Protein container, polynucleotide, vector, expression cassette, cell, method for producing container, method for pathogen recognition or disease diagnosis, use of container, and diagnostic kit

Abstract

The present invention relates to protein containers, polynucleotides, vectors, expression cassettes, cells, methods for producing containers, pathogen recognition or disease diagnosis methods, uses of containers, and diagnostic kits. The present invention relates to a protein container into which various exogenous polyamino acid sequences can be inserted simultaneously for expression in various systems and for various uses. The present invention relates to polynucleotides that can produce protein containers mentioned above. The invention also relates to vectors and expression cassettes comprising the above mentioned polynucleotides. The invention also relates to a cell comprising the vector or the expression cassette mentioned above. The invention also relates to methods for producing said protein container and identifying pathogens or diagnosing diseases in vitro. The invention also relates to the use of said protein container and a kit comprising said protein container for diagnosis or as a vaccine composition.

Description

[0001] This application is a divisional application of the patent application "Protein containers, polynucleotides, vectors, expression cassettes, cells, container production methods, pathogen identification or disease diagnosis methods, uses of containers, and diagnostic kits" with an application date of August 27, 2020 and application number 202080058664.7 (international application number PCT / BR2020 / 050341). Technical Field

[0002] The present invention falls within the application fields of chemistry, pharmacy, medicine, and biotechnology, and more specifically, within the field of preparations for biopharmaceutical purposes. The present invention relates to a protein receptacle that can concomitantly accept multiple exogenous polyamino acid sequences for expression in various systems and for different uses. The present invention relates to polynucleotides that can produce the above-mentioned protein receptacles. The present invention also relates to vectors and expression cassettes comprising the aforementioned polynucleotides (one or more). The present invention further relates to cells comprising the aforementioned vectors or expression cassettes. The present invention further relates to methods for producing the protein receptacles and for pathogen identification or in vitro disease diagnosis. The present invention further relates to the use of the protein receptacles and kits comprising the protein receptacles for diagnostic purposes or as vaccine compositions. Background Art

[0003] There are several references throughout the text herein, which are indicated in parentheses. The information disclosed in these references is included herein to better describe the state of the art in the field to which this invention pertains.

[0004] Green fluorescent protein (GFP), produced by the cnidarian jellyfish Aequorea victoria, emits high fluorescence in the green region of the visible spectrum (Prasher et al., Gene 15, 111(2):229-33, 1992(1)), and is classically used as a marker for gene expression and localization, and is therefore considered a reporter protein. After the initial observation that the GFP protein could emit fluorescence, several uses of the protein were described.

[0005] The use of GFP as a reporter protein has been patented for different purposes and in different systems. Patent application US2018298058 uses GFP as a protein production and purification system. Patent application CN108303539 uses GFP as an internal reference (input) for cancer detection tests; application CN108220313 proposes a high-throughput GFP fusion and expression method. Application CN108192904 requests protection for a GFP fusion protein that can insert itself into a biological membrane; application CN107703219 has emphasized the use of GFP in metabolomics studies of mesenchymal cells. In addition, patent application US2018016310 proposes a variant of "superfolder" fused GFP. There are several patents, such as these examples of GFP with mutations to increase fluorescence expression or modify fluorescence wavelength peaks: US6054321, US6096865, US6027881, US6025485. It is therefore clear that since its description, the GFP protein has been used and patented for various applications as a reporter protein.

[0006] Reporter molecules are commonly used in biological systems to monitor gene expression. The GFP protein represents a significant innovation in this context by eliminating the need for any substrates or cofactors, meaning it doesn't require the addition of any other reagents for visualization, as most other reporter proteins do. Another advantage of GFP is its ability to exhibit autofluorescence, eliminating the need for fluorescent markers. For various reasons, fluorescent markers lack the appropriate sensitivity or specificity for their intended use.

[0007] Due to its ability to self-produce detectable green fluorescence, GFP has been widely used to study gene expression and protein localization and is considered one of the most promising reporter proteins in the literature.

[0008] In its use as a reporter protein, a gene encoding GFP can be used in the production of fusion proteins, that is, a specific gene of interest is fused to a gene encoding GFP. The fusion gene cassette can be inserted into a living system to allow expression of the fusion gene and monitoring of the intracellular localization of the protein of interest (Santos-Beneit & Errington, Archives Microbiology, 199(6):875-880, 2017; Belardinelli & Jackson, Tuberculosis (Edinb), 105:13-17, 2017; Wakabayashi et al., International Journal Food Microbiology, 19, 291:144-150, 2018; Caì et al., Viruses, 20; 10(11), 2018).

[0009] In both yeast and mammalian cell systems, GFP protein has also been used as a framework for peptide display or even peptide libraries (Kamb et al., Proc. Natl. Acad Sci. USA, 95:7508-7513, 1998; WO 2004005322). GFP protein as a framework protein for display of random peptides can be used to define the properties of peptide libraries.

[0010] Progress in the development of new GFP variants seeks to achieve improvements in the properties of the protein to produce new reagents for a wide range of research purposes. Through mutations, new forms of GFP have been developed that include DNA sequences optimized for increased production in human cell systems, i.e., humanized GFP proteins (Cormack, et al., Gene 173, 33-38, 1996; Haas, et al., Current Biology 6, 315-324, 1996; Yang, et al., Nucleic Acids Research 24, 4592-4593, 1996).

[0011] Among these forms, the enhanced green fluorescent protein (eGFP) has been described (Heim & Cubitt, Nature, 373, pp. 663-664, 1995).

[0012] GFP, which originates from the cnidarian jellyfish Aequorea victoria and is encoded by the gfp10 gene, is a 238-amino acid protein. The protein has the ability to absorb blue light (with a main excitation peak at 395 nm) and emit green light (main emission peak at 509 nm) from a chromophore in the center of the protein (Morin & Hastings, Journal Cell Physiology, 77(3):313-8, 1971; Prasher et al., Gene 15, 111(2):229-33, 1992). The chromophore is composed of a hexapeptide, starting at amino acid 64, and derived from the primary amino acid sequence by cyclization and oxidation of serine, tyrosine, and glycine (positions 65, 66, and 67) (Shimomura, 104(2), 1979; Cody et al., Biochemistry, 32(5):1212-8, 1993). The light emitted by GFP is independent of the cell biological species in which it is expressed and does not require any substrates, cofactors, or other gene products from the victoria jellyfish (Chalfie et al., Science, 263(5148):802-5, 1994). This property of GFP allows its fluorescence to be detected in living cells other than the victoria jellyfish because it can be processed in the protein expression system of the cell (Ormo et al., Science 273:1392-1395, 1996; Yang et al., Nature Biotech 14:1246-1251, 1996).

[0013] The basic structure of GFP consists of 11 antiparallel folded beta chains that are intertwined to form a tertiary structure in the shape of a beta barrel. Each chain is connected to the next by a loop domain that projects onto the upper and lower surfaces of the barrel to interact with the environment. By convention, each chain and loop are identified by a number to better describe the protein.

[0014] Directed mutagenesis experiments have shown that certain biochemical properties of the GFP protein are caused by this barrel structure. Therefore, amino acid changes in the primary structure are responsible for accelerating protein folding, reducing aggregation of translation products, and increasing protein stability in solution.

[0015] Specific loops move to the cavity of the protein barrel, forming an α-helix, which is the reason for the fluorescent properties of the protein (Crone et al., GFP-Based Biosensors, InTech, 2013). Some interventions in the protein structure can interfere with the ability to emit fluorescence. Mutations in certain amino acids can change the intensity of the fluorescence emission to the wavelength, thereby changing the emission color. Mutations in Tyr66, which is an internal residue involved in the fluorescent chromophore, can produce a large number of fluorescent protein variants with altered chromophores or surrounding structures. These changes interfere with the absorption and emission of light at different wavelengths, producing a wide range of different emission colors (Heim & Tsien, Current Biology, 6 (2): 178-82, 1996).

[0016] Changes in pH can also interfere with fluorescence intensity. At physiological pH, GFP exhibits a maximum absorption at 395 nm, while it absorbs less light at 475 nm. However, increasing the pH to about 12.0 results in a maximum absorption at 475 nm, while it has reduced absorption at 395 nm (Ward et al., Photochemistry and Photobiology, 35(6):803-808, 1982).

[0017] The compact structure of the core protein makes GFP highly stable even under adverse conditions, such as treatment by proteases, making this protein very useful as a reporter protein in general.

[0018] There are different forms of GFP, but there is always a search for ways to improve the protein by adding new functions or removing some limitations. eGFP itself appears to be an enhanced version, which gives the protein greater flexibility when faced with the modification of the F64L and S65T amino acids (Heim et al., Nature 373: 663-664, 1995; Li et al., Journal Biology Chemistry, 272 (45): 28545-9, 1997). This enhancement allows GFP to achieve both its desired three-dimensional shape and its ability to express fluorescence, even when it has heterologous sequences in its protein sequence (Pedelacq et al., Nat Biotechnology, 24 (1): 79-88, 2006).

[0019] GFAb is a protein modified form that accepts exogenous sequences in two ring domains. In its development, several rounds of directed evolution are needed to select and support the insertion of exogenous peptides into three mutant protein clones of two proximal regions (i.e., Glu-172-Asp-173 and Asp-102-Asp-103). The author uses unmutated protein to prove that the insertion of two exogenous peptides prevents GFP fluorescence production and protein expression on the yeast cell surface. After a series of mutations and selections, it is possible to insert in only two regions at the same time, but still cause the inherent activity of greatly losing GFP, sometimes making the production or expression of its insert impossible (Pavoor et al., PNAS 106 (29): 11895-11900, 2009).

[0020] Mutations in the N- and C-terminal loops also showed that eGFP can be manipulated in the coding sequence without affecting the structural aspects of the core protein (Topell et al., FEBS Letters 457(2):283-289, 1999). However, analysis of 20 protein variants with loops showed that the protein had a low tolerance to the insertion of new termini and, in most cases, lost the ability to form a chromophore. This fact suggests that manipulation of a protein sequence can strongly interfere with its properties or even its cellular expression.

[0021] Some attempts have been made to simultaneously insert multiple epitopes into the loop region of GFP with the goal of enabling the use of the protein for target-specific binding reactions. However, all of these efforts have shown limited success given the structural sensitivity of GFP and its chromophore.

[0022] Other mutant proteins of GFP protein show improved forms that emit other types of fluorescence spectra. For example, Heim et al. (Proc Natl Acad Sci USA, 91 (26): 12501-4, 1994) described a mutant protein that emits blue fluorescence by containing histidine instead of tyrosine at amino acid 66. Thereafter, Heim et al. (Nature, 373 (6516): 663-4, 1995) also described a mutant GFP protein that has a spectrum similar to that obtained from the marine animal Renilla reniformis by replacing serine with threonine at amino acid 65, and the extinction coefficient per monomer is more than 10 times the wavelength peak of the natural GFP from the genus Aequorea. Other patent documents describe mutant GFP proteins that display other light emission spectra other than green, such as blue and red (US 5625048, WO 2004005322).

[0023] In addition, other GFP mutant proteins have optimized excitation spectra, particularly for use in certain argon laser flow cytometers (FACS) equipment (US 5804387). There are also descriptions of mutant GFP proteins modified to better express in plant cell systems (WO1996027675). Patent literature US5968750 proposes a humanized GFP adapted for expression in mammalian cells, including humans. Humanized GFP incorporates the preferred codon for reading into the human cell gene expression system.

[0024] In the prior art, as is apparent from the patent literature listed above, GFP is capable of containing genes at its 5' or 3' end without interfering with expression, three-dimensional tangling, and fluorescence generation. Furthermore, GFP has been used as a vector for in vivo peptide display or even peptide libraries. In the case of peptide libraries, GFP can facilitate the display of random peptides and thereby help define the characteristics of the peptide library (Kamb et al., Proc. Natl. Acad. Sci. USA, 95:7508-7513, 1998; WO2004005322).

[0025] For example, Abedi et al. (1998, Nucleic Acids Res. 26: 623-300) inserted a peptide into the region of the ring exposed in the GFP protein of the victoria jellyfish and showed that the GFP molecule retained autofluorescence when expressed in yeast and Escherichia coli. The authors further explained that the fluorescence of the GFP framework can be used to monitor the diversity of peptides and the presence or expression of specified peptides in specified cells. However, compared with native GFP, the fluorescence rate of the GFP framework molecule is relatively low. Kamb and Abedi (US 6025485) prepared a library of GFP arrays from enhanced green fluorescent protein (eGFP) to enhance fluorescence intensity.

[0026] In addition, Peele et al. (Chem. & Bio. 8:521-534, 2001) tested a peptide library with different structural deviations in mammalian cells using eGFP as a framework. Anderson et al. further enhanced fluorescence intensity by inserting peptides into the GFP loop with a tetraglycine ligand (US20010003650). Happe et al. described a humanized GFP that can be expressed in large quantities in mammalian cell systems, tolerates peptide insertion, and maintains autofluorescence (WO 2004005322).

[0027] However, there remains a need in the art for a GFP molecular framework that not only exhibits fluorescence of suitable intensity, but is also expressed at high levels in cellular systems.

[0028] There is variability in the ability of GFP molecules to tolerate peptide display while retaining autofluorescence. Therefore, there is a need in the art to develop GFP that can express at high levels and tolerate insertions while retaining autofluorescence.

[0029] In addition to its ability to support gene expression at the terminal end, GFP allows for the insertion of epitopes into surface loops where the molecule is exposed to a medium. Several attempts have been made to simultaneously insert multiple peptides into the loop region of GFP, allowing proteins to be used in target-specific binding reactions. However, given the structural sensitivity of GFP and its chromophore, all of these efforts have had limited success.

[0030] Pavoor and colleagues have worked hard to develop a protein modified to accept exogenous sequences in two loop domains. In this development, several rounds of directed evolution are needed to select three mutant protein clones that support the insertion of exogenous peptides in two proximal regions (i.e., Glu-172-Asp-173 and Asp-102-Asp-103). The authors used unmutated proteins to prove that the insertion of two exogenous peptides prevented GFP fluorescence production and protein expression on the yeast cell surface. After a series of mutations and selections, simultaneous insertion in only two regions was possible, but still resulted in a significant loss of the intrinsic activity of GFP, sometimes making the production or expression of its insert impossible (Pavoor et al., PNAS 106 (29): 11895-11900, 2009).

[0031] Abedi et al. (Nucleic Acids Research 26(2):623-30, 1998) proposed 10 protein positions in the loop region, 8 of which are between β-sheets, for peptide expression. Chimeric proteins can be used for experiments requiring intracellular expression, so fluorescence uninterruptedness will be a limiting factor. In this study, only three chimeric proteins (which have insertion sites at amino acids 157-158, 172-173, and 194-195) showed fluorescence (reduced to a quarter of the original); and only two insertion sites (studied separately) could have peptides without losing fluorescence. The authors of the document further concluded that "it is curious how GFP is so sensitive even to structural perturbations in the β-sheet".

[0032] Li et al. (Photochemistry and Photobiology, 84(1):111-9, 2008) reported on a chimeric protein (red fluorescent protein - RFP) in which they showed that six genetically distinct sites were located in three different loops where a five-residue sequence could be inserted without interfering with the protein's ability to fluoresce. However, the authors did not demonstrate the simultaneous use of these sites for the insertion of different peptides.

[0033] Patent application WO02090535 proposes a fluorescent GFP that does not insert peptides simultaneously in 5 different rings of a protein. This patent application shows in its descriptive report the possibility of inserting peptides in more than one ring of a protein simultaneously, which increases the complexity of the library and allows displaying proteins on the same surface. However, patent documentation does not prove this possibility, because it only proposes a peptide insertion test once in 5 different protein rings. It should be noted that document further emphasizes that ring 1 and ring 5 itself are not presented as good insertion sites, because the peptide insertion at these sites stops protein expression. In addition, other patent documentation proposes GFP variants for peptide expression in protein rings, but these studies do not prove the feasibility (WO02090535, US2003224412, WO200134824) of expressing more than 4 kinds of peptides in different insertion sites in the GFP protein ring without losing any of its basic characteristics. Summary of the Invention

[0034] To address the aforementioned issues, the present invention provides significant advantages. Because container proteins can express a large number of different polyamino acid sequences, they are characterized as multivalent container proteins, expanding their use for vaccine compositions, as internal controls for research and technology development, or for disease diagnosis. There remains a real need in the art to develop container proteins that not only exhibit sufficient fluorescence intensity, but can also be expressed in large quantities in production cell systems, and furthermore, tolerate the concomitant display of multiple exogenous polyamino acid sequences while still exhibiting detectable autofluorescence.

[0035] In one aspect, the present invention relates to a protein container capable of simultaneously displaying multiple exogenous polyamino acid sequences at more than four different sites on the container protein.

[0036] In another aspect, the present invention relates to a polynucleotide capable of producing the aforementioned protein container.

[0037] In another aspect, the present invention relates to a vector comprising the aforementioned polynucleotide.

[0038] In another aspect, the present invention relates to an expression cassette comprising the aforementioned polynucleotide.

