibilidade. Dentre eles, as combinações 1 e 3 apresentaram também 100% de especificidade. A combinação 3 apresentou a maior capacidade discriminatória entre os soros, com a maior distância entre as MFIs (mediana de fluorescência) para os soros negativos e positivos. Para essa combinação, o IR (índice de reatividade) foi de até 140 vezes maior que o IR obtido para os soros negativos. Esse ensaio demonstrou também o menor desvio padrão para os soros negativos.  [00141] It was observed that the combination of antigens 2 and 3 (combination 1), 2, 3, 4 and 5 (combination 2) and 1, 2, 3, 4 and 5 (combination 3) showed 100% sensitivity. Among them, combinations 1 and 3 also showed 100% specificity. Combination 3 showed the highest discriminatory capacity among sera, with the longest distance between MFIs (median fluorescence) for negative and positive sera. For this combination, the IR (reactivity index) was up to 140 times higher than the IR obtained for negative sera. This assay also demonstrated the lowest standard deviation for negative sera.
[00142] Tanto no ensaio ELISA, quanto no LUMINEX, os sinais de fundo ou backgrounds foram considerados baixos. O sinal de fundo acusa a eficiência ou não das etapas de lavagens ou do bloqueio. Este sinal também pode estar relacionado à pureza dos antígenos utilizados na presença de componentes heterólogos que podem causar aumento no sinal de fundo, como por exemplo, proteínas contaminantes, provenientes da E. coli. Uma vez que os ensaios realizados apresentaram sinais de fundo baixos, isto permite concluir que a obtenção dos antígenos a partir de uma única etapa cromatográfica nesta invenção, foi suficiente para obtenção de proteínas com um grau de pureza alto, confirmou eficiência das etapas de lavagens durante os ensaios e do bloqueio adicional realizado com o extrato de E. coli. [00142] Both in the ELISA assay and in the LUMINEX, 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.
[00143] Semelhantemente à avaliação dos ensaios realizados em formato singleplex, a aplicação de conjuntos de antígenos permitiu a obtenção de 100% de sensibilidade e especificidade. No entanto, a probabilidade de positividade e capacidade de diferenciação da infecção dos soros positivos pelos soros negativos, obtidos pelo SNR foi consideravelmente superior nos ensaios em combinação de antígenos.  [00143] Similar to the evaluation of the tests performed in singleplex format, the application of sets of antigens allowed to obtain 100% sensitivity and specificity. However, the probability of positivity and differentiation of infection from positive sera by negative sera, obtained by SNR was considerably higher in assays in combination of antigens.
[00144] Dentre todos os conjuntos testados, a combinação composta pelos cinco antígenos demonstrou a melhor performance, apresentando uma capacidade discriminatória equivalente a 36 vezes a capacidade de separação obtida pelo antígeno quimérico comercial utilizado como controle do ensaio, (ProsPec), o menor desvio padrão dentre as amostras negativas e a maior separação entre os valores de MFI e IR entre as amostras negativas e as positivas. O ensaio também não demonstrou nenhuma reação cruzada após testes realizados com soros positivos para HIV, HTLV, HBV, Sífilis e Chagas.  [00144] Among all the tested sets, 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.
[00145] Além disto, a condição ótima do conjunto de proteínas foi alcançada fazendo uso de baixas concentrações de proteínas para o acoplamento. As concentrações utilizadas na presente invenção são, consideravelmente, inferiores às concentrações de acoplamentos utilizadas para os antígenos comerciais como, por exemplo, a proteína comercial utilizada como controle do ensaio. [00145] In addition, the optimal condition of the protein set was achieved by making use of low concentrations of proteins for coupling. The 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.
[00146] A capacidade discriminatória dos ensaios através da plataforma LUMINEX foi superior ao ELISA em todos os aspectos AUC (95% IC), capacidade de separação entre as amostras e probabilidade de positividade através do IR e SNR. O sistema LUMINEX apresenta muitas vantagens dentre as quais a cinética de reação na superfície caboxilada das microesferas magnéticas de poliestireno é superior à cinética de reação de outros métodos. Dentre as vantagens que o ensaio LUMINEX apresenta sobre o ELISA, a capacidade de multiplexação, é considerada na técnica como a mais atrativa. [00146] 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.
[00147] Apesar do desafio de padronizar um ensaio multiplex do ponto de vista de desenvolvimento de imunodiagnósticos esta característica fornece economia de tempo, de processamento de um número maior de amostras e volumes menores de reagente e soros. Uma vez que no ELISA isto é realizado em triplicata, enquanto no LUMINEX, cada microesfera lida, corresponde a uma amostragem diferente e são lidas 100 microesferas por poço, não havendo a necessidade de repetições para o ensaio. Portanto, esta invenção fornece antígenos com características compatíveis para aplicação no desenvolvido de imunoensaios dentro de uma plataforma moderna e automatizada. [00147] Despite the challenge of standardizing a multiplex assay from the point of view of developing immunodiagnostics, this feature provides time savings, processing a larger number of samples and smaller volumes of reagent and sera. Since in ELISA this is done in triplicate, while in LUMINEX, each microsphere read corresponds to a different sample and 100 microspheres are read per well, with no need for repetitions for the assay. Therefore, this invention provides antigens with compatible characteristics for application in the development of immunoassays within a modern and automated platform.
[00148] Os marcadores sorológicos utilizados em bancos de sangue devem ser altamente confiáveis, apresentarem boa reprodutibilidade e máxima sensibilidade e especificidade. Um outro fator importante para a implementação de novos marcadores neste tipo de laboratório é que o cut-off para os ensaios empregados devem ser mais baixos, com o objetivo de aumentar a sensibilidade. Esta estratégia visa garantir a segurança do receptor, pois objetiva evitar a transfusão de bolsas de sangue de doadores infectados que apresentem baixos títulos para os marcadores. O conjunto de proteínas da combinação 4, foi o conjunto que apresentou o menor ponto de corte dentre todos os ensaios realizados, cut-off igual a 50, com uma probabilidade de positividade superior às demais combinações ou às avaliações das proteínas individualmente.  [00148] 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.
[00149] A presente invenção revelou antígenos que promoveram altos níveis de discriminação sorológica, obtendo-se ensaios com 100% de especificidade para um total de 165 soros negativos testados para HCV e sem a presença de reação cruzada, o que conta positivamente para a aplicação diagnóstica dos mesmos. [00149] 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.
[00150] Os exemplos citados a seguir são meramente ilustrativos, devendo ser empregados somente para uma melhor compreensão dos desenvolvimentos constantes na presente invenção, não devendo, contudo, serem utilizados com o intuito de limitar os objetos descritos.  [00150] The examples cited below are merely illustrative, and should be used only for a better understanding of the developments contained in the present invention, however, they should not be used in order to limit the objects described.
EXEMPLOSEXAMPLES
EXEMPLO 1: Clonagem das sequências codificadoras em vetor de expressão EXAMPLE 1: Cloning of the coding sequences into an expression vector
[00151] As sequências nucleotídicas de interesse foram sintetizadas e sofreram um processo de otimização para a substituição dos códons raros, distribuição de conteúdo GC e remoção de sequências repetitivas visando a transcrição e estabilidade e tradução do mRNA visando a obtenção de altos níveis de expressão em sistema procarioto. Foram também projetadas para receberem duas combinações de sítios de restrição Bam HI/Hind III e Nco I/Eco RI em suas extremidades visando a subclonagem no vetor de expressão pET28a de modo a permitir o posicionamento da hexa-histidina na região N- terminal ou C-terminal.  [00151] The 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.
[00152] Entre os sítios de restrição foram adicionados nucleotídeos espaçadores que colocam a sequência codificante da proteína em fase de leitura e um códon de terminação entre os sítios das enzimas Bam Hl e Hind III a fim de proporcionar alternativas para a subclonagem. Desta forma, se a clonagem for realizada com estas enzimas, a finalização da transcrição é assegurada e se a subclonagem for realizada com as enzimas Nco I e Eco RI, o término de transcrição será determinado pelo códon de terminação do pET28a. As sequências foram clonadas separadamente em vetores de pET28a, sob comando do promotor T7 polimerase, utilizando as enzimas de restrição Bam Hl e Hind III posicionando a hexa-histidina na região N-terminal. [00153] Especificamente com relação aos clones 4.1, 4.5, 4.7 (antígeno 1), 4.8 e 4.9, foi realizada subclonagem por transferência de vetor. Para isolar o inserto, 5pg do vetor pUC57, contendo o polinucleotídeo sintético, foram linearizados com as enzimas Bam Hl e Hind III a 37°C, conforme recomendação do fabricante, considerando o volume final da reação de 50 pL. Após visualização e excisão das bandas no gel de agarose 0,8%, os fragmentos foram purificados usando o kit QIAquick Gel Extraction® (Qiagen). [00152] Between the restriction sites spacer nucleotides were added that put the protein coding sequence in reading phase and a termination codon between the Bam Hl and Hind III enzyme sites in order to provide alternatives for subcloning. In this way, if the cloning is performed with these enzymes, the transcription is completed and if the subcloning is performed with the enzymes Nco I and Eco RI, the transcription termination will be determined by the pET28a termination codon. The sequences were cloned separately in pET28a vectors, under the command of the T7 polymerase promoter, using the restriction enzymes Bam Hl and Hind III, positioning the hexa-histidine in the N-terminal region. [00153] Specifically with respect to clones 4.1, 4.5, 4.7 (antigen 1), 4.8 and 4.9, vector transfer subcloning was performed. To isolate the insert, 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).
[00154] O vetor pET28a foi linearizado por digestão com as enzimas Bam Hl e Hind III e teve as extremidades defosforiladas pela Fosfatase Alcalina (FastAp - ThermoFischer ) a 37°C, conforme protocolo do fabricante e a ligação dos insertos contendo os genes sintéticos foi realizada usando a enzima Ligase T4 ( Thermo Scientific ), a 16°C, por 16h. Uma alíquota do conteúdo da reação de ligação foi utilizado para transformação em E. coli DH5a (Promega) por choque térmico e confirmada por análise dos fragmentos no gel SDS-PAGE. O controle da reação de ligação constituiu na adição apenas do pET28a desfosforilado e da enzima T4 DNA ligase sem a presença do inserto num tubo. O Tag de hexa-histidina foi posicionado na região N- terminal.  [00154] 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.
[00155] As sequências codificadoras dos antígenos 2 a 5 foram obtidas a partir de amplificação por PCR das regiões NS4 e NS5. Para isso, foram utilizados iniciadores diretos (forward ) e reversos ( reverse ) (SEQ ID Nos: 11 a 16).  [00155] 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.
