BRPI0705968A2 - mimetic recombinant peptides and protein motifs of dengue virus antigens and their diagnostic and therapeutic applications - Google Patents

Abstract

MIMETIC RECOMBINANT PEPTIDS AND PROTEIN REASONS FOR DENGUE VIRUS ANTIGENS AND THEIR DIAGNOSTIC AND THERAPEUTIC APPLICATIONS. The present invention relates to the use of the Phage Display technique for the selection, characterization and use of novel recombinant peptides and protein motifs which mimic polyprotein antigenic regions of the four dengue virus types by determining functional epitopes of viral proteins through of polyclonal antibodies obtained from chickens immunized with dengue total protein (IgY). The peptides have been specifically immunoreactive with the serum of dengue virus infected individuals and can potentially be used in immunodiagnostics and vaccine compositions for the control of viral infection.

Description

"MIMETIC RECOMBINANT PEPTIDES AND PROTEIN REASONS FOR DENGUE VIRUS ANTIGENS AND THEIR DIAGNOSTIC AND THERAPEUTIC APPLICATIONS"

The present invention relates to the selection, characterization and use of recombinant peptides and protein motifs that mimic polyprotein antigenic regions of the four dengue virus types. The peptides have been specifically immunoreactive with the serum of dengue virus infected individuals and can potentially be used in immunodiagnostics and vaccine compositions for the control of viral infection.

Dengue is today the most important arbovirus that affects man and is a serious public health problem in the world, especially in tropical climate countries, where environmental conditions favor the development and proliferation of Aedes aegypti, the main transmitter of the disease. (Marques GRAM et al. Archives of the Biological Institute, 71: 462-465, 2004). Over the past two decades, the incidence of dengue infection has grown considerably in endemic areas, particularly in the Americas region, where dengue virus has shown a characteristic of hyperendicity as a result of the circulation of multiple serotypes in many countries of the continent (from Simone TS, Royal Society of Tropical Medicine and Hygiene, 98: 553-562, 2003). According to the World Health Organization (WHO), two-fifths of the world's population is at risk of being infected with the disease, and more than 100 countries have been hit by dengue or dengue hemorrhagic epidemics. WHO estimates that more than 50 million cases of dengue and dengue hemorrhagic contagion occur annually, of which half a million are hospitalized, with about 20,000 deaths (www.who.int).

Dengue virus is spherical with a relatively smooth surface except in regions where glycoprotein molecules associate. It has a diameter of approximately 500 A, and an icosahedral capsid covered by a lipid envelope. The RNA of this virus is single stranded, with approximately 10,700 nucleotides. The genome is 11 Kb and encodes 10 proteins, 3 structural proteins (Capsid C, Membrane M and Envelope E), where the last two are on the surface of the virus, and 7 non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a , NS4b, NS5) (Guzmán MG et al.

The Lancet Infectious Diseases, 2: 33-42, 2002 and Kuhn RJ et al. Cell, 108: 717-725, 2002). During virus replication, the positive sense strand of viral genomic RNA is translated as a 5'-C-prM (M) -E-NS1-NS2a-NS2b-NS3-NS4a-NS4b-NS5-3 'polyprotein, where prM represents the membrane precursor protein (Wu CF et al. Journal of Virological Methods, 114: 45-54,2003).

Four antigenically similar serotypes called dengue virus type 1 (DENV-1), dengue virus type 2 (DENV-2), dengue virus type 3 (DENV-3) and dengue virus type 4 (DENV-4), according to Ligon ( Ligon BL Seminars in Pediatric Infectious Diseases 15: 60-65, 2004).

The infectious process triggered by dengue virus has a wide clinical picture, ranging from a simple inapparent (asymptomatic) infection to more severe cases, in which there are hemorrhages, abrupt increase in vascular permeability and development of hypovolemic shock. The disease manifests clinically in two forms: classical dengue fever (CD), also described as dengue fever (DH) and hemorrhagic dengue fever (DH) or dengue hemorrhagic fever (DHF) (Lee MS et al. J. Mierobiol Immunol Infeet ., 39: 121-9, 2006).

A primary infection with any of the four dengue virus serotypes typically results in some asymptomatic disease or dengue fever. Epidemiological studies suggest that individuals who have been infected a second time with a different serotype have a significantly higher risk of developing dengue hemorrhagic fever (Simmons CP et al. Journal of Virology, 79: 5665-5675, 2005).

The clinical characteristics of dengue often depend on the age of the patient. Infants and young children may suffer from undifferentiated fever with maculopapular rash. Older children and adults have a benign fever syndrome or disabling classic disease with abrupt onset and high fever, severe headache, retro-orbital pain, muscle and joint pain. The case mortality rate is extremely low. Many dengue epidemics are accompanied by complications involving bleeding, such as epistaxis, gingival bleeding, gastrointestinal bleeding, hematuria, and hypermenorrhea. In some cases, a singularly severe bleeding can lead the patient to death (Guzmán MG et al. The Lancet Infectious Diseases, 2: 33-42, 2002).

Typical cases of Hemorrhagic Dengue are characterized by four main clinical manifestations: high fever, hemorrhagic phenomena, hepatomegaly, and often circulatory failure (Guzmán MG et al. The Lancet Infectious Diseases, 2: 33-42, 2002). Autopsies of dengue hemorrhagic cases, in order of frequency, show bleeding in the skin and subcutaneous tissues, mucous membranes of the gastrointestinal tract, and in the heart and liver. In general, subarachnoid or cerebral hemorrhages occur infrequently. The hemorrhagic volume, however, is not excessive (WHO. Dengue haemorrhagic fever: diagnosis, treatment, prevention and control. 2nd edition. Geneva: World Health Organization, 1997).

The four dengue viruses are capable of producing cases of HDF, and DENV-2 and DENV-3 are most often associated with severe disease (Guzmán MG et al. The Laneet Infeetious Diseases, 2: 33-42, 2002).

Methods commonly used to confirm dengue infection involve virus isolation, detection of virus antigen or RNA in plasma or serum or tissues, and the presence of virus-specific antibodies in serum and other fluids.

Recently, several techniques have been developed for rapid laboratory diagnosis of dengue virus: amplification for an increased rate of isolated virus; the flow cytometry method for rapid virus detection; detection of viral nucleic acid by PCR-NESTED, RT-PCR, sequencing based on nucleic acid amplification and real-time PCR; detection of free viral antigens, dengue virus reactive M (IgM) or G (IgG) immunoglobulins by ELISA (Enzyme Linked Immunosorbent Assay); and differentiation of primary from secondary ELISA infection (Kao CL et al. Journal of Microbiology, Immunology and Infection, 38: 5-16, 2005).

Knowing that many infectious diseases, such as dengue, lack more reliable, faster and more accurate diagnoses, the following patents were developed with applications of new diagnostic modalities, allowing both qualitative and quantitative detection of the virus in vitro: W02005014627 (Phage display), US2006099573 (anti IgE ELISA), US2002155435 (RT-PCR), W00179546 (RT-PCR), US6197568 (virus interaction with target cells).

W09909414 consists of the use of polypeptides containing from 2 to 8 amino acids derived from proteins of different flavivirus serotypes, in particular dengue virus, in order to diagnose virus infection by ELISA and DotBIot method. The present patent discloses the use of Phage Display methodology for selection of peptides containing 7 amino acid residues, derived from proteins of different dengue virus serotypes, characterized by an efficient selection method, confirmed by bioinformatics.

In the present invention novel recombinant and mimetic highly immunoreactive dengue virus polyprotein peptides for the detection of anti-dengue antibodies for the various viral types are identified and characterized which recognize both IgM and IgG response.

The four dengue virus serotypes have very similar antigens but are sufficiently different to cause partial protection against the others after infection with one of them. A natural dengue virus infection induces protective immunity to only the same (homologous) serotype, and short-term protection (months) against another serotype (Rothman AL. The Journal of Clinical Investigation, 113: 946-951 , 2004). After an incubation period of 4-6 days (minimum of three, maximum of ten), the virus is present in patients' blood during the acute phase of the disease (WHO. Dengue haemorrhagic fever: diagnosis, treatment, prevention and control. Geneva: http://www.who.int/csr/resources/publications/denque/024-33.pdf (World Health Organization 1997).