[0039] In another aspect, the present invention relates to a method for producing said protein container, and for use in pathogen identification or in vitro disease diagnosis.

[0040] In another aspect, the present invention relates to the use of said protein container for diagnostic purposes or as a vaccine composition.

[0041] In another aspect, the invention relates to a kit comprising a container of said protein for diagnostic purposes or as a vaccine composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 -Purification of PlatCruzi protein by affinity chromatography. (A) Using Elution profile of container proteins from a nickel column in a liquid chromatography system. (B) Analysis of eluates from 13 to 26 by polyacrylamide gel electrophoresis (SDSPAGE). Elution was performed in ascending order using buffer B. PM - molecular weight marker.

[0043] Figure 2 - Reactivity of PlatCruzi with sera from the WHO International Standard Biological Reference for Trypanosoma cruzi by ELISA. (A) Pool of patient sera that recognize the TcI strain, designated IS 09 / 188. (B) Pool of patient sera that recognize the TcII strain, designated IS 09 / 186.

[0044] Figure 3 - Determination of antibody titers in sera from patients with chronic Chagas disease using PlatCruzi receptacle protein as antigen. Sera were provided by LACENS at a concentration of 500 ng / well and serum dilutions of 1:50-1:1000 for ELISAs.

[0045] Figure 4 - Expression of PlatCruzi antigen by ELISA on sera from patients with different diseases. PlatCruzi antigen was used at a concentration of 500 ng / well and sera were diluted 1:250.

[0046] Figure 5 - Detection of rabies virus-specific epitopes by rabbit anti-RxRabies2 serum. Rabbit antibodies immunized with RxRabies2 were purified by RxRabies2 affinity chromatography and used as primary antibodies by immunoblotting to detect crude extracts (*; 2nd column) or semi-purified extracts at two different concentrations of 1x and 0.5x. RxRabies2 in extracts (columns 4 and 6, respectively). Negative controls: RxContainer protein (column 3) and PlatCruzi (at two concentrations: 1x and 0.5x in columns 5 and 7, respectively).

[0047] Figure 6 Analysis of RxHoIgG3 protein by polyacrylamide gel electrophoresis (SDS PAGE). A, Soluble extract of E. coli that does not produce RxHoIgG3. B, Water-insoluble fraction of RxHoIgG3-producing bacteria. C, Soluble fraction of RxHoIgG3-producing bacteria. Arrows indicate the location of RxHoIgG3 protein.

[0048] Figure 7 - Detection of IgM anti-RxOro antibodies by ELISA. C-: Negative control (serum from a patient without Oropouche virus infection); C+: Standard positive serum for Oropouche virus infection. Patient: Suspected case of Oropouche virus infection. Oro+: Positive for Oropouche virus infection (detection of IgM anti-RxOro antibodies). Oro- (negative control): No IgM response to RxOro. Protein concentration: 0.288 μg / μL. Cutoff: 0.0613.

[0049] Figure 8 - Polyacrylamide gel electrophoresis (SDS-PAGE) showing the production of PlatCruzi, RxMayaro_IgG, and RxMayaro_IgM proteins. Columns 1 to 8 represent: 1) molecular weight; 2) total bacterial extract without recombinant protein induction; 3) total bacterial extract after induction of PlatCruzi production; 4) total bacterial extract after induction of RxMayaro_IgG production; 5) total bacterial extract after induction of RxMayaro_IgM production; 6) insoluble bacterial protein after induction of PlatCruzi production; 7) insoluble bacterial protein after induction of RxMayaro_IgG production; 8) insoluble bacterial protein after induction of RxMayaro_IgM production. Arrows indicate the bands representing PlatCruzi (columns 3 and 6), RxMayaro_IgG (columns 4 and 7), and RxMayaro_IgM (columns 5 and 8).

[0050] Figure 9 - Reactivity of sera from a Mayaro virus positive patient (S MAY) and a healthy individual (SN) by ELISA using the RxMayaro_IgG protein. Visualization was performed with anti-IgG immunoglobulin conjugated to alkaline phosphatase (cutoff = 0.0210).

[0051] Figure 10 - Reactivity of sera from individuals considered healthy (SN) and positive for Mayaro virus (S MAY) by ELISA using RxMayaro_IgM protein. Visualization was performed with anti-IgM immunoglobulin conjugated to alkaline phosphatase (cutoff = 0.0547).

[0052] Figure 11 - Polyacrylamide gel electrophoresis (SDS-PAGE) showing the yield of insoluble (I) and soluble (S) proteins from PlatCruzi, TxCruzi, RxPtx, TxNeuza, and RxYFIgG. Columns 1 to 10 include: 1) insoluble protein from bacteria induced to produce PlatCruzi; 2) soluble protein from bacteria induced to produce PlatCruzi; 3) insoluble protein from bacteria induced to produce TxCruzi; 4) soluble protein from bacteria induced to produce TxCruzi; 5) insoluble protein from bacteria induced to produce RxPtx; 6) soluble protein from bacteria induced to produce RxPtx; 7) insoluble protein from bacteria induced to produce TxNeuza; 8) soluble protein from bacteria induced to produce TxNeuza; 9) insoluble protein from bacteria induced to produce RxYFIgG; 10) soluble protein from bacteria induced to produce RxYFIgG. Arrows indicate bands representing PlatCruzi (lanes 1 and 2), TxCruzi (lanes 3 and 4), RxPtx (lanes 5 and 6), TxNeuza (lanes 7 and 8), and RxYFIgG (lanes 9 and 10).

[0053] Figure 12- A patterned cellulose membrane (pictorial cellulose membrane) with polyamino acids of SARS-CoV-2 that react with IgM antibodies from Covid-19 positive patient serum, visualized as spots of various shades of gray in areas delineated by a grid in the form of a checkerboard. Each square contains a reactive spot in an area of cellulose membrane where different polypeptide sequences synthesized in linear form are covalently bound to the membrane surface. The relationship between the physical position in the membrane and the sequence of the polyamino acids is listed in Table 18. The combined polyamino acid sequence represents the coding sequence of the following: spike protein SARS-CoV-2 (S1: aa1-1273, A7-K19), protein ORF3a (OF3: aa 1-275, K22-N2), membrane glycoprotein (M: aa1-222, N5-O23); ORF6 (OF6: aa 1-61, P2-P12); ORF7 protein (OF7: aa1-121, P15-Q13), ORF8 protein (OF8: aa 1-121, Q16-R17), nucleocapsid protein (N: aa1-419, R20-V17), envelope protein (E: aa 1-75, W1-W13), ORF10 protein region (OF10: aa1-38, W15-W20). Each polyamino acid has a length of 15 amino acids and an adjacent, continuous overlap of 10 amino acids.

[0054] Figure 13 A patterned cellulose polyamino acid film of SARS-CoV-2 that reacts with IgG antibodies from Covid-19 patient sera is visualized as spots of various shades of gray within a grid-like area. Each square contains a reactive spot within the cellulose membrane area, where different peptide sequences synthesized in linear form are covalently bound to the membrane surface. The relationship between the physical location in the membrane and the polyamino acid sequence is listed in Table 19. The combined polyamino acid sequence represents the following coding sequences: SARS-CoV-2 ORF3a protein (ORF3: aa 1-275, A7-C11), membrane glycoprotein (G: aa 1-61, C14-E8); ORF6 protein (ORF6: aa 1-61, E11-E21); ORF7 protein (OF7: aa1-121, E24-F22), ORF8 protein (ORF8: aa1-121, G1-G23), spike protein (S: aa 1-1273, H1-R13), nucleocapsid protein (N: aa1-419, R16-V1), envelope protein (E: aa 1-75, W1-W13), ORF10 (ORF10: aa 1-38, W15-W20). Each polyamino acid has a length of 15 amino acids and an adjacent, continuous overlap of 10 amino acids.

[0055] Figure 14 A patterned cellulose membrane with polyamino acids from SARS-CoV-2 that react with IgA antibodies from Covid-19 patient sera, visualized as spots of various shades of gray within areas delineated in a grid pattern. Each square contains a reactive spot on the cellulose membrane area, where different peptide sequences synthesized in linear form are covalently bound to the membrane surface. The relationship between the physical location in the membrane and the polyamino acid sequence is listed in Table 20. The polyamino acid sequences of these combinations represent the following coding sequences: SARS-CoV-2 spike protein (S: aa1-1273, A6-K18), ORF3a (ORF3: aa1-275, K21-N1), membrane glycoprotein (M: aa 1-61, N4-O22); ORF6 (ORF6: aa 1-61, P1-P11); ORF7 (ORF7: aa1-121, P14-Q12), ORF8 (ORF8: aa 1-121, Q15-R13), nucleocapsid protein (N: aa1-419, R16-V1), envelope protein (E: aa 1-75, region V4-V16), ORF10 (ORF10: aa 1-38, V19-V24). Each polyamino acid has a length of 15 amino acids and an adjacent, continuous overlap of 10 amino acids.

[0056] 15A to 15I - Reactivity of sera from patients with COVID to SARS-CoV-2 peptides synthesized on cellulose membranes, visualized by alkaline phosphatase-labeled anti-human IgM antibodies ( Figure 12 15A, surface glycoprotein; 15B, ORF 3a; 15C, membrane glycoprotein; 15D, ORF 6; 15E, ORF 7; 15F, ORF 8; 15G, nucleoprotein; 15H, E protein; 15I, ORF 10.

[0057] 16A to 16H - Reactivity of sera from patients with COVID to SARS-CoV-2 peptides synthesized on cellulose membranes, developed with alkaline phosphatase-labeled anti-human IgG antibodies ( Figure 13 ). 16A: ORF 3a; 16B: membrane glycoprotein; 16C: ORF 6; 16D: ORF 7; 16E: ORF 8; 16F: nucleoprotein; 16G: E protein; 16H: ORF 10.

[0058] 17A to 17I - Reactivity of sera from patients with COVID to SARS-CoV-2 peptides synthesized on cellulose membranes, developed with alkaline phosphatase-labeled anti-human IgG antibodies ( Figure 14). 17A, surface glycoprotein; 17B, ORF 3a; 17C, membrane glycoprotein; 17D, ORF 6; 17E, ORF 7; 17F ORF 8; 17G, nucleoprotein; 17H, E protein; 17I, ORF 10.

[0059] Figure 18 - ELISA of sera from hospitalized patients (n=36) (Group 3) using branched synthetic peptides (SARS-X1-SARS-X8) developed with an anti-IgM secondary antibody.

[0060] Figure 19 - ELISA of sera from hospitalized patients (n=36) using branched synthetic peptides (SARS-X1-SARS-X8) and developed with an anti-IgG secondary antibody.

[0061] Figure 20 - ELISA of sera from hospitalized patients (n=36) using branched synthetic peptides (SARS-X4-SARS-X8) developed with anti-IgA secondary antibodies.

[0062] Figure 21 - ELISA of sera from four patient groups (Group 1: asymptomatic, 2: suspected; 3: hospitalized, and 4: immune protected) using SARS-X3 branched synthetic peptides, developed with anti-IgM secondary antibodies.

[0063] Figure 22 - ELISA of sera from four patient groups (Group 1: asymptomatic, 2: suspected; 3: hospitalized, and 4: immune protected) using SARS-X8 branched synthetic peptides, developed with anti-IgG secondary antibodies.

[0064] Figure 23 - ELISA of sera from four patient groups (Group 1: asymptomatic, 2: suspected; 3: hospitalized, and 4: immune protected) using the synthetic peptide SARS-X7, developed with an anti-IgA secondary antibody.

[0065] Figure 24- Electrophoresis on polyacrylamide gel (SDS-PAGE) showed the production of Ag-Covid19, Ag-COVID19 protein with a six-histidine tail, Tx-SARS-IgM, Tx-SARS2-IgG, Tx-SARS2-G / M, Tx-SARS2-IgA, Tx-SARS2-Universal and Tx-SARS2-G5. Columns 1 to 10 represent: 1) molecular weight; 2) total bacterial extracts induced without recombinant protein; 3) total bacterial extracts after induction of Ag-COVID19 production; 4) total bacterial extracts after induction of Ag-COVID19 protein with six histidine tails; 5) total bacterial extracts after induction of Tx-SARS2-IgM protein production; 6) total bacterial extracts after induction of Tx-SARS2-IgG protein production; 7) total bacterial extracts after induction of Tx-SARS2-G / M protein production; 8) total bacterial extracts after induction of Tx-SARS2-IgA protein production; 9) total bacterial extracts after induction of Tx-SARS2-Universal protein production; and 10) total bacterial extracts after induction of Tx-SARS2-G5 protein production. The letters representing the molecular weight standards are A) 250 kDa; B) 130 kDa; C) 100 kDa; D) 70 kDa; E) 55 kDa; F) 35 kDa and G) 25 kDa.

[0066] Figure 25 - Purification of Ag-COVID19 protein by affinity chromatography was demonstrated by electrophoresis on polyacrylamide gel (SDS-PAGE). (Total) Spectrum of total bacterial extract after induction of production; (FT) Spectrum of protein not bound to the nickel column; (200) elution profile of Ag-COVID19 protein after addition of 200 mM imidazole; (75) elution profile of Ag-COVID19 protein after addition of 75 mM imidazole and (500) elution profile of Ag-COVID19 protein after addition of 500 mM imidazole.

[0067] Figure 26 - Polyacrylamide gel (SDS-PAGE) electrophoresis shows the purification of Tx-SARS2-G5 protein by affinity chromatography. (Total) Spectrum of total bacterial extract after induction of production; (FT) Spectrum of protein not bound to the nickel column; (200) elution profile of Tx-SARS2-G5 protein after addition of 200 mM imidazole; (75) elution profile of Tx-SARS2-G5 protein after addition of 75 mM imidazole; and (500) elution profile of Tx-SARS2-G5 protein after addition of 500 mM imidazole.

[0068] Figure 27- ELISA of serum from seven groups of patients infected with malaria, dengue fever, or SARS-CoV-2, who were still hospitalized, recovered, suspected, or asymptomatic. As a control, a pool of serum from healthy individuals collected before the epidemic was used. Ag-COVID19 protein was used in the ELISA and the bound antibodies were visualized with an anti-human IgG secondary antibody.

[0069] Figure 28 - ELISA testing of sera from six groups of patients with syphilis, malaria, dengue fever, or hospitalized or suspected SARS-CoV-2. As a control, a pool of sera from healthy individuals collected before the epidemic was used. The Tx-SARS2-G5 protein was used in the ELISA, and the bound antibodies were visualized using an anti-human IgG secondary antibody.

[0070] Figure 29 - Antibody titration against Ag-COVID19 by ELISA in mice 2 or 4 weeks after immunization with Ag-COVID19 protein.

[0071] Figure 30 - Antibody purification using Ag-COVID19 protein. DETAILED DESCRIPTION

[0072] While the invention is susceptible to different embodiments, preferred embodiments are shown in the drawings and discussed in detail below, it should be understood that the description is to be considered as an exemplification of the principles of the invention and is not intended to limit the invention which has been illustrated and described herein.

[0073] In this article, some abbreviations are used. The following is a list of abbreviations:

[0074] About nitrogenous bases:

[0075] C = cytosine; A = adenine; T = thymine; G = guanine

[0076] About amino acids:

[0077] I = isoleucine; L = leucine; V = valine; F = phenylalanine; M = methionine; C = cysteine; A = alanine; G = glycine; P = proline; T = threonine; S = serine; Y = tyrosine; W = tryptophan; Q = glutamine; N = asparagine; H = histidine; E = glutamic acid; D = aspartic acid; K = lysine; R = arginine.

[0078] Protein Container

[0079] The present invention relates to the production and use in various methods and compositions of protein containers based on the sequence of the green fluorescent protein, referred to herein as GFP, which methods and compositions exploit the ability of such protein containers to simultaneously display multiple different or identical exogenous polyamino acid sequences at more than four different protein sites, and further the ability to exhibit sufficient fluorescence intensity, the ability to be efficiently expressed in cellular protein production systems, and the ability to be used as reagents for research, diagnostics, or in vaccine compositions.

