BR102022017576B1 - SYNTHETIC PEPTIDES P.SC2.ORF9B.27 AND P.SC2.ORF8.50 AND THEIR USE IN IMMUNOLOGICAL DIAGNOSIS OF COVID-19 - Google Patents

Abstract

SYNTHETIC PEPTIDES P.SC2.ORF9B.27 and P.SC2.ORF8.50AND THEIR USE IN IMMUNOLOGICAL DIAGNOSIS OF COVID-19. The present invention deals with two new synthetic peptide antigens and their use in the immunological diagnosis of COVID-19. The P.SC2 peptides. ORF9b.27 and P.SC2.ORF8.50 come from the ORF9b and ORF8 proteins, respectively, of the SARS-CoV-2 virus. Both antigens were immunoreactive with biological samples from individuals who had the disease. Therefore, the peptides were applied in enzyme-linked immunosorbent assay tests (indirect ELISA) to detect the presence of specific anti-SARS-CoV-2 antibodies of the IgA, IgM and IgG isotypes. The tests make it possible to differentiate individuals infected with the SARS-CoV-2 virus from those who are not infected, even in asymptomatic cases of the disease. The assay using the P.SC2.ORF9b.27 peptide as antigen showed 95.5% sensitivity and 93.3% specificity, and the assay with the P.SC2.ORF8.50 peptide showed 90.9% sensitivity and 86.7% specificity. This invention emerged in response to existing technical problems and the need to discover new immunological diagnostic markers for COVID-19.

Description

Field of Invention

[001]. The present invention deals with two new synthetic peptide antigens and their use in the immunological diagnosis of COVID-19. The peptide P.SC2.ORF9b.27 (Seq ID N°1) and P.SC2.ORF8.50 (Seq ID N° °2) come from the ORF9b and ORF8 proteins, respectively, of the SARS-CoV-2 virus. Both antigens were immunoreactive with biological samples from individuals who had the disease. Therefore, the peptides were chemically synthesized with the addition of amino acids to improve solubility and applied in enzyme-linked immunosorbent assay tests (indirect ELISA) to detect the presence of anti-SARS-CoV-2 antibodies of the IgA, IgM and IgG isotypes. The tests make it possible to differentiate individuals infected with the SARS-CoV-2 virus from those who are not infected, even in asymptomatic cases of the disease. This invention emerged in response to existing technical problems and the need to discover new immunological diagnostic markers for COVID-19.

Fundamentals of the Invention and State of the Art

[002]. In December 2019, a new virus belonging to the coronavirus (CoV) family and genus e-covonavirus was identified in Wuhan, China. This virus has been designated as SARS-CoV-2 or new coronavirus. Previously, two other highly pathogenic e-coronaviruses had been identified: SARS-CoV in 2003 and MERS- CoV in 2012. In addition to these, there are also four other low pathogenic β-coronaviruses: HCoV-OC43, HCoVHKU1, HCoV- NL63 and HCoV-229E. Viruses in this family are known to infect vertebrates such as bats and companion animals, and thanks to the zoonotic cycle they start to infect humans and cause severe diseases (GORBALENYA, Alexander E. et al. Severe acute respiratory syndrome- related coronavirus: The species and its viruses-a statement of the Coronavirus Study Group. BioRxiv, 2020).

[003]. The SARS-CoV-2 genome is made up of positive-sense single-stranded RNA of approximately 30 kb, and shares approximately 80% sequence identity with SARS-CoV. Two-thirds of the genome is translated into the ORF1a and ORF1b polyproteins, which play a role in the replication of viral genetic material. The remaining third of the genome has overlapping ORFs, encoding four structural proteins: Spike (S), Membrane (M), Envelope (E), and Nucleocapsid (N), and some accessory proteins, including ORF3a, ORF6, ORF7a, ORF7b, ORF8, ORF9b and ORF10 (ARYA, Rimanshee et al. Structural insights into SARS- CoV-2 proteins. Journal of molecular biology, v. 433, n. 2, p. 166725, 2021).

[004]. Infection with the new coronavirus causes the disease called COVID-19. The most common symptoms are: fever, cough, nausea/vomiting and tiredness. Around 80% of cases are considered mild, and have a recovery period of one to two weeks. Severe cases can progress to pneumonia, difficulty breathing and even death. Infected individuals can transmit two to three times more than the common flu and can transmit the virus even if they are asymptomatic. As the clinical manifestations of infected patients are nonspecific, tests are needed to confirm the diagnosis of COVID-19 as quickly as possible, so that these patients can be adequately isolated and treated (LARSEN, Joseph R. et al. Modeling the onset of symptoms of COVID-19. Frontiers in public health, v. 8, p.