An effective vaccine must protect against all four dengue serotypes, ie be tetravalent (Rothman AL. The Journal of Clinical Investigation, 113: 946-951, 2004). In addition, it must be safe for human use and economically viable (Halstead SB et al. The Lance, t 360: 1243-1245, 2002). To this end, a number of techniques have been studied, including attenuated virus and recombinant virus vaccines, recombinant subunit vaccines, and DNA vaccines (WHO. Vaccine, 23: 2689-2695, 2005). Tetravalent vaccines containing live attenuated viruses appear to be the most suitable to prevent disease due to the possibility of protection for all serotypes and for long periods of time. Already live vaccines have the advantage of stimulating both humoral immunity (high neutralizing antibody titers) and cellular response (Bricks LF. Pediatria (São Paulo) 26: 268-281, 2004).

According to W09718311, the polyvalent flavivirus vaccine, and particularly the dengue virus, comprises recombinant peptides emitted from each serotype, the carboxylated end of which is substituted by a peptide containing from 2 to 8 histidines, tryptophans and cysteines. The present invention proposes the use of novel recombinant peptides, which function as antigenic epitopes and are potential targets for generating immune response to various types of dengue virus, comprising sequences that recognize common dengue polyprotein protein motifs in the four viral types. .

Other patents have also used recombinant purified protein particles to provide protection to susceptible individuals, such as: US2004022811 (DENV-2 M / pM protein expressed in baculovirus with protective intent in mice), EP1159968 (vaccines containing attenuated strains dengue virus), W09915692 (use of antigenic epitopes for vaccine purposes), US2004077022 (fusion of Fc domains with biologically active peptides) and W09718311 (use of recombinant peptides emitted from each serotype).

Also in order to develop a vaccine capable of producing protective immunity, W09963095 proposes the use of a flavivirus vaccine whose active agent is nucleic acid, while US6254873 uses inactivated dengue viruses which are non-infectious, however. have preserved immunogenicity.

The Phage Display methodology, or phage biomolecule exposure, was developed by Smith (Smith GP. Science, 228: 1315-1317, 1985) by achieving expression of the restriction enzyme EcoRI by fusion with protein three (plll ) of the phage capsid. Phage display allows selection of peptides and proteins, including antibodies, with high affinity and specificity for various targets. The crucial advantage of this technology lies in the direct link that exists between the experimental phenotype and the encapsulated genotype, showing the evolution of selected ligands to optimized molecules (Azzazy HM et al. Clinical biochemistry, 35: 425-445, 2002).

Bacteriophages, or simply phages, are viruses that infect a variety of Gram-negative bacteria using the sexual pilus as a receptor. The filamentous phage particles (strains M13, f1 and fd) that infect E. coli via pilus F consist of a single strand of DNA that is encased in a protein capsule (Russell M. Filamentous phage assembly. Molecular Microbiology, 5: 1607 -1613, 1991). The bacteriophage is composed of five structural proteins present in the capsid: p11, pVI, pVII, pVIII and pIX. The pVIII protein forms the cylindrical body of the capsid. At the ends of this capsid are three to five copies of the other structural proteins. The distal end contains the pVII and pIX proteins, while the proximal end is composed of proteins encoded by gene 3 (plll) and pVI itself. The incorporation of exogenous proteins on the surface of filamentous phages is done by fusing these peptides to the structural proteins of the viral particles. The two main proteins used for this purpose are pVII and plll protein. (Brigido MM et al. Biotechnology Science & Development, 26: 44-51, 2002).

Phage display has proven to be a very powerful technique in obtaining libraries containing millions or even billions of different peptides or proteins. The most widely used library methodology is based on the use of filamentous phages (Smith GP. Science, 228: 1315-1317, 1985).

The DNA sequences of interest are inserted at a location in the filamentous bacteriophage genome, so that the encoded protein is expressed on the surface of the filamentous phage as a fusion product to one of the phage surface proteins (Azzazy HM et al. Clinical biochemistry, 35: 425-445, 2002). The peptide or protein expressed on the surface of the phage enables the selection of binding affinity-based sequences for a target molecule by an in vitro selection process called biopanning (Parmley S et al. Gene, 73: 305-318, 1988).

Although the Phage Display technique was first introduced approximately 15 years ago, the applications and development of this technology are just beginning to be explored. This exploration of Phage Display technology will lead to the production of a huge range of ligands, including recombinant antibodies and peptides, with predefined specificities. In addition, emerging Phage Display-based technologies will benefit diagnostics through the production of molecules impossible to obtain by traditional methods. Specifically, recombinant antibodies against toxic antigens or conserved sequences and carbohydrates can be isolated (Soderlind E et al. Nature Biotechnology, 18: 852-856, 2000).

Over the past 10 years, phage antibody exposure has become a well-accepted technique and in a short time has generated very high quality molecules. Phage Display-derived antibodies have been shown to be safe and effective in clinical trials. Selection procedures adapted to this technique, in combination with easily assembled antibodies, open the door to sophisticated antibody development as medicines. In addition, these Phage Display-selected antibodies offer the greatest speed and yield advantages for searching and targeting / validating. Phage Display is already part of a modern way of discovering new drugs (Kretzschmar T et al. Current Opinion in Biotechnology, 13: 598-602, 2002).

US7083945, US2006068421, US2005202512, GB2408332, US2003104604, EP1452599, US2006035223 use the Phage Display technique to express recombinant ligand-affinity polypeptides and propose methodologies that make the technique more efficient.

The use of chicken immunoglobulin (IgY) for proteomic research was termed IgY technology by Zhang (Zhang WW. Drug Discovery Today, 8: 364-371, 2003), having antibody and serum sources (Gassmann M et al. (FASEB J., 4: 2528-2532, 1990). According to Lemamy (Lemamy GJ. International Journal of Cancer; 80: 896-902, 1999), birds have the ability to produce antibodies with high titer and high persistence of immune response to bacterial and mammalian proteins. In addition, antibodies produced in chickens have biochemical advantages such as extreme pH resistance (Lee et al. J Biochem Mol Biol, 35: 488-493, 2002), high temperature resistance (Jensenius et al. J. Immunol Methods, 46). : 63-68, 1988), high affinity (Ikemori et al. Poult Sci, 72: 2361-2365, 1993) and greed (Stuart et al. Anal Biochem, 173: 142-150, 1988), which is why they have have been used efficiently in immunological analyzes (Schade et al. ALTEX, 13: 5-9, 1996), as exemplified by the patenting processes below.

JP8188599 proposes a method of separating IgY antibodies such that they react specifically to an immune antigen. Some patents refer to the use of immunologically reactive IgY to Alzheimer's disease (GR1005016), the prevention of intestinal infection by Escherichia coli (US2004086513) and gynecological infection by pathogenic bacteria (N1454901), against venereal diseases (CN1485343), among others. In addition, W02006093080 relates to the combined use of IgY and Phage Display to produce divalent chicken antibodies.

In the search for a fast, efficient and capable methodology to provide a diagnosis of dengue virus infection, as well as a preventive and / or therapeutic vaccine, the present invention proposes the use of Phage Display technique for the identification and selection of specific peptides. with dengue virus, determining functional epitopes of viral proteins by polyclonal antibodies obtained from chickens immunized with total dengue proteins (IgY).

The invention may be better understood by the following detailed description by way of examples for the obtained experimental results, presented in Tables 1 to 5. The experimental procedures involved are detailed at the end of this descriptive report. The methodology employed for the selection, characterization and use of recombinant peptides and protein motifs that mimic polyprotein antigenic regions was applied to the four types of dengue virus and could be applied to other flavivirus types efficiently.

Example 1:

This example relates to the selection and characterization of the mimetic recombinant peptides and protein motifs of the present invention. Several parameters can be used to indicate the successful selection of each peptide, as shown in Table 1. These same parameters were previously used by Rodi et al. (Rodi DJ et al. Journal of Molecular Biology, 322: 1039-1052, 2002 ).