[00156] Especificamente com relação ao antígeno 2, a sequência codificadora do mesmo foi obtida a partir da região“NC+NS4” presente em clone em pUC57 (clone 4.5). Posteriormente, o produto de amplificação foi inserido no vetor pET28a utilizando as enzimas Bam Hl e Hind III. Foram utilizados os iniciadores de SEQ ID NOs: 11 e 12 para amplificar a região NC+NS4. [00157] Especificamente com relação ao antígeno 3, a sequência codificadora do mesmo foi obtida por amplificação a partir da região NS5 (clone 4.5) e fusionada no C-terminal de plasmídeo pET28a+NS3 (clone 4.1) utilizando as enzimas Eco RI e Hind III. Foram utilizados os iniciadores de SEQ ID Nos: 13 e 14. [00156] Specifically with respect to antigen 2, its coding sequence was obtained from the “NC + NS4” region present in a clone in pUC57 (clone 4.5). Subsequently, the amplification product was inserted into the pET28a vector using the enzymes Bam Hl and Hind III. Primers of SEQ ID NOs: 11 and 12 were used to amplify the NC + NS4 region. [00157] Specifically with respect to antigen 3, its coding sequence was obtained by amplification from the NS5 region (clone 4.5) and fused at the C-terminal of plasmid pET28a + NS3 (clone 4.1) using the enzymes Eco RI and Hind III. Primers of SEQ ID Nos: 13 and 14 were used.
[00158] Especificamente com relação ao antígeno 4, a sequência codificadora do mesmo foi obtida pelo isolamento da região NS4 de plasmídeo pUC57 (clone 4.5 ou 4.11) e o produto amplificado e digerido foi ligado a plasmídeo pET28a+NS3 (clone 4.1) utilizando as enzimas EcoR I e Hind III. Foram utilizados os iniciadores de SEQ ID Nos: 15 e 16 para isolar a região NS4.  [00158] Specifically with respect to antigen 4, 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.
[00159] Especificamente com relação ao antígeno 5, a sequência codificadora do mesmo foi obtida pela utilização dos iniciadores de SEQ ID Nos: 15 e 16 para amplificar a região NS5 (clone 4.5) e fusioná-la no C-terminal de plasmídeo pET28a+NC (clone 4.2) utilizando as enzimas EcoRI e Hind III.  [00159] Specifically with respect to antigen 5, its coding sequence was obtained by using the primers of SEQ ID Nos: 15 and 16 to amplify the NS5 region (clone 4.5) and fuse it in the C-terminal of plasmid pET28a + NC (clone 4.2) using the enzymes EcoRI and Hind III.
[00160] A reação de amplificação foi realizada com 1U da Taq Platinum High Fidelity (Invitrogen) e com 20 a 40ng de DNA plasmidial template. A reação foi realizada atentando-se para a temperatura de dissociação (Tm) dos primers. Confirmados os tamanhos dos fragmentos amplificados por gel de agarose 0,8%, estes foram purificados usando o kit de purificação para produto da PCR da Qiagen. A seguir, as extremidades das sequências foram digeridas com as enzimas Bam Hl e Hind III para gerarem extremidades coesivas e obter a ligação no vetor pET28A linearizado na presença da T4 Ligase, a 16°C por 16h. [00160] 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.
1.1. Seleção dos clones positivos 1.1. Selection of positive clones
[00161] Com relação ao antígeno 1, após a transformação foi necessário confirmar a positividade dos clones para as colónias que cresceram em meio LB com canamicina (25pg/mL). Aleatoriamente, foram selecionadas dez colónias que, com o auxílio de uma alça de platina, foram transferidas para 5mL de meio líquido LB contendo o antibiótico. Após a propagação bacteriana, por 16h, a 37°C, a biomassa foi coletada e o DNA plasmidial extraído por lise alcalina. A presença do inserto foi confirmada por digestão com as enzimas de restrição Bam Hl e Hind III e análise das digestões em gel de agarose 0,8 % após digestão enzimática. [00161] Regarding antigen 1, after transformation it was necessary to confirm the positivity of the clones for colonies that grew in LB medium with kanamycin (25pg / mL). At random, ten colonies that, with the aid of a platinum loop, were transferred to 5mL of LB liquid medium containing the antibiotic. After bacterial propagation, for 16h, at 37 ° C, the biomass was collected and the plasmid DNA extracted by alkaline lysis. The presence of the insert was confirmed by digestion with restriction enzymes Bam Hl and Hind III and analysis of digestions in 0.8% agarose gel after enzymatic digestion.
[00162] A seleção das colónias positivas para os antígenos 2 a 5 foi realizada por PCR de colónia. Para isto, as colónias escolhidas de forma aleatória foram coletadas com o auxílio de um palito estéril e transferidas para um tubo do tipo Eppendorf contendo 20 pL de água. Os tubos foram incubados por 10 minutos a 95°C para a lise das células e apenas lpL deste material foi adicionado a um tubo do tipo Eppendorf contendo uma solução MIX para a reação da PCR no volume final de 20pL. As condições de amplificação foram as mesmas determinadas para a obtenção da sequência do inserto.  [00162] Selection of positive colonies for antigens 2 to 5 was performed by colony PCR. For this, the colonies chosen at random were collected with the aid of a sterile toothpick and transferred to an Eppendorf tube containing 20 pL of water. The tubes were incubated for 10 minutes at 95 ° C for lysis of the cells and only 1pL of this material was added to an Eppendorf tube containing a MIX solution for the PCR reaction in the final 20pL volume. The amplification conditions were the same as those determined for obtaining the insert sequence.
1.2. Estoque de colónias positivas transformadas 1.2. Stock of transformed positive colonies
[00163] As colónias positivas foram cultivadas em 5mL de meio líquido LB contendo canamicina (25pg/ml), crescidas sob agitação a 37°C por 16h. Foram realizadas alíquotas deste material em tubos do tipo Eppendorf c ontendo 200pL de glicerol (60%) na proporção 1 :2. Os tubos foram identificados e armazenados em freezer a -80°C. Adicionalmente, placas de Petri contendo as colónias do clone positivo foram mantidas a 4°C por até um mês para uso imediato. Quando necessário, os tubos armazenados a -80°C, contendo as células em glicerol, foram retirados para a manutenção das células no refrigerador.  [00163] Positive colonies were cultured in 5mL of LB liquid medium containing kanamycin (25pg / ml), grown under agitation at 37 ° C for 16h. Aliquots of this material were carried out in Eppendorf tubes containing 200pL of glycerol (60%) in a 1: 2 ratio. The tubes were identified and stored in a freezer at -80 ° C. In addition, Petri dishes containing the colonies of the positive clone were kept at 4 ° C for up to one month for immediate use. When necessary, the tubes stored at -80 ° C, containing the cells in glycerol, were removed to keep the cells in the refrigerator.
EXEMPLO 2: Expressão dos polipeptídeos recombinantes EXAMPLE 2: Expression of recombinant polypeptides
[00164] Os polipeptídeos demonstraram altos níveis de expressão na cepa E. coli BL21 Star (DE3) cultivada em meio líquido LB (Luria Bertani - 10g Triptona, 5g Extrato de Levedura e 5g NaCl/L). [00164] 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).
[00165] Estabelecidas as condições ideais para a expressão das proteínas, foram realizados cultivo em volumes acima de 1L de cultura que foram obtidos a partir de 25mL de um pré-inóculo do transformante positivo crescido por 16h a 37°C, sob agitação (250 rpm) e transferido para um frasco Erlenmeyer de 2L contendo 500mL de meio LB suplementado com antibiótico. As células foram cultivadas até a obtenção da densidade óptica de 0,6-0, 8 nm quando foi adicionado lmM de IPTG. O cultivo prosseguiu por mais quatro horas. Ao final da expressão, a biomassa foi gentilmente coletada por centrifugação a 5.000 rpm, 15 minutos, sob refrigeração (4°C) e o sobrenadante descartado. A células foram lavadas com lOOmL de PBS 1 X e novamente centrifugada. O sobrenadante foi descartado e a biomassa pesada. O peso da biomassa foi anotado a fim de avaliar o rendimento da expressão para cada proteína após a etapa de purificação. O material pôde ser armazenado a -20°C até o momento do uso.  [00165] Established the ideal conditions for the expression of proteins, culture was performed in volumes above 1L of culture that were obtained from 25mL of a positive transformant pre-inoculum grown for 16h at 37 ° C, under agitation (250 rpm) and transferred to a 2L Erlenmeyer flask containing 500mL of LB medium supplemented with antibiotics. The cells were cultured until the optical density of 0.6-0.8 nm was obtained when 1 mM IPTG was added. Cultivation continued for another four hours. At the end of the expression, the biomass was gently collected by centrifugation at 5,000 rpm, 15 minutes, under refrigeration (4 ° C) and the supernatant discarded. 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.
[00166] Os polipeptídeos foram expressos na forma de corpos de inclusão, cujas análises por gel SDS-PAGE 13% revelam que as posições das bandas, após a eletroforese, correspondem aos tamanhos esperados para as construções (Figura 01).  [00166] 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).
EXEMPLO 3: Lise das células e tratamento dos corpos de inclusão EXAMPLE 3: Lysis of cells and treatment of inclusion bodies
[00167] A biomassa, foi adicionado Tampão de Lise contendo 50mM de fosfato de sódio pH 7,4, 300mM de NaCl, 5% de glicerol, 0,05% Tween 20, 7mM b-mercaptoetanol (b-ME) e inibidores de proteases (lmM PMSF (phenylmethanesulfonyl fluo ride) e coquetel inibidor de proteases da Roche (1 comprimido/ 10ml de tampão)) na proporção de 3-4mL de tampão para cada lg de célula. Os tampões foram avaliados de acordo com o pi de cada proteína, bem como a necessidade da adição de agentes redutores, detergentes ou glicerol na tentativa de manter a estabilidade ou a solubilização das mesmas. A lise das células para volumes pequenos de cultura (25-50mL) foi realizada usando o reagente FastBreak Cell Lysis Reagent da Promega de acordo com a descrição do fabricante, seguida de dois ciclos de sonicação usando o equipamento ultrassónico Cole Parmer processor a 40% de amplitude, 30 segundos on e um minuto off ou adicionando 200pg/mL de Lisozima aos tubos contendo as células e incubando-os por até 30 minutos em tampão contendo 50mM fosfato de sódio pH 7,4, 300mM NaCl, 5% glicerol, 0,05% Tween 20 e 7mM b- mercaptoetanol (b-ME). [00167] Biomass, Lysis Buffer containing 50mM sodium phosphate pH 7.4, 300mM NaCl, 5% glycerol, 0.05% Tween 20, 7mM b-mercaptoethanol (b-ME) and inhibitors of proteases (1mM PMSF (phenylmethanesulfonyl fluo ride) and Roche protease inhibitor cocktail (1 tablet / 10ml of buffer)) in the proportion of 3-4mL of buffer for each lg of cell. 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 (25-50mL) 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).