[005]. From the confirmation of the first case of COVID-19 in December 2019 until 07/19/2022, 559.5 million cases and 6.3 million deaths were confirmed around the world. In Brazil, there were 33.3 million cases and 675 thousand deaths (WHO, World Health Organization. WHO Coronavirus Disease (COVID-19) Dashboard. 2020. Available at: https://covid19.who.int/. Acesso em 19 /07/2022; BRAZIL. COVID-19: Coronavirus Panel. Ministry of Health, 2020. Available at: https://covid.saude.gov.br/, accessed on 07/19/2022).

[006]. Preventive measures against COVID-19 include vaccination and strategies to limit the spread of the virus. Early screening, diagnosis, isolation and treatment are necessary to prevent the spread of the virus. Therefore, preventive strategies focus on identifying active cases, isolating these patients and carefully controlling the evolution of the infection. Furthermore, it is important to map the true extent of outbreaks, identify populations at risk and have greater control over the transmission rate in each region (GÜNER, Hatice Rahmet; HASANOGLU, imran; AKTA§, Firdevs. COVID-19: Prevention and control measures in community. Turkish Journal of medical sciences, v. 50, n. 9, p.

[007]. Serological tests are tools used in the management of infectious diseases. They are based on the detection of antibodies produced by the host in response to infection by a pathogenic agent. The detection of different antibody isotypes (IgA, IgM and IgG) can be carried out in body fluids such as blood, serum, tears, saliva, urine, among others. By using in vitro techniques, serological tests are easy to operate, require minimal training and equipment and the result is issued within a few hours. Examples of serological testing platforms are enzyme-linked immunosorbent assays (ELISA), immunochromatography (lateral flow), western blot, immunoelectrochemistry, among others. Thus, antibody detection tests have been used to diagnose COVID-19 since the beginning of the pandemic. They provide an effective means of screening symptomatic or asymptomatic individuals in community settings, enable appropriate case management, and guide public health measures such as quarantine or isolation (PEELING, Rosanna W. et al. Serology testing in the COVID-19 pandemic response.

[008]. The immune response against SARS-CoV-2 normally takes 40 days with variations in the dynamics of antibody production depending on the severity of the disease. In most studies of laboratory-confirmed COVID-19 cases, IgM antibodies begin to be detected about 5 to 10 days after the onset of symptoms and increase rapidly, so it is an indicator of acute infection. In contrast, IgG immunoglobulins are produced later and are maintained for a longer period of time, indicating active infection and past infection. Seroconversion generally occurs within the first three weeks, with an average time of 9 to 11 days after the onset of symptoms for total antibodies, 10 to 12 days for IgM and 12 to 14 days for IgG (KRAMMER, Florian; SIMON, Viviana. Serology assays to manage COVID-19. Science, v. 368, n. 6495, p.

[009]. The enzyme-linked immunosorbent assay (ELISA) is a simple and rapid technique, and can be used to detect and quantify antibodies or antigens. It is one of the most sensitive immunoassays, has been used in several diagnostic tests for various diseases and has allowed the development of a multibillion-dollar industry. Indirect ELISA is one of the ways to perform the assay, and is so called when the antigen remains adhered to the surface of a solid support and binds to the antibodies present in the biological sample. Subsequently, a secondary antibody is added. As it is conjugated to an enzyme, this secondary antibody will be responsible for the color change of the substrate, which is related to the amount of antibodies present in the original sample (LIN, Alice V. Indirect Elisa. In: ELISA. Humana Press, New York, NY, 2015. p. 51-59).

[010]. The antigens used in indirect ELISA assays can be crude extracts of the pathogen; in addition, soluble extract, whole proteins, protein fragments, peptides, among others. The appropriate choice of antigen is what guarantees diagnostic performance. Therefore, selecting the best antigen is a crucial step in developing a test (THOMAZ-SOCCOL, Vanete; PANDEY, Ashok; RESENDE, Rodrigo R. (Ed.). Current developments in biotechnology and bioengineering: Human and animal health applications . Elsevier, 2016). The two SARS-CoV-2 proteins most used in serological diagnostic tests are the Spike protein and the Nucleocapsid. These proteins play an important role in pathogenesis and are very antigenic, which activates the immune response and generates antibodies against these regions. However, other viral proteins also have the potential to be used as antigens in serological tests, as is the case with the ORF9b and ORF8 proteins.