Table 1 shows the identity of the selected amino acid sequences, as well as their respective observed frequencies (FO); expected frequencies (EF), which corresponds to the probability of the random sequence; amplification of peptides resulting from the selection process in relation to the expected frequency of peptides in the original library; degree of information of each peptide (I (M)), which is given by -In (random sequence probability); and the probable number of independent clones within the library (λ). The greater the degree of information, the more effective the selection of the peptide, hence the rarer it is in the library.

Sixteen clones with different sequences were obtained (Seq. ID N - 1 to 16), and these presented their respective inverted sequences (Seq. ID N - 17 to 32). For example, Seq. ID # 1 has the reverse sequence Seq. ID No. 17, Seq. ID N-2 has the inverse sequence Seq. ID No. 18, a and so on, as shown in Table 2. Peptide Seq. ID No. 3 and its inverse Seq. ID No. 19 was the most frequent in the selection and the fourth with the highest information index, becoming one of the main selected mimotopes, especially when considering the number of predicted independent clones in the library with this same sequence, ie in this case. only a single clone is expected. On the other hand, the peptide with the highest information index was Seq. ID No. 6 and its inverse Seq. ID No. 22, which presents the main protein motif Seq. 36. Peptides that exhibit high information are less likely to occur at random than those with low information (Rodi DJ et al. Journal of Molecular Bioiogy, 322: 1039-1052, 2002).

The arginine (Arg) and cysteine (Cys) amino acids in random peptide sequences act on the secretion of Protein III and may interfere with phage infectivity. Consequently, clones with peptides containing these amino acids may be less frequent during selection (Noren KA et al. Methods, 23: 169-178, 2001). According to the results obtained, only one peptide has Cys (represented by Seq. ID No. 16), while 22 have Arg (represented by Seq. ID No. 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31), demonstrating not only their importance in protein motifs, but also their low interference on the frequency of peptides with this protein. amino acid. It was also observed that there was a great enrichment of all selected peptides in relation to their expected frequencies in the phage library, suggesting that the phages are specific for dengue. Table 1

<table> table see original document page 12 </column> </row> <table>

Table 3 shows the peptides obtained in the selection process aligned by the Blast program with the complete Dengue-specific protein sequences deposited in GeneBank and the access to these sequences, as well as the motifs that are similar to such peptides. Also included are motifs that have a very short amino acid sequence (3 amino acids) with activity. The sequences have similarities with dengue-related proteins being found in proteins of all viral types. Because of the conserved representation in certain regions along the peptides, motifs could represent important continuous or discontinuous antigen epitopes (Cortese R et al. Current Opinion in Biotechnology, 6: 73-80, 1995 and Yang WJ et al. Journal of Immunological Methods, 276: 175-183, 2003). In addition, Table 3 also shows the sequence ELISA (IE) indices, which prove the high reactivity of clones to the viral antigen.

Table 2

<table> table see original document page 13 </column> </row> <table>

Looking at the results presented in Table 3, we notice the similarity of some clones with DENV polyproteins, which were presented in the following order: the peptides and their reversals (given by Seq. ID No. 1 to 16 and Seq. ID N 17 to 32, respectively) and the protein motifs (in parentheses), where:

- have similarities with DENV-1 peptides and their protein motifs Seq. ID No. 1 (Trp Lys Lys), Seq. ID No. 7 (Seq. ID No. 39), Seq. ID No. 9 (Seq. ID No. 67 and Seq. ID No. 68), Seq. ID No. 11 (Seq. ID No. 47, Seq. ID No. 70, Seq. ID No. 72), Seq. ID No. 12 (Seq. ID No. 73), Seq. ID No. 14 (Seq. ID No. 51 and Seq. ID No. 73), Seq. ID No. 17 (Trp Lys Lys), Seq. ID No. 25 (Seq. ID No. 67 and Seq. ID No. 68), Seq. ID No. 27 (Seq. ID No. 70) and Seq. ID No. 30 (Seq. ID No. 73);

- have similarities with DENV-2 peptides and their protein motifs Seq. ID No. 1 (Seq. ID No. 53), Seq. ID No. 2 (Seq. ID No. 57), Seq. ID No. 4 (Seq. ID No. 36), Seq. ID No. 6 (Seq. ID No. 64), Seq. ID No. 8 (Seq. ID No. 66), Seq. ID No. 10 (Seq. ID No. 47), Seq. ID No. 11 (Seq. ID No. 47, Seq. ID No. 48 and Seq. ID No. 72), Seq. ID No. 12 (Seq. ID No. 49), Seq. ID No. 17 (Seq. ID No. 53), Seq. ID No. 18 (Seq. ID No. 57), Seq. ID No. 22 (Seq. ID No. 63 and Seq. ID No. 64), Seq. ID No. 24 (Seq. ID No. 66);

- have similarities with DENV-3 peptides and their respective protein motifs Seq. ID No. 1 (Seq. ID No. 55 and Seq. ID No. 56), Seq. ID No. 2 (Seq. ID No. 58), Seq. ID No. 3 (Seq. ID No. 35), Seq. ID No. 5 (Seq. ID No. 62), Seq. ID No. 7 (Seq. ID No. 38, Seq. ID No. 65 and Pro Asn Met), Seq. ID No. 9 (Seq. ID No. 43 and Seq. ID No. 44), Seq. ID No. 11 (Seq. ID No. 47 and Seq. ID No. 73), Seq. ID No. 12 (Seq. ID No. 49), Seq. ID No. 13 (Seq. ID No. 50), Seq. ID No. 17 (Seq. ID No. 55 and Seq. ID No. 56), Seq. ID No. 18 (Seq. ID No. 58), Seq. ID No. 21 (Seq. ID No. 62), Seq. ID No. 23 (Seq. ID No. 65 and Pro Asn Met);

- present similarities with DENV-4 motifs and their respective protein motifs, Seq. ID No. 9 (Seq. ID No. 67), Seq. ID No. 11 (Seq. ID No. 48, Seq. ID No. 71 and Seq. ID No. 72), Seq. ID No. 14 (Seq. ID No. 51), Seq. ID No. 25 (Seq. ID No. 67), Seq. ID No. 27 (Seq. ID No. 71).

These polyproteins are encoded by the virus genome and are processed into 10 polypeptides, 3 structural and 7 nonstructural, as described above. It was also observed that the peptides and their respective motifs were similar to DENV precursors:

- have similarities with the DENV-4 polyprotein precursor peptides and their corresponding motifs (in parenthesis): Seq. ID No. 1 (very short sequence Trp Lys Lys), Seq. ID No. 5 (Seq. ID No. 37), Seq. ID No. 6 (Seq. ID No. 37), Seq. ID No. 7 (Seq. ID No. 37), Seq. ID No. 8 (Seq. ID No. 37), Seq. ID No. 9 (Seq. ID No. 67), Seq. ID No. 10 (Seq. ID No. 47), Seq. ID No. 11 (Seq. ID No. 48, Seq. ID No. 71, Seq. ID No. 72), Seq. ID No. 12 (Seq. ID No. 49) and Seq. ID No. 14 (Seq. ID No. 51), Seq. ID No. 17 (Trp Lys Lys), Seq. ID No. 25 (Seq. ID No. 67), Seq. ID No. 27 (Seq. ID No. 71);

- have similarities with the DENV-1 polyprotein precursor peptides and their corresponding motifs, represented by the sequences Seq. ID No. 1 (very short amino acid sequence Trp Lys Lys) and Seq. ID No. 12 (Seq. ID No. 73), Seq. ID No. 28 (Seq. ID No. 73).

All virus proteins are derived from a large precursor polyprotein by co-translational and post-translational proteolytic processing (Tibaire Montes, M. Revista de la Sociedad Venezolana de Microbiologia, 21: 39-45, 2001), hence the importance of the peptides are similar to this polyprotein.

Clones that show similarity to envelope proteins (E) of different viral types are: Seq. ID No. 11 (Seq. ID No. 47) for DENV-1 envelope glycoprotein; Seq. ID No. 8 (Seq. ID No. 41), for DENV-2 envelope protein; and Seq. ID No. 2 (Seq. ID No. 34), Seq. ID No. 4 (Seq. ID No. 60) and Seq. ID No. 7 (Seq. ID No. 38), Seq. ID No. 18 (Seq. ID No. 58), Seq. ID No. 20 (Seq. ID No. 60) for DENV-3 envelope protein. This protein (E) is 53 kDa, and contains important antigenic determinants (Alisson SL et al. Journal of Virology, 75: 4268-4275, 2001) and is therefore used for the development of a Dengue control vaccine.