[00168] Além disto, aos tampões foram adicionados inibidores de proteases como o lmM de PMSF e o coquetel inibidor de proteases da Roche. Após incubação, a ruptura da parede das células se deu por sonicação utilizando o equipamento ultrassónico da Cole Parmer processor por seis ciclos de lise a uma amplitude de 40%, 30 segundos de pulso e 60 segundos de intervalo a 4°C. O extrato foi clarificado por centrifugação (20 minutos, 4°C, a 20.000 g).  [00168] In addition, to the buffers were added protease inhibitors such as PMSF lmM and Roche's protease inhibitor cocktail. After incubation, 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).
[00169] A lise de biomassa a partir de cultivos para volumes acima de 1L foi realizada no equipamento microfluidificador (Microfluidics Corporation, USA) utilizando uma pressão de aproximadamente 80 Psi em banho de gelo. As células foram ressuspensas em Tampão de Lise (50 mM fosfato de sódio pH 7,4, 300mM NaCl, 5% glicerol, 0,05% Tween 20, 7mM b- mercaptoetanol (b-ME) e inibidores de proteases) utilizando 3-4mL de tampão para cada lg de células. [00169] 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.
3.1: Tratamento dos polipeptídeos expressos na forma de corpos de inclusão e solubilização 3.1: Treatment of polypeptides expressed as inclusion and solubilization bodies
[00170] Após a lise das células, os sedimentos das centrifugações contendo os corpos de inclusão foram coletados e o material tratado para a clarificação dos corpos de inclusão. Para isto, foi adicionado aproximadamente 7mL de tampão contendo 50mM de fosfato de sódio pH 7,4, 300mM NaCl, 5% glicerol, 1% de Tween 20, 2% de Triton-X 100, 7mM de b-ME e inibidores de proteases para cada 1 grama de biomassa. O material foi sonicado utilizando o equipamento ultrassónico com amplitude de 40% em 6 ciclos de 30 segundos e intervalo de 30 segundos em banho de gelo. Na sequência, o material foi centrifugado a 20.000g por 30 minutos e, novamente, levado ao equipamento ultrassónico com tampão composto por 50mM de fosfato de sódio pH 7,4, 300mM NaCl, 7mM de b-ME com o objetivo de retirar resquícios do Triton X- 100 presente no tampão anterior. Os corpos de inclusão foram recuperados por centrifugação e armazenados a -20°C até o momento do uso. [00170] After the lysis of the cells, the sediments from the centrifuges containing the inclusion bodies were collected and the material treated to clarify the inclusion bodies. For this, approximately 7mL of buffer containing 50mM sodium phosphate pH 7.4, 300mM NaCl, 5% glycerol, 1% Tween 20, 2% Triton-X 100, 7mM b-ME and inhibitors of proteases for every 1 gram of biomass. The material was sonicated using the ultrasonic equipment with 40% amplitude in 6 cycles of 30 seconds and an interval of 30 seconds in an ice bath. Then, 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.
[00171] Para a solubilização, aos corpos de inclusão, descongelados, foi adicionado tampão de solubilização composto por 8M uréia, 7mM b-ME, 150mM NaCl, l-20mM Ditiotreitol (DTT), lmM PMSF ou 6M Guanidina, 7mM b-ME, 150mM NaCl, l-20mM Ditiotreitol (DTT) e lmM PMSF na proporção de 3 a 7mL de tampão por grama de material que foi incubado à temperatura ambiente por até 16h fazendo uso de um agitador do tipo orbital (20 rotações por minuto). Ao final da incubação, seguiu-se com uma curta sonicação de três ciclos, usando 40% de amplitude por 10 segundos de pulso e 30 segundos de intervalo em banho de gelo e centrifugação a 20.000 x g por 30 minutos. O pellet foi descartado e o sobrenadante recolhido e encaminhado para a purificação.  [00171] For solubilization, the inclusion bodies, thawed, were added 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). At the end of the incubation, it was followed by a short sonication of three cycles, using 40% amplitude for 10 seconds of pulse and 30 seconds of interval in ice bath and centrifugation at 20,000 x g for 30 minutes. The pellet was discarded and the supernatant collected and sent for purification.
[00172] Foi obtida a solubilização das proteínas em tampão contendo 6M Guanidina, o que permitiu reverter a afinidade das proteínas à coluna e obter as amostras purificadas (vide Figura 6.4, gel C ou D) e as eluições foram obtidas em tampão contendo 4M de uréia e 500 mM de imidazol.  [00172] 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.
3.2: Retirada da ureia ou guanidina das frações das proteínas solúveis 3.2: Removal of urea or guanidine from fractions of soluble proteins
[00173] Uma vez que a ureia e a guanidina são agentes incompatíveis com as técnicas de SDSPAGE e Western Blotting, fez-se uso do protocolo descrito por Palmer e Wingfield (2004), no qual 20 pL das alíquotas de proteínas solubilizadas ou frações purificadas receberam 180pL de etanol 100%, previamente resfriado (4°C). A mistura foi agitada em um vórtex e incubada por 10 minutos a -20°C ou por 5 minutos a - 80°C. Seguiu-se com a centrifugação dos tubos por 5 minutos a 15.000 g, 4°C. O sobrenadante foi retirado gentilmente e o pellet foi ressuspendido em 200 pL de etanol 90% gelado. O tubo foi vigorosamente agitado usando o vórtex e uma nova centrifugação de 5 minutos a 15.000g foi realizada a 4°C. Nesta etapa, as proteínas são precipitadas pela ação do etanol e o sobrenadante deve ser descartado. [00173] Since urea and guanidine are agents incompatible with the techniques of SDSPAGE and Western Blotting, the protocol described by Palmer and Wingfield (2004) was used, in which 20 pL of the aliquots of solubilized proteins or purified fractions received 180pL of ethanol 100%, previously cooled (4 ° C). The mixture was vortexed and incubated for 10 minutes at -20 ° C or for 5 minutes at - 80 ° C. This was followed by centrifuging the tubes for 5 minutes at 15,000 g, 4 ° C. The supernatant was gently removed and the pellet was resuspended in 200 pL of cold 90% ethanol. The tube was vigorously shaken using the vortex and a new 5 minute centrifugation at 15,000g was performed at 4 ° C. In this step, proteins are precipitated by the action of ethanol and the supernatant must be discarded.
[00174] O tubo contendo a proteína precipitada foi mantido à temperatura ambiente por alguns minutos para a completa evaporação do etanol. A ressolubilização das proteínas foi realizada após a adição de 6pL de água destilada e 4pL de tampão de corrida (IX) aos tubos que foram aquecidos a 95°C e cujas frações foram analisadas por gel SDS-PAGE ou Western Blotting.  [00174] 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.
EXEMPLO 4: Purificação dos polipeptídeos EXAMPLE 4: Purification of Polypeptides
[00175] A purificação foi realizada por cromatografia de afinidade em condições desnaturantes em tampão A contendo 6M de guanidina, lOOmM de fosfato, lOmM de Tris-HCl (pH 7.5), 7mM de b-mercaptoetanol, lmM de fluoreto de fenilmetilsulfonila (PMSF) para ligação e tampão B contendo 4M de ureia, lOOmM de Fosfato, lOmM de Tris-HCl (pH 7.5), 7mM de b- mercaptoetanol, 500mM de Imidazol, lmM de PMSF para a diluição, utilizando a coluna Ni-NTA de 1 ou 5mL da GE no equipamento ÀKTA purifier 100, cromatógrafo semi-automatizado.  [00175] Purification was performed by affinity chromatography under denaturing conditions in buffer A containing 6M guanidine, 100mM phosphate, 10mM Tris-HCl (pH 7.5), 7mM b-mercaptoethanol, 1mM phenylmethylsulfonyl fluoride (PMSF) for binding and buffer B containing 4M urea, 100mM Phosphate, 10mM Tris-HCl (pH 7.5), 7mM b-mercaptoethanol, 500mM Imidazole, 1mM PMSF for dilution using the Ni-NTA column of 1 or 5mL from GE in the ÀKTA purifier 100 equipment, semi-automated chromatograph.
[00176] Os procedimentos de purificação empregados foram:  [00176] The purification procedures employed were:
Fluxo: l mL/mim  Flow: l mL / m
Etapa 1 : Tampão A, 10 VC  Step 1: Buffer A, 10 VC
Gradiente: Etapa 2: 0 a 10% de tampão B, 10 VC, Gradient: Step 2: 0 to 10% buffer B, 10 VC,
Etapa 3: 10 a 100% de B Step 3: 10 to 100% B
[00177] A composição dos tampões (mostrados na Tabela 2) e parâmetros de purificação utilizados resultaram em maiores rendimentos e na obtenção de proteínas mais puras. A solubilização das proteínas ocorreu em tampão A, com a adição de 1 a 20mM de DTT, overnight sob agitação (29 rotações/minuto), temperatura ambiente. [00177] The composition of the buffers (shown in Table 2) and Purification parameters used resulted in higher yields and in obtaining purer proteins. Protein solubilization occurred in buffer A, with the addition of 1 to 20mM DTT, overnight under stirring (29 revolutions / minute), at room temperature.
Tabela 2: Composição dos tampões utilizados para a padronização de purificação de proteínas. Todos os tampões receberam inibidores de proteases como o 1 mM de PMSF ( Phenylmethanesulfonyl fluoride ), coquetel inibidor de proteases (Roche) e 7 mM de b-mercaptoetanol (b-ME). 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).
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000044_0001
Figure imgf000045_0001
[00178] Antes de ser aplicada à resina de níquel, os antígenos foram diluídos até concentrações inferiores à 5mM de DTT (por questão de incompatibilidade com a técnica cromatográfica por afinidade) e filtrados. [00178] Before being applied to nickel resin, the antigens were diluted to concentrations below 5mM DTT (due to incompatibility with the affinity chromatographic technique) and filtered.
[00179] As proteínas não foram purificadas na presença de tampão contendo ureia 8M. A ligação entre as proteínas solubilizadas e a resina contendo níquel foi obtida apenas na presença de tampão contendo 6M de guanidina. A substituição do agente desnaturante foi fundamental para a eficiência de ligação das proteínas à resina de níquel, o que permitiu a purificação das mesmas. O motivo pelo qual isto ocorreu pode estar relacionado à falta de exposição do Tag de hexa-histidina. [00179] 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.