[0080] In a first embodiment, the present invention relates to a stable protein structure that supports the simultaneous insertion of four or more exogenous polyamino acid sequences at different sites. In another embodiment, the protein container comprises the amino acid sequence shown in SEQ ID NO: 1. In another embodiment, the protein container comprises the amino acid sequence shown in SEQ ID NO: 3. In another embodiment, the protein container comprises the amino acid sequence shown in SEQ ID NO: 77. In another embodiment, the protein container presents insertion sites for exogenous polyamino acid sequences in a protein loop facing the external environment. In another embodiment, the simultaneous insertion of exogenous polyamino acid sequences does not interfere with the production conditions of the container protein. In another embodiment, the protein container simultaneously comprises exogenous polyamino acid sequences for use in vaccine compositions, for diagnostics, or for the development of laboratory reagents. In another embodiment, upon simultaneous insertion of exogenous polyamino acid sequences into the protein loops of the container protein, the exogenous polyamino acid sequences do not lose their immunogenic properties. In another embodiment, the protein container simultaneously comprises the exogenous polyamino acid sequences of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. In another embodiment, the protein container simultaneously comprises the amino acid sequence set forth in SEQ ID NO: 18. In another embodiment, the protein container simultaneously comprises the exogenous polyamino acid sequences of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29. In another embodiment, the protein container simultaneously comprises the amino acid sequence set forth in SEQ ID NO: 20. In another embodiment, the protein container simultaneously comprises multiple copies of the exogenous polyamino acid sequence of SEQ ID NO: 30. In another embodiment, the protein container comprises the amino acid sequence set forth in SEQ ID NO: 31. In another embodiment, the protein container comprises the exogenous polyamino acid sequences of SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40. In another embodiment, the protein container comprises the amino acid sequence shown in SEQ ID NO: 33.In another embodiment, the protein container comprises the exogenous polyamino acid sequences of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44. In another embodiment, the protein container comprises the amino acid sequence set forth in SEQ ID NO:45. In another embodiment, the protein container comprises the exogenous polyamino acid sequences of SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50. In another embodiment, the protein container comprises the amino acid sequence set forth in SEQ ID NO:51. In another embodiment, the protein container comprises the exogenous polyamino acid sequences of SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:95, and SEQ ID NO:96. In another embodiment, the protein container comprises the amino acid sequence set forth in SEQ ID NO:64. In another embodiment, the protein container comprises the exogenous polyamino acid sequences of SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 97. In another embodiment, the protein container comprises the amino acid sequence set forth in SEQ ID NO: 75. In another embodiment, the protein container comprises the exogenous polyamino acid sequences of SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, and SEQ ID NO: 98. In another embodiment, the protein container comprises the amino acid sequence set forth in SEQ ID NO: 88.In another embodiment, the protein container comprises the exogenous polyamino acid sequences of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, and SEQ ID NO: 99. In another embodiment, the protein container comprises the amino acid sequence set forth in SEQ ID NO: 90. In another embodiment, the protein container comprises an exogenous polyamino acid sequence as defined in SEQ ID NOs: 100, 124, 125, and 126; or, comprises an exogenous polyamino acid sequence as defined in SEQ ID NOs: 101, 127, 128, 129, 130, 131, 132, 133, 134, and 135; or, comprises an exogenous polyamino acid sequence as defined in SEQ ID NOs: 136, 137, 138, 139, 140, 141, 142; or, comprises an exogenous polyamino acid sequence as defined in SEQ ID NOs: 129, 133, 135, 137, 140, 141, 142, 143, 144, 146, 147, and 148; or, comprises an exogenous polyamino acid sequence as defined in SEQ ID NOs: In another embodiment, the protein container comprises an exogenous polyamino acid sequence as defined in SEQ ID NOs: 103, 149, 150, 151, 152, 153, 154, 155; or, in combination with an exogenous polyamino acid sequence as defined in SEQ ID NOs: 104, 156, 157, 158, 159, 160, 161, 162, and 163; or, in combination with an exogenous polyamino acid sequence as defined in SEQ ID NOs: 136, 139, 140, 141, 142, 143, 144, 146, and 147. In another embodiment, the protein container comprises any of the amino acid sequences shown in SEQ ID NOs: 334-341.

[0081] Another embodiment of the present invention relates to the efficient expression of the protein container described herein in a cell system, based on the sequence of GFP accompanied by one, two, more than two, three, four, or more than ten exogenous polyamino acid sequences at 10 different sites on the container protein. Specifically, the present invention relates to the efficient expression of the protein container described herein that simultaneously displays exogenous polyamino acid sequences at up to 10 different protein sites. More specifically, the present invention relates to the efficient expression of the container described herein that carries the exogenous polyamino acid sequences inserted into 10 different protein sites, without losing its inherent properties, such as autofluorescence.

[0082] The present invention also relates to the production and use of protein containers, "Platform," "Rx," and "Tx," and their amino acid sequences (described in SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 77, respectively), their nucleotide sequences (described in SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 78, respectively), and their amino acid sequences including selected exogenous polyamino acid sequences (described in SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 45, SEQ ID NO: 51, SEQ ID NO: 64, SEQ ID NO: 75, SEQ ID NO: 88, and SEQ ID NO: 90). Container proteins can also be modified as necessary to develop their uses by inserting accessory elements. Sequences that aid in purification processes can also be added to container proteins, such as, but not limited to, polyhistidine tails, chitin binding proteins, maltose binding proteins, calmodulin binding proteins, strep-tags, and GST. Sequences for stabilization, such as thiorodixin, can also be incorporated into the container protein. Sequences that can facilitate any antibody detection process, such as V5, Myc, HA, Spot, and FLAG sequences, can also be added to the container protein.

[0083] Furthermore, for the purposes of the present invention, sequences that are at least about 85%, more preferably at least about 90%, 95%, 96%, 97%, 98% or 99% identical to the proteins and polyamino acid acceptors described herein, as determined by well-known sequence identity evaluation algorithms such as FASTA, BLAST or Gap, are included.

[0084] The sequence of the target cleavage site (catalytic site) for protease can also be added to the protein container, allowing the main protein to be separated from the above-mentioned auxiliary elements, including the purpose of strictly optimizing the production or purification of protein, but not contributing to the proposed end-use. These sequences comprising the site for the target of protease can be inserted anywhere, and include but are not limited to thrombin, factor Xa, enteropeptidase, PreScission, TEV (Kosobokova et al., Biochemistry, 8:187-200,2015). And the auxiliary sequence (accessorysequence) of another kind of marker protease can be AviTag, which allows specific biotinylation at a single point during or after protein expression. In this case, the protein (Wood, Current Opinion in Structural Biology, 26:54-61,2014) of the marker can be produced by combining different elements.

[0085] The isolated container proteins can be further modified in vitro for different uses.

[0086] polynucleotides

[0087] In a first embodiment, the present invention relates to a polynucleotide comprising any one of SEQ ID NOs: 2, 4, 78, 17, 19, 32, 34, 46, 52, 63, 76, 89, 91, 326-333 and degenerate sequences thereof, capable of producing a polypeptide defined by SEQ ID NOs: 1, 3, 77, 18, 20, 31, 33, 45, 51, 64, 75, 88, 90, 334-341, respectively.

[0088] The present invention also provides isolated container proteins produced by any expression system from a DNA molecule comprising regulatory elements comprising a nucleotide sequence encoding a selected container protein.

[0089] The DNA sequences encoding the container proteins differ from the DNA sequences of naturally occurring forms of GFP by deletion, addition, or substitution of amino acids with respect to the identity or position of one or more amino acid residues. However, they still retain some or all of the characteristics inherent to the naturally occurring form, such as, but not limited to, fluorescence production, characteristic three-dimensional shape, ability to be expressed in different systems, and ability to accept exogenous peptides.

[0090] The DNA sequence encoding the container protein of the present invention includes: incorporating preferred codons for expression by certain expression systems; inserting cleavage sites for restriction enzymes; inserting optimized sequences for facilitating the construction of expression vectors; inserting enhancer (facilitator) sequences to contain the selected polyamino acid sequence to be introduced into the container protein. All of these strategies are known in the art.

[0091] In addition, the present invention further provides genetic elements that, for example, have a nucleotide sequence encoding a container protein added to the sequence described in SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 78. Furthermore, elements are provided that comprise a nucleotide sequence encoding a container protein to which DNA encoding a selected exogenous polyamino acid sequence has been added. Such genetic elements include the sequences described in SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 46, SEQ ID NO: 52, SEQ ID NO: 63, SEQ ID NO: 76, SEQ ID NO: 89, and SEQ ID NO: 91.

[0092] Regulatory elements required for container protein expression include a promoter sequence for binding to RNA polymerase and a translation initiation sequence for binding to ribosomes. For example, bacterial expression vectors must include a start codon, promoter, and Shine-Dalgarno sequence appropriate for the cell system and translation initiation. Similarly, eukaryotic expression vectors include a promoter, a start codon, a downstream polyadenylation signal, and a stop codon. Such vectors can be commercially available or constructed from known prior art sequences.

[0093] carrier

[0094] In a first embodiment, the present invention relates to a vector comprising a polynucleotide as defined above.

[0095] Conversion from one plasmid to another can be achieved by altering the nucleotide sequence without modifying the amino acid sequence by adding or deleting restriction sites, which can be accomplished by nucleic acid amplification techniques.

[0096] Expression cassette

[0097] In a first embodiment, the present invention relates to an expression cassette comprising a polynucleotide as defined above.

[0098] Optimized expression in other systems can be achieved by altering the nucleotide sequence to add or delete restriction sites, and also optimizing codons for alignment with the preferred codons of the new expression system without changing the final amino acid sequence.

[0099] cell

[0100] In a first embodiment, the invention relates to a cell comprising a vector or an expression cassette as defined above.

[0101] The present invention further provides cells containing nucleotide sequences encoding container proteins, or container proteins derived from DNA encoding a selected exogenous polyamino acid sequence, to serve as expression systems for container proteins. The cells can be bacterial, fungal, yeast, insect, plant, or even animal cells. The DNA sequence encoding the container protein derived from DNA encoding the selected exogenous polyamino acid sequence can be inserted into a virus, which can be used for the expression and production of container proteins as a delivery system, such as, but not limited to, baculovirus, adenovirus, adeno-associated virus, alphavirus, herpes virus, poxvirus, retrovirus, or lentivirus.

[0102] There are various methods for introducing exogenous genetic material into cells, all of which are known in the art. For example, exogenous DNA material can be introduced into cells by calcium phosphate precipitation. Other techniques can be used in the development of the present invention, such as the use of electroporation, lipofection, microinjection, retroviral vectors, and other viral vector systems such as adeno-associated virus systems.

[0103] The present invention provides a living organism comprising a cell containing at least a DNA molecule comprising a regulatory element for the expression of a sequence encoding a container protein. The present invention can be used to produce container proteins in vertebrates, invertebrates, plants and microorganisms.

[0104] The expression of the container protein can be carried out in, but is not limited to, Escherichia coli cells, Bacillus subtilis, Saccharomyces cerevisiae, Pichia pastoris, Pichia methanolica, Candida boidinii, Pichia angusta, mammalian cells such as CHO cells, HEK293 cells, or insect cells such as Sf9 cells. All prokaryotic or eukaryotic protein expression systems known in the art can be used to produce the container protein.

[0105] In one development of the present invention, viruses or bacteriophages carrying coding sequences for container proteins can infect specific bacterial or eukaryotic cell types and provide for the expression of container proteins in these cell systems. Infection can be readily detected by detecting expression of the container protein. Similarly, eukaryotic plant or animal cell viruses carrying sequences encoding container proteins can infect specific cell types and result in the expression of container proteins in eukaryotic cell systems.

[0106] Method for producing protein container

[0107] In a first embodiment, the present invention relates to a method for producing a protein container, comprising introducing a polynucleotide as defined above into competent cells of interest; culturing the competent cells and isolating the protein container containing the selected exogenous polyamino acid. In another embodiment, the protein container is not disturbed by the insertion of various exogenous polyamino acid sequences.

[0108] Container proteins can also be produced by a variety of synthetic biological systems. The generation of fully synthetic genes is essentially related to three ligation-based synthesis systems widely described in the prior art.

[0109] The present invention provides a method for producing a container protein using a protein expression system comprising the following steps: introducing a DNA sequence encoding the container protein plus a DNA encoding a selected exogenous polyamino acid sequence into competent cells of interest, culturing the cells under conditions conducive to the production of the container protein containing the selected polyamino acid sequence, and isolating the container protein containing the selected exogenous polyamino acid sequence.

[0110] The present invention further provides a technique for producing container proteins containing selected exogenous polyamino acids. The present invention demonstrates an efficient method for expressing container proteins containing selected exogenous polyamino acids, which facilitates mass production of the protein of interest. The production method of container proteins can be performed in various cell systems, such as yeast, plants, plant cells, insect cells, mammalian cells, and transgenic animals. Each system can be utilized by integrating a codon-optimized nucleic acid sequence that produces the desired amino acid sequence into a plasmid suitable for a particular cell system. The plasmid can contain elements that confer numerous properties to the expression system, including, but not limited to, sequences that promote retention and replication, selectable markers, promoter sequences for transcription, sequences that stabilize the transcribed RNA, and ribosome binding sites.

[0111] Methods for isolating expressed proteins are known in the art, and in this regard, container proteins can be readily isolated by any technique. The presence of a polyhistidine tail allows purification of recombinant proteins after expression in bacterial systems (Hochuli et al. Bio / Technology 6:1321-25; Bornhorst and Falke, Methods Enzymology 326:245-54).

[0112] The present invention further contemplates the choice and selection of exogenous polyamino acids. The container protein can comprise different polyamino acid sequences from different sources, ranging from vertebrates including mammals, invertebrates, plants, microorganisms, or viruses, to facilitate their expression, display, or use in different media. Different polyamino acid sequence selection methods can be used, for example, by specific selection for binding affinity to antibodies or other binding proteins, or by epitope mapping, or other techniques known in the art.

[0113] A polyamino acid sequence is a sequence of 5 to 30 amino acids that sensitively or specifically represents an organism for any of the purposes described herein. Polyamino acid sequences can represent, but are not limited to, the following examples: (i) linear B-cell epitopes; (ii) T-cell epitopes; (iii) neutralizing epitopes; (iv) protein regions that are specific for pathogens or non-pathogen sources; (v) regions adjacent to the active site of an enzyme that is not normally the target of an immune response. These epitope regions can be identified by various methods, including but not limited to spot synthesis analysis, random peptide libraries, phage display, software analysis, use of X-ray crystallography data, epitope databases, or other state-of-the-art methods.

[0114] Inserting an exogenous polyamino acid sequence into a container protein at a site defined above surprisingly does not disrupt or interfere with the genetic properties of the protein. Eight sites for the introduction of exogenous polyamino acid sequences have been identified in container proteins (Kiss et al. Nucleic Acids Res 34:e132, 2006; Pavoor et al. Proc Natl Acad Sci USA 106:11895-900, 2009; Abedi et al. Nucleic Acids Res 26:623-30, 1998; Zhong et al. Biomol Eng 21:67-72, 2004).

[0115] These introduction sites can contain one, two, or more different exogenous polyamino acid sequences in tandem at the same insertion site, greatly expanding the expression of different polyamino acid sequences.

[0116] In a first embodiment, the present invention relates to a method for pathogen identification or in vitro disease diagnosis, characterized in that the method uses a container protein as defined above. In another embodiment, the method is used to diagnose Chagas disease, rabies, pertussis, yellow fever, Oropouche virus infection, Mayaro, IgE hypersensitivity, house dust mite (D. pteronyssinus) allergy, or COVID-19.

[0117] Uses of Protein Containers

[0118] In a first embodiment, the present invention relates to the use of the protein container as a laboratory reagent. In a further embodiment, the present invention relates to the use of the protein container for the production of a vaccine composition for immunization against Chagas disease, rabies, pertussis, yellow fever, Oropouche virus infection, Mayaro, IgE hypersensitivity, house dust mite allergy, or COVID-19.

[0119] Furthermore, the present invention relates to a system for the concomitant expression of multiple polyamino acid sequences using a single GFP-based protein container for various applications, such as as research reagents, diagnostics, or vaccine compositions. Specifically, the expression system can serve as a useful research reagent for purifying antibodies by binding to epitopes. Furthermore, the expression system can also serve as an immunological and / or molecular technique for diagnosing chronic and infectious diseases. Furthermore, the expression system can be advantageously used in vaccine compositions containing multiple antigens for animal and human immunization.

[0120] Furthermore, one embodiment of the present invention is a method for the concomitant production of multiple polyamino acid sequences using a single protein container based on GFP for different uses, such as as reagents for research, for diagnostics, or for vaccine compositions.

[0121] The present invention further discloses certain uses of container proteins. These uses include, but are not limited to, the use of protein containers as: (i) as reporter molecules in cell-based screening assays, including intracellular assays; (ii) as proteins for displaying random or selected peptide libraries; (iii) as antigen-presenting proteins, as reagents for developing in vitro immunological diagnostic tests, typically for infectious, parasitic, or other immunological diseases; (iv) as antigen-presenting proteins for selecting, capturing, screening, or purifying binding substances such as antibodies; (v) as antigen-presenting proteins for vaccine compositions; (vi) as proteins containing antibody sequences for binding to antigens; and (vii) as antigen-presenting proteins with passive immunization activity.

[0122] The container protein can be used as a vaccine composition by specifically having: (a) a large, simultaneous immune response inducing polyamino acid sequence, and (b) a non-immune response inducing core protein.

[0123] diagnostic kits

[0124] In a first embodiment, the present invention relates to a diagnostic kit comprising a container of a protein as defined above.

[0125] Finally, the present invention is described in detail by means of the examples given below. It should be emphasized that the present invention is not limited to these examples and that it also encompasses variations and modifications within the scope of possible development. It is also worth noting that the authorized use of all biological sequences of the Brazilian genetic heritage is registered with SISGEN under registration number AC53976.