Analyzing the data in Table 3, it is concluded that peptides are sharing antigenic similarities between motifs and between proteins. Table 3

<table> table see original document page 16 </column> </row> <table> Continued from Table 3

<table> table see original document page 17 </column> </row> <table> Continued from Table 3

<table> table see original document page 18 </column> </row> <table>

Because peptides (Seq. ID N-1 to 32) share antigenic similarities between motifs and proteins, it is very important to assess the feasibility of producing a vaccine using such compounds. Thullier and colleagues (Thullier P et al. Journal of General Virology, 82: 1885-1892, 2001), using a monoclonal antibody that neutralizes all envelope glycoprotein-binding dengue serotypes, identified the clone Trp Ser Leu Phe Leu Asn His Ala Glu similar to the protein sequence. According to the authors, this is a critical segment for the infectivity of all four serotypes. Interestingly, this critical sequence has some similarities to the selected peptides described in this invention that aligned with the envelope protein, specifically considering the Leu Val or Leu Asn motifs.

The NS1 protein has identity with the Seq peptides. ID No. 9 (Seq. ID No. 67) and Seq. ID No. 25 (Seq. ID No. 68) from DENV-1; Seq. ID No. 26 (Seq. ID No. 63), Seq. ID No. 28 (Seq. ID No. 42) and Seq. ID NO 22 (Seq. ID NO 63) from DENV-2; and Seq. ID No. 11 (Seq. ID No. 48) from DENV-4. The 40 kDa NS1 protein has activity in viral maturation and is found on the surface, linked to the infected cell membrane, and can also be secreted. This protein has immunizing capacity, and antibodies act as mediators of lymphocyte cytotoxicity phenomena through their receptors for the Fc portion of immunoglobulins (Figueiredo, LTM. Dengue Vaccines. Symposium: Medical Virology I, 32: 21- 25, 1999). In addition, Wu and colleagues (Wu HC et al. Journal of Clinical Microbiology, 39: 977-982, 2001) using a DENV-1 serotype-specific monoclonal antibody, identified a phage-expressed random peptide epitope (cell B) , with consensus motif His Tyr Trp, which mimics the His Lys Tyr Ser Trp Lys sequence of the same viral type NS1 protein. In another paper, the same authors (Wu HC et al. Journal of General Virology, 84: 2771-2779, 2003), using the same methodology focusing on DENV-2, confirmed that the His Arg Leu motif was crucial for binding. antibody peptide. An important fact was that this reason presented partial identity to the Arg Leu linear sequences observed in the various clones selected in this invention, indicating a high specificity with viral proteins. The peptide Seq. ID No. 12 shows similarity to DENV-4 protein NS2a having the Seq motif. ID No. 49. The clones Seq. ID No. 5, Seq. ID No. 9 and Seq. ID N0 21 through reasons Seq. ID No. 62, Seq. ID No. 43 and Seq. ID NO. 62 are similar to the DENV-3 NS2b protein and the Seq sequences. ID No. 11, Seq. ID N2 14 through reasons Seq. ID No. 71 and Seq. ID No. 51, show similarities to DENV-4 NS2b protein. Both are proteins little known immunologically in Dengue, suggesting further research involving these proteins.

The peptides Seq. ID No. 5, Seq. ID No. 9, Seq. ID No. 13, Seq. ID No. 21, Seq. ID No. 25 and Seq. ID No. 29 show similarities with DENV-3 NS3 protein through the Seq motif. ID No. 61, and peptide Seq. ID No. 10 shows similarity to DENV-1 and DENV-2 NS3 protein through the Seq motif. 47. This 69 kDa protein is a bifunctional nucleotide enzyme, triphosphatase / viral helicase enzyme. NS3 protein that is in contact with or secreted from the cell surface has immunizing capacity. The presence of NS3 stimulates the destruction of cells infected by cytotoxic T lymphocytes (Figueiredo, LTM. Symposium: Virology Medical I, 32: 21-25, 1999).

The peptide Seq. ID No. 1 is similar to NS5 protein in that it has the very short amino acid sequences Trp Lys Lys (DENV-1 and DENV-4), Seq. ID No. 33 and Ile Asn Trp (DENV-2); the peptide Seq. ID No. 17 is similar to the NS5 protein of DENV-1 and DENV-4 for the very short amino acid sequence Trp Lys Lys, and with DENV-1 for the Ile Asn Trp motif; already the peptide Seq. ID No. 23 has similarity to DENV-3 NS5 for the very short Pro Asn Met amino acid sequence motif. NS5 protein is the largest of the ten proteins and the most conserved at 104 kDa. It is a protein that has domains that contain enzymatic activity that are crucial for the replicative cycle of the virus (Chão DY et al. Virology Journal, 72: 1-10, 2005). The similarity of this peptide to NS5 protein justifies the occurrence of cross-reactivity of the other peptides to multiple viral serotypes (Rothman AL. Clin. Invest., 113: 946-951, 2004).

The peptides Seq. ID No. 26, Seq. ID No. 28, Seq. ID No. 9, Seq. ID No. 11, similar to NS1; and Seq. ID No. 5, Seq. ID No. 9, Seq. ID No. 10, Seq and ID No. 13, similar to NS3, become important vaccine targets because they are highly immunogenic and important targets for dengue antibodies.

The mimetic peptides Seq. ID No. 15 and Seq. ID No. 16 as well as their respective inverted sequences Seq. ID No. 31 and Seq. ID Nos. 32 have no similar protein motifs (which is why they were not cited in Table 3).

Example 2:

This example relates to confirming the specificity of the peptides and protein motifs object of the present invention.

Table 4 presents sequences with similarities to proteins of known biological communities. Where Sl refers to the identification sites, TA to the taxonomic amplitude and P to the probability of random occurrence of the peptides. Of the 16 selected peptides (Seq. ID N— 1 to 16), only six sequences showed similarities with proteins from biological communities and with very low chance of occurrence, suggesting that the phages were selected specifically for the target sites.

The peptides Seq. ID No. 3 and ID No. 19, Seq. ID No. 4 and Seq. ID No. 20 show similarities with the eukaryotic virus protein kinase C phosphorylation site through the Ser Ala Lys and Thr Tyr Lys identification sites, respectively, with Ser or Thr being the phosphorylation site. Protein kinase C is involved in cellular processes including secretion, exocytosis, gene expression, modulation of ion conduction, cell proliferation and downregulation of extracellular receptors (Kapczinski F et al. Brazilian Journal of Psychiatry 26: 17-21, 2004). In vivo, Kinase C protein exhibits a preference for phosphorylation of serine or threonine residues found near a C-terminal basic residue (Kishimoto A et al. Journal of Biological Chemistry, 260: 12492-12499, 1985). The presence of additional basic residues at the N or C-terminus of the target amino acid increases the rate of the phosphorylation reaction.

The peptides Seq. ID No. 1 and Seq. ID No. 17, Seq. ID No. 9 and Seq. ID No. 25, Seq. ID No. 12 and Seq. ID No. 28 showed similarities with the N-glycosylation site of eukaryotic viruses through the Asn Ile Ser Ser, Asn Ala Ser Thr and Asn Tyr Thr Ser identification sites, respectively, with Asn being the glycosylation site. N-glycosylation is one of the major post-translational modifications, being responsible for changes in conformation, stability and consequently in protein functionality in eukaryotes (Maia IG et al. Genetics and molecular biology, 24: 231-234, 2001).

The presence of known eukaryotic virus domains suggests that the selected peptides are similar to dengue virus and may be associated with the virus modulation process in the host.

Table 4

<table> table see original document page 22 </column> </row> <table>

Table 5 refers to the ELISA test of phage immunoreactivity against sera from patients previously typed with DENV-1 and DENV-3 for both IgG and IgM. The results demonstrate that the peptides were not specific for either of the two viral types as well as for the immunoglobulin types. All ELISA results were significant, albeit with different phage profiles.

Recombinant peptides were superior in immunoreactivity compared to ELISA index (IE) obtained with total dengue proteins.