[00180] Os rendimentos obtidos com os sistemas de expressão e purificação da presente invenção corresponderam à obtenção de 10 mg/L para o Antígeno 1 , 8,4mg/mL para o Antígeno 2, 24mg/L para o Antígeno 3, 18mg/L para o Antígeno 4 e 9,6 mg/mL para o Antígeno 5 que apresentaram pureza acima de >95%, conforme mostra a Figura 1. [00180] 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.
[00181] As estimativas para os rendimentos de cada proteína foram determinadas a partir da quantificação final, obtida através das dosagens das frações mais puras e aproveitáveis, em gramas, após a purificação. Frações impuras com presença de contaminantes não foram utilizadas para o cálculo de rendimento.  [00181] The estimates for the yields of each protein were determined from the final quantification, obtained by measuring the purest and most usable fractions, in grams, after purification. Impure fractions with contaminants were not used to calculate yield.
[00182] Estas informações foram registradas e os rendimentos relativos por litro de cultura foram estipulados: Antígeno 1 (lOmg), Antígeno 2 (8,4mg), Antígeno 3 (24mg), Antígeno 4 (18mg), Antígeno 5 (9,6mg). As proteínas demonstraram altos níveis de pureza (> 95%).  [00182] This information was recorded and the relative yields per liter of culture were stipulated: Antigen 1 (10mg), Antigen 2 (8.4mg), Antigen 3 (24mg), Antigen 4 (18mg), Antigen 5 (9.6mg ). The proteins demonstrated high levels of purity (> 95%).
EXEMPLO 5: Análise dos polipeptídeos EXAMPLE 5: Analysis of Polypeptides
[00183] Análises in silico ProtParam das sequências de SEQ ID NOs: 1, 3, 5, 7 e 9 identificaram parâmetros teóricos bioquímicos, conforme demonstrado na Tabela 3. Para essas análises, foram consideradas a sequência de 34 aminoácidos codificados pela sequencia de nucleotídeos do vetor pET28a em fase com o a sequencia do linker e a sequencia dos antígenos (sequencia traduzida: MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGS + poly- linker MAGS).  [00183] ProtParam in silico analyzes of the sequences of SEQ ID NOs: 1, 3, 5, 7 and 9 identified theoretical biochemical parameters, as shown in Table 3. For these analyzes, the sequence of 34 amino acids encoded by the nucleotide sequence were considered of the pET28a vector in phase with the linker sequence and the antigen sequence (translated sequence: MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGS + poly-linker MAGS).
Tabela 3: Parâmetros obtidos a partir da análise in silico das sequências aminoacídicas para os polipeptídeos.  Table 3: Parameters obtained from the in silico analysis of the amino acid sequences for the polypeptides.
Figure imgf000046_0001
Figure imgf000046_0001
n° de aa = número de aminoácidos; MW = massa molecular; pi = ponto isoelétrico; Coef. de extinção = coeficiente de extinção. EXEMPLO 6: Formulação dos tampões de armazenamento para estabilização das proteínas aa number = number of amino acids; MW = molecular weight; pi = isoelectric point; Coef. extinction = extinction coefficient. EXAMPLE 6: Formulation of storage buffers for protein stabilization
[00184] O tampão de armazenamento foi definido após experimentos com o objetivo de prevenir perdas por precipitação, manter a estabilidade das proteínas e retirar resquícios de ureia, guanidina ou imidazol e diminuir a concentração do sal, uma vez que estes agentes podem agir como interferentes para as técnicas de imunoensaios a serem empregadas. Assim, o tampão de armazenamento para os antígenos, após purificação foi composto por 4M de ureia, lOmM de Fosfato de sódio (pH 7,5), 20mM de NaCl, 7mM de B- mercaptoetanol, lmM de PMSF.  [00184] 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. Thus, 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.
[00185] As análises de estabilidade e integridade das proteínas foram realizadas pós-descongelamentos, durante o período de 12 meses, dos quais, periodicamente, tubos contendo 200uL de proteínas em concentrações que variaram 1,5 a 3 mg/mL foram mantidas a -20°C em tampão de estoque.  [00185] 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.
[00186] Durante este período, foram realizados três descongelamentos pontuais, a cada três meses, em banho de gelo a fim de avaliar a integridade destas proteínas em gel SDS-PAGE (13%), o que demonstrou a preservação das mesmas. Adicionalmente, após este período e descongelamentos, as proteínas foram avaliadas quando a reprodutibilidade de ensaios (sensibilidade e especificidade) por teste em microesferas, o que acusou desempenho semelhante ao obtido logo após a purificação. Podendo inferir que o tempo de vida das proteínas podem ser superiores ao tempo testado. [00186] During this period, three point defrostings were performed, every three months, in an ice bath in order to assess the integrity of these proteins in SDS-PAGE gel (13%), which demonstrated their preservation. Additionally, after this period and thawing, proteins were evaluated when the reproducibility of assays (sensitivity and specificity) by testing on microspheres, which showed a performance similar to that obtained immediately after purification. It can be inferred that the lifetime of proteins may be longer than the time tested.
[00187] Devido à presença da ureia no tampão de armazenamento, não foi possível quantificar as proteínas por métodos como Bradford, BCA (SIGMA, USA) ou através do Qubit® (Qiagen) devido à necessidade de diluições em tampões específicos sem a presença de ureia. Assim, a quantificação das proteínas foi determinada por absorbância a 280 nm no espectrofotômetro Nanodrop 2000 quando as amostras foram diluídas no tampão de armazenamento, contendo ureia. [00187] Due to the presence of urea in the storage buffer, it was not possible to quantify proteins by methods such as Bradford, BCA (SIGMA, USA) or through Qubit® (Qiagen) due to the need for dilutions in specific buffers without the presence of urea. Thus, protein quantification was determined by absorbance at 280 nm in the Nanodrop 2000 spectrophotometer when the samples were diluted in the storage buffer, containing urea.
[00188] Paralelamente, foi realizado uma análise qualitativa, como controle, através de uma curva contendo concentrações conhecidas de BSA. As medidas foram realizadas utilizando a massa molecular e o coeficiente de extinção molar reduzido de cada proteína, A absorbância do branco foi subtraída pela absorbância das amostras, cujo valor foi multiplicado pelo fator de diluição.  [00188] In parallel, a qualitative analysis was performed, as a control, through a curve containing known concentrations of BSA. The measurements were performed using the molecular weight and the reduced molar extinction coefficient of each protein. The absorbance of the blank was subtracted by the absorbance of the samples, whose value was multiplied by the dilution factor.
[00189] A análise da integridade das proteínas após 12 meses de uma sequência de descongelamentos que ocorreu a cada três meses e a avaliação da reprodutibilidade do imunoensaio no final deste período foi realizada. O descongelamento das alíquotas de proteínas foi realizado conforme descrito na metodologia. O gel de SDS-PAGE (13%) para avaliação qualitativa dos antígenos pode ser visualizado na Figura 2 e apresenta pouca presença de degradação, revelando integridade das proteínas. O acoplamento foi realizado após descongelamentos. O imunoensaio demonstrou reprodutibilidade quanto à sensibilidade e especificidade obtidos previamente, com intervalo de confia a [00189] The analysis of the integrity of proteins after 12 months of a sequence of thawing that occurred every three months and the evaluation of the reproducibility of the immunoassay at the end of this period was carried out. Thawing of protein aliquots was performed as described in the methodology. The SDS-PAGE gel (13%) for qualitative evaluation of the antigens can be seen in Figure 2 and has little presence of degradation, revealing protein integrity. Coupling was performed after thawing. The immunoassay demonstrated reproducibility in terms of sensitivity and specificity previously obtained, with a confidence interval at
95%. Esta avaliação, embora não tenha seguido os padrões do guia de estabilidade para reagentes de diagnósticos in vitro chamado de Clinicai and laboratorial Standards Institute publicado pelo CDC, USA, sugere que os antígenos desenvolvidos podem se manter estáveis por período até superior ao avaliado, mantendo sua capacidade reativa. E possível que a composição do tampão de estoque tenha favorecido esta estabilidade devido ao uso de inibidores de proteases ou pela presença do agente desnaturante ou, até mesmo, devido à sequência de aminoácidos das mesmas. Logo, antígenos estáveis e resistentes a proteases também são atrativos quando existe interesse de aplicação diagnóstica. 95%. This evaluation, although it did not follow the standards of the stability guide for in vitro diagnostic reagents called the Clinicai and laboratorial Standards Institute published by CDC, USA, suggests that the developed antigens can remain stable for a period even longer than the one evaluated, maintaining their reactive capacity. It is possible that the composition of the stock buffer has favored this stability due to the use of protease inhibitors or the presence of the denaturing agent or even due to their amino acid sequence. Therefore, stable and protease-resistant antigens are also attractive when there is interest for diagnostic application.
EXEMPLO 7: Imunoensaios EXAMPLE 7: Immunoassays
[00190] Foi aplicado o modelo de ensaio indireto que consiste no acoplamento dos antígenos às microesferas ou no revestimento de antígenos às cavidades das microplacas de poliestireno visando a interação destes com anticorpos específicos presentes no soro de indivíduos infectados. [00190] 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.
[00191] Foram utilizados controles positivos para as imunorreações usando as proteínas comerciais quiméricas para HCV da Fapon (GECHCV1303, specificity Core, NS3, NS4, NS5) e da ProSpec (#HCV-207 specificity Core, NS3, NS4 e NS5) ou ainda uma proteína TpN17 para Sífilis.  [00191] Positive controls for immunoreactions were used using the commercial chimeric proteins for HCV from Fapon (GECHCV1303, specificity Core, NS3, NS4, NS5) and ProSpec (# HCV-207 specificity Core, NS3, NS4 and NS5) or even a TpN17 protein for syphilis.
[00192] Os ensaios passaram por etapas de otimização até a completa padronização com a avaliação da melhor concentração de proteína a ser utilizada no acoplamento ou a massa ideal de antígenos para o revestimento das superfícies das microesferas. Foram avaliados também o tipo de bloqueio e concentração, a diluição de trabalho do soro e do anticorpo secundário, além do tempo estimado para reação. [00192] 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.