[011]. The ORF9b protein of SARS-CoV-2 is an alternative open reading frame within the nucleocapsid gene. This protein interacts with TOM70, located on the outer membrane of mitochondria, and can significantly inhibit the production of IFN-I. This inhibition is a mechanism of evasion of the immune system by SARS-CoV-2, since the induction of IFN-I is a central mechanism of immune defense against viral infection. Furthermore, antibodies against ORF9b were found in the sera of patients convalescent from SARS-CoV and SARS-CoV-2 infection. This indicates that this protein may play a critical role in the interactions of coronaviruses with the host (JIANG, He-wei et al. SARS-CoV-2 Orf9b suppresses type I interferon responses by targeting TOM70. Cellular & molecular immunology, v. 17, n . 9, p. 998-1000, 2020).

[012]. The ORF8 accessory gene stands out for showing a series of specific characteristics. A single continuous copy of this gene is found in the genome, and is only present in lineage B β-coronaviruses (SARS-CoV and SARS-CoV-2). In the reference genome of SARS-CoV-2 (NC_045512.2), ORF8 has 366 nucleotides (positions 27.89428.259) and encodes the protein of the same name with 121 amino acids (PEREIRA, Filipe. Evolutionary dynamics of the SARS-CoV- 2 ORF8 accessory gene. The ORF8 protein proved to be highly immunogenic in patients infected with SARS-CoV-2, who showed early seropositivity for IgM, IgG and IgA anti-ORF8. The secretion of ORF8 during the first 48 hours of infection may promote an early B cell response against the viral antigen before or shortly after the onset of symptoms, leading to the detection of anti-ORF8 antibodies during the initial stage of infection. Furthermore, anti-ORF8 antibodies were detected in symptomatic and asymptomatic individuals. For this reason, the ORF8 protein is a viable target for the development of early and accurate serological diagnostic tests for COVID-19 (WANG, Xiaohui et al. Accurate diagnosis of COVID-19 by a novel immunogenic secreted SARS-CoV-2 orf8 protein . MBio, v. 11, n. 5, p.

[013]. Working with the whole or inactivated virus as an antigen for diagnostic tests is a risk, as a specific structure is needed to guarantee the safety and containment of this virus in the antigen production process. To resolve this limitation, recombinant proteins and synthetic peptides derived from viral proteins are highly explored alternatives (MA, Li-na et al. An overview on ELISA techniques for FMD. Virology journal, v. 8, n. 1, p. 1- 9, 2011). The production of viral proteins by recombinant means, mainly in Escherichia coli, presents some limitations. Among them is the difficulty of expression in the correct structure in the folding stage, as well as the glycosylation and purification of these proteins. In addition to increasing the cost associated with its production and storage. On the other hand, synthetic peptides can be produced chemically and with high purity, in addition to not depending on three-dimensional conformation. Peptides identify the response directed to a single dominant epitope, which guarantees the sensitivity of the diagnostic test and increases its specificity. Therefore, synthetic peptides are viable and convenient substrates for use as antigens in immunoassays (GOMARA, M. J.; HARO, I. Synthetic peptides for the immunodiagnosis of human diseases. Current Medicinal Chemistry, v. 14, n. 5, p. 531-546, 2007).

[014]. The rapid spread of COVID-19 across the globe has led to a global shortage of reagents and healthcare supplies. This high demand for inputs caused prices to increase and there was an international dispute. Brazil became dependent on foreign countries and had to import items such as diagnostic tests and vaccines. Therefore, efforts are needed to develop national solutions to combat the pandemic, to reduce dependence on international markets and consolidate national technologies.

[015]. To study the state of the art, a search was made for technological solutions published in patent documents related to the diagnosis of COVID-19. As the disease emerged in 2019, the search was carried out from December 2019 to July 2022.

[016]. Some technological solutions deal with RT-qPCR tests for diagnosing COVID-19. Such as the inventions: RU2720713C9, US20220090186A1, US10689716B1, WO2022018685A1 and CN111118228A. Others deal with lateral flow type tests, such as: US20210373018A1, US20210325388A1, EP3904877A1, WO2021216276A1 and CN111024954A. Some inventions describe ELISA tests for serological diagnosis of COVID-19 using recombinant proteins as antigen, such as documents KR20220024452A, CN111983226A and US8541003B2, US10948490B1, US20210340189A1, KR20210123234A and CN112881681A. Some of these inventions have the Spike protein as an antigen and others use the nucleocapsid protein, different from the present invention.

[017]. Patented inventions also exploit peptides for COVID-19 diagnosis by ELISA, such as WO2021227364A1, WO2022077613A1, and CN111978378B, but the antigens correspond to protein S and N epitopes, respectively. There are also inventions that deal with ELISA tests using combined peptides: US20220120737A1, WO2022015912A1 and CN111423496B, but they also correspond to epitopes of the S and N protein, having no relation to peptides designed here.