These results corroborate previous ELY tests with IgY and cultures as well as bioinformatics results.

Table 5

<table> table see original document page 23 </column> </row> <table> All combined results make the 16 selected peptides and their respective reverse sequences (Seq. ID N-1 to 32) as potential vaccine targets, thus as well as probable biomarkers for diagnostic assays, since they presented high specificity to dengue proteins of all viral types.

The main advantages of this invention are:

- use of humoral response in chickens to characterize dengue antigens;

- selection of specifically immunoreactive peptides for the detection of anti-dengue antibodies for all viral types;

- Use of these peptides in immunodiagnostic procedures;

- Use of these peptides in vaccine compositions for the control of viral infection.

The present invention is of great importance, since dengue virus infections represent a major public health problem worldwide, especially in tropical countries such as Brazil, where climate and urban habits offer optimal conditions for development and development. proliferation of its transmitting mosquito, the Aedes aegypti.

Dengue has become endemic in the country since the middle of the last decade, causing repeated epidemics in many cities, including the occurrence of the dreaded hemorrhagic form. The World Health Organization (WHO) estimates that between 50 and 100 million people are infected annually in more than 100 countries on all continents except Europe. About 550,000 patients require hospitalization and 20,000 die as a result of dengue.

In this scenario, the need for protein peptides in the diagnosis and treatment of dengue, points to a great advance in the clinical investigation of dengue. Thus, health policies aimed at detection and beyond effective vaccine treatment should be prioritized.

The invention may be better understood by means of the detailed description, in accordance with the attached Figures, where: FIGURE 1 shows a histogram of the Y1 immunoglobulin ELISA reactivity generated after four type 3 dengue virus total protein immunizations (DENV-3), tested against total protein from DENV-1, DENV-2 and DENV-3 cultures, where (a) corresponds to immune IgY and (b) corresponds to control IgY.

FIGURE 2 shows the three-dimensional structure of the DENV-2 type viral capsid for alignment in conformational epitopes. Some amino acids are indicated where (N) is an aspargine or Ans; (R) is an arginine or Arg; (V) is a valine or Val and (L) is a Ieucine or Leu.

According to FIGURE 1, its lack of specificity regarding dengue viral types was confirmed, but a high sensitivity to dengue virus was found. According to FIGURE 2, as the motifs seq. ID N-75 and Leu Arg Asn did not align with the linear structure of the capsid protein, the motif search was done directly on the tertiary protein structure by superposition by the Cn3D program.

Epitopes can be classified as conformational (discontinuous sequences) or non-conformational (linear sequences). Linear epitopes are short stretches of the primary protein structure composed of continuous amino acid residues of the primary sequence. Conformational epitopes consist of many discrete amino acid residues in the primary sequence that assemble antigenic determinants in the form of the tertiary structure of proteins (Barlow DJ et al. Nature, 322: 747-748, 1986).

Consensus sequences generally show no similarity to the natural antigen sequence (Felici F et al. Gene, 128: 21-27, 1993). In this case, epitopes can mimic natural epitopes (mimotopes) or conformational epitopes. This justifies the fact that the probable reasons were not found in the linear sequence of the capsid protein. DENV-2 protein C is a symmetric four-helix dimer arranged in two antiparallel pairs (Jones CT et al. Journal of Virology, 7: 7143-7149, 2003). Each dimer has one of the target sequences analyzed.

It can be observed that both motifs are located in the outermost portion of the molecule, providing greater surface contact, thus becoming a candidate region for epitope localization.

For a better understanding of the examples used, below is a detailed description of the experimental procedures adopted.

In the immunization process, inactivated antigens of serotype DENV-3, produced in mouse brains and extracted by sucrose-acetone method were used. The immunization scheme was that described by Barbas et al. (Barbas CF et al. New York: Cold Spring Harbor Laboratory Press, p.8.4-8.7, 2001), with some modifications. Six intramuscular applications of total viral proteins emulsified in adjuvant were performed, and the amount of protein was 200.0 μg in the first immunization, followed by subsequent immunizations of 100.0 pg, with intervals of 15 days. Two chickens were immunized and two kept as controls.

After 3 months the blood was collected and the specific antibody titer was monitored by ELISA.

IgY purification was performed using the HiTrap IgY Purification HP 5 mL column (Amersham Biosciences). The peristaltic pump was filled with elution buffer (20 mM Na 2 HPO 4, pH 7.5), and then connected to the column. The column was washed with 5 volumes (25.0 mL) of each buffer: binding (20 mM Na 2 HPO 4, 0.5 M K 2 SO 4, pH 7.5), elution and cleaning (20 mM Na 2 HPO 4, 30% volume / volume isopropanol, pH 7.5). Then the column was equilibrated with 5 volumes of binding buffer, and sample (6.0 mL of serum) was injected. The column was washed with 10 volumes of it (50.0 mL) of binding buffer. The IgY were then eluted with 10 column volumes of elution buffer, and 1.0 mL samples were collected and quantified on the spectrophotometer at 280 nm. The column was regenerated with 8 volumes (40.0 mL) of cleaning buffer, and rebalanced with 5 volumes of binding buffer. As samples were filtered on the purification column, optical readings (OD 280nm) of aliquots were obtained.

For dialysis of the obtained samples a Fisherbrand® cellulose membrane (regenerated cellulose-DYALYSIS tubing) was used. Purified serum samples were placed in dialysis tubes (tubes 1 to 8), concentrated in sucrose for 1 hour at 4 ° C and then dialyzed in PBS (137 mM NaCl, 2.7 mM KCl, 12 mM Na 2 HPO 4, 1.2 mM KH 2 PO 4, pH 7.4) for 12 hours at 4 ° C. The samples were quantified in the spectrophotometer at 280nm.

After obtaining the purified antibody, it was necessary to confirm the specificity of IgY in relation to the other viral types. A high affinity plate (NUNC) was sensitized with 20 pg / mL total protein of each viral type, diluted in 100.0 pL / well carbonate buffer (60 mM NaHCO 3, pH 9.6) and incubated for 12 hours. under stirring at 4 ° C. After 3 washes with 0.05% PBS-T (PBS + 0.05% volume / volume Tween 20), 250.0 µl blocking buffer (0.05% PBS-T - 3% BSA) was added, the plate being incubated for 1 hour at room temperature. The plate was then washed 3 more times and 100.0 µl of solution containing serum from the immunized chickens at a concentration of 1: 600, plus 50.0 µl blocking (one clone in each well) was added. After incubation for 1 hour at 37 ° C, an additional 6 washes were performed. Peroxidase-labeled conjugated Anti-IgY antibody (Amersham Biosciences, 1: 5000) diluted in blocking solution was added, incubated for 1 hour at 37 ° C. The plate was then washed an additional 6 times and then developed with 100.0 µl / well of a solution containing OPD (O-Phenylenediamine dihydrochrloide - Sigma Chemical), H2O2, and citrate buffer (0.1 Μ). The reaction was stopped by the addition of 10.0 pL of 4 M H2SO4. Absorbance reading was performed on a Multiscan Plus version 2.03 reader with a 492nm filter.

For the selection of peptides (expressed as filamentous phage capsid protein III) reactive with the antibodies described above, a random 7-amino acid peptide library (Ph.D-7 mer - New England Biolabs) was used.

A 96-well microtiter plate (Maxisorp - NUNC) was adsorbed to the well of 150.0 μΙ_ IgY-specific purified column IgY (100 μg / mL in 0.1 M NaHCO3, pH 8.6) and incubated overnight at 4 ° C in a container containing humidified paper towels. The plate was blocked with 150.0 μl of a blocking buffer (0.1 M NaHCO3, pH 8.6, 5 mg / mL BSA) for 1h while stirring at 4 ° C, and washed six times with TBST (TBS (Tris-HCI 50 mM pH 7.5, 150 mM NaCl, water, 0.1% volume / volume of Tween 20.) 10.0 μΐ_ of the peptide library diluted in 100.0 μΙ_ of TBST was added to the plate and the plate was stirring for 1 hour at room temperature Non-ligating phages were removed by washing the plate ten times with TBST Ligating phages were eluted with 100.0 μ of elution buffer (0.2 M glycine-HCl, pH 2 , Containing 1 mg / mL BSA) for 10 min at room temperature and immediately neutralized with 15.0 μΙ_ neutralization buffer (1 M Tris-HCl, pH 8.0). of the title.