[00193] No ensaio LUMINEX, foram realizados os bloqueios dos sítios reativos com tampão PBS/TBN utilizado para o armazenamento das microesferas. Este tampão contém 1 % de albumina de soro bovino (BSA) que foi responsável por preencher sítios ainda expostos na superfície das microesferas não recobertos pelo antígeno. No ensaio ELISA, o bloqueio foi realizado com 2% de caseína (peso/volume) a partir de leite em pó desnatado.  [00193] In the LUMINEX assay, the reactive sites were blocked with PBS / TBN buffer used for the storage of the microspheres. This buffer contains 1% bovine serum albumin (BSA) which was responsible for filling sites still exposed on the surface of microspheres not covered by the antigen. In the ELISA test, blocking was performed with 2% casein (weight / volume) from skimmed-milk powder.
[00194] Adicionalmente, tanto o ensaio ELISA quanto o LUMINEX receberam 2% do extrato de Escherichia coli como um bloqueio adicional, uma vez que as proteínas desenvolvidas haviam sido obtidas por expressão em E. coli. [00194] Additionally, both the ELISA assay and LUMINEX received 2% of Escherichia coli extract as an additional block, since the proteins developed had been obtained by expression in E. coli.
[00195] Em ambos os ensaios, os valores obtidos para o sinal de fundo (ruído) foram subtraídos dos valores obtidos a partir da leitura dos soros.  [00195] In both tests, the values obtained for the background signal (noise) were subtracted from the values obtained from the reading of the sera.
[00196] Os ensaios imunodiagnósticos tanto pela plataforma de microarranjo quanto por ELISA foram realizados utilizando as proteínas provenientes da purificação pelo equipamento ÀKTA frente ao painel completo de Análise Externa de Qualidade (AEQ), que tem como principal característica a alta reatividade aos marcadores das doenças. [00196] The immunodiagnostic assays both by the microarray platform and by ELISA were performed using the proteins from the purification by the ÀKTA equipment in front of the complete panel of External Quality Analysis (AEQ), whose main characteristic is high reactivity to disease markers.
[00197] O painel AEQ é caracterizado pela FIOCRUZ, INCQS e composto por 192 soros, dos quais 23 são reativos para HCV, 26 para HIV 1/2, 30 HTLV 1/2, 31 HBV, 30 Sífilis, 28 Chagas e 21 soros são negativos para todos os patógenos que compões o painel (Figura 3).  [00197] 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).
7.1. Preparo do extrato de Escherichia coli 7.1. Preparation of Escherichia coli extract
[00198] Partindo do descongelamento de um tubo do tipo Eppendorf contendo as células de E. coli DH5-a acondicionado a -80°C, foi realizado um pré-cultivo em tubo de ensaio a partir de 2mL de meio líquido LB cultivados a 37°C por 24 horas sob agitação, 250 rpm. O conteúdo do tubo de ensaio foi transferido para um Erlenmeyer contendo 1 litro do meio LB com antibiótico que foram mantidos em cultura até atingir 0,6 a 0,8 de absorbância na densidade óptica (280 nm no espectrofotômetro Ultrospec II®). A biomassa foi centrifugada sob baixa rotação em centrífuga Sorvall® sob refrigeração e lavadas com PBS IX, pH 7,2. O sobrenadante foi desprezado e o pellet recuperado em 10 ml de tampão PBS. A biomassa foi lisada utilizando o Sonicador Ultrasonic Homogenizer® a 40% de amplitude por três ciclos de 20 segundos on e 1 minuto q/ em banho de gelo. O extrato lisado foi centrifugado a 10.000 rpm por 20 minutos e o sobrenadante foi filtrado em membrana Millipore de 0,45 pm, seguida de uma nova filtração em membrana de 0,22 pm. O pellet foi descartado e ao extrato foi adicionado 0,1 % de uma solução de azida sódica para preservação. Após a quantificação, o material foi aliquotado em microtubos que foram armazenados a -20°C. A solução de trabalho foi utilizada na concentração de 10 mg/mL diluídos em tampão PBS pH 7.2 e mantidos a 4°C.  [00198] Starting from defrosting an Eppendorf tube containing E. coli DH5-a cells stored at -80 ° C, a pre-culture in a test tube was carried out from 2 ml of LB liquid medium grown at 37 ° C. ° C for 24 hours under agitation, 250 rpm. The contents of the test tube were transferred to an Erlenmeyer containing 1 liter of LB medium with antibiotics, which were kept in culture until reaching 0.6 to 0.8 absorbance in the optical density (280 nm in the Ultrospec II® spectrophotometer). The biomass was centrifuged at low speed in a Sorvall® centrifuge under refrigeration and washed with PBS IX, pH 7.2. 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.
7.2. Ensaios do tipo ELISA 7.2. ELISA-type assays
7.2.1. Ensaios Simples ( Singleplex ) [00199] Inicialmente, foram avaliadas as condições ótimas para realização dos ensaios, com o objetivo de avaliar a massa mínima de antígeno (em ng) e obter uma boa separação entre as amostras positivas e negativas. Para cada antígeno, foi realizada uma curva de onze pontos em triplicata onde os poços receberam 2,5, 5, 10, 20, 50, 75, 100, 150, 200, 300, 500 e l .OOOng/poço em tampão carbonato-bicarbonato 50mM, pH 9.6 que foi testado contra um pool de amostras reativas para HCV. 7.2.1. Simple Tests (Singleplex) [00199] Initially, the optimal conditions for carrying out the tests were evaluated, with the objective of evaluating the minimum antigen mass (in ng) and obtaining a good separation between the positive and negative samples. For each antigen, an eleven point triplicate curve was performed where the wells received 2.5, 5, 10, 20, 50, 75, 100, 150, 200, 300, 500 and .OOOng / well in carbonate-bicarbonate buffer 50mM, pH 9.6 which was tested against a pool of HCV reactive samples.
[00200] Posteriormente, os ensaios foram realizados com a adição de 50pL do tampão carbonato/bicarbonato pH 9,6 contendo 20ng da proteína de interesse para adesão. A placa foi vedada com filme selante e armazenada a 4°C por 16 horas. Na sequência, o excesso da solução de revestimento foi removido por lavagem com 150pL de PBS-T (3X) e foram adicionados aos poços 100pL de uma solução de bloqueio PBS-T contendo 2% de caseína, a placa foi incubada durante 1 hora a 37°C, sob leve agitação (200rpm).  [00200] Subsequently, 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. Then, 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).
[00201] Após a incubação, as placas foram lavadas com 150pL de tampão PBS-T (5X) seguida da adição de 50pL do soro previamente diluído (1 : 100) em tampão PBS-T contendo 2% do extrato de E. coli. A placa foi novamente incubada por 40 minutos a 37 °C sob agitação e lavada com 150pL de PBS-T (5X). Por fim, 50pL do anticorpo monoclonal anti-IgG humana conjugado a HRP (SIGMA, USA) diluído 1 :5.000 foi adicionado aos poços e a placa incubada, mais uma vez, a 37°C por lh, 200 rpm. Procedeuse as etapas de lavagens por 5X, seguida da adição de 50pL do substrato TMB (KPL, USA).  [00201] After incubation, the plates were washed with 150pL of PBS-T buffer (5X) followed by the addition of 50pL of the previously diluted serum (1: 100) in PBS-T buffer containing 2% of the E. coli extract. The plate was again incubated for 40 minutes at 37 ° C with shaking and washed with 150 µl of PBS-T (5X). Finally, 50pL of HRP-conjugated anti-human IgG monoclonal antibody (SIGMA, USA) diluted 1: 5,000 was added to the wells and the plate incubated again at 37 ° C for 1h, 200 rpm. The washing steps were carried out for 5X, followed by the addition of 50pL of the TMB substrate (KPL, USA).
[00202] A reação enzimática ocorreu por 10 minutos e a neutralização foi obtida pela adição de 50pL de ácido sulfúrico (2M H2S04). As placas foram lidas por um leitor automático (Multiscan, Suécia) no comprimento de onda de 450nm. [00202] The enzymatic reaction occurred for 10 minutes and neutralization was obtained by adding 50pL of sulfuric acid (2M H 2 S0 4 ). The plates were read by an automatic reader (Multiscan, Sweden) at a wavelength of 450nm.
[00203] Os resultados obtidos com a avaliação individual dos antígenos pelo formato ELISA foram plotados em um gráfico de dispersão usando o índice de reatividade (Figura 4 e podem ser observados na Tabela 4 abaixo, onde demonstrou-se que as maiores AUCs ( Area Uder Curve) foram apresentadas pelo Antígeno 3, com 100% de sensibilidade e especificidade, com IC 95% de 100 (1-1.000). [00203] The results obtained with the individual evaluation of antigens by the ELISA format were plotted on a scatter plot using the reactivity index (Figure 4 and can be seen in Table 4 below, where it was demonstrated that the largest AUCs (Area Uder Curve) were presented by Antigen 3, with 100% sensitivity and specificity, with 95% CI of 100 (1-1,000).
Tabela 4: Parâmetros gerados através das análises de desempenho individual dos antígenos por ensaio ELISA simples. Table 4: Parameters generated through analysis of individual antigens performance by simple ELISA assay.
Figure imgf000052_0001
Figure imgf000052_0001
7.2.2. Ensaios em Combinação ( Multiplex ) 7.2.2. Combination Assays (Multiplex)
[00204] No ensaio ELISA em combinação de antígenos, as combinações de antígenos foram realizadas em um tubo do tipo Eppendorf em tampão carbonato/bicarbonato e cada poço recebeu 50pL desta solução de antígenos contendo 20ng de massa total. [00205] Foram avaliadas combinações de antígenos mais simples (Antígenos 2, 3, 4 e 5), bem como combinação dos mesmos com o antígeno mais completo (Antígeno 1), conforme mostrado na Tabela 5. [00204] In the ELISA assay in combination of antigens, the combinations of antigens were carried out in an Eppendorf tube in carbonate / bicarbonate buffer and each well received 50pL of this antigen solution containing 20ng of total mass. [00205] Combinations of simpler antigens (Antigens 2, 3, 4 and 5) were evaluated, as well as their combination with the most complete antigen (Antigen 1), as shown in Table 5.
[00206] Enquanto no ensaio ELISA simples apenas um antígeno único recobriu o fundo da microplaca, no ensaio ELISA para avaliar as combinações de antígenos, o fundo da microplaca foi revestido com 50pL de tampão carbonato/bicarbonato pH 9.6 contendo um total de 20ng de uma mistura de antígenos. Os ensaios foram realizados de acordo com a descrição do item 7.2.1.  [00206] While in the simple ELISA assay only a single antigen covered the bottom of the microplate, in the ELISA assay to evaluate the antigen combinations, the bottom of the microplate was coated with 50pL of carbonate / bicarbonate buffer pH 9.6 containing a total of 20ng of a mixture of antigens. The tests were performed according to the description in item 7.2.1.