[018]. The ORF8 protein is used in an ELISA test for diagnosing COVID-19 in the invention WO2021219029A1, however it is used in association with other proteins. No invention was found on the Google Patents platform that dealt with the use of peptides from the ORF8 and ORF9b proteins in ELISA tests for the serological diagnosis of COVID-19.

[019]. A search was also carried out for scientific articles on the Google Scholar, Science Direct and PubMed platforms that dealt with the subject of the present invention. No work was found with the same content as the present invention. Some works are related to the topic, but they are not identical to the present invention, which has the novelty of using only antigenic epitopes.

[020]. Vedova-Costa, et al (2021) found that, in Brazil, only 7.6% of tests approved for the diagnosis of COVID-19 by the National Health Surveillance Agency (ANVISA) until February 2021 were ELISA tests. The two main viral proteins used as antigens in approved tests are the S and N proteins. Furthermore, the sensitivity and specificity provided by manufacturers range from 55 to 100% and 85 to 100%, respectively (VEDOVA-COSTA, Jean Michel Dela et al. A review on COVID-19 diagnostic tests approved for use in Brazil and the impact on pandemic control.

[021]. Wang, et al (2020) recombinantly produced the ORF8 protein in mammalian cells and used it in an ELISA test. The authors concluded that patients infected with SARS-CoV-2 had early seropositivity for IgM, IgG and IgA anti-ORF8. Therefore, the serological test that detects the anti-ORF8 IgG antibody can be used for the early and accurate diagnosis of COVID-19. In the study, all 29 patients with COVID-19 presented anti-ORF8 antibodies (WANG, Xiaohui et al. Accurate diagnosis of COVID-19 by a novel immunogenic secreted SARS-CoV-2 orf8 protein. MBio, v. 11, n. 5, p. e02431-20, 2020), however the authors used the complete protein as the antigen, and the sequence claimed here is not included.

[022]. Meinberger, et al (2021) produced recombinant ORF8 and used it in immunoblot and ELISA tests, with 12 samples positive for COVID-19 and expressed it in HEK293 cells for production. The authors describe that IgM isotype antibodies directed against the ORF8 protein were detected in the majority of patients (66.7%), but other isotypes had lower performance, such as IgA (16%) or IgG (32%) isotypes (MEINBERGER, Denise et al. Analysis of IgM, IgA, and IgG isotype antibodies directed against SARS-CoV-2 spike glycoprotein and ORF8 in the course of COVID-19, v. . In this case, it also deals with a complete protein as an antigen and the sequence claimed here is not contained 100% in the protein.

[023]. Hachim, et al (2020) produced recombinant ORF8 protein in Cos1 cells. The authors concluded that the ORF8 protein was the one that showed the greatest difference in signal between positive and negative samples for COVID-19 among the ORFs they tested. Furthermore, only the N, ORF3b and ORF8 antigens showed high sensitivity levels of 86.6 to 100%, for a panel of 61 individuals (HACHIM, Asmaa et al. ORF8 and ORF3b antibodies are accurate serological markers of early and late SARS -CoV-2 infection. Nature immunology, v. 21, n. 10, p. Likewise, the complete protein was used and not just an antigenic region, in addition to the sequence not being identical.

[024]. Wang, et al (2021) characterized the immunodominant regions in the ORF8 protein that were reactive in the serum of 40 patients infected with SARS-CoV-2. The main immunogenic epitopes found by the author are located in (1) the N-terminal alpha-helix region, (2) the residues covering beta sheets 2 and 3 and (3), the loop between beta sheets 4 and 5. According to authors, regions 10, 11 and 12 were not immunoreactive, and regions 13 and 14 showed low reactivity. (WANG, Xiaohui et al. Mining of linear B cell epitopes of SARS- CoV-2 ORF8 protein from COVID-19 patients. Emerging microbes & infections, v. 10, n. 1, p. 1016-1023, 2021). In the present invention, the P.SC2.ORF8.50 peptide was immunoreactive with serum from patients with COVID-19.

[025]. Li, et al (2021) evaluated the antibody response profile against the SARS-CoV-2 proteome. The authors concluded that the patterns and dynamics of IgG production against non-structural/accessory proteins are different from those produced against proteins S and N. The non-structural proteins investigated, including ORF9b, produced a strong IgG response (LI, Yang et al. Antibody landscape against SARS-CoV-2 reveals significant differences between non-structural/accessory and structural proteins. JIANG, et al. (2020) also concluded that in addition to the N and S1 proteins, there are significant antibody responses to the ORF9b and NSP5 proteins (JIANG, He-wei et al. Global profiling of SARS- CoV-2 specific IgG/IgM responses of convalescents using a proteome microarray. MedRxiv, 2020). The sequence claimed here is not 100% similar to the protein described by the authors, in addition to being a synthetic peptide and not a recombinant protein.