[0126] Example

[0127] Example 1 -Container protein construction

[0128] The amino acid sequences of different examples of green fluorescent proteins, including eGFP (GenBank: L29345.1; UniProtKB-P42212), Cycle-3 (GenBank: CAH64883.1), SuperFolder (GenBank: AOH95453.1), Split (Cabantous et al., Science Reports 3: 2854, 2013), and Superfast (Fisher & DeLisa, PLoS One 3: e2351, 2008), were used to construct the novel proteins of the present invention. Sequence alignment and comparison were performed using Intaglio software (Purgatory Design, V3.9.4). From these data, certain changes were made to achieve the desired properties of the container protein.

[0129] The modifications are made to create restriction enzyme sites. The insertion of these sites is designed so that the physicochemical properties of the GFP protein are not altered and therefore the properties or qualities described in this patent application are not affected. Furthermore, the insertion of these restriction sites will allow genetic manipulation of these proteins by allowing potential use in genetic engineering methods and processes to incorporate various peptides into different regions of the protein, thereby adding further properties to the container protein.

[0130] The nucleotide sequence of the GFP protein was manipulated to introduce or replace nucleotides, thereby creating new restriction enzyme sites. This resulted in the production of two new container proteins, the "Platform" protein and the "Rx" protein.

[0131] Based on the changes in the nucleotide sequence of the eGFP protein, the following amino acid changes were caused in the container protein:

[0132] "Platform" proteins

[0133] Position 16, amino acid 1;

[0134] position 28, amino acid F;

[0135] Position 30, amino acid R;

[0136] Position 39, amino acid 1;

[0137] Position 43, amino acid S;

[0138] position 72, amino acid S;

[0139] position 99, amino acid Y;

[0140] position 105, amino acid T;

[0141] position 111, amino acid E;

[0142] position 124, amino acid V;

[0143] Position 128, amino acid 1;

[0144] position 145, amino acid F;

[0145] position 153, amino acid T;

[0146] position 163, amino acid A;

[0147] position 166, amino acid T;

[0148] position 167, amino acid V;

[0149] position 171, amino acid V;

[0150] position 205, amino acid T;

[0151] Position 206, amino acid 1;

[0152] Position 208, amino acid L.

[0153] "Rx" proteins

[0154] position 16, amino acid V;

[0155] position 28, amino acid S;

[0156] Position 30, amino acid R;

[0157] Position 39, amino acid 1;

[0158] Position 43, amino acid T;

[0159] position 72, amino acid A;

[0160] Position 99, amino acid S;

[0161] Position 105, amino acid K;

[0162] position 111, amino acid V;

[0163] position 124, amino acid V;

[0164] position 128, amino acid T;

[0165] position 145, amino acid F;

[0166] position 153, amino acid T;

[0167] position 163, amino acid A;

[0168] position 166, amino acid T;

[0169] position 167, amino acid V;

[0170] position 171, amino acid V;

[0171] position 205, amino acid T;

[0172] position 206, amino acid V;

[0173] Position 208, amino acid S.

[0174] For all container proteins, optional amino acid substitutions may still be selected at the following positions:

[0175] Position 39, amino acid N;

[0176] position 72, amino acid S;

[0177] Position 99, amino acid S;

[0178] Position 105, amino acid Y or K;

[0179] Position 206, amino acid 1;

[0180] Position 208, amino acid L.

[0181] The presence of some mutations can affect the biochemical properties of the protein. The S30R mutation positively affects the helical properties of the protein; the Y145F and I171V mutations prevent the translation of undesirable intermediates; and the A206V or I mutations reduce the possibility of aggregation of the nascent protein.

[0182] Other changes made to the container proteins "Platform" and "Rx," ranging from the inclusion of new nucleotide codons to the creation of new restriction sites, are shown in Table 1 (below).

[0183] Table 1

[0184] Amino acid sequence changes Substituted nucleotides Introduced nucleotides Restriction enzyme to be used D102_D103insV - GTC AatII G116_D117insT - ACC KpN L137_G138insK - AAG AfIII D191_P192insG - GGT RsrII E213_K214insL - CTC SacI

[0185] In addition, the nucleotide sequences of the container proteins "Platform" and "Rx" have two additional restriction sites for NdeI and NheI, located at the 5' amino terminus of the protein, resulting from the insertion of the sequence CATATGGTGGCTAGC (SEQ ID NO: 5), and two additional restriction sites for EcoRI and XhoI, located at the 3' carboxyl terminus, resulting from the insertion of the sequence GAATTCTAATGACTCGAG (SEQ ID NO: 6). In addition, two stop codons and a polyhistidine tail at the amino terminus have been incorporated into the container proteins.

[0186] The amino acid sequence of the "platform" protein can be shown in SEQ ID NO: 1 and its corresponding nucleotide sequence is described in SEQ ID NO:2.

[0187] The amino acid sequence of the "Rx" protein can be shown in SEQ ID NO:3 and its corresponding nucleotide sequence is described in SEQ ID NO:4.

[0188] By creating restriction sites without changing the three-dimensional structure of the original protein, 10 new insertion sites for foreign polyamino acid sequences are allowed to appear in the protein. Each new insertion site will be referred to herein as position 1 to position 10.

[0189] The locations of positions 1 to 10 of the container proteins "Platform" and "Rx" in the nucleotide and amino acid sequences are shown in Table 2 (below).

[0190] Table 2

[0191] Position in the protein container Position in the amino acid sequence 1 MVAS 2 TTGKLPVP 3 FKDVDG 4 FEGTDTL 5 TDFKEDGNILKGHKL 6 DKQKN 7 ED 8 PIGDGPVLLPDN 9 SKDPNELKRD 10 DELYKEF

[0192] From the comparison of the amino acid sequences of different GFP proteins, the consensus amino acid sequence was designated as CGP (Dai et al. Protein Engineering, Design and Selection 20(2):69-79 2007). Despite its high stability, this fluorescent protein was improved by directed evolution to exhibit better stability relative to CGP (Kiss et al. Protein Engineering, Design & Selection 22(5):313-23, 2009). However, due to the presence of three mutations, the enhanced protein is prone to aggregation. Based on the analysis of its crystal structure, other mutations were also incorporated, resulting in the elimination of aggregation and the production of a protein called Thermal Green Protein (TGP) (Close et al. Proteins 83(7):1225-37, 2015). When used as a protein container, the sequence is referred to as "Tx". The amino acid sequence of the "Tx" protein can be shown in SEQ ID NO:77, and the sequence in its corresponding nucleotides is described in SEQ ID NO:78.

[0193] The nucleotide and amino acid sequence positions for positions 1 through 13 of the "Tx" container protein are shown in Table 3 (below). Two polyamino acid sequences can be inserted consecutively at both ends of the container protein's amino and carboxyl termini. Thus, insertion sites 1a and 1b and 13a and 13b are characterized by being located in the amino and carboxyl terminal regions, respectively.

[0194] Table 3

[0195]

[0196]

[0197] Example 2 -Structure of PlatCruzi protein

[0198] The "platform" container protein is genetically engineered to possess a Trypanosoma cruzi epitope, which we refer to herein as the PlatCruzi platform. The gene corresponding to the PlatCruzi protein, referred to herein as the PlatCruzi gene, is described in nucleotide sequence SEQ ID NO: 17.

[0199] Taking into account experimental data on the specificity and sensitivity of diagnostic tests for Chagas disease, polyamino acid sequences derived from Trypanosoma cruzi were selected from the available prior art literature (Peralta JM et al. J Clin Microbiol 32:971-974, 1994; Houghton RL et al. J Infect Dis 179:1226-1234, 1999; Thomas et al. Clin Exp Immunol 123:465-471, 2001; Rabello et al., 1999; Gruber & Zingales, Exp Parasitology, 76(1):1-12, 1993; Lafaille et al., Molecular Biochemistry Parasitology, 35(2):127-36, 1989). Ten polyamino acid sequences, referred to herein as TcEp1 to TcEp10, were selected for insertion into the 10 insertion sites of the "platform" protein, as shown in Table 4 (below).

[0200] After selecting the Trypanosoma cruzi polyamino acid sequence, a synthetic gene corresponding to the PlatCruzi protein was generated by chemical synthesis and ligation gene synthesis methods and inserted into a plasmid for use in experiments. The amino acid sequence corresponding to the PlatCruzi gene encompassing epitopes TcEp1 to TcEp10 is described in SEQ ID NO: 18.

[0201] Table 4

[0202]

[0203] Example 3 - Development of PlatCruzi protein

[0204] The synthetic gene was introduced into the pET28a plasmid using restriction sites for enzymes NdeI and Xhol by molecular biology techniques of the prior art. To identify whether the synthetic gene matched the sequence designed for PlatCruzi, the DH5α strain of E. coli was transformed and the plasmid material was analyzed by restriction enzyme digestion techniques followed by sequencing.

[0205] The sequencing method used is an enzymatic method, a dideoxy method or a chain termination method, which is based on the enzymatic synthesis of complementary chains, and the growth of the complementary chain is stopped by adding dideoxynucleotides (Sanger et al., Proceeding National Academy of Science, 74 (12): 5463-5467, 1977). The method consists of the following steps: sequencing reaction (DNA replication in 25 cycles in a thermal cycler), DNA precipitation by isopropanol / ethanol, denaturation of the double strand (95°C, 2 minutes) and reading of the nucleotide sequence in an ABI 3730XL automatic sequencer (ThermoFischer SCIENTIFIC) (Otto et al., Genetics and Molecular Research 7: 861-871, 2008). The analysis of the obtained sequence is completed with the help of the 4Peaks program (Nucleobytes; Mac OS X, 2004). Primers from the pET-28a vector (T7 promoter and T7 terminator) are used for the reaction.

[0206] The plasmid clone with the correct sequence of PlatCruzi analyzed was transferred to E. coli strain BL21 to produce PlatCruzi protein. When induced by isopropyl β-D-1-thiogalactopyranoside (IPTG), BL21 strain can express T7 RNA polymerase. The strain was grown overnight in LB medium and then inoculated in the same medium supplemented with kanamycin (30 μg / ml) on a shaking table at 200 rpm until it reached an optical density of turbidity of 0.6-0.8 (600 nm). Then, IPTG (qsp1mM) was added to the culture and the same culture conditions were maintained for another 3 hours.

[0207] The culture is centrifuged and the precipitation is resuspended in urea buffer (100mM NaH PO , 10mM Tris-base, 8M urea, pH 8.0). The solution is passed through a HisTrap™ affinity column, 1mL, GE Healthcare Life Sciences, and chromatography is performed, which allows the high-resolution purification of histidine-tagged proteins. Supernatant is applied to a nickel affinity column (HisTrap™, 1mL, GE Healthcare Life Sciences) at a flow rate of 0.5mL per minute, and the post is previously balanced in buffer A (50mM Tris-HCl, pH 8.0, 100mM NaCl, and 5mM imidazoles). After binding, the resin is washed with 10mL of buffer A. Protein is eluted 45 minutes in a buffer B (50mM Tris-HCl, pH 8.0, 100mM NaCl, and 500mM imidazoles) at a flow rate of 0.7mL / minute in 100% gradient. PlatCruzi was then purified (black line) and shown at 280 nm. Figure 1 A. The percentage of imidazole is marked in red. The protein was eluted in a volume of approximately 19 to 25 ml.

[0208] Recombinant protein samples (1 μg / well) were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, Nature 227:680-685, 1970). Stacking gels and running gels were prepared at 4% and 11% acrylamide concentrations, respectively (Table 5, below). Samples were prepared under denaturing conditions in 62.5 mM Tris-HCl buffer, pH 6.8, 2% SDS, 5% β-mercaptoethanol, 10% glycerol and boiled at 95°C for 5 minutes (Hames BD, Gel electrophoresis of proteins: a practical approach. 3. ed. Oxford. 1998). After electrophoresis, proteins were detected by staining with Coomassie Brilliant Blue R250 (Bio-Rad, USA). Kaleidoscope™ Prestained Standards markers were used as molecular weight references (Bio-Rad, USA). Figure 1 B, showing the use of Purification of PlatCruzi protein by affinity chromatography using a nickel-agarose column on a liquid chromatography system.

[0209] Table 5: Volumes and concentrations of reagents used to prepare 4% sample stacking gel and 11% separating gel

[0210]

[0211] Example 4 - Development of ELISA from PlatCruzi

[0212] The performance of the PlatCruzi protein was evaluated against a panel of reference biological samples and individuals infected with Trypanosoma cruzi. PlatCruzi protein in carbonate / bicarbonate buffer (50 mM, pH 9.6) was added to a 96-well ELISA plate at 0.1, 0.25, 0.5, and 1.0 μg / well and incubated at 4°C for 12-18 hours. The wells were washed with a saline-phosphate buffered saline (PBS) solution supplemented with Tween 20 (PBS-T, 10 mM sodium phosphate-Na3PO4, 150 mM sodium chloride-NaCl, and 0.05% Tween-20, pH 7.4), and then incubated at 37°C for 2 hours with 1x PBS buffer containing 5% (weight / volume) dehydrated skim milk.

[0213] The wells were then washed three times with PBS-T buffer and incubated at 37°C for 1 hour with 2, 4, 8, 16, 32, and 64-fold diluted reference biological samples TC1 (IS 09 / 188) or TCII (IS 09 / 186) (World Health Organization). After incubation, the wells were washed three times with PBS-T and incubated at 37°C for 1 hour with alkaline phosphatase-labeled human IgG antibodies diluted 1:5000. The wells were washed three times with PBS-T and the substrate p-nitrophenyl phosphate (PNPP, 1 mg / mL, ThermoFischer SCIENTIFIC) was added. After 30 minutes in the dark, the absorbance at 405 nm was measured in an ELISA plate reader.

[0214] Regardless of the amount of PlatCruzi used, the results showed satisfactory responses in all dilutions of the TCI and TCII reference biological samples used ( Figure 2 The results strongly support the use of PlatCruzi for identifying infections caused by any of the six DTUs (discrete typing units or different typing units), covering the complete geographic range of circulating T. cruzi strains. The same behavior was observed when sera from Chagas disease patients with low or high antibody test titers were used ( Figure 3 ).

[0215] An Elisa plate containing 500 ng of PlatCruzi (in 0.3 M urea, pH 8.0) was prepared as described above, and after washing three times with PBS-T, sera from four patients with high anti-T. cruzi antibody indices (6C-CE, 9C-CE, 15C-CE, and 12-SE) and from four patients with low anti-T. cruzi antibody indices (3C-PB, 6C-PB, 16C-PB, and 17C-PB) were used at different dilutions of 1:50, 1:100, 1:250, 1:500, and 1:1000, and the Elisa plate was incubated at 37°C for 1 hour. Thereafter, the wells were washed three times with PBS-T and incubated with alkaline phosphatase-labeled human IgG antibody diluted 1:5000 for 1 hour. The wells were washed three times with PBS-T buffer, and the substrate p-nitrophenyl phosphate (PNPP, 1 mg / mL, ThermoFischerSCIENTIFIC) was added. After 30 minutes, the absorbance at 405 nm was measured in an ELISA plate reader.

[0216] The results showed that for sera with low antibody titers, the reading signals at 1:50, 1:100 and 1:250 dilutions were clearly above the threshold reached by the negative control, demonstrating the potential of the PlatCruzi platform for detecting anti-T. cruzi antibodies in both high and low antibody patient sera.

[0217] The use of patient serum samples for experimental purposes was approved by the Ethics Committee of Fiocruz under authorization letter CEP / IOC-CAAE: 52892216.8.0000.5248.

[0218] Example 5 -Sensitivity and specificity of PlatCruzi ELISA

[0219] 71 sera from patients diagnosed with Trypanosoma cruzi, plus 18 sera from patients diagnosed with leishmaniasis (negative for Trypanosoma cruzi), 20 sera from patients diagnosed with dengue fever (negative for Trypanosoma cruzi), and 39 negative sera (other infectious diseases and uninfected individuals) were used to incubate an Elisa plate developed in the above example containing 500 ng of PlatCruzi (0.3 M urea, pH 8.0) at a dilution of 1:250 at 37°C for 1 hour. The plate was then washed and labeled with antibodies, and the color development and reading process were performed as described above.

[0220] The results from the receiver operating characteristic (ROC) curve analysis indicated that the PlatCruzi platform had excellent sensitivity and specificity ( Figure 4 No false negative results were observed for sera previously identified as positive for Trypanosoma cruzi, and no false positives were observed for other sera known to be negative for Trypanosoma cruzi, including sera positive for other infectious diseases. Both the sensitivity and specificity indices were 100%.

[0221] Example 6 -Development of RxRabies2 protein

[0222] "Rx" proteins are tested for their performance and ability to express epitopes from other microorganisms, including viruses. The literature indicates that a large number of specific polyamino acid sequences can serve as targets for neutralizing antibodies. However, small variations in sequence observed between viral strains can interfere with neutralization. Therefore, a thorough investigation of the optimal polyamino acid sequences requires extensive knowledge of the biology of the virus and the epidemiology of its interactions with its host.