Tabela 5: Combinações usadas para a realização dos imunoensaios Table 5: Combinations used to perform immunoassays
Figure imgf000053_0001
Figure imgf000053_0001
[00207] Os resultados obtidos com os ensaios em combinação de antígenos pelo formato ELISA foram plotados em um gráfico de dispersão usando o índice de reatividade conforme a Figura 5 e podem ser observados na Tabela 6 abaixo. [00207] The results obtained with the assays in combination of antigens by the ELISA format were plotted on a scatter plot using the reactivity index according to Figure 5 and can be seen in Table 6 below.
Tabela 6: Parâmetros gerados através das análises de desempenho dos antígenos por ensaios ELISA em combinação. Table 6: Parameters generated through the analysis of antigen performance by ELISA assays in combination.
Figure imgf000053_0002
Figure imgf000053_0002
Figure imgf000054_0001
Figure imgf000054_0001
[00208] Sensibilidade e especificidade foram obtidas pela análise da curva ROC, revelando que as combinações 1, 2 e 4 apresentaram sensibilidade máxima (100%). Dentre elas, a combinação 4 apresentou sensibilidade e especificidade de 100% com desempenhos superiores aos obtidos quando os antígenos foram avaliados individualmente no ensaio ELISA. [00208] Sensitivity and specificity were obtained by analyzing the ROC curve, revealing that combinations 1, 2 and 4 showed maximum sensitivity (100%). Among them, combination 4 showed sensitivity and specificity of 100% with performances superior to those obtained when the antigens were evaluated individually in the ELISA assay.
[00209] Além disto, os resultados obtidos concordam com os dados dos ensaios LUMINEX em formato multiplex dentre os quais, as maiores AUC foram obtidas pela combinação 4 que possui epítopos específicos para a os genótipos lb e 3a.  [00209] In addition, the results obtained agree with the data from the LUMINEX assays in multiplex format, among which, the largest AUC were obtained by the combination 4 that has specific epitopes for the lb and 3a genotypes.
7.3. Ensaio de Microarranjo Líquido 7.3. Liquid Microarray Assay
7.3.1. Acoplamento 7.3.1. Coupling
[00210] Primeiramente, foram realizados ensaios de acoplamento dos antígenos purificados em pequena escala às microesferas a partir de 25, 50 e 100pg/mL em tampão PBS pH 7,4, MES pH 5.5 e Carbonato de Sódio lOOmM PH 9.0 com o objetivo de avaliar o potencial das proteínas desenvolvidas frente às variáveis apresentadas. [00210] Firstly, tests were carried out for coupling the purified antigens on a small scale to microspheres from 25, 50 and 100pg / mL in PBS buffer pH 7.4, MES pH 5.5 and 100mM P H 9.0 Sodium Carbonate with the objective to evaluate the potential of the proteins developed in view of the variables presented.
[00211] Devido à presença dos agregados no momento da diluição, optou-se por trabalhar com concentrações inferiores a 25pg/mL e a realização do acoplamento em tampão contendo ureia, o que foi fundamental para conferir estabilidade às proteínas. [00211] Due to the presence of aggregates at the time of dilution, it was decided to work with concentrations below 25pg / mL and to perform the coupling in a buffer containing urea, which was essential to check protein stability.
[00212] Confirmado o potencial de uso destes antígenos, os ensaios foram realizados com os antígenos purificados obtidos pelo equipamento ÀKTA purifier cujas concentrações de acoplamento variaram entre 7.5 e 15pg/mL de proteínas.  [00212] Confirming the potential use of these antigens, the tests were performed with the purified antigens obtained by the ÀKTA purifier equipment whose coupling concentrations varied between 7.5 and 15pg / mL of proteins.
[00213] Foi realizado o acoplamento das proteínas às microesferas na presença dos tampões: (1) 1M de ureia em PBS pH 7,4, (2) Tampão de estocagem contendo 4M de ureia, 20 mM Fosfato pH 7,5, 50mM NaCl, 7mM B-mercaptoetanol, (3) PBS pH 7,4 e (4) Carbonato de Sódio lOOmM, pH 9,0. Estes ensaios foram realizados frente ao painel completo AEQ contendo os 188 soros.  [00213] 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.
[00214] Com relação ao Antígeno 1, foram acoplados na concentração de 15pg/mL. O acoplamento na presença de tampão contendo 4M de ureia não apresentou precipitação, também não houve diminuição na contagem do número de microesferas. Os parâmetros obtidos a partir da análise da curva ROC para as microesferas acopladas no tampão contendo 4M de ureia demonstraram uma performance superior quando comparados aos resultados obtidos com os acoplamentos em tampões contendo 1M de ureia, PBS ou Carbonato de sódio. Ensaios realizados com concentrações inferiores a 15pg/mL para não favoreceu o desempenho dos ensaios para estas proteínas e houve perda de sensibilidade.  [00214] Regarding 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.
[00215] No acoplamento para os antígenos mais simples (Antígenos 2 e 3), o acoplamento em concentração inferior a 15pg/mL favoreceu o desempenho do ensaio e foi possível padronizar o acoplamento na concentração de 7,5pg/mL, na presença de tampão contendo 4M de ureia.  [00215] In the coupling for the simplest antigens (Antigens 2 and 3), the coupling at a concentration below 15pg / mL favored the assay performance and it was possible to standardize the coupling at a concentration of 7.5pg / mL, in the presence of buffer containing 4M urea.
[00216] Nestas condições foram obtidos os melhores valores para as áreas de abaixo da curva ROC, 100% de sensibilidade e aumento na especificidade, além de maiores capacidades de separação entre as amostras negativas e positivas, observadas através dos valores do signal to noise ratio (SNR). [00216] Under these conditions, the best values were obtained for the areas below the ROC curve, 100% sensitivity and increased specificity, in addition to greater separation capabilities between negative and positive samples, observed through the values of the signal to noise ratio (SNR).
[00217] Já a padronização para os antígenos 3 e 4 foi realizada em tampão PBS e carbonato de sódio a 15pg/mL, respectivamente (Figuras 4.16 e 4.17). Estes resultados forneceram ótimas informações a respeito dos antígenos desenvolvidos, uma vez que a produção em larga escala com aplicabilidade diagnóstica requerem ensaios que proporcionem maior performances como o aumento da probabilidade de positividade, a máxima sensibilidade e especificidade, além da utilização de baixas concentrações de antígenos, com o objetivo de diminuir custos.  [00217] The standardization for antigens 3 and 4 was performed in PBS buffer and sodium carbonate at 15pg / mL, respectively (Figures 4.16 and 4.17). These results provided excellent information regarding the antigens developed, since large-scale production with diagnostic applicability requires assays that provide greater performance such as increased probability of positivity, maximum sensitivity and specificity, in addition to the use of low concentrations of antigens , in order to reduce costs.
[00218] Em resumo, as condições ótimas para realização dos acoplamentos para cada um dos antígenos são descritas na Tabela 7.  [00218] In summary, the optimal conditions for performing the couplings for each of the antigens are described in Table 7.
Tabela 7: Melhores parâmetros para o acoplamento dos antígenos às microesferas Table 7: Best parameters for coupling antigens to microspheres
Figure imgf000056_0001
Figure imgf000056_0001
[00219] O acoplamento foi realizado diluindo-se os antígenos para o volume final de 100pL. Os antígenos foram diluídos e mantidos refrigerados até o momento do uso. Paralelamente, as microesferas foram homogeneizadas no frasco matriz com o auxílio de um agitador do tipo vórtex, seguida de um banho de ultrassom no Cole-Parmer (Vernon Hills-IL, EUA). O frasco matriz contendo a solução com as microesferas (12.500.000 microesferas/mL) foi agitado seguidamente no vórtex e no banho de ultrassom por 30 segundos por até três vezes. Na sequência, o volume de 80m1 contendo 1.000.000 microesferas foi transferido para um poço da microplaca de 96 poços, fundo chato de baixa adesão. As microesferas foram lavadas 2 vezes na lavadora de placas Hydroflex (TECAN, Durham - NC) utilizando o programa MAGSOAK. Aos poços contendo as microesferas, foram adicionados 80pL de Tampão de Ativação (0,1M NaH2P04, pH 6,2) seguida da adição de 10pL de soluções recém-preparadas de 50mg/mL Sulfo-NHS ( N-hydroxysulfosuccinimide ) e 50mg/mL EDC ( l-etil-3-[3 - dimetilaminopropil] carbodiimida). Os poços foram homogeneizados com o auxílio de uma pipeta e a placa foi incubada a 25°C por 20 minutos, sob agitação (250rpm) ao abrigo da luz. Dado o período de incubação, a solução foi aspirada automaticamente selecionando o programa ASP da lavadora, seguida da adição de 100pL do Tampão de Acoplamento desejado com o objetivo de equilibrar o ambiente onde as microesferas se encontram para receber o antígeno previamente diluído. As placas foram seladas com um filme, revestidas em papel alumínio e incubadas à temperatura de 37°C por 2 horas, sob agitação (250rpm). Por fim, o programa MAGSOAK 2 foi acionado para a última lavagem utilizando 100pL de Tampão de Bloqueio PBS/TBN (Tampão PBS pH 7.2, 1 % de albumina bovina, Tween 20 0,02%, NaN3 0,01%). As microesferas foram transferidas para um tubo do tipo Eppendorf de baixa aderência ( low-binding - USA Scientific ) e mantidas em Tampão de Bloqueio por um tempo mínimo de 24 h a 4°C por recomendação da Luminex Corporation. [00219] The coupling was performed by diluting the antigens to the final volume of 100pL. The antigens were diluted and kept refrigerated until use. In parallel, 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. The microspheres were washed twice in the Hydroflex plate washer (TECAN, Durham - NC) using the MAGSOAK program. To the wells containing the microspheres, 80pL of Activation Buffer (0.1M NaH 2 P0 4 , pH 6.2) was added followed by the addition of 10pL of freshly prepared solutions of 50mg / mL Sulfo-NHS (N-hydroxysulfosuccinimide) and 50mg / mL EDC (1-ethyl-3- [3 - dimethylaminopropyl] carbodiimide). 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). Finally, 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%). 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.