[026]. The present invention deals with two new synthetic peptide antigens originating from the ORF9b and ORF8 proteins of SARS-CoV-2 (Seq ID N°1 and Seq ID N°2, respectively). In addition to two serological diagnostic tests (indirect ELISA) for COVID-19 using these peptides as antigens. Both peptides correspond to artificial sequences, produced from laboratory modifications of natural sequences. The examples presented in this report demonstrated the immunoreactivity of these peptides with sera from individuals infected with SARS-CoV-2. The indirect ELISA assay using the P.SC2.ORF9b.27 peptides as antigen shows 95.5% sensitivity and 93.3% specificity for total antibodies (IgA, IgM and IgG), in a panel of 96 sera. Likewise, the indirect ELISA assay using the P.SC2.ORF8.50 peptide as antigen showed 90.9% sensitivity and 93.3% specificity. The sensitivity and specificity of the present tests are in line with the test parameters reported in the literature using the S and N proteins.

[027]. The present invention innovates by making use of peptides from accessory proteins, which represent a minority in relation to tests that use proteins S or N. At the current time of the pandemic, there is a demand for second generation tests, which serve to complement the existing tests, ensuring greater variability. This is because any mutations suffered by the virus in structural proteins would result in a loss of sensitivity of the available tests. This problem can be overcome by using accessory protein antigens. Based on the above, this work offers an original and promising serological test option for diagnosing COVID-19.

Description of the approach to the technical problem

[028]. At the current stage of the COVID-19 pandemic, the tools available for control are vaccines and diagnostic tests. Early diagnosis, followed by treatment and isolation of patients is a key point in controlling the spread of the disease. Our invention emerged in response to existing technical problems and the need to discover new immunological diagnostic markers for COVID-19.

[029]. Molecular diagnostic techniques, especially RT-qPCRs, are considered the gold standard for diagnosing COVID-19. However, these techniques have limitations regarding the virus detection period, which is only possible in the first weeks of the viral infection. Furthermore, developing countries are at a disadvantage due to lack of quality laboratory setup, adequate sample handling tools, safety equipment, and well-trained laboratory personnel. Furthermore, the RT-qPCR test is expensive, which makes access difficult for low- or medium-income populations. Finally, the technique presents a moderate rate of false-negative results (ADNAN, Nihad et al. Detection of SARS-CoV-2 by antigen ELISA test is highly swayed by viral load and sample storage condition. Expert Review of Anti-infective Therapy, v. 20, n. 3, p. 473-481, 2022).

[030]. Serological tests are used to identify the presence of antibodies against the pathogen, which is why they are considered an indirect diagnosis. IgM and IgA antibodies are produced first, and indicate the acute phase of the infection. IgG antibodies remain detectable for periods longer than seven weeks, so they allow diagnosis even after a long period of infection. Furthermore, both symptomatic and asymptomatic cases can be diagnosed using this technique. Serological tests also help with vaccination policies and allow monitoring the incidence of the disease in the population.

[031]. The ELISA technique overcomes the limitations presented by other techniques and is mainly indicated for testing in large quantities due to its relative low cost. Furthermore, it is a well-established technique, well disseminated among diagnostic laboratories and capable of automation, and presents a variety of possible antigens to be used. Antigens can be the inactivated pathogen, its fragments, recombinant proteins or even protein fractions called antigenic epitopes.

[032]. In the present invention, synthetic peptides were used instead of recombinant proteins as antigens, thus exploiting the antigenic potential of a few, or just one, epitope. Restricting the epitopes available for antigen-antibody interaction can offer an advantage in specificity to the test, without compromising sensitivity.

[033]. Synthetic peptides are commonly produced quickly on a large scale using automated equipment. In this way, enabling its use as an antigen in ELISA tests, which are also used for large-scale testing.

[034]. In this context, the present invention describes the use of two new antigens, synthetic peptides with artificial sequences P.SC2.ORF9b.27 and P.SC2.ORF8.50 for use in serological diagnosis. They are produced by chemical synthesis, on a large scale, and can be used in a COVID-19 testing platform. The indirect ELISA method allows the identification of IgA, IgM and IgG immunoglobulins, and can be used for diagnosis at different stages of virus infection, for symptomatic and asymptomatic cases. Therefore, the set of the present invention is a viable option for serological testing of COVID-19 and presents a difference between the vast majority of available tests by using peptides from little explored accessory proteins. In this way, more input options are available to control the pandemic.