[0223] Considering the experimental data on specificity and sensitivity for diagnosing diseases caused by rabies virus, rabies virus polyamino acid sequences were selected from available prior art literature (Kuzmina et al., J Antivir Antiretrovir 5:2:37-43, 2013; Cai et al., Microbes Infect 12:948-955, 2010).

[0224] Ten polyamino acid sequences, referred to herein as RaEp1 to RaEp10, were selected for insertion into ten insertion sites within the Rx protein, as described below. Combining these polyamino acid sequences with the sequence of the Rx protein yields the RxRabies2 protein. The gene corresponding to the RxRabies2 protein, referred to herein as the RxRabies2 gene, is described in SEQ ID NO: 19. The amino acid sequence corresponding to the RxRabies2 gene comprising the polyamino acid sequences RaEp1 to RaEp10 is described in SEQ ID NO: 20.

[0225] Table 6

[0226] Polyamino acid sequence Location in protein Original epitope protein sequence SEQ ID no. RaEp 1 1 Antigenic site 1 CKLKLCGVLGL SEQ ID no. 21 RaEp 2 2 Antigenic site 1 CKLKLCGCSGL SEQ ID no. 22 RaEp 3 3 Antigenic site 1 CKLKLCGVPGL SEQ ID no. 23 RaEp 4 4 - VDERGLYK SEQ ID no. 24 RaEp 5 5 - WVAMQTSN SEQ ID no. 25 RaEp 6 6 Antigenic site III KSVRTWNEI SEQ ID no. 26 RaEp 8 8 g5 antigenic site LHDFHSD SEQ ID no. 27 RaEp 9 9 g5 antigenic site LHDFRSD SEQ ID no. 28 RaEp 10 10 g5 antigenic site LHDLHSD SEQ ID no. 29

[0227] A synthetic gene comprising a sequence encoding the Rx protein and a sequence encoding the polyamino acid sequence described in Table 6 above has been synthesized.

[0228] The synthetic gene was introduced into the pET28a plasmid using restriction sites for the enzymes NdeI and Xhol by state-of-the-art molecular biology techniques. To identify whether the synthetic gene matched the sequence designed for RxRabies2, the DH5α strain of E. coli was transformed and the plasmid material was analyzed by restriction enzyme digestion followed by sequencing, in the same manner as described in PlatCruzi.

[0229] The analyzed plasmid clone with the correct sequence of RxRabies2 was transferred to the E. coli strain BL21 to produce the RxRabies2 protein. When induced by isopropyl β-D-1-thiogalactopyranoside (IPTG), the BL21 strain can express T7 RNA polymerase. The BL21 strain was grown overnight in LB medium and then inoculated in the same medium with kanamycin (30 μg / ml) on a shaker at 200 rpm until it reached an optical density of turbidity of 0.6-0.8 (600 nm). Then, IPTG (qsp1 mM) was added to the culture and the same culture conditions were maintained at 37°C for another 3 hours.

[0230] The culture was centrifuged and the pellet was resuspended in urea buffer (100 mM NaH PO , 10 mM Tris-base, 8 M urea, pH 8.0). The solution was chromatographed over a HisTrap™ affinity column (1 mL, GE Healthcare Life Sciences) at a flow rate of 0.5 mL per minute. This allowed high-resolution purification of histidine-tagged proteins, which were previously balanced in buffer A (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 5 mM imidazole). After binding, the resin was washed with 10 mL of buffer A. Protein was eluted 45 minutes at a flow rate of 0.7 mL / minute in a 100% gradient of buffer B (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 500 mM imidazole).

[0231] RxRabies2 production was analyzed in three different culture volumes: 3, 25 and 50 ml. As shown in Table 7 (below), the expression of RxRabies2 was an average of 123 μg / ml.

[0232] Table 7

[0233]

[0234] Example 7 -RxRabies2 protein as a vaccine composition

[0235] RxRabies2 protein was produced as described in the previous examples. 100 μg of protein was suspended in Freud's incomplete adjuvant (0.5 mL) and inoculated intramuscularly into the quadriceps of two 6-month-old male New Zealand rabbits. Seven and 14 days after the initial inoculation, the rabbits were re-inoculated with RxRabies protein (100 μg / 0.5 mL) suspended in PBS.

[0236] 21 days after the first dose of the vaccine composition, blood was collected from the animals. Plasma was collected by centrifugation and affinity purified by binding to Rx-Rabies2 protein adsorbed to a nitrocellulose membrane.

[0237] A nitrocellulose membrane containing Rx-Rabies2 protein was prepared as described below to separate the Rx-Rabies2 protein from potential contaminants. After electrophoresis, the protein was transferred to the nitrocellulose membrane using state of the art immunoblotting techniques.

[0238] 11% SDS-PAGE gels were prepared as described in the above examples and Table 5;

[0239] 10 μg of Rx-Rabies2 protein was applied to an 11% SDS-PAGE gel (Table 5) and subjected to an electrophoretic current of 100 volts for approximately 2 hours;

[0240] Transfer of proteins to nitrocellulose membranes: Proteins were transferred to nitrocellulose membranes using a Trans-Blot Cell (Bio-Rad, USA) with transfer buffer (25 mM Tris base, 192 mM glycine, and 20% methanol) at 100 V for 1 h.

[0241] The presence of the recombinant protein was confirmed by staining with Ponceau S red (Ponceau S 0.1%, acetic acid 5%).

[0242] Cut the membrane so as to specifically obtain a piece having only the RxRabies2 protein;

[0243] The membranes were then destained in distilled water and placed in TBS (0.1%) for 12 to 18 h (overnight);

[0244] The membranes were incubated overnight with blocking solution (25 mM Tris-HCl, 125 mM NaCl pH 7.4 (TBS) containing 0.05% (v / v) Tween 20 (TBS-T) and 5% (w / v) nonfat dry milk) and then incubated again in blocking solution for 1 hour. The membranes were then washed three times with TBS-T for 5 minutes each and three more times with TBS for 5 minutes each.

[0245] The serum from the immunized rabbit was diluted in TBS at 1:500, and then 10 ml was placed in contact with a nitrocellulose membrane fragment containing RxRabies2 protein for 1 hour under stirring, and then washed 3 times with TBS-T for 5 minutes each time, and again washed 3 times with TBS for 5 minutes each time. The specifically bound antibody was then released by adding 1 ml of 100 mM glycine (pH 3.0). The pH of the solution was raised to 7 to purify the rabbit antibody by adding 100 μl of 1 M Tris (pH 9.0). The purification of antibodies that specifically bind to antigens is an important step in using these antibodies for therapy because it allows a significant reduction in the amount to be administered while minimizing potential adverse reactions.

[0246] Different extracts were used to demonstrate the specific ability of the generated rabbit antibodies to bind to RxRabies2. The following were used:

[0247] Crude bacterial extracts with Rx protein expression were obtained using the same conditions as mentioned in PlatCruzi;

[0248] RxRAbies2 protein in crude bacterial extracts and purified at 1x and 0.5x concentrations;

[0249] Purified PlatCruzi protein at 1x and 0.5x concentrations.

[0250] Potential ligands were subjected to polyacrylamide gel electrophoresis (11% SDS-PAGE, Table 5) as described above, and then transferred to nitrocellulose membranes and immunoblotted, the details of which were described for RxRabies2.

[0251] The membrane was incubated with the purified anti-RxRabies2 serum as described above for 1 hour under stirring. Then, it was washed 3 times with TBS-T for 5 minutes each and 3 times with TBS for 5 minutes each. Thereafter, the membrane was incubated with peroxidase and anti-rabbit IgG secondary antibody diluted 1:10,000 for 1 hour. After incubation with the secondary antibody, it was washed 3 times with TBS-T for 5 minutes each and 3 times with TBS for 5 minutes each. With the help of SigmaFast TM DAB Peroxidase Substrate Tablet was used for color development.

[0252] The results showed that rabbit antibodies generated by vaccination with RxRabies2 specifically bound to ligands containing rabies virus 2 protein. Figure 5It is shown that bands were read only in lanes 2, 4, and 6, which contain crude bacterial extracts of RxRabies2 protein, and purified and diluted RxRabies2 protein at 1x and 0.5x concentrations, respectively.

[0253] The specificity of the immune response can also be observed by the absence of bands in lanes 3, 5, and 7, containing (i) Rx container protein without epitope introduction and (ii) 1x and 0.5x diluted Platcruzi platform, respectively. These results confirm that the immune response is limited to rabies virus epitopes, indicating that the Rx protein itself is not immunogenic.

[0254] Example 8 -Development of RxHolgG3 protein

[0255] From mapping studies of the polyamino acid sequences of horse immunoglobulins (De-Simone et al., Toxicon 78:83-93, 2014; Wagner et al., Journal of Immunology 173:3230-3242, 2004), the polyamino acid sequence of horse IgG3 recognized by human IgG and IgE was identified, which is suitable for laboratory testing to diagnose horse serum allergy.

[0256] The polyamino acid sequence DVLFTWYVDGTEV (SEQ ID NO: 30) was incorporated into the Rx protein at positions 1, 5, 6, 8, 9, and 10 to produce the RxHolgG3 protein. The amino acid sequence of the RxHolgG3 protein is set forth in SEQ ID NO: 31. The nucleotide sequence of the RxHolgG3 protein is set forth in SEQ ID NO: 32. A synthetic gene comprising a sequence encoding the Rx protein and a sequence encoding the polyamino acid sequence (SEQ ID NO: 30) described above was synthesized.

[0257] The synthetic gene was introduced into the pET28a plasmid using restriction sites for the enzymes NdeI and Xhol by state-of-the-art molecular biology techniques. To identify whether the synthetic gene matched the sequence designed for the RxHoIgG3 protein, the DH5α strain of E. coli was transformed and the plasmid material was analyzed by restriction enzyme digestion followed by sequencing, as described in detail in PlatCruzi et al.

[0258] The plasmid clone with the correct sequence of RxHoIgG3 analyzed was transferred to E. coli strain BL21 to produce RxHoIgG3 protein. When induced by isopropyl β-D-1-thiogalactopyranoside (IPTG), the BL21 strain can express T7 RNA polymerase. The BL21 strain was grown overnight in LB medium and then inoculated in the same medium with kanamycin (30 μg / ml) on a shaker at 200 rpm until it reached an optical density of turbidity of 0.6-0.8 (600 nm). Then, IPTG (qsp1mM) was added to the culture and the same culture conditions were maintained for another 3 hours.

[0259] The culture was centrifuged and the pellet was resuspended in urea buffer (100 mM NaH PO , 10 mM Tris-base, 8 M urea, pH 8.0). The solution was chromatographed using a nickel affinity column (HisTrap™, 1 mL, GE Healthcare Life Sciences) with a flow rate of 0.5 mL per minute, which had previously been balanced in buffer A (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 5 mM imidazole). After binding, the resin was washed with 10 mL of buffer A. The protein was eluted for 45 minutes in a 100% gradient of buffer B (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 500 mM imidazole) at a flow rate of 0.7 mL / min. The production of RxHolgG3 protein was confirmed in the eluate after 11% SDS-PAGE electrophoresis (Table 5). The results are shown in Table 5. Figure 6 , and showed the expression of RxHolgG3 as a recombinant protein. In the uninduced soluble bacterial extract ( Figure 6 , column 1), no band was detected, and a band was detected in each of the insoluble (column 2) and soluble (column 3) fractions.

[0260] Example 9 -Development of RxOro proteins

[0261] From polyamino acid sequence mapping studies of oropouche virus (strains Q71MJ4 and Q9J945, Uniprot) (Acrani et al., Journal of General Virology 96:513-523, 2014 Tilston-Lunel et al., Journal of General Virology 96(Pt 7):1636-1650, 2015), we selected polyamino acid sequences from spot synthesis or peptide microarray technology available in the prior art, considering their diagnostic potential. Six polyamino acid sequences, referred to herein as OrEp1 to OrEp7, were selected for insertion into the nine insertion sites of the Rx protein, as shown in Table 8 below:

[0262] Table 8

[0263]

[0264] The combination of these polyamino acid sequences with the Rx protein produces the RxOro protein. The amino acid sequence corresponding to the RxOro gene comprising the polyamino acid sequences OrEp1 to OrEp6 is described in SEQ ID no. 33. The gene corresponding to the RxOro protein, referred to herein as the RxOro gene, is described in nucleotide sequence SEQ ID no. 34.

[0265] The synthetic RxOro gene was introduced into the pET28a plasmid using restriction sites for the enzymes NdeI and Xhol by state of the art molecular biology techniques. To identify whether the synthetic gene matched the sequence designed for RxOro, a DH5α strain of E. coli was transformed and the plasmid material was analyzed by restriction enzyme digestion followed by sequencing techniques, as described in PlatCruzi.

[0266] The analyzed plasmid with the correct sequence for the RxOro protein was transferred to the E. coli strain BL21. When induced by isopropyl β-D-1-thiogalactopyranoside (IPTG), the BL21 strain can express T7 RNA polymerase. The BL21 strain was grown overnight in LB medium and then inoculated in the same medium with kanamycin (30 μg / ml) on a shaker at 200 rpm until it reached an optical density of 0.6-0.8 (600 nm) of turbidity. IPTG (1 mM) was then added to the culture and the same culture conditions were maintained for another 3 hours.

[0267] The culture was centrifuged and the pellet was resuspended in urea buffer (100 mM NaH2PO4, 10 mM Tris-base, 8 M urea, pH 8.0). The solution was chromatographed using a nickel affinity column (HisTrap™, 1 mL, GE Healthcare Life Sciences) with a flow rate of 0.5 mL per minute, which had previously been balanced in buffer A (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 5 mM imidazole). After binding, the resin was washed with 10 mL of buffer A. The protein was eluted in a 100% gradient of buffer B (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 500 mM imidazole) at a flow rate of 0.7 mL / min for 45 minutes. RxOro production was tested in three different bacterial growth volumes: 3, 25, and 50 ml. The expression level of RxOro was an average of 203 μg / ml, and the results are shown in Table 9.

[0268] Table 9

[0269]

[0270] Example 10 - Development of ELISA from RxOro

[0271] The performance of RxOro protein was evaluated against sera from individuals infected with Oropouche virus. RxOro protein in a solution (0.3 M urea, pH 8.0) was added to a 96-well ELISA plate at 500 ng / well for 12-18 hours at 4°C. The wells were washed with saline-phosphate buffered saline (PBS) supplemented with Tween 20 (PBS-T, 10 mM sodium phosphate-Na3PO4, 150 mM sodium chloride-NaCl and 0.05% Tween-20, pH 7.4) and then incubated at 37°C for 2 hours with 1x PBS buffer containing 5% (weight / volume) skim milk powder.

[0272] The wells were then washed three times with PBS-T buffer and incubated at 37°C for 1 hour with 98 serum samples from patients suspected of Oropouche virus infection and 51 serum samples from healthy patients diluted 1:100. After incubation, the wells were washed three times with PBS-T and then incubated at 37°C for 1 hour with alkaline phosphatase-labeled human IgG antibody diluted 1:5000. The wells were washed three times with PBS-T buffer again and p-nitrophenyl phosphate (PNPP, 1 mg / mL, ThermoFischerSCIENTIFIC) was added. After 30 minutes in the dark, the absorbance at 405 nm was measured in an ELISA plate reader.

[0273] The results indicated excellent sensitivity and specificity using RxOro ( Figure 7 The results strongly support the use of RxOro for the detection of Oropouche virus infection.

[0274] The use of patient serum samples for experimental purposes was approved by the Ethics Committee of Fiocruz under authorization letter CEP / IOC-CAAE: 52892216.8.0000.5248.

[0275] Example 11 -Development of RxMayaro_IgG protein

[0276] Based on the polyamino acid sequence mapping study of Mayaro virus (strains Q8QZ73 and Q8QZ72, Uniprot) (Espósito et al., Genome Announcement 3:e01372-15, 2015), we selected polyamino acid sequences from the available prior art literature, considering their diagnostic potential. Four polyamino acid sequences, referred to herein as MGEp1 to MGEp4, were selected for insertion into the nine insertion sites of the Rx protein, as shown in Table 10 below:

[0277] Table 10

[0278] Polyamino acid sequence Location in protein Original epitope protein sequence SEQ ID no. MGEp 1 1 nsP2 KLSATDWSAI SEQ ID no.41 MGEp 2 3 Capsid KPKPQPEK SEQ ID no.42 MGEp 3 4 nsP1 KKMTPSDQI SEQ ID no.43 MGEp 4 5 nsP3 VELPWPLETI SEQ ID no.44 MGEp 2 6 Capsid KPKPQPEK SEQ ID no.42 MGEp 1 7 nsP2 KLSATDWSAI SEQ ID no.41 MGEp 4 8 nsP3 VELPWPLETI SEQ ID no.44 MGEp 3 9 nsP1 KKMTPSDQI SEQ ID no.43 MGEp 1 10 nsP2 KLSATDWSAI SEQ ID no.41

[0279] The combination of these polyamino acid sequences with the sequence of the Rx protein produces the RxMayaro_IgG protein. The amino acid sequence corresponding to the RxMayaro_IgG gene comprising the polyamino acid sequences MGEp1 to MGEp4 is described in SEQ ID no. 45. The gene corresponding to the RxMayaro_IgG protein, referred to herein as the RxMayaro_IgG gene, is described in nucleotide sequence SEQ ID no. 46.