7.3.2. Ensaios no Formato Singleplex 7.3.2. Tests in Singleplex Format
[00220] No formato singleplex, considerou-se que cada poço receberia 50pL de uma solução contendo 2.500 microesferas acopladas ao antígeno de interesse. O frasco matriz foi agitado por vórtex e banho ultrassom e o volume de trabalho foi transferido para um tubo do tipo Falcon de 15 ou 20mL contendo tampão PBS/TBN e 2% de uma solução de extrato de E. coli (lOmg/mL). Os poços receberam as amostras sorológica previamente diluída 1 :200 em tampão PBS/TBN (Tampão PBS pH 7.2, 1 % de albumina bovina, Tween 20 0,02 %, NaN3 0,01%) e após a adição das microesferas, as placas de 96 poços foram incubadas por 20 minutos a 37°C a 250 rpm, seguida de lavagens com Tampão de Bloqueio PBS/TBN. A reação dos antígenos acoplados com os anticorpos específicos presentes no soro do paciente ocorreu através da interação de anticorpos IgG anti-HCV. As placas foram lavadas através do programa MAGSOAK O anticorpo secundário de cabra anti-IgG humano conjugado à ficoeritrina (R-PE) (Moss Inc., Pasadena-CA, EUA) foi diluído 1 : 1000 em tampão de bloqueio PBS/TBN e 50pL desta diluição foram adicionados aos poços. A placa foi novamente incubada por 20 minutos a 37 °C sob proteção da luz e sob agitação (250rpm) seguida de uma sequência de lavagens onde, no final a lavadora desprezou 100 pL de um tampão chamado SheathFluid em cada poço. O SheathFluid é um tampão específico desenvolvido e comercializado pela Luminex Corporation para uso na leitura das microesferas pela plataforma xMAP 200, adquirindo 100 eventos independentes por poço. Por fim, a placa foi levada para a leitura da reação por identificação dos códigos das microesferas e detecção da presença do anticorpo secundário. A análise dos resultados foi realizada considerando os sinais da Intensidade Mediana de Fluorescência (MFI) e do código da microesfera acoplada no equipamento Luminex 200® para avaliação de 100 eventos por poço. [00220] In the singleplex format, it was considered that 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 wells received the serological samples previously diluted 1: 200 in buffer PBS / TBN (PBS buffer pH 7.2, 1% bovine albumin, Tween 20 0.02%, NaN3 0.01%) and after adding the microspheres, the 96-well plates were incubated for 20 minutes at 37 ° C at 250 rpm, followed by washes with PBS / TBN Blocking Buffer. The reaction of the antigens coupled with the specific antibodies present in the patient's serum occurred through the interaction of anti-HCV IgG antibodies. The plates were washed using the MAGSOAK program. 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. Finally, 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.
[00221] As Figuras 6 a 10 mostram o desempenho individual dos Antígenos 1 a 5, respectivamente, apresentados na forma de gráfico através da normatização obtida pelo índice de reatividade (IR). A linha pontilhada representa o ponto de corte e a área sombreada representa a zona cinza ( cut off ± 10%). A análise dos resultados foi realizada considerando os sinais medianos da imunofluorescência (MFI) e intervalo de confiança a 95%.  [00221] 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.
[00222] Nas Figuras 6 a 10, é possível verificar o desempenho das proteínas em diferentes condições de acoplamento e os dados referentes às análises estatísticas a partir da curva ROC para diferentes condições de acoplamento dos Antígenos 1 a 5 são apresentados nas Tabelas 8-12. [00222] In Figures 6 to 10, it is possible to check the performance of proteins under different coupling conditions and data related to Statistical analyzes from the ROC curve for different conditions for coupling Antigens 1 to 5 are shown in Tables 8-12.
Tabela 8: Parâmetros obtidos a partir do desempenho do Antígeno 1 para avaliação das melhores condições de acoplamento  Table 8: Parameters obtained from the performance of Antigen 1 to evaluate the best coupling conditions
Figure imgf000059_0001
Figure imgf000059_0001
Tabela 9: Parâmetros obtidos a partir do desempenho do Antígeno 2 para avaliação das melhores condições de acoplamento Table 9: Parameters obtained from the performance of Antigen 2 to evaluate the best coupling conditions
Figure imgf000059_0002
Figure imgf000060_0001
Figure imgf000059_0002
Figure imgf000060_0001
Tabela 10: Parâmetros obtidos a partir do desempenho do Antígeno 3 para avaliação das melhores condições de acoplamento Table 10: Parameters obtained from the performance of Antigen 3 to assess the best coupling conditions
Figure imgf000060_0002
Figure imgf000060_0002
Figure imgf000061_0001
Figure imgf000061_0001
Tabela 11: Parâmetros obtidos a partir do desempenho do Antígeno 4 para avaliação das melhores condições de acoplamento Table 11: Parameters obtained from the performance of Antigen 4 to evaluate the best coupling conditions
Figure imgf000061_0002
Figure imgf000062_0001
Figure imgf000061_0002
Figure imgf000062_0001
Tabela 12: Parâmetros obtidos a partir do desempenho do Antígeno 5 para avaliação das melhores condições de acoplamento Table 12: Parameters obtained from the performance of Antigen 5 to evaluate the best coupling conditions
Figure imgf000062_0002
Figure imgf000063_0001
Figure imgf000062_0002
Figure imgf000063_0001
7.3.3. Ensaios no Formato Multiplex 7.3.3. Tests in Multiplex Format
[00223] Em vista das condições de acoplamento e concentração de antígenos padronizadas pelos ensaios singleplex no LUMINEX, os antígenos foram acoplados individualmente a um código diferente de microesfera que foram utilizados no mesmo poço, ao mesmo tempo, para testar um único soro. Os ensaios foram realizados segundo o protocolo descrito acima, onde cada poço recebeu 2.500 microesferas/código. A identificação dos códigos das microesferas e a presença da fluorescência do anticorpo secundário conjugado a ficoeritrina foi realizada no equipamento LUMINEX.  [00223] In view of the coupling conditions and concentration of antigens standardized by the singleplex assays in LUMINEX, the antigens were individually coupled to a different microsphere code that were used in the same well, at the same time, to test a single serum. The tests were performed according to the protocol described above, where each well received 2,500 microspheres / code. The identification of the microsphere codes and the presence of the secondary antibody conjugated to phycoerythrin fluorescence was performed on the LUMINEX equipment.
[00224] Os ensaios no formato multiplex aplicaram as mesmas combinações identificadas na Tabela 3.  [00224] The tests in the multiplex format applied the same combinations identified in Table 3.
[00225] Nos ensaios multiplexados, as análises individuais dos antígenos apresentaram real perda de sensibilidade em todos os experimentos. Estes resultados foram esperados, uma vez que o ensaio multiplex trabalha com a adição de um número maior de antígenos (2.500/código) dentro de um único poço onde haviam os mesmos números de anticorpos competindo por sítios semelhantes.  [00225] In multiplexed assays, individual analyzes of antigens showed a real loss of sensitivity in all experiments. These results were expected, since the multiplex assay works with the addition of a larger number of antigens (2,500 / code) within a single well where there were the same numbers of antibodies competing for similar sites.
[00226] Através do índice de reatividade e dos dados obtidos a partir dos valores gerados pela curva ROC foi observado que as combinações 1, 3, e 4 apresentaram 100% de sensibilidade (Tabela 13 e Figura 11). A Figura 11 mostra Scatter plot para o IR dos soros para diferentes condições de acoplamento. A linha pontilhada representa o ponto de corte e a área sombreada representa a zona cinza ( cut ojf ± 10%). A análise dos resultados foi realizada considerando os sinais medianos da imunofluorescência (MFI) e intervalo de confiança a 95%. Os dados da análise do controle foram obtidos a partir do ensaio singleplex utilizando a proteína comercial quimérica ProSpec #HCV- 207. [00227] Dentre eles, os conjuntos de antígenos para as combinações 1 e 4 apresentaram 100% de sensibilidade e de especificidade. Entretanto, a combinação 4 foi a que, claramente, apresentou a maior capacidade discriminatória entre os soros, com a maior distância entre as MFIs para os soros negativos e positivos. Para esta combinação, o IR foi de até 140 vezes maior que o IR obtido para os soros negativos. Este ensaio demonstrou também, o menor desvio padrão para os soros negativos. [00226] Through the reactivity index and the data obtained from the values generated by the ROC curve, it was observed that combinations 1, 3, and 4 showed 100% sensitivity (Table 13 and Figure 11). Figure 11 shows a Scatter plot for the IR of the sera for different coupling conditions. The dotted line represents the cutoff point and the shaded area represents the gray zone (cut ojf ± 10%). The analysis of the results was performed considering the median signs of immunofluorescence (MFI) and a 95% confidence interval. Control analysis data were obtained from the singleplex assay using the commercial chimeric protein ProSpec # HCV-207. [00227] Among them, the antigen sets for combinations 1 and 4 showed 100% sensitivity and specificity. However, 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. For this combination, 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.
Tabela 13: Análise dos ensaios no formato multiplex via LUMINEX Table 13: Analysis of tests in multiplex format via LUMINEX
Figure imgf000064_0001
Figure imgf000064_0001
EXEMPLO 8: Análise da Relação Sinal Ruído (SNR) EXAMPLE 8: Signal to Noise Ratio Analysis (SNR)
[00228] Na avaliação do desempenho individual através da relação sinal/ruído (SNR) para os antígenos no ensaio ELISA simples, foi demonstrado que os maiores valores foram apresentados pelo Antígeno 3, com 23,6 vezes a capacidade de separação dos soros positivos e negativos (Figura 12).  [00228] In the evaluation of individual performance through the signal / noise ratio (SNR) for antigens in the simple ELISA assay, it was demonstrated that the highest values were presented by Antigen 3, with 23.6 times the separation capacity of positive and negative (Figure 12).
[00229] No ensaio LUMINEX singleplex, as relações sinal/ruído ou SNR demonstraram baixas performances para o Antígeno 1 , com probabilidade de positividade de 21,5. Estes valores foram superiores ao valor apresentado para a proteína comercial ProSpec #HCV-207, cuja probabilidade de positividade foi de 13 (Figura 13). A média dos sinais para os soros concordam com os valores apresentados. [00229] In the LUMINEX singleplex test, the signal / noise ratios or SNR demonstrated low performances for Antigen 1, with a probability of positivity of 21.5. These values were higher than the value presented for the commercial protein ProSpec # HCV-207, whose probability of positivity was 13 (Figure 13). The mean of the signals for the sera agree with the values presented.
[00230] Para os demais antígenos, a probabilidade de positividade ou a capacidade de separação entre os soros foi superior ao valor obtido para o antígeno comercial. Dentre eles, os maiores valores alcançados foram para os ensaios realizados com o Antígeno 4, que apresentou SNR de 652.5, respectivamente (Figura 13).  [00230] For the other antigens, the probability of positivity or the separation capacity between the sera was higher than the value obtained for the commercial antigen. Among them, the highest values reached were for the tests carried out with Antigen 4, which presented SNR of 652.5, respectively (Figure 13).