Detailed description of the Invention

[035]. The present invention concerns two new synthetic peptides P.SC2.ORF9b.27 and P.SC2.ORF8.50. The P.SC2.ORF9b.27 peptide is characterized by containing the artificial sequence (SEQ ID NO. 1). This sequence is composed of base fragments from the SARS-CoV-2 ORF9b protein (code P0DTD2 from the NCBI platform - https://www.ncbi.nlm.nih.gov/), plus a hydrophilic tail. This tail is an artificial sequence of amino acids that were added at the N-terminal end in order to provide greater physicochemical stability and hydrophilicity to the molecule, in order to make the peptide more soluble in water. Furthermore, it has a positive charge, which increases the binding strength of the antigen with the solid support during the coating phase of the ELISA test.

[036]. Likewise, the peptide P.SC2.ORF8.50 is characterized by containing the artificial sequence (SEQ ID NO. 2). The base fragment of this sequence comes from the ORF8 protein of SARS-CoV-2 (code P0DTC8 from the NCBI platform - https://www.ncbi.nlm.nih.gov/). An artificial amino acid sequence was also added to the N-terminal region of the peptide.

[037]. Both antigens were synthesized in automated MultiPep RS equipment (Intavis Bioanalytical Instruments), using the chemical synthesis technique on a solid support. In summary, the α-carboxyl covalently bonds to a polymeric support or resin. Synthesis generally occurs in the C to N-terminal direction. The amino acids are protected by Fmoc groups, which are removed after the coupling step (MACHADO, Alessandra et al. Chemical and enzymatic synthesis of peptides: basic principles and applications. Química nova, v. 27, p. 781-789, 2004) . After synthesis, the peptides were purified by washing three times with tert-butyl-methyl ether. They were then frozen in an ultrafreezer at -80°C and lyophilized. After lyophilization, the peptides were in a solid state, which allows the antigens to be stored for long periods. Then, the peptides were hydrated and the protein concentration in solution was determined using the Micro BSA BCA Protein Assay Kit (ThermoFisher Scientific).

[038]. The P.SC2.ORF9b.27 and P.SC2.ORF8.50 peptides are immunoreactive with serum from individuals infected with SARS-CoV-2, and can be used to diagnose COVID-19 on various platforms, such as: ELISA, test lateral flow, immunoblotting, immunofluorescence, but are not limited to these.

[039]. The present invention also describes indirect ELISA assays for serological diagnosis of COVID-19, using the peptides P.SC2.ORF9b.27 and P.SC2.ORF8.50 as antigen. The two tests have the same steps, but differ in the antigen used. The steps are:

[040]. Coating at 4°C overnight of 96-well flat-bottom high-adhesion plates (Greiner Bio-one) with the peptides diluted in coating solution (PBS with 53% Na2CO3 and 42% NaHCO3, pH 9.6). Followed by blocking with blocking solution (0.05% Tween 20, 1-10% BSA in PBS) for one hour at 37°C. Followed by diluted serum samples (1:50 to 1:400) bound for 1 h at 37 °C. Followed by an HRP-labeled anti-human IgTotal (IgA, IgM, and IgG) secondary antibody (Invitrogen) at dilution ranging from 1:5,000 to 1:30,000 in dilution buffer (1% BSA, in PBS) for 1 h at 37° W. The development is done with TMB (3,3', 5,5'-tetramethylbenzidine), but is not restricted to this solution. After the incubation period, which varies from 5 to 45 minutes, the reaction is stopped with hydrochloric acid (HCl 5N) (Anidrol Produtos para Laboratórios). Finally, the absorbance is read at 450 nm. Between each step, 4 to 6 washes are performed with washing solution (0.138 M NaCl, 0.05% Tween 20). The method described here is detailed in example 2.

[041]. This invention is not limited to the specific reagents, methodology and protocols described herein, as these may vary. Furthermore, the terminologies used herein are only for the purpose of describing the procedures and are not intended to limit the scope of the present invention and will be limited by the appended claims. The present invention will be better understood by describing the results obtained described in examples 1, 2 and 3 that make up this descriptive report.