[0280] The synthetic RxMayaro_IgG gene was introduced into the pET28a plasmid using restriction sites for the enzymes NdeI and Xhol by molecular biology techniques known in the art. To identify whether the synthetic gene matched the sequence designed for RxMayaro_IgG, the DH5α strain of E. coli was transformed and the plasmid material was analyzed by restriction enzyme digestion followed by sequencing, as cited in PlatCruzi.

[0281] The plasmid with the correct sequence of RxMayaro_IgG protein analyzed is transferred to Escherichia coli strain BL21.When induced by isopropyl β-D-1-thiogalactopyranoside (IPTG), BL21 bacterial strain can express T7 RNA polymerase.BL21 bacterial strain is grown overnight in LB substratum, and then inoculated in the same substratum with kanamycin (30 μ g / ml), on the shaking table under 200rpm until it reaches the optical density of the turbidity of 0.6-0.8 (600nm).Then, IPTG (qsp1mM) is added to culture and maintains the other 3 hours of identical culture conditions.

[0282] The culture is centrifuged and the precipitation is resuspended in urea buffer (100mM NaH PO , 10mM Tris-base, 8M urea, pH 8.0). On nickel affinity column (HisTrap™, 1mL, GE Healthcare Life Sciences), solution is chromatographed at a flow rate of 0.5mL per minute, and the post is previously balanced in buffer A (50mM Tris-HCl, pH 8.0, 100mM NaCl and 5mM imidazoles). After binding, the resin is washed with 10mL of buffer A. Protein is eluted 45 minutes in buffer B (50mM Tris-HCl, pH 8.0, 100mM NaCl and 500mM imidazoles) of 100% gradient at a flow rate of 0.7mL / minute. The production of RxMayaro_IgG protein can be confirmed in the eluent from carrying out 11% SDS-PAGE electrophoresis (table 5). The RxMayaro_IgG protein was also examined by SDS-PAGE to confirm its production and determine its distribution between the soluble and insoluble fractions. Figure 8 As shown in , columns 4 and 7 (arrows) show that RxMayaro_IgG was produced as soluble and insoluble.

[0283] RxMayaro_IgG production was tested in 3 different growth volumes: 3, 25 and 50 ml. As shown in Table 11 (below), the expression level of RxMayaro_IgG was an average of 130 μg / ml.

[0284] Table 11

[0285]

[0286] Example 12 - Development of ELISA from RxMayaro_IgG

[0287] The performance of RxMayaro_IgG protein was evaluated for a group of serum samples from individuals affected by Mayaro virus infection. RxMayaro_IgG protein in a solution (0.3 M urea, pH 8.0) was added to a 96-well ELISA plate at 500 ng / well for 12-18 hours at 4°C. The wells were washed with saline-phosphate buffered saline (PBS) (PBS-T, 10 mM sodium phosphate-Na PO , 150 mM sodium chloride-NaCl and 0.05% Tween-20, pH 7.4) added with Tween 20, and then incubated at 37°C for 2 hours with 1x PBS buffer containing 5% (w / v) skim milk powder.

[0288] The wells were then washed with PBS-T buffer and incubated at 37°C for 1 hour with 6 samples of serum from patients suspected of Mayaro virus infection and 29 samples of serum from healthy patients diluted 1:100. After incubation, the wells were washed 3 times with PBS-T and then incubated at 37°C for 1 hour with alkaline phosphatase-labeled human IgG antibodies diluted 1:5000. The wells were washed again 3 times with PBS-T buffer and p-nitrophenyl phosphate (PNPP, 1 mg / mL, ThermoFischerSCIENTIFIC) was added. After 30 minutes in the dark, the absorbance at 405 nm was measured in an ELISA plate reader.

[0289] The results indicated excellent sensitivity and specificity using RxMayaro_IgG ( Figure 9 The results strongly support the use of RxMayaro_IgG for the detection of Mayaro virus infection.

[0290] The use of patient serum samples for experimental purposes was approved by the Ethics Committee of Fiocruz under authorization letter CEP / IOC-CAAE: 52892216.8.0000.5248.

[0291] Example 13 -Development of RxMayaro_IgM protein

[0292] Based on a mapping study of the polyamino acid sequences of Mayaro virus (strains Q8QZ73 and Q8QZ72, Uniprot) (Espósito et al., Genome Announcement 3:e01372-15, 2015), we selected polyamino acid sequences from the available prior art literature, considering their diagnostic potential. Four polyamino acid sequences, referred to herein as MMEp1 to MMEp4, were selected for insertion into the nine insertion sites of the Rx protein, as shown in Table 12 (below):

[0293] Table 12

[0294]

[0295] The combination of these polyamino acid sequences with the sequence of the Rx protein produces the RxMayaro_IgM protein. The amino acid sequence corresponding to the RxMayaro_IgM gene comprising the polyamino acid sequences MMEp1 to MMEp4 is described in SEQ ID no. 51. The gene corresponding to the RxMayaro_IgM protein, referred to herein as the RxMayaro_IgM gene, is described in nucleotide sequence SEQ ID no. 52.

[0296] The synthetic RxMayaro_IgM gene was introduced into the pET28a plasmid using restriction sites for the enzymes NdeI and Xhol by molecular biology techniques known in the art. To identify whether the synthetic gene matched the sequence designed for RxMayaro_IgM, the DH5α strain of E. coli was transformed and the plasmid material was analyzed by restriction enzyme digestion followed by sequencing, as previously described in PlatCruzi.

[0297] The plasmid with the correct sequence of RxMayaro_IgM protein analyzed was transferred to Escherichia coli strain BL21. When induced by isopropyl β-D-1-thiogalactopyranoside (IPTG), BL21 bacterial strain can express T7 RNA polymerase. BL21 bacterial strain was grown overnight in LB medium and then inoculated in the same medium with kanamycin (30 μg / ml), on a shaking table under 200 rpm until it reaches an optical density of turbidity of 0.6-0.8 (600 nm). Then, IPTG (qsp1mM) was added to the culture and maintained for another 3 hours under the same culture conditions.

[0298] The culture is centrifuged and the precipitation is resuspended in urea buffer (100mM NaH2PO4, 10mM Tris-base, 8M urea, pH 8.0). With a flow rate of 0.5mL per minute, the solution is chromatographed on a nickel affinity column (HisTrap™, 1mL, GE Healthcare Life Sciences), which was previously balanced in buffer A (50mM Tris-HCl, pH 8.0, 100mM NaCl, and 5mM imidazole). After binding, the resin is washed with 10mL of buffer A. Protein is eluted 45 minutes in a 100% gradient of buffer B (50mM Tris-HCl, pH 8.0, 100mM NaCl, and 500mM imidazole) at a flow rate of 0.7mL / minute.

[0299] The production of RxMayaro_IgM protein can be confirmed in the eluate by performing SDS-PAGE electrophoresis, and its distribution between the soluble and insoluble fractions can be observed. Figure 8 As shown in FIG, columns 5 and 8 (arrows) show that RxMayaro_IgM was produced as soluble and insoluble.

[0300] RxMayaro_IgM production was also examined in three different growth volumes: 3, 25 and 50 ml. As shown in Table 13 (below), the expression level of RxMayaro_IgM was an average of 205 μg / ml.

[0301] Table 13

[0302]

[0303] Example 14 - Development of ELISA based on RxMayaro_IgM

[0304] The performance of RxMayaro_IgM protein was evaluated for a group of serum from individuals affected by Mayaro virus infection. The RxMayaro_IgM protein in a solution (0.3M urea, pH 8.0) was added to a 96-well ELISA plate at 4°C for 12-18 hours at 500 ng / well. The wells were washed with saline-phosphate buffered saline (PBS) (PBS-T, 10 mM sodium phosphate-Na PO , 150 mM sodium chloride-NaCl and 0.05% Tween-20, pH 7.4) to which Tween 20 was added, and then incubated at 37°C for 2 hours with 1x PBS buffer containing 5% (w / v) skim milk powder.

[0305] The wells were then washed three times with PBS-T buffer and incubated at 37°C for 1 hour with 6 serum samples from patients suspected of Mayaro virus infection and 29 serum samples from healthy patients diluted 1:100. After incubation, the wells were washed three times with PBS-T and then incubated at 37°C for 1 hour with alkaline phosphatase-labeled human IgM antibodies diluted 1:5000. The wells were washed three times with PBS-T buffer again and p-nitrophenyl phosphate (PNPP, 1 mg / mL, ThermoFischerSCIENTIFIC) was added. After 30 minutes in the dark, the absorbance at 405 nm was measured in an ELISA plate reader.

[0306] The results indicated excellent sensitivity and specificity using RxMayaro_IgM ( Figure 10 The results strongly support the use of RxMayaro_IgM for the detection of Mayaro virus infection.

[0307] The use of patient serum samples for experimental purposes was approved by the Ethics Committee of Fiocruz under authorization letter CEP / IOC-CAAE: 52892216.8.0000.5248.

[0308] Example 15 -Protein RxPtx Development

[0309] Polyamino acid sequences were selected from available prior art literature based on their diagnostic potential from mapping studies of polyamino acid sequences of the bacterial toxin proteins of Bordetella pertussis (P04977; P04978; P04979; P0A3R5 and P04981: Uniprot), which cause pertussis. Ten polyamino acid sequences, designated herein as PtxEp1 to PtxEp10, were selected for insertion into nine insertion sites in the Rx protein, as shown in Table 14 below. In this example, a spacer region (SEQ ID NO: 95: SYWKGS) was used between the two epitopes to position them at position 1. Another spacer region (SEQ ID NO: 96: EAAKEAAK) was also used to insert the two epitopes at position 10. The purpose of introducing these spacers is to create inert physical space between consecutive polyamino acids, thereby helping to prevent binding competition between adjacent antibodies.

[0310] Table 14

[0311]

[0312] These polyamino acid sequences are combined with the sequence of the Rx protein to produce the protein RxPtx. The amino acid sequence corresponding to the RxPtx gene encompassing epitopes PtxEp1 to PtxEp10 is described in SEQ ID no. 64. The gene corresponding to the RxPtx protein, referred to herein as the RxPtx gene, is described in nucleotide sequence SEQ ID no. 63.

[0313] The synthetic gene RxPtx was introduced into the pET28a plasmid using restriction sites for the enzymes NdeI and Xhol by state of the art molecular biology methods. To identify whether the synthetic gene matched the sequence designed for RxPtx, the DH5α strain of E. coli was transformed and the plasmid material was analyzed by restriction enzyme digestion and subsequent sequencing techniques, as previously described in PlatCruzi.

[0314] The analyzed plasmid with the correct sequence for the RxPtx protein was transferred to the E. coli strain BL21. When induced with isopropyl β-D-1-thiogalactopyranoside (IPTG), the BL21 strain can express T7 RNA polymerase. The BL21 strain was grown overnight in LB medium and then inoculated into the same medium supplemented with kanamycin (30 μg / ml) and shaken at 200 rpm until it reached an optical density of 0.6-0.8 (600 nm). IPTG (1 mM) was then added to the culture and the same culture conditions were maintained for an additional 3 hours.

[0315] The culture was centrifuged and the pellet was resuspended in 2 mL of PBS with CelLytic (0.5x) for 1 hour at 4°C. After another centrifugation, the supernatant was collected and the pellet was resuspended in the same volume of 8 M urea solution (pH 8.0) as the supernatant. Equal volumes were loaded onto SDS-PAGE gels (11%, Table 5).

[0316] The RxPtx protein was examined by SDS-PAGE electrophoresis to demonstrate its production and determine its distribution between the soluble and insoluble fractions. Figure 11 As shown in FIG, columns 5 and 6 (arrows) show that RxPtx is produced as both soluble and insoluble fractions, with a higher proportion in the insoluble fraction.

[0317] Example 16 -Development of RxYFIgG protein

[0318] Based on a mapping study of the polyamino acid sequences of yellow fever virus (strain 17DD and sequences in the p03314-Uniprot archive), polyamino acid sequences were selected from available prior art literature for their diagnostic potential. Ten polyamino acid sequences, designated herein as YFIgGEp1 to YFIgGEp10, were selected for insertion into the nine insertion sites of the Rx protein, as shown in Table 15 below. A spacer region (SEQ ID NO: 97: TSYWKGS) was used between the two epitopes, placing them at position 10. The spacer region functions to create physical space between consecutive epitopes, helping to prevent interaction with antibodies.

[0319] Table 15

[0320]

[0321] Combining these polyamino acid sequences with the Rx protein produces the RxYFIgG protein. The amino acid sequence corresponding to the RxYFIgG gene encompassing epitopes YFIgGEp 1 to YFIgGEp 10 is depicted in SEQ ID No. 75. The gene corresponding to the RxYFIgG protein, referred to herein as the RxYFIgG gene, is depicted in the nucleotide sequence of SEQ ID No. 76.

[0322] The synthetic gene RxYFIgG was introduced into the pET28a plasmid by state-of-the-art molecular biology methods using restriction sites for the enzymes NdeI and Xhol. To identify whether the synthetic gene matched the sequence designed for RxYFIgG, the DH5α strain of E. coli was transformed and the plasmid material was analyzed by restriction enzyme digestion and subsequent sequencing techniques, as previously described in PlatCruzi.

[0323] The analyzed plasmid with the correct sequence for RxYFIgG was transferred to the E. coli strain BL21 to produce the RxYFIgG protein. The BL21 strain can express T7 RNA polymerase when induced with isopropyl β-D-1-thiogalactopyranoside (IPTG). The BL21 strain was grown overnight in LB medium at 37°C and then inoculated in the same medium with kanamycin (30 μg / ml) on a shaker at 200 rpm until it reached an optical density of 0.6-0.8 (600 nm) of turbidity. IPTG (1 mM) was then added to the culture and the same culture conditions were maintained at 37°C for an additional 3 hours.

[0324] The culture was centrifuged and the pellet was resuspended in 2 mL of PBS with CelLytic (0.5x) at 4°C for 1 hour. After another centrifugation, the supernatant was collected and the pellet was resuspended in the same volume of 8 M urea solution (pH 8.0) as the supernatant. Equal volumes were loaded onto SDS-PAGE gels (11%, Table 5). Protein RxYFIgG was tested by SDS-PAGE electrophoresis to confirm its production and determine its distribution between the soluble and insoluble fractions. Figure 11 As shown in , columns 9 and 10 (arrows) show that RxYF IgG is produced as both soluble and insoluble fractions, with a higher proportion in the insoluble fraction.

[0325] Example 17 -Development of TxNeuza protein

[0326] The container protein "Tx" is genetically manipulated to have a T-cell epitope from the house dust mite (Dermatophogoides pteronyssinus), a major cause of respiratory allergy in humans, which we refer to herein as the TxNeuza platform. The gene corresponding to the TxNeuza protein, referred to herein as the TxNeuza gene, is described in nucleotide sequence SEQ ID NO: 89.

[0327] From mapping studies of T cell polyamino acid sequences of house dust mites, polyamino acid sequences were selected from available prior art literature (Hinz et al., Clin Exp Allergy 45:1601-1612, 2015; Oseroff et al., Clin Exp Allergy 47:577-592, 2017). Nine polyamino acid sequences, designated herein as NeuzaEp1 to NeuzaEp9, were selected for insertion into the nine insertion sites of the Tx protein, as shown in Table 16 below. In this example, a spacer region (SEQ ID NO: 98: GGSG) was used between the two epitopes to position the two epitopes at position 12.

[0328] Table 16

[0329]

[0330] The combination of these epitopes with the sequence of the Tx protein produces the TxNeuza protein. The amino acid sequence corresponding to the TxNeuza gene comprising epitopes NeuzaEp1 to NeuzaEp9 is described in SEQ ID NO:88.

[0331] The synthetic gene TxNeuza was introduced into the pET28a plasmid using restriction sites for the enzymes NdeI and Xhol by state of the art molecular biology methods. To identify whether the synthetic gene matched the sequence designed for TxNeuza, the DH5α strain of E. coli was transformed and the plasmid material was analyzed by restriction enzyme digestion and subsequent sequencing techniques, as previously described in PlatCruzi.

[0332] The analyzed plasmid with the correct sequence of TxNeuza was transferred to the E. coli strain BL21 to produce the TxNeuza protein. When induced by isopropyl β-D-1-thiogalactopyranoside (IPTG), the BL21 strain can express T7 RNA polymerase. The BL21 strain was grown overnight in LB medium at 37°C and then inoculated in the same medium with kanamycin (30 μg / ml) on a shaker at 200 rpm until it reached an optical density of turbidity of 0.6-0.8 (600 nm). Then, IPTG (qsp1 mM) was added to the culture and the same culture conditions were maintained at 37°C for another 3 hours.