[00231] Enquanto a capacidade de separação para o Antígeno 1 no formato simples por ELISA foi a segunda mais baixa dentre os antígenos testados, no formato combinado por ELISA este antígeno foi fundamental para aumentar o desempenho e a probabilidade de positividade para a combinação 4 (Figura 14).  [00231] While the separation capacity for Antigen 1 in the simple format by ELISA was the second lowest among the tested antigens, in the format combined by ELISA this antigen was fundamental to increase the performance and the probability of positivity for combination 4 ( Figure 14).
[00232] A Figura 15 mostra o gráfico de barras onde a combinação 4 apresenta a maior capacidade de separação dentre a média dos sinais para as amostras positivas e negativas, cuja probabilidade de positividade para esta combinação foi de 1.053,7 vezes. De longe, este foi o melhor desempenho alcançado na presente invenção. Além disto, capacidades de separação para as demais combinações no ensaio multiplex foram altas quando comparada à performance do antígeno controle.  [00232] 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.
EXEMPLOEXAMPLE
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Claims

REIVINDICAÇÕES
1. Polipeptídeo, caracterizado pelo fato de compreender pelo menos duas regiões antigênicas do vírus da hepatite C (HCV) selecionadas a partir do grupo de região do nucleocapsídeo (NC) e regiões não estruturais NS3, NS4 e NS5.  1. Polypeptide, characterized by the fact that it comprises at least two hepatitis C virus (HCV) antigenic regions selected from the group of nucleocapsid region (NC) and non-structural regions NS3, NS4 and NS5.
2. Polipeptídeo de acordo com a reivindicação 1, caracterizado pelo fato de compreender a sequência de aminoácidos tendo pelo menos 90% de identidade com qualquer uma das SEQ ID NOs: 1, 3, 5, 7 e 9.  2. Polypeptide according to claim 1, characterized in that it comprises the amino acid sequence having at least 90% identity with any of SEQ ID NOs: 1, 3, 5, 7 and 9.
3. Polipeptídeo de acordo com a reivindicação 1, caracterizado pelo fato de consistir na sequência de aminoácidos de qualquer uma das SEQ ID NOs: 1, 3, 5, 7 e 9.  Polypeptide according to claim 1, characterized in that it consists of the amino acid sequence of any of SEQ ID NOs: 1, 3, 5, 7 and 9.
4. Polipeptídeo de acordo com qualquer uma das reivindicações 1 a 3, caracterizado pelo fato de ser para uso na triagem imunológica de Vírus da Hepatite C (HCV).  Polypeptide according to any one of claims 1 to 3, characterized in that it is for use in the immunological screening of Hepatitis C Virus (HCV).
5. Polinucleotídeo, caracterizado pelo fato de que codifica o polipeptídeo como definido em qualquer uma das reivindicações 1 a 4.  5. Polynucleotide, characterized by the fact that it encodes the polypeptide as defined in any one of claims 1 to 4.
6. Polinucleotídeo de acordo com a reivindicação 5, caracterizado por compreender a sequência de ácidos nucleicos de qualquer uma das SEQ ID NO: 2, 4, 6, 8 e 10 e suas degenerações.  Polynucleotide according to claim 5, characterized in that it comprises the nucleic acid sequence of any of SEQ ID NO: 2, 4, 6, 8 and 10 and their degenerations.
7. Cassete de expressão, caracterizado pelo fato de compreender um polinucleotídeo como definido na reivindicação 5 ou 6 operacionalmente ligado a um promotor e a um terminador de transcrição.  7. Expression cassette, characterized in that it comprises a polynucleotide as defined in claim 5 or 6 operatively linked to a promoter and a transcription terminator.
8. Vetor de expressão, caracterizado pelo fato de compreender um polinucleotídeo como definido na reivindicação 5 ou 6 ou um cassete de expressão como definido na reivindicação 7.  8. Expression vector, characterized in that it comprises a polynucleotide as defined in claim 5 or 6 or an expression cassette as defined in claim 7.
9. Célula hospedeira, caracterizada pelo fato de compreender um cassete de expressão como definido na reivindicação 7 ou um vetor de expressão como definido na reivindicação 8.  9. Host cell, characterized by the fact that it comprises an expression cassette as defined in claim 7 or an expression vector as defined in claim 8.
10. Método para produzir um polipeptídeo, caracterizado pelo fato de que compreende: 10. Method for producing a polypeptide, characterized by fact that you understand:
(a) transformar uma célula hospedeira com o polinucleotídeo como definido na reivindicação 5 ou 6,  (a) transforming a host cell with the polynucleotide as defined in claim 5 or 6,
(b) cultivar dita célula para a produção do polipeptídeo;  (b) cultivating said cell for the production of the polypeptide;
(c) isolar dito polipeptídeo de dita célula ou do meio de cultura circundando dita célula.  (c) isolating said polypeptide from said cell or culture medium surrounding said cell.
11. Composição, caracterizada pelo fato de que compreende um polipeptídeo como definido em qualquer uma das reivindicações 1 a 3 ou uma combinação de dois ou mais polipeptídeos como definidos em qualquer uma das reivindicações 1 a 3.  11. Composition, characterized by the fact that it comprises a polypeptide as defined in any one of claims 1 to 3 or a combination of two or more polypeptides as defined in any one of claims 1 to 3.
12. Composição de acordo com a reivindicação 11, caracterizada pelo fato de ser utilizada na triagem imunológica de HCV.  12. Composition according to claim 11, characterized by the fact that it is used in the immunological screening of HCV.
13. Kit para triagem imunológica de HCV ou diagnóstico de hepatite C, caracterizado pelo fato de compreender pelo menos um polipeptídeo como definido em qualquer uma das reivindicações 1 a 3 ou uma composição como definida na reivindicação 11 ou 12.  13. Kit for immunological screening of HCV or diagnosis of hepatitis C, characterized in that it comprises at least one polypeptide as defined in any one of claims 1 to 3 or a composition as defined in claim 11 or 12.
14. Kit de acordo com a reivindicação 13, caracterizado pelo fato de compreender ainda um meio de detecção para detectar o complexo antígeno-anticorpo.  Kit according to claim 13, characterized in that it also comprises a detection means to detect the antigen-antibody complex.
15. Kit de acordo com a reivindicação 13 ou 14, caracterizado pelo fato de compreender ainda um controle de reação.  15. Kit according to claim 13 or 14, characterized by the fact that it also includes a reaction control.
16. Kit de acordo com qualquer uma das reivindicações 13 a 15, caracterizado pelo fato de ser desenvolvido para ensaios do tipo indireto.  16. Kit according to any one of claims 13 to 15, characterized in that it is developed for tests of the indirect type.
17. Uso de pelo menos um polipeptídeo como definido em qualquer uma das reivindicações 1 a 3, da composição como definida na reivindicação 11 ou 12 ou do kit como definido em qualquer uma das reivindicações 13 a 16, caracterizado pelo fato de ser para triagem imunológica de HCV ou para diagnóstico de hepatite C.  Use of at least one polypeptide as defined in any one of claims 1 to 3, the composition as defined in claim 11 or 12 or the kit as defined in any one of claims 13 to 16, characterized in that it is for immunological screening HCV or for diagnosis of hepatitis C.
18. Método para triagem imunológica do HCV, caracterizado pelo fato de compreender as etapas de: 18. Method for immunological screening of HCV, characterized by understanding the steps of:
(a) prover um ou mais polipeptídeos como definidos em qualquer uma das reivindicações 1 a 4 ou uma composição como definida na reivindicação 11 ou 12;  (a) providing one or more polypeptides as defined in any one of claims 1 to 4 or a composition as defined in claim 11 or 12;
(b) contatar os ditos um ou mais peptídeos ou a dita composição com a amostra biológica a ser testada por um tempo suficiente e sob condições suficientes para formação de complexos anticorpo/antígeno; e  (b) contacting said one or more peptides or said composition with the biological sample to be tested for a sufficient time and under conditions sufficient to form antibody / antigen complexes; and
(c) detectar o complexo antígeno/anticorpo formado na etapa (b), pela adição de um meio de detecção, capaz de gerar um sinal detectável na presença do referido complexo antígeno/anticorpo.  (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.
19. Método para diagnóstico de hepatite C, caracterizado pelo fato de compreender as etapas de:  19. Method for the diagnosis of hepatitis C, characterized by the fact of understanding the steps of:
(a) prover um ou mais polipeptídeos como definidos em qualquer uma das reivindicações 1 a 4 ou uma composição como definida na reivindicação 11 ou 12;  (a) providing one or more polypeptides as defined in any one of claims 1 to 4 or a composition as defined in claim 11 or 12;
(b) contatar os ditos um ou mais peptídeos ou a dita composição com a amostra biológica a ser testada por um tempo suficiente e sob condições suficientes para formação de complexos anticorpo/antígeno; e  (b) contacting said one or more peptides or said composition with the biological sample to be tested for a sufficient time and under conditions sufficient to form antibody / antigen complexes; and
(c) detectar o complexo antígeno/anticorpo formado na etapa (b), pela adição de um meio de detecção, capaz de gerar um sinal detectável na presença do referido complexo antígeno/anticorpo.  (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.
20. Uso ou métodos de acordo com qualquer uma das reivindicações 17 a 19, caracterizado pelo fato de ser um imunoensaio do tipo ELISA ou LUMINEX.  20. Use or methods according to any of claims 17 to 19, characterized in that it is an immunoassay of the ELISA or LUMINEX type.
21. Uso ou métodos de acordo com a reivindicação 20, caracterizado pelo fato de o imunoensaio ser em formato singleplex ou multiplex.  21. Use or methods according to claim 20, characterized by the fact that the immunoassay is in a singleplex or multiplex format.
22. Uso ou métodos de acordo com a reivindicação 21, caracterizado pelo fato de o ensaio em formato multiplex compreender o uso combinado de pelo menos dois dos polipeptídeos de SEQ ID NO: 1, 3, 5, 7 e 9. 22. Use or methods according to claim 21, characterized in that the multiplex format assay comprises the use combination of at least two of the polypeptides of SEQ ID NO: 1, 3, 5, 7 and 9.
PCT/BR2019/050454 2018-10-22 2019-10-22 Polypeptide, expression cassette, expression vector, host cell, kit for immunological screening for hcv and/or for diagnosing hepatitis c, composition, use of at least one polypeptide and methods for producing a polypeptide, for immunological screening for hcv and for diagnosing hepatitis c WO2020082145A1 (en)

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