A small sample of this eluate (10.0 μΙ_) was titrated and the reagent was used for reamplification, performed on 20.0 mL of early growing E. coli (ER2537) culture (OD600≤0.3) containing tetracycline by incubating the culture for 4-5 hours on a shaker at 37 ° C before phage precipitation and subsequent titration.

From the third cycle, two separate selection processes were performed, one positive (against total DENV-3 antigens) and the other negative (against total uninfected mouse brain cell proteins). It is important to emphasize that in negative selection the supernatant that was kept for the next selection cycle, thus excluding nonspecific binding phages.

The culture was transferred to an Oakridge tube (Hitachi, 50 mL) and centrifuged (10 min, 10,000 rpm). Residual cells were discarded and the supernatant transferred to a clean, recentrifuged tube. 80% of the supernatant was pipetted into a sterile tube where 20% of the volume of PEG / NaCl (20% weight / volume of Polyethylene glycol-8000, 2.5M NaCl, water) was added, and the mixture was allowed to stand for 12 hours. at 4 ° C.

The next day, the precipitation was centrifuged 15 minutes at 10,000 rpm at 4 ° C. The supernatant was discarded and the tube was briefly centrifuged so that residual supernatant could be removed. The precipitate was resuspended in 1.0 mL TBS, transferred to a microtube and centrifuged for 5 minutes, 10,000 rpm at 4 ° C to precipitate cell debris. The supernatant was transferred to a new microtube for the addition of PEG / NaCl (1/6 volume). The mixture was incubated on ice for 1 hour and then centrifuged (10 min, 14,000 rpm, 4 ° C). The supernatant was discarded, and centrifugation was repeated to remove residual supernatant. The precipitate was resuspended in 200.0 µl TBS to give the amplified eluate ready for titration.

Reamplified phages from the first selection cycle were used in a second cycle and subsequently thereafter for a total of four cycles, and from the second cycle the stringency of the wash buffer was increased from 0.1% to 0, 5% Tween-20 in all washes.

For all titrations, 10.0 µl of the phages, diluted in 90.0 µl of LB culture medium, were used. Dilutions (10 ~ 1 to 10 ~ 4 for unamplified eluate and 10 "8 to 10" 11 for amplified phages) were incubated with 200.0 µl E. coli (ER2738) in early growth for 5 min. and plated in LB medium containing IPTG (0.5 mM) and X-gal (40 pg / mL), along with 3.0 mL Top Agarose (10 g Bacto-Tryptone, 5 g yeast extract, 5 g NaCl, 1 g MgCl 2 .6H 2 O / liter).

After overnight incubation at 37 ° C, blue colonies were counted for entry and exit titers for all selection cycles (number of blue colonies χ dilution factor).

Blue-stained colonies showing breakdown of X-Gal substrate and phage β-galactosidase gene expression by ER2738 bacteria were separately reamplified in Deepwell plates for storage of selected clones. For this, each isolated colony obtained from the unamplified 4th cycle was dispensed into a Deepwell well containing 1.0 mL of E. coli culture medium in the initial growth phase. The plate was incubated for 12 hours at 37 ° C under shaking. The Deepwell plate was centrifuged for 60 minutes at 3,700 rpm for removal of the supernatant from the culture. Its contents were transferred to another Deepwell plate and then 1/6 volume of PEG / NaCl (20% polyethylene glycol-8000, 2.5 M NaCl) was added by incubating it for 12 hours at 4 ° C. The plate was centrifuged for 1 hour at 3,700 rpm, the supernatant discarded, and the precipitate suspended in 200.0 μL PBS.

ELISA tests were performed to determine the reactivity of recombinant peptides (expressed in selected phages) against antibodies present in the serum of chickens immunized with dengue type 3 total viral proteins.

A high affinity plate (NUNC) with 1 pg / well of purified polyclonal anti-dengue IgY antibody diluted in 100.0 pL / well of carbonate buffer (60 mM NaHCO3, pH 9.6) and incubated for 12 hours was sensitized. under stirring at 4 ° C. After 3 washes with 0.05% PBS-T (PBS + 0.05% volume / volume Tween 20), 250.0 µL blocking buffer (0.05% PBS-T - 3% BSA) was added, the plate being incubated for 1 hour at room temperature. The plate was then washed 3 more times and added 100.0 μL of solution containing 50.0 μL of phage plus 50.0 μL block (one clone in each well). After incubation for 1 hour at 37 ° C, an additional 6 washes were performed. For detection, peroxidase labeled anti-M13 conjugated antibody (1: 500) diluted in blocking solution was used, incubated for 1 hour at 37 ° C. The plate was then washed an additional 6 times and then developed with 100.0 µl / well of a solution containing OPD (O-Phenylenediamine dihydrochrloide - Sigma Chemical), H2O2, and citrate buffer (0.1 Μ). The reaction was stopped by the addition of 10.0 pL of 4 M H2SO4. Absorbance reading was performed on a Multiscan Plus reader (Thermo Plate) version 2.03 with a 492nm filter.

To obtain phage DNA, 10.0 μί of Deepwell plate purified phages were added to 1.0 mL of ER2738 culture in initial growth phase 5 in 15 mL FALCON tubes where they remained under vigorous shaking for 6 hours. at 37 ° C. After growth, the cultures were transferred to microtubes for sedimentation of the bacteria which were centrifuged for 10 minutes at 4,000 rpm. 500.0 μL of the supernatant was transferred to another tube, and 200.0 μl PEG-NaCl was added to this tube, then incubated at room temperature for 10 minutes. After centrifuging for 10 minutes at 14,000 rpm, the supernatant was discarded, and the precipitate was resuspended with 100.0 μL Iodide Buffer (10 mM Tris-HCI, 1 mM EDTA, 4 M NaH), and then 250 µl was added. 0 μL absolute ethanol, incubating the tube for 10-20 minutes at room temperature. Tube 15 was centrifuged for 10 minutes at 14,000 rpm, discarding the supernatant, and then the precipitate was washed with 500.0 μL 70% ethanol, centrifuging again for 10 minutes at 14,000 rpm. The supernatant was discarded and the precipitate suspended in 20.0 µl sterile ultra pure water.

The sequencing was performed using the Big Dye Terminator 20 Kit (Amersham Biosciences) and a MegaBace 1000 automated sequencer (.Amersham Biosciences. For the sequencing reaction, the primer - 96 M13 - (5'-HoCCCTCATAGTTAGCGTAACG -3'-Amersham) was used. Biosciences), which amplifies the region of the coding amino acids of the fused random peptides in recombinant M13 phages.

The analysis of the DNA sequences from the automatic sequencer were processed in the equipment's own software (Sequence Analyzer, BASE CALLER, Cimarron 3.12, Phred 15). Soon after this pre-analysis, the vector sequences were removed and only those inserts with 16 perfect residues were then translated.

The DNA sequences generated by sequencing were analyzed using bioinformatics programs available online: - The amino acid sequences obtained from sequencing were translated using the DNA2PR07 program (http://relic.bio.anl.gov/dna2pro7). aspx);

- Calculating the frequency and measurement of information for each peptide within the original phage library was done by the INFO program (https://relic.bio.anl.gov/info.aspx);

- the protein motifs generated were analyzed for dengue protein homologies deposited in "GENBANK" by the BLAST - Basic Local Alignment Search Tool program (http://www.ncbi.nlm.nih.gov/BLAST);

- To find protein patterns and taxonomic lineage referring to protein families and domains common in species, genera or taxa to which the peptides belong, the PROSCAN (PROSITE SCAN) program was used. This program examines peptides against PROSITE, a protein family and domain database consisting of biologically significant sites, patterns, and profiles that help to reliably identify the known protein family, if one exists (http: // npsa-

pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_prosite.html).

To confirm the specificity of the clones obtained, the ELISA test was performed with serum from 2 patients individually contaminated with DENV-1 and DENV-3. These sera were previously typed by PCR. The negative control was a well without phage and serum, and the positive controls were the total culture antigens specific for DENV-1, -2, and -3.