Example 1: Method of selection and production of synthetic peptides and in silico evaluation of physicochemical properties

[042]. The amino acid sequences of the peptides P.SC2.ORF9b.27 and P.SC2.ORF8.50 (SEQ ID No. 1 and SEQ ID No. 2, respectively) were defined from a prediction of B cell epitopes performed by bioinformatics. For this, the amino acid sequence of both proteins was downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/), whose codes are P0DTD2 and P0DTC8. Next, the protein sequence was scanned using four software programs: 1. Bepipred linear epitope prediction; 2. BepiPred-2.0: Sequential B-Cell Epitope Predictor; 3. RelativeSurfaceAccessilibity; 4. Exposed surface. (LARSEN, Jens Erik Pontoppidan; LUND, Ole; NIELSEN, Morten. Improved method for predicting linear B-cell epitopes. Immunome research, v. 2, n. 1, p. 1-7, 2006. JESPERSEN, Martin Closter et al . BepiPred- 2.0: improving sequence-based B-cell epitope prediction using conformational epitopes research, v. chains in proteins. Journal of molecular biology, v. 125, n.

[043]. Each protein residue received a predicted value for each of the four parameters obtained from the software listed above. The highest values for each parameter (20%) received a score of 0.25, the remainder received a score of zero for that parameter. At the end, the scores obtained for each parameter were added together. In this way, each residue can have a total score ranging from 0 to 1. With zero corresponding to non-significant in any parameter and one corresponding to significant values in the four parameters. In this way, it was possible to identify the dominant epitope regions within the ORF9b (Figure 1) and ORF8 (Figure 2) proteins. These regions were selected as the basis for the construction of the peptides P.SC2.ORF9b.27 and P.SC2.ORF8.50

[044]. Once the base fragments were identified, unnatural amino acids were added, thus composing SEQ ID No. 1 and SEQ ID No. 2 (Table 1). The objective of these insertions is to increase the solubility of the peptides, so that they can be manipulated in aqueous solution. The positively charged tail added to the N-terminal region allows better binding to the high affinity plate used in the ELISA assay. The physicochemical properties of the peptides were evaluated using the PepCalc online platform (https://pepcalc.com/). Table 1. Physicochemical properties of the new peptides P.SC2.ORF9b.27 and P.SC2.ORF8.50.

[045]. Next, the BLASTp tool was used to evaluate the coverage and identity of the peptide sequences with sequences from the organisms SARS-CoV-2 (taxid:2697049), MERS (1335626), H1N1 (taxid:114727) and humans (taxid:9606 ) (ALTSCHUL, Stephen F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research, v. 25, n. 17, p. 3389-3402, 1997).

[046]. The sequences SEQ ID No. 1 and SEQ ID No. 2 showed coverage of 84% and 87% respectively with the SARS-CoV-2 virus. Therefore, the peptides P.SC2.ORF9b.27 (SEQ ID No. 1) and P.SC2.ORF8.50 (SEQ ID No. 2) show similarity with the pathogen, which contributes to the high specificity of the test. However, the amino acid sequences of the peptides do not correspond to natural sequences. Furthermore, both peptides showed less similarity to MERS, H1N1 and human organisms, reducing the risk of cross-reaction and contributing to greater specificity of the diagnostic test.

[047]. Once the amino acid sequences of the peptides were defined, they were synthesized. The process was carried out in automated MultiPep RS equipment (Intavis Bioanalytical Instruments), using the solid matrix-anchored synthesis technique. After synthesis, the peptides were purified with successive washes with tert-butyl-methyl ether solvent. It was then frozen in a freezer at -80°C and went through a freeze-drying process in Modulyod Freeze Dryer (ThermoFischer) freeze-drying equipment until solidification. Finally, they were resuspended in ultrapure water, the concentration of the final solution was determined using the Micro BCA Protein Assay kit (MERRIFIELD, R. B. Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. Journal of the American Chemical Society, v. 85 , n. 14, p. 2149-2154, 1963).

Example 2: Reactivity of peptides with anti-ORF9b and anti-ORF8 antibodies and standardization of indirect ELISA assay to detect IgA, IgM and IgG antibodies

[048]. 66 biological samples from patients who tested positive for COVID-19 by the RT-qPCR test were collected. The collection and use of the material was approved by the Research Ethics Committee (CEP) No. 4,421,856. In addition, 30 negative serum samples collected from healthy individuals before December 2019 were used.

[049]. To perform the ELISA test, it is necessary to optimize the test conditions. This implies varying the mass of antigen per well, concentration of the blocking solution, serum dilution, dilution of the conjugated secondary antibody and reaction stopping time (Table 2). For the stage of standardizing the optimal reaction conditions, pools of positive and negative sera were created. Table 2. Parameters evaluated in the standardization of indirect ELISA assays.