[0333] The culture was centrifuged and the pellet was resuspended in 2 mL of PBS with CelLytic (0.5x) at 4°C for 1 hour. After another centrifugation, the supernatant was collected and the pellet was resuspended in the same volume of 8 M urea solution (pH 8.0) as the supernatant. Equal volumes were loaded onto SDS-PAGE gels (11%, Table 5). The TxNeuza protein was examined by SDS-PAGE to demonstrate its production and to determine its distribution between the soluble and insoluble fractions. Figure 11 As shown in , columns 7 and 8 (arrows) show that TxNeuza is produced as insoluble.

[0334] Example 18 -Development of TxCruzi protein

[0335] Considering the experimental specificity and sensitivity of the diagnostic test for Chagas disease, the Trypanosoma cruzi polyamino acid sequence was selected from the available prior art literature (Balouz, et al., Clin Vaccine Immunol 22, 304-312, 2015; Alvarez, et al., Infect Immun 69, 7946-7949, 2001; Fernandez-Villegas, et al., JAntimicrob Chemother 71, 2005-2009, 2016; Thomas, et al., Clin Vaccine Immunol 19, 167-173, 2012). Ten polyamino acid sequences were selected for insertion into the ten insertion sites of the "Tx" protein, as shown in Table 17 below, and are referred to herein as TcEp 1, TcEp 3, TcEp 4, TcEp 6, TcEp 8, TcEp 9, TcEp 10, TcEp 11, TcEp 12, and TcEp 13. A spacer (SEQ ID NO: 99: GGASG) was used between the two epitopes to place them at position 12.

[0336] Table 17

[0337]

[0338] After selecting multiple amino acid sequences from Trypanosoma cruzi, synthetic genes corresponding to the TxCruzi protein were generated by chemical synthesis and gene synthesis by ligation, and inserted into plasmids for use in experiments. The nucleotide sequence corresponding to the TxCruzi gene encompassing epitopes TcEp 1, TcEp 3, TcEp 4, TcEp 6, TcEp 8, TcEp 9, TcEp 10, TcEp 11, TcEp 12, and TcEp 13 is set forth in SEQ ID No. 91.

[0339] The combination of the sequences of these epitopes with the sequences of the Tx proteins produces the TxCruzi protein. The amino acid sequence corresponding to the TxCruzi gene comprising epitopes TcEp 1, TcEp 3, TcEp 4, TcEp 6, TcEp 8, TcEp 9, TcEp 10, TcEp 11, TcEp 12, and TcEp 13 is described in SEQ ID NO: 90.

[0340] The synthetic gene was introduced into the pET28a plasmid by state-of-the-art molecular biology methods using restriction sites for the enzymes NdeI and Xhol. To identify whether the synthetic gene matched the sequence designed for the TxCruzi protein, the DH5α strain of E. coli was transformed and the plasmid material was analyzed by restriction enzyme digestion followed by sequencing, as described in detail in PlatCruzi.

[0341] The plasmid with the correct sequence of TxCruzi analyzed was transferred to the E. coli strain BL21 to produce the TxCruzi protein. When induced by isopropyl β-D-1-thiogalactopyranoside (IPTG), the BL21 strain can express T7 RNA polymerase. The BL21 strain was grown overnight in LB medium at 37°C and then inoculated in the same medium with kanamycin (30 μg / ml) on a shaking table at 200 rpm until it reached an optical density of turbidity of 0.6-0.8 (600 nm). Then, IPTG (qsp1mM) was added to the culture and the same culture conditions were maintained at 37°C for another 3 hours.

[0342] The culture was centrifuged and the pellet was resuspended in 2 mL of PBS with CelLytic (0.5x) for 1 hour at 4°C. After another centrifugation, the supernatant was collected and the pellet was resuspended in the same volume of 8 M urea solution (pH 8.0) as the supernatant. Equal volumes were loaded onto SDS-PAGE gels (11%, Table 5).

[0343] TxCruzi protein was examined by SDS-PAGE to demonstrate its production and to determine its distribution between the soluble and insoluble fractions. Figure 11 As shown in , columns 3 and 4 (arrows) show that TxCruzi is produced as soluble and insoluble proteins. Compared to PlatCruzi (columns 1 and 2), TxCruzi shows an increase in the proportion of soluble protein produced, which can be attributed to the use of Tx as a container protein.

[0344] Example 19 - Synthesis of SARS-CoV-2 Peptide Library on Cellulose Membranes and Reactivity with Sera from SARS-CoV-2 Positive and Negative Individuals

[0345] Based on the genome sequence of SARS-CoV-2 isolated in Wuhan, China and published in the GenBank database (https: / / www.ncbi.nlm.nih.gov / nuccore / MN908947.3?report=genbank), a peptide library covering all protein coding regions of ORF3a, ORF6, ORF7, ORF8, ORF10, N, M, S, and E of the SARS-CoV-2 virus was synthesized and annotated as follows:

[0346] Four polypeptides not encoded by the SARS-CoV-2 virus are included in the peptide library list to represent positive controls for reactivity with human serum. In Table 18, A1, V5 (IHLVNNESSEVIVHK, Clostridium tetani precursor peptide), A2, V6 (GYPKDGNAFNNLDR, Clostridium tetani), A3, V7 (KEVPALTAVETGATG, human poliovirus), A4, V8 (YPYDVPDYAGYPYD, triple hemagglutinin peptide) are used as such controls. In Tables 19 and 20, A1, V4 (IHLVNNESSEVIVHK, Clostridium tetani precursor peptide), A2, V5 (GYPKDGNAFNNLDR, Clostridium tetani), A3, V6 (KEVPALTAVETGATG, human poliovirus), A4, V8 (YPYDVPDYAGYPYD, trihemagglutinin peptide) were used as such controls.

[0347] As negative controls, no peptide spot reactions were used for A5, A6, K20, K21, N3, N4, O24, P1, P13, P14, Q14, Q15, R15, R16, V3, V4, V9-V24, W14 in Table 18, and A5, K19, K20, N2, N3, O23, O24, P12, P13, Q13, Q14, R15, R15, V2, V3, V17, V18 in Tables 19 and 20.

[0348] The relationships of the synthetic linear polyamino acids are shown in Tables 18, 19, and 20.

[0349] Table 18 - For Figure 12 List of synthetic SARS-CoV-2 polyamino acids mapped to IgM reactive epitopes from patient sera.

[0350]

[0351]

[0352]

[0353] Table 19 - For Figure 13 List of synthetic SARS-CoV-2 polyamino acids mapped to IgG reactive epitopes from patient sera.

[0354]

[0355]

[0356]

[0357] Table 20 - For Figure 14 List of synthetic SARS-CoV-2 polyamino acids mapped to IgA-reactive epitopes from patient sera.

[0358]

[0359]

[0360]

[0361] Example 20 -Synthesis of a multi-amino acid library of SARS-CoV-2 on cellulose membrane

[0362] The polypeptide library described in Example 19 was prepared using the standard spot synthesis technique on an Amino-PEG500-UC540 cellulose membrane using an Auto-Spot Robot ASP-222 according to the manufacturer's instructions. Polyamino acids with a length of 15 residues and an overlap of 10 adjacent residues were synthesized, covering the entire length of the protein.

[0363] After synthesis, the free sites of the membrane were blocked with 1.5% BSA (bovine serum albumin) prepared in TBS-T buffer (50mM Tris, NaCl; 136mM, 2mM KCl; 0.05%, Tween-20; pH 7.4) for 90 minutes. Then, the membrane was incubated with patient serum (n=3; 1:100, diluted in TBS-T containing 0.75% BSA) and washed 4 times with TBS-T. Thereafter, the membrane was incubated with goat IgG anti-IgM (mu, KPL), anti-IgG (H+L chain, Thermo Scientific) or anti-human IgA (α chain specific, Calbiochem) (1:5000, prepared in TBS-T) for 1.5h and then washed with TBS-T and CBS (sodium citrate buffer containing 50mM NaCl, pH 7.0). Then, the membrane was added Chemiluminescent substrate (0.25 mM) and Nitro-Block-II TM Enhancer (Applied Biosystems, USA) was used to complete the reaction.

[0364] Chemiluminescent signals were detected on an Odyssey FC instrument (LI-COR Bioscience) and the intensity of the signals was quantified using TotalLabTL100 software (v 2009, Nonlinear Dynamics, USA). Data were analyzed using Microsoft Excel, and only spots with a signal intensity (SI) greater than or equal to 30% of the highest value obtained in the pool of spots on each membrane were included in the polyamino acid signature. As a negative control, the background signal intensity of each membrane was used.

[0365] Example 21 - Synthesis of branched-chain polyamino acids from SARS-CoV-2

[0366] Multiple branched-chain polyamino acids (SARS-X1-SARS-X8) were synthesized using the F-moc solid-phase polyamino acid synthesis strategy on a Schimadzu synthesizer model PSS8 according to the manufacturer's instructions. Wang Kcore resin (dilysine core, K4) (Novabiochem) was used as a solid support for the synthesis of branched-chain polyamino acids. The first amino acid to be conjugated was the amino acid located at the C-terminal portion of the polypeptide sequence, and the last was located at the N-start. After completing all cycles of the synthesis of the branched-chain polyamino acid, the branched-chain polyamino acid was detached from the solid support by treatment with a cleavage cocktail (trifluoroacetic acid, triisopropylsilane, and ethylene glycol) according to the standard procedures used in the prior art for the production of synthetic polyamino acids and the deprotection of the protecting groups of the amino acid side chains (Guy and Fields, Methods Emzymol 289, 67-83, 1997). For quality control of the synthesis, each polyamino acid was analyzed by HPLC and MALDI-TOF.

[0367] The synthetic polyamino acids X1, X2, and X5 of the SARS-CoV-2 S protein comprise SEQ ID NOs: 100, 101, and 104, respectively. The synthetic polyamino acids X3 and X6 of the SARS-CoV-2 N protein comprise SEQ ID NOs: 102 and 105, respectively. The synthetic polyamino acids X4 of the SARS-CoV-2 E protein comprise SEQ ID NO: 103. The synthetic polyamino acids X7 and X8 of the SARS-CoV-2 protein encoded by the open reading frame (ORF8) between the S and Se genes comprise SEQ ID NOs: 106 and 107, respectively.

[0368] The gene encoding the X8 protein is found in a small open reading frame (ORF) between the S and Se genes. The genes encoding the ORF6, X4 and X5 proteins are found within the ORF between the M and N genes.

[0369] The list of synthetic branched polypeptides is shown in Table 21.

[0370] Table 21 - Synthetic branched peptides of SARS-CoV-2 proteins

[0371]

[0372] Example 22 - Human serum sample set

[0373] 134 human serum samples were divided into 5 groups. The groups are:

[0374] - Group 0: 10 serum samples from healthy individuals obtained before 2016 from a blood bank (HEMORIO) (sera #1-#10);

[0375] - Group 1: 26 serum samples (#1-#26) from “asymptomatic SARS patients” determined according to the WHO case definition;

[0376] - Group 2: 24 serum samples from “suspected patients” (#27-#50);

[0377] - Group 3: 38 serum samples (#51-#88) from “SARS hospitalized patients (severe illness)” defined according to the WHO case definition;

[0378] - Group 4: 36 serum samples from "patients immune-protected against SARS" (#89-#124).

[0379] For sera #1-#26, the high body temperature returned to normal temperature, and they were RT-PCR positive for SARS-CoV-2, and the SD rapid test for anti-SARS-CoV-2 antibodies (Standard diagnostic Inc.) was negative.

[0380] #27-#50: Patients had a diagnosis of SARS-CoV-2 positive by RT-PCR and negative by SD rapid test for anti-SARS-CoV-2 antibodies.

[0381] #51-#88: Hospitalized individuals with worsening signs and symptoms of SARS-CoV-2, rapid RT-PCR test+, SD positive for anti-SARS-CoV-2 antibodies.

[0382] #89-#124: Immune-protected individuals are defined as recovered patients, hospitalized or not hospitalized, who were diagnosed as SARS-CoV-2 positive or not diagnosed as SARS-CoV-2 positive, sometimes not diagnosed by RT-PCR+, but showed characteristic symptoms.

[0383] Example 23 -Recognition of SARS-CoV-2 related IgM polyamino acids

[0384] To identify potential polyamino acids that can be specifically recognized by anti-SARS-CoV-2 IgM antibodies, serum samples from infected patients were analyzed by synthetic arrays of a SARS-CoV-2 spotted peptide library encompassing all regions of S, ORF3a, M, ORF6, ORF7, ORF8, N, E, ORF10, and control polyamino acids.

[0385] Peptide arrays were used to test human sera from infected individuals for potential binding activity against polyamino acids. The peptide arrays covered peptide sequences 15 amino acids long, with 10 amino acid overlaps between adjacent spots. These linear polyamino acids included the following SARS-CoV-2 proteins:

[0386] - Spike protein (S): aa 1-1273 (speck A7-K19),

[0387] -ORF3a protein (ORF3): aa 1-275 (spots K22-N2),

[0388] -Membrane glycoprotein (M): aa 1-222 (spot N5-O23),

[0389] -ORF6 protein (ORF6): aa 1-61 (spots P2-P12),

[0390] -ORF7 protein (ORF7): aa 1-121 (spots P15-Q13),

[0391] -ORF8 protein (ORF8): aa 1-121 (spots Q16-R17),

[0392] - Nucleocapsid protein (N): aa 1-419 (spots R20-V17),

[0393] -Envelope protein (E): aa 1-75 (spots W1-W13)

[0394] -ORF10 protein (ORF10): aa 1-38 (spots W15-W20)

[0395] - Positive control polyamino acids: A1 and V5 (Clostridium tetani precursor peptide), A2 and V6 (Clostridium tetani precursor peptide), A3 and V7 (human poliovirus peptide), A4 and V8 (trihemagglutinin epitope)

[0396] - Non-reactive spots served as negative controls.

[0397] The serological immune responses of human anti-IgM antibodies to various SARS-CoV-2 (S, ORF3a, M, ORF6, ORF7, ORF8, N, E, ORF10) synthetic polyamino acids and control polyamino acids (Examples 19 and 20) were analyzed using covalently synthesized polyamino acids on cellulose membranes (spots) and serum pools (n=3) from patients #55, #60, and #74 (Group 3), as shown in Figure 2. Figure 12 As shown in , supplemented by Table 18.

[0398] 15A to 15IShown are quantifications of signals from membrane blots developed with goat anti-human IgM secondary antibody and incubated with human serum.

[0399] When developed with anti-human IgM antibodies, human serum has shown significant reactivity with multiple amino acids from different viral proteins, such as 15A to 15I As shown in , a large number of different polyamino acids have great potential for diagnosis of diseases even in the early stages of infection.

[0400] Example 24 -Recognition of SARS-related IgG epitopes

[0401] To identify potential epitopes that can be specifically recognized by anti-SARS-CoV-2 IgG antibodies, serum samples from infected patients were analyzed by synthetic arrays of a SARS-CoV-2 spotted polyamino acid library including all regions of S, ORF3a, M, ORF6, ORF7, ORF8, N, E, ORF10, and control polyamino acids.

[0402] Peptide arrays were used to test human sera from infected individuals for potential binding activity against polyamino acids. The peptide arrays covered peptide sequences 15 amino acids long, with 10 amino acid overlaps between adjacent spots. These linear polyamino acids included the following SARS-CoV-2 proteins:

[0403] -ORF3a protein (OF3a): aa 1-275 (spots A7-C11),

[0404] -Membrane glycoprotein (M): aa 1-222 (spots C14-E8),

[0405] -ORF6 protein (OF6): aa 1-61 (spots E11-E21),

[0406] -ORF7 protein (OF7(aa 1-121 (spots E24-F22),

[0407] -ORF8 protein (OF8): aa 1-121 (spots G1-G23),

[0408] - Spike protein (S): aa 1-1273 (speck H1-R13),

[0409] - Nucleocapsid protein (N): aa 1-419 (spot R16-V1),

[0410] - Envelope protein (E): aa 1-75 (spots W1-W13),

[0411] -ORF10 protein (OF10): aa 1-38 (spots W15-W20),

[0412] - Positive control peptides: A1 and V4 (Clostridium tetani precursor peptide), A2 and V5 (Clostridium tetani precursor peptide), A3 and V6 (human poliovirus peptide), A4 and V7 (trihemagglutinin epitope)

[0413] - No reaction spot as negative control

[0414] Serological immune responses of IgG antibodies to various SARS-CoV-2 (ORF3a, M, ORF6, ORF7, ORF8, S, N, E, ORF10) synthetic polyamino acids and control polyamino acids were analyzed using peptides covalently synthesized on cellulose membranes (spots) and serum pools (n=3) from patients #55, #60, and #74 (Group 3), as shown. Figure 13 As shown in , supplemented by Table 19.

[0415] 16A to 16H Shown are quantifications of signals from membrane blots developed with goat anti-human IgG secondary antibody and incubated with human serum.

[0416] When developed with anti-human IgG antibodies, human serum has shown significant reactivity with multiple amino acids from different viral proteins, such as 16A to 16H As shown in , a large number of different polyamino acids have great potential for diagnosis of diseases even in the stage after the acute phase of infection.