Four ELISA tests were performed, two human DENV-1 sera and 2 human DENV-3 sera. In both viral types, anti-IgG antibody was applied to one plate, peroxidase conjugated and in the other, anti-IgM antibody, also conjugated to peroxidase. In the negative control used, the phages and serum tested were not placed. Plaque sensitization was performed with phages at a concentration of 5x109 viral particles and also with total proteins of DENV-1 and DENV-3 at the concentration of 20 Mg / mL, both diluted in sodium bicarbonate. It was incubated for 12 hours under shaking at 4 ° C.

After 3 washes with 0.05% PBS-T (PBS + 0.05% volume / volume Tween 20), 250 μL blocking buffer (0.05% PBS-T - 3% BSA) was added, with plate incubated for 1 hour at room temperature. The plates were then washed an additional 3 times and sera from DENV-1 and DENV-3 infected patients were added. In the plates where peroxidase-conjugated anti-IgG would be applied, the serum concentration was 1: 500, and in the plates where peroxidase-conjugated anti-IgM would be applied, the serum concentration was 1:50. After incubation for 1 hour at 37 ° C, an additional 6 washes were performed. Peroxidase-labeled conjugated Anti-IgG and Anti-IgM antibodies (Amersham Biosciences, 1: 5000) diluted in blocking solution were added and incubated for 1 hour at 37 ° C. The plate was then washed an additional 6 times, and then developed with 100 µl / well of a solution containing OPD (O-Phenylenediamine dihydrochrloide - Sigma Chemical), H2O2, and citrate buffer (0.1M). The reaction was stopped by the addition of 10 μL H2So4 4 Μ. The absorbance reading was made in a Multiscan Plus reader (Thermo Plate) version 2.03, with a 492nm filter. LIST OF BIOLOGICAL SEQUENCES

(1) General Patent Application Information

(1) Applicant Data:

a) Name: Federal University of Uberlândia

b) Address: Av. João Naves de Avila, 2121 - Block 5L, Santa Monica Neighborhood, Zip Code: 38400-902, Uberlândia - MG.

(ii) Title of the invention: "MIMETIC RECOMBINANT PEPTIDES AND PROTEIN REASONS FOR DENGUE VIRUS ANTIGENS AND THEIR DIAGNOSTIC AND THERAPEUTIC APPLICATIONS"

(iii) Number of sequences contained in the order: 75 (seventy-five)

(iv) Computer readable format: Microsoft Word; Windows XP PC type computer.

(2) General sequence information Characteristics of sequenced molecules:

a) Type: PROTEIN

b) Protein Name: Dengue Virus Polyprotein: DENV-1, DENV-2, DENV-3 and DENV-4.

c) Sequence Identity: Seq. ID No. 1 to Seq ID No. 74.

d) Original source of molecules: GenBank (www.ncbi.nlm.nih.gov/GenBank/)

3- Information for Seq. ID # 1 1

Sequence Features:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID # 1:

Lys Leu Trp Asn Ile Ser Ser 1 5 7

3- Information for Seq. ID # 2 2

Sequence Characteristics: a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID # 2:

Lys Leu Phe Asn Wing Asn Pro 1 5 7

3- Information for Seq. ID # 3

Sequence Features:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID # 2: Tyr Glx Asx Ser Wing Lys Thr 1 5 7

3- Information for Seq. ID # 4

Sequence Features:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID # 4:

Thr Tyr Lys Leu Pro Pro Pro 1 5 7

3- Information for Seq. ID # 5

Sequence Features:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID # 5:

Val Leu Arg Asn Pro Pro Wing 1 5 7

3- Information for Seq. ID N2 6 String Features:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID # 6:

Val Leu Arg Asn Val His Asn 1 5 7

3- Information for Seq. ID # 7

Sequence Features:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID # 7:

Val Leu Arg Asn Met Asn Pro 1 5 7

3- Information for Seq. ID # 8

Sequence Features:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID # 8:

Val Leu Arg Asn Ser Pro Thr 1 5 7

3- Information for Seq. ID # 9 9

Sequence Features:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID # 9:

Leu Leu Arg Asn Ala Ser Thr 1 5 7 3- Information for Seq. ID No. 10 Sequence Characteristics:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 10 Met Leu Arg Asn Leu Pro Pro 1 5 7

3- Information for Seq. ID N2 11 String Features:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID # 11

Wing Read Arg Asn Read Gly Pro 1 5 7

3- Information for Seq. ID No. 12 Sequence Characteristics:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID N2 12 Wing Read Arg Asn Tyr Thr Ser 1 5 7

3-Information for Seq. ID No. 13 Sequence Characteristics:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID # 13 Glx Leu Arg Asn Ala Pro Pro 1 5 7

3- Information for Seq. ID N2 14 String Features:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID # 14:

Gly Leu Glx Be Read Be Arg 1 5 7

3- Information for Seq. ID N-

Sequence Features:

a) Size: 7 amino acids

b) Type: amino acid 15 c) Topology: linear

Description of sequence: Seq. ID # 15:

Asn Leu Arg Asn Val His Trp 1 5 7

3- Information for Seq. ID # 16

Sequence Features:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 16:

His Cys Glx Read Ala Lys Cys 1 5 7

3- Information for Seq. ID N2 17 String Features:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID # 17:

Ser Ser Ile Asn Trp Leu Lys 1 5 7

3- Information for Seq. ID No. 18

Sequence Features:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID # 18:

Pro Asn Wing Asn Phe Leu Lys 1 5 7

3- Information for Seq. ID No. 19 Sequence Characteristics:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 19: Thr Lys Ala Ser Asx Glx Tyr 1 5 7

3- Information for Seq. ID # 20

Sequence Features:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID # 20:

Pro Pro Pro Read Lys Tyr Thr 1 5 7

3- Information for Seq. ID No. 21 Sequence Characteristics:

a) Size: 7 amino acids

b) Type: amino acid c) Topology: linear Sequence description: Seq. ID N2 21: Pro Pro Wing Asn Arg Leu Val 1 5 7

3- Information for Seq. ID # 22 22

Sequence Features:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N- 22:

Asn His Val Asn Arg Leu Val 1 5 7

3- Information for Seq. ID N2 23 String Features:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID # 23:

Pro Asn Met Asn Arg Leu Val 1 5 7

3- Information for Seq. ID N2 24 String Features:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 24: Thr Pro Ser Asn Arg Leu Val 1 5 7

3- Information for Seq. ID No. 25

Sequence Features:

a) Size: 7 amino acids b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 25:

Thr Ser Wing Asn Arg Leu Leu 1 5 7

3- Information for Seq. ID No. 26 Sequence Characteristics:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID # 26: Pro Pro Leu Asn Arg Leu Met 1 5 7

3- Information for Seq. ID N- 27

Sequence Features:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 27:

Pro Gly Leu Asn Arg Leu Wing 1 5 7

3- Information for Seq. ID No. 28 Sequence Characteristics:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 28: Ser Thr Tyr Asn Arg Leu Wing 1 5 7

3- Information for Seq. ID No. 29 Sequence Characteristics: a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID # 29:

Pro Pro Wing Asn Arg Leu Glx 1 5 7

3- Information for Seq. ID No. 30 Sequence Characteristics:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 30: Arg Ser Leu Ser Glx Leu Gly 1 5 7

3- Information for Seq. ID No. 31

Sequence Features:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 31:

Trp His Val Asn Arg Read Le Asn 1 5 7

3- Information for Seq. ID No. 32 Sequence Characteristics:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 32:

Cys Lys Wing Read Glx Cys His 1 5 7

3- Information for Seq. ID No. 33 Sequence Characteristics:

a) Size: 5 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 33:

Trp Xaa Xaa Ser Ser 1 5

3- Information for Seq. ID No. 34 Sequence Characteristics:

a) Size: 6 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID N- 34:

Read Phe Xaa Wing Asn Pro 1 5 6

3- Information for Seq. ID No.

Sequence Features:

a) Size: 4 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 35: Asx Ser Ala Lys 1 4

3- Information for Seq. ID No. 36

Sequence Features:

a) Size: amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 36:

Tyr Lys Xaa Pro Pro 1 5 3- Information for Seq. ID No. 37 Sequence Characteristics:

a) Size: 4 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 37:

Val Leu Arg Asn 1 4

3- Information for Seq. ID No. 38

Sequence Features:

a) Size: 4 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 38:

Read Arg Xaa Met 1 4

3- Information for Seq. ID No. 39 Sequence Characteristics:

a) Size: 6 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No 39: Val Leu Xaa Xaa Met Asn 1 5 6

3- Information for Seq. ID No. 40 Sequence Characteristics:

a) Size: 6 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N- 40: Val Leu Xaa Xaa Pro Thr 1 5 6

3- Information for Seq. ID No. 41 Sequence Characteristics:

a) Size: 5 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID N-41:

Arg Asn Xaa Pro Thr 1 5

3- Information for Seq. ID No. 42 Sequence Characteristics:

a) Size: 6 amino acids

b) Type: amino acid

c) Topology: linear

B. Description of sequence: Seq. ID N- 42 Leu Arg Xaa Xaa Pro Thr 1 5 6

3- Information for Seq. ID No. 43 Sequence Characteristics:

a) Size: 4 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 43:

Leu Leu Arg Asn 1 4

3- Information for Seq. ID N2 44 String Features:

a) Size: 5 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 44: Leu Arg Xaa Ala Ser 1 5

3- Information for Seq. ID No. 45

Sequence Features:

a) Size: 4 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 45:

Asn Leu Pro Pro 1 4

3- Information for Seq. ID No. 46 Sequence Characteristics:

a) Size: 5 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 46: Leü Arg Xaa Leu Pro 1 5

3- Information for Seq. ID No. 47 Sequence Characteristics:

a) Size: 5 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 47:

Wing Leu Xaa Asn Leu 1 5

3- Information for Seq. ID No. 48 Sequence Characteristics:

a) Size: 5 amino acids

b) Type: amino acid c) Topology: linear Sequence description: Seq. ID N2 48: Leu Arg Xaa Leu Gly 1 5

3- Information for Seq. ID No. 49 Sequence Characteristics:

a) Size: amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 49:

Read Arg Xaa Thr Ser 1 5

3- Information for Seq. ID No. 50 Sequence Characteristics:

a) Size: 5 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 50:

Glx Leu Xaa Asn Wing 1 5

3- Information for Seq. ID No. 51 Sequence Characteristics:

a) Size: 5 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 51:

Gly Leu Xaa Ser Leu 1 5

3- Information for Seq. ID No. 52 Sequence Characteristics:

a) Size: 5 amino acids b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID N-52:

Glx Leu Xaa Wing Lys 5 1 5

3- Information for Seq. ID N- 53 Sequence Characteristics:

a) Size: 4 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No 53: Asn Trp Leu Lys 1 4

3- Information for Seq. ID No. 54

Sequence Features:

a) Size: 6 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 54:

20 Ser Xaa Ile Xaa Trp Read 1 5 6

3- Information for Seq. ID No. 55 Sequence Characteristics:

a) Size: 4 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 55: Ser Ser Ile Asn 1 4

3- Information for Seq. ID No. 56 Sequence Characteristics: a) Size: 6 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 56:

5 Asn Ile Asn Xaa Leu Lys 15 6

3- Information for Seq. ID No. 57 Sequence Characteristics:

a) Size: 4 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 57: Asn Phe Leu Lys 1 4

3- Information for Seq. ID No. 58

Sequence Features:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 58:

Asn Ala Xaa Phe Xaa Leu Lys 1 5 7

3- Information for Seq. ID N2 59 String Features:

a) Size: 4 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 59:

Thr Lys Wing Ser 301 4

3- Information for Seq. ID N2 60 String Features:

a) Size: 4 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 60

Read Lys Tyr Thr 1 4

3- Information for Seq. ID N2 61 String Features:

a) Size: 4 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 61

Wing Asn Arg Lys 1 4

3- Information for Seq. ID N2 62 String Features:

a) Size: 6 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 62

Pro Wing Xaa Xaa Lys Val 1 5 6

3- Information for Seq. ID N- 63 String Features:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID N- 63

Asn Xaa Val Asn Xaa Lys Val 1 5 7 3- Information for Seq. ID N- 64 String Features:

a) Size: 6 amino acids

b) Type: amino acid 5 c) Topology: linear

Description of sequence: Seq. ID No. 64: His Val Xaa Xaa Lys Val 1 5 6

3- Information for Seq. ID No. 65

Sequence Features:

a) Size: 6 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 65:

Pro Asn Met Xaa Arg Lys 1 5 6

3- Information for Seq. ID N2 66 String Features:

a) Size: 6 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No 66: To Be Xaa Xaa Lys Val 1 5 6

3- Information for Seq. ID No. 67

Sequence Features:

a) Size: 4 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N2 67: Thr Ser Wing Asn 1 4

3- Information for Seq. ID No. 68 Sequence Characteristics:

a) Size: 5 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 68

Ser Ala Xaa Arg Lys 1 5

3- Information for Seq. ID No. 69 Sequence Characteristics:

a) Size: 5 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 69

Pro Pro Xaa Asn Arg 1 5

3- Information for Seq. ID No. 70 Sequence Characteristics:

a) Size: 4 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 70

Pro Gly Lys Asn 1 4

3- Information for Seq. ID No. 71 Sequence Characteristics:

a) Size: 5 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID No. 71:

Gly Lys Xaa Lys Wing 1 5

3- Information for Seq. ID No. 72 Sequence Characteristics:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID N- 72:

Pro Gly Xaa Xaa Xaa Lys Wing 1 5 7

3- Information for Seq. ID No. 73 Sequence Characteristics:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID N- 73

Ser Thr Xaa Xaa Xaa Lys Wing 1 5 7

3- Information for Seq. ID Ne 74 Sequence Characteristics:

a) Size: 6 amino acids

b) Type: amino acid

c) Topology: linear Sequence description: Seq. ID N2 74

Ser Lys Xaa Xaa Lys Gly 1 5 6

3- Information for Seq. ID No. 75 Sequence Characteristics:

a) Size: 4 amino acids

b) Type: amino acid c) Topology: linear Sequence description: Seq. ID No. 75:

Val Xaa Arg Asn 1 4

Claims (8)

1. Dengue virus antigen mimetic recombinant peptides and their reverse sequences, characterized by the sequences Seq ID N-1 to Seq ID N ° 32. Dengue virus antigen protein motifs according to claim 1, characterized in that they comprise the sequences Seq ID No. 33 to Seq ID No. 75, including the three amino acid protein motifs: Trp Lys Lys, Ile Asn Trp and Pro Asn Met. Peptides, their reverse sequences and protein motifs according to claim 1 or 2, characterized in that they are used as probes for in vitro detection of the presence of circulating antibodies or other flavivirus binding molecules, preferably dengue virus. Peptides, their reverse sequences and synthetic or recombinant protein motifs according to any one of claims 1 to 3, used in in vitro immunodiagnostic procedures as probes to detect the presence of linker molecules, such as circulating antibodies, that react against the virus. dengue fever, characterized by detection by immunoassays, such as immunoenzymatic tests (such as ELISA, Western Blot, immunofluorescence, immunohistochemistry, EIA and others), immunoagglutination tests, electrochemical sensors, or any other form of detection directly or indirectly related in body fluid samples such as saliva, urine, blood, preferably blood. Use of the peptides, their reverse sequences and protein motifs according to claim 1 or 2, characterized in that they are in the preparation of a vaccine composition, to stimulate the human or animal immune system as a vaccine therapeutic process against flavivirus, mainly dengue virus. 6 Vaccine composition according to claim 5, characterized in that it comprises one or more compounds described by the sequences Seq ID No. 1 to Seq ID No. 75, preferably those related to Seq ID No. 11, Seq ID No. 47, Seq ID No. 8, Seq ID No. 41, Seq ID No. 2, Seq ID No. 34 Seq ID No. 58, Seq ID No. 4, Seq ID No. 7, Seq ID No. 38 Seq ID No. 18, Seq ID No. 20, Seq ID No. 60.