[0417] Example 25 -Recognition of polyamino acid IgA associated with SARS

[0418] To identify potential polyamino acids that can be specifically recognized by anti-SARS-CoV-2 IgA antibodies, serum samples from infected patients were analyzed by synthetic arrays of a SARS-CoV-2 spotted polyamino acid library including all regions of S, ORF3a, M, ORF6, ORF7, ORF8, N, E, ORF10, and control polyamino acids.

[0419] Peptide arrays were used to test human sera from infected individuals for potential binding activity against polyamino acids. The peptide arrays covered peptide sequences 15 amino acids long, with 10 amino acid overlaps between adjacent spots. These linear polyamino acids included the following SARS-CoV-2 proteins:

[0420] - Spike protein (S): aa 1-1273 (speck A6-K18),

[0421] -ORF3a protein (ORF3): aa 1-275 (spots K21-N1),

[0422] -Membrane glycoprotein (M): aa 1-222 (N4-O22),

[0423] -ORF6 protein (ORF6): aa 1-61 (P2-P12),

[0424] -ORF7 protein (ORF7): aa 1-121 (P15-Q13),

[0425] -ORF8 protein (ORF8): aa 1-121 (Q16-R17),

[0426] - Nucleocapsid protein (N): aa 1-419 (spots R20-V17),

[0427] - Envelope protein (E): aa 1-75 (spots W1-W-13),

[0428] -ORF10 protein (ORF10): aa 1-38 (spots W15-W20),

[0429] - Positive control peptides: A1 and V4 (Clostridium tetani precursor peptide), A2 and V5 (Clostridium tetani precursor peptide), A3 and V6 (human poliovirus peptide), A4 and V7 (trihemagglutinin epitope),

[0430] -No reactant spot served as negative control.

[0431] The B cell immune responses of IgA antibodies to various synthetic polyamino acids of SARS-CoV-2 (ORF3a, M, ORF6, ORF7, ORF8, S, N, E, ORF10) and control polyamino acids were analyzed using peptides covalently synthesized on cellulose membranes (spots) and serum pools (n=3) from patients #55, #60, and #74 (Group 3), as shown. Figure 14 As shown in , supplemented by Table 20.

[0432] 17A to 17I Shown are quantifications of signals from membrane blots developed with goat IgG anti-human IgA secondary antibody and incubated with human serum.

[0433] When developed with anti-human IgA antibodies, human serum has shown significant reactivity with multiple amino acids of different viral proteins, such as 17A to 17I As shown in , a large number of different polyamino acids have great potential for diagnosis of diseases through such antibodies that are mainly present in mucosal membranes.

[0434] Example 26 - ELISA for detecting anti-SARS-CoV-2 antibodies

[0435] Enzyme-linked immunosorbent assay (ELISA) was used to screen for anti-SARS-CoV-2 antibodies in patient serum. ELISA was performed by coating 96-well polystyrene plates with 1 μg / well of branched polyamino acids. To compare the results, the experiments of each group were performed in parallel and simultaneously. To reduce possible differences in performance between the experiments, the reactivity index (RI) was used, which is defined as the OD450 of the target minus the cutoff OD450. The original human serum was diluted 100 times (100X) in PBS / BSA1% and the secondary antibody goat anti-human IgM (Merck-Sigma) and 8000x biotin-labeled goat anti-human IgG (Merck-Sigma), followed by incubation with HRP-labeled high-sensitivity neutravidin (Thermo Fisher Scientific). The anti-IgA response was visualized by alkaline phosphatase-labeled goat anti-human IgA (KPL). TMB (3,3',5,5'-tetramethylbenzidine) was used as a substrate (Thermo Fisher Scientific), and the immune response was defined as significantly increased when the reactivity index was greater than 1.

[0436] The results showed that the branched-chain polyamino acids SARS-X1 to SARS-X8 had different reactivities against anti-SARS-CoV2 antibodies, and these differences were related to the type of human antibodies detected (IgM, IgG, or IgA) and the status of the patients diagnosed with SARS-CoV2, such as Figures 18 to 23 Observing such differences allows for the design of diagnostic tests that can provide more accurate or reliable information than simply diagnosing a positive or negative diagnosis for anti-SARS-CoV2 antibodies. Therefore, in addition to detecting IgM, IgG, or IgA antibodies, diagnostic tests can also be designed to indicate whether an individual should be hospitalized, even in the absence of symptoms.

[0437] Example 27 - Development of container proteins for SARS-CoV-2

[0438] The "Tx" container protein was genetically manipulated to harbor SARS-CoV-2 epitopes. Reactive epitopes from sera of individuals infected with SARS-CoV-2 were selected for the construction of eight Tx proteins: Ag-COVID19, Ag COVID19(H), Tx-SARS2-IgM, Tx-SARS2-IgG, Tx-SARS2-G / M, Tx-SARS2-IgA, Tx-SARS2-Universal, and Tx-SARS-G5 (non-RBD).

[0439] The genes corresponding to Ag-COVID19, Ag COVID19 (H), Tx-SARS2-IgM, Tx-SARS2-IgG, Tx-SARS2-G / M, Tx-SARS2-IgA, Tx-SARS2-Universal, and Tx-SARS-G5 (non-RBD) proteins are respectively referred to herein as Ag-COVID19 gene, Ag-COVID19 (H) gene, Tx-SARS2-IgM gene, Tx-SARS2-IgG gene, Tx-SARS2-G / M gene, Tx-SARS2-IgA gene, Tx-SARS2-Universal gene, and Tx-SARS-G5 (non-RBD) gene, and are described in the nucleotide sequences of SEQ ID NO: 108 to SEQ ID NO: 115. The amino acid sequences corresponding to Ag-COVID19, Ag COVID19(H), Tx-SARS2-IgM, Tx-SARS2-IgG, Tx-SARS2-G / M, Tx-SARS2-IgA, Tx-SARS2-Universal and Tx-SARS-G5 (without RBD) proteins are described in SEQ ID NOs 116 to 123, respectively.

[0440] From the SARS-CoV-2 epitope sequence mapping study, the above 8 proteins having multiple amino acids are shown in Tables 22 to 28 in consideration of their diagnostic potential as illustrated in Examples 19 to 26.

[0441] Table 22-Ag-COVID19 and Ag COVID19 protein (H)

[0442]

[0443] Table 23-Tx-SARS2-IgM

[0444]

[0445] Table 24-Tx-SARS2-IgG

[0446]

[0447] Table 25-Tx-SARS2-G / M

[0448]

[0449] Table 26-Tx-SARS2-IgA

[0450]

[0451] Table 27-Tx-SARS2-Universal

[0452]

[0453] Table 28-Tx-SARS2-G5

[0454]

[0455] Example 28 Expression of container protein with multiple amino acids from SARS-CoV-2

[0456] Ag-COVID19, Ag-COVID19(H), Tx-SARS2-IgM, Tx-SARS2-IgG, Tx-SARS2-G / M, Tx-SARS2-IgA, Tx-SARS2-Universal, and Tx-SARS-G5 (non-RBD) proteins were expressed using pET24 plasmids with restriction sites for BamHI and XhoI enzymes and genes encoding each protein. Each plasmid containing the gene for a specific protein was transferred to the E. coli BL21 strain to promote the expression of the eight different proteins listed above.

[0457] The strain was grown overnight in LB medium and then inoculated into the same medium supplemented with kanamycin (30 μg / ml) on a shaker at 200 rpm until it reached an optical density of turbidity of 0.6-0.8 (600 nm). The BL21 strain expresses T7 RNA polymerase when induced by isopropyl β-D-1-thiogalactopyranoside (IPTG). IPTG (1 mM) was then added to the culture and the same culture conditions were maintained at 37°C for another 3 hours.

[0458] Cultures of each bacterial strain were centrifuged and the pellets were resuspended in 10% CelLytic A in 150 mM NaCl and 50 mM Tris, pH 8.0. TM(Sigma, BR). Samples of the recombinant protein (1 μg / well) were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, Nature 227: 680-685, 1970). Stacking gels (stacking gel) and separating gels (separating gel) were prepared at 4% and 11% acrylamide concentrations, respectively (Table 5, below). Under denaturing conditions, samples were prepared in 62.5 mM Tris-HCl buffer, pH 6.8, 2% SDS, 5% β-mercaptoethanol, 10% glycerol and boiled at 95°C for 5 minutes (Hames BD, Gel electrophoresis of proteins: a practical approach. 3. Ed. Oxford. 1998). After electrophoresis, proteins were detected by staining with Coomassie Brilliant Blue Simply Blue R250 (ThermoFisher, BR). The marker PageRuler Plus Prestained Standards was used as a molecular weight reference (ThermoFisher, BR). Figure 24 , showing bands for Ag-COVID19 (column 3), Ag-COVID19(H) (column 4), Tx-SARS2-IgM (column 5), Tx-SARS2-IgG (column 6), Tx-SARS2-G / M (column 7), Tx-SARS2-IgA (column 8), Tx-SARS2-Universal (column 9), and Tx-SARS-G5 (non-RBD) (column 10). Column 1 shows molecular weight markers: A) 250 kDa; B) 130 kDa; C) 100 kDa; D) 70 kDa; E) 55 kDa; F) 35 kDa; and G) 25 kDa. Column 2 shows total extracts of uninduced bacteria.

[0459] The elution of 40min is carried out under the condition of 1% dextrose.Alternatively, culture is also centrifuged and precipitation is resuspended in urea buffer (100mM NaH pO , 10mMTris-base, 8M urea, pH 8.0).By nickel affinity column (HisTrap , 1mL, GE Healthcare LifeSciences) mL per minute, solution is carried out chromatography, and described post was previously balanced in buffer A (50mM Tris-HCl, pH 8.0,100mM NaCl and 5mM imidazoles).After combining, use the buffer A washing resin of 10mL.With the flow rate of 0.7mL / minute in buffer B (50mM Tris-HCl, pH 8.0,100mM NaCl) with 75mM, 200mM and 500mM imidazoles, gradient elution protein 45 minutes. Figure 25A pattern corresponding to the Ag-COVID19 protein with six histidine tails is shown, indicating a 200 mM elution concentration. Figure 26 Shown is a pattern corresponding to SARS2-G5 protein purified by affinity using 200 mM imidazole (elution from 75 mM shows contaminants).

[0460] The results show that Tx container proteins can be easily used to generate new and different proteins. Furthermore, the same expression protocol can be used to express different container proteins with different polyamino acids, greatly saving investment, time, and infrastructure. The inclusion of a six-histidine tail has been shown to be a potential facilitator for purification at high purity levels.

[0461] Example 29 - Enzyme-linked immunosorbent assay (ELISA) for detecting anti-SARS-CoV-2 antibodies using Ag-COVID19 and SARS2-G5 proteins

[0462] Enzyme-linked immunosorbent assay (ELISA) was used to screen for the presence of anti-SARS-CoV-2 antibodies. The performance of Ag-COVID19 and SARS2-G5 proteins was evaluated against a panel of sera from individuals affected by SARS-CoV-2 virus infection.

[0463] By using Ag-COVID19 protein ( Figure 27 ) or Tx-SARS2-G5 protein ( Figure 28 ELISA was performed by coating 96-well polystyrene plates with 1 μg / well of a solution of 0.3 M urea, pH 8.0 for 12-18 hours. The wells were washed with a saline-phosphate buffered saline (PBS) solution supplemented with Tween 20 (PBS-T, 10 mM sodium phosphate-Na3PO4, 150 mM sodium chloride-NaCl and 0.05% Tween-20, pH 7.4), and then incubated at 37°C for 2 hours with 1× PBS buffer containing 5% (weight / volume) skim milk powder.

[0464] The wells were then washed three times with PBS-T buffer and incubated for 1 hour at 37°C with human serum samples diluted 1:100 in PBS / BSA 1%. After incubation, the wells were washed three times with PBS-T and then incubated for 1 hour at 37°C with a biotinylated goat anti-human IgG antibody (Merck-Sigma) diluted 1:8000. Subsequently, HRP-labeled high-sensitivity neutravidin (Thermo Fisher Scientific) was added. The wells were washed again with PBS-T buffer three times and TMB substrate (3.3', 5.5' tetramethylbenzidine, Thermo Fisher Scientific) was used. After 30 minutes in the dark, the absorbance at 405 nm was measured in an ELISA plate reader.

[0465] The results showed that Ag-COVID19 protein ( Figure 27 ) and Tx-SARS2-G5 protein ( Figure 28 ) has proven to be advantageous for detecting antibodies against SARS-CoV-2. Figure 27 and Figure 28 As can be seen, antibodies to proteins with multiple amino acids from SARS-CoV-2 were not detected in sera from individuals collected before the SARS-CoV-2 epidemic, or from individuals who were healthy or affected by diseases such as dengue fever, malaria, and syphilis. In contrast, antibodies to the protein were detected in sera from individuals diagnosed positive for SARS-CoV-2 (symptomatic or asymptomatic), hospitalized patients, or recovered patients.

[0466] Example 30 -Ag-COVID19 protein as a vaccine composition

[0467] Ag-COVID19 protein was produced and purified according to the protocol described in this patent application. On days 0, 14, 21, and 28, three mice were inoculated with 10 μg of Ag-COVID19 protein suspended in 25 μl of PBS in Freund's incomplete adjuvant (25 μl). Negative controls were performed using animals inoculated with PBS. Blood samples from animals were collected before each reinoculation and subjected to ELISA. Plasma was separated from the collected blood by centrifugation and serially diluted for antibody assay ( Figure 29 ). The results showed excellent antibody production against Ag-COVID19 four weeks after the first injection.

[0468] Example 31 -Use of Ag-COVID19 protein for purification of anti-SARS-CoV-2 antibodies

[0469] Anti-SARS-CoV-2 antibodies were purified from serum of patients diagnosed with COVID-19 using the antibody affinity principle. Ag-COVID-19 protein was conjugated to Sepharose activated by CNBr (GE Healthcare, USA) TM 4B. A 10 mL serum sample from a SARS-CoV-2 positive patient was diluted in 10 mL PBS and subjected to Sepharose-Ag-COVID19 for 1 hour. The mixture was then placed on a chromatography column. After the solution passed through the column, 10 mL of PBS was added to the chromatography system, followed by 5 mL of 100 mM sodium citrate buffer at pH 4. As the antibody was recovered from the column, 0.5 mL fractions were collected sequentially and the presence of antibodies was quantified by spectrophotometry at 280 nm. The absorbance of each fraction was converted to protein concentration and plotted as a function of fraction volume.

[0470] The results indicate that Ag-COVID19 protein can be useful as an internal control (input) for affinity purification of antibodies from patients previously infected with SARS-CoV-2 ( Figure 30 ), indicating its importance in generating a usable internal reference for passive immunization against the COVID-19 epidemic.

Claims

1. A protein container, characterized in that It includes a stable protein structure that supports the simultaneous insertion of four or more exogenous polyamino acid sequences at different sites. The amino acid sequence of the stable protein structure is shown in SEQ ID NO: 77, and the amino acid sequence of the protein container is shown in SEQ ID NO: 90, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122 or SEQ ID NO:

123.

2. The protein container according to claim 1, characterized in that It has an insertion site for foreign polyamino acid sequences in a protein loop facing the external environment.

3. The protein container according to claim 1 or 2, characterized in that The simultaneous insertion of exogenous polyamino acid sequences does not interfere with the production conditions of the container protein.

4. The protein container according to claim 1, characterized in that It also contains foreign polyamino acid sequences and is used for vaccine compositions, diagnosis, or development of laboratory reagents.

5. The protein container according to claim 1, characterized in that The foreign polyamino acid sequences do not lose their immunogenic properties when simultaneously inserted into the protein loops of the protein container.

6. A polynucleotide, characterized in that It includes any one of SEQ ID NO: 78, 91, 110, 111, 112, 113, 114, 115, which can produce the polypeptide defined by SEQ ID NO: 77, 90, 118, 119, 120, 121, 122, 123, respectively.

7. A carrier, characterized in that It comprises a polynucleotide as defined in claim 6.

8. An expression cassette, characterized in that It comprises a polynucleotide as defined in claim 6.

9. A cell, characterized in that It comprises a vector as defined in claim 7 or an expression cassette as defined in claim 8 .

10. A method for producing a protein container, characterized in that The method comprises introducing a polynucleotide as defined in claim 6 into competent cells of interest; culturing the competent cells and isolating a container protein containing selected exogenous polyamino acids.

11. The method for producing a protein container according to claim 10, characterized in that It is protected from interference by the insertion of various foreign polyamino acid sequences.

12. Use of the container protein as defined in any one of claims 1 to 5 in the preparation of a medicament for pathogen identification or in vitro disease diagnosis by the following method, characterized in that: The method uses a container protein as defined in any one of claims 1 to 5, said method facilitating the diagnosis of Chagas disease or Sars2 infection.

13. Use of a protein container as defined in any one of claims 1 to 5, characterized in that The protein container is a laboratory reagent.

14. Use of a protein container as defined in any one of claims 1 to 5 for the preparation of a medicament for diagnosing Chagas disease or infection by Sars2 virus.

15. A diagnostic kit, characterized in that It comprises a protein container as defined in any one of claims 1 to 5.

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