[050]. The indirect ELISA assay begins with the dilution of the peptide in carbonate buffer (0.05M, pH 9.6) and other buffers can be used. The peptide concentration in the diluted solution ranges from 0.1 to 3.0 ng/μL. Then, the solution is pipetted into the wells of the high-adherence microplate (solid support) so that the peptide mass per well varies from 10 to 300 ng. The microplate remains incubated at 4°C overnight, allowing the peptide to bind and remain adhered to its surface. Solid supports include high-affinity microplates, which can be made of nitrocellulose, nylon, latex, polypropylene or polystyrene, but are not restricted to these.

[051]. Subsequently, the wells are washed four times with 1x washing solution (100 mM PBS, 0.05% Tween 20, pH 7.4). Bovine serum albumin (BSA) blocking solution ranging from 1 to 10% in PBS buffer, pH 7.4, is added. And another four to six washes are carried out.

[052]. Then, biological fluid samples (blood, serum, plasma, saliva, urine or tears) are added to the solid support. In this example the biological sample used was serum. Biological samples must be diluted and dilution rates ranged from 1:50 to 1:400. The sample diluent is the incubation buffer composed of casein at a concentration between 1.6 and 4.4% (w/v) in phosphate-saline buffer solution (PBS) pH 7.4. Molecules present in the samples that do not specifically interact with the immobilized antigen are removed by six washes.

[053]. Solutions of secondary antibodies conjugated with radioactive isotopes, enzymes or fluorophores, anti-human total Ig (IgA, IgM and IgG) are added. Secondary antibodies vary according to the target antibody of the test, and must be diluted in incubation buffer at dilutions of 1:5000 to 1:30000. The wells are washed six times with washing solution.

[054]. Subsequently, a developing solution is added to the solid support, such as TMB (3,3',5,5'-Tetramethylbenzidine Liquid Substrate) and OPD (o-phenylenediamine dihydrochloride), but not restricted to these. The incubation time varies from 5 to 60 minutes, after this time the reaction is stopped by adding a stop solution (such as 5N HCL). The signal emitted after the addition of enzyme substrates can be colorimetric, chemiluminescent or fluorescent, and quantification of this signal indicates the presence or absence of anti-SARS-CoV-2 antibodies.

[055]. Once this optimization stage was completed, it was possible to determine the optimal working conditions. This was used to evaluate the reactivity of a pool of positive and negative sera for the peptides P.SC2.ORF9b.27 and P.SC2.ORF8.50 (Figure 3). From these results, it was possible to conclude that both antigens showed stronger reactivity towards the pool of positive sera than the pool of negative sera. This indicates the immunoreactivity of these peptides with anti-ORF9b and anti-ORF8 antibodies present in the serum of individuals infected with SARS-CoV-2.

Example 3: Validation of the use of the peptides P.SC2.ORF9b.27 and P.SC2.ORF8.50 as antigens for diagnosing COVID-19

[056]. For antigen validation, the complete panel of 66 positive and 30 negative sera was used. These serum panels were evaluated by indirect ELISA using the peptides P.SC2.ORF9b.27 and P.SC2.ORF8.50. The protocol used was that described in example 2. Among the positive sera, there are symptomatic and asymptomatic cases of COVID-19.

[057]. The reading results were analyzed using the MedCalc software (https://www.medcalc.org/). From the analysis of the ROC curve, it was possible to determine the AUC indexes (area under the ROC curve), sensitivity, specificity, Youden's J index and the cut off value of the reaction between positive and negative (cut off) (Table 3).

[058]. The assay using the P.SC2.ORF9b.27 antigen showed a sensitivity of 95.5% and a specificity of 93.3% (Figures 4 and 5). The assay using the P.SC2.ORF8.50 antigen showed a sensitivity of 90.9% and a specificity of 86.7% (Figures 6 and 7). Both tests were able to differentiate groups of positive from negative individuals. Thus, the potential of antigens to detect the presence of anti-SARS-CoV-2 antibodies through a serological assay is demonstrated, both for symptomatic and asymptomatic cases of the disease. Table 3. Parameters of ELISA tests using the peptides P.SC2.ORF9b.27 and P.SC2.ORF8.50 as antigens for diagnosing COVID-19.

Description of Figures

[059]. Figure 1 - Predicted value for each residue of the SARS-CoV-2 ORF9b protein from the analysis of four in silico bioinformatics epitope prediction parameters. The blue area represents the identified immunodominant region that gave rise to the P.SC2.ORF9b.27 peptide. Residue number 1 corresponds to the N-terminus and 121 corresponds to the C-terminus.

[060]. Figure 2 - Predicted value for each residue of the SARS-CoV-2 ORF8 protein from the analysis of four in silico bioinformatics epitope prediction parameters. The blue area represents the identified immunodominant region that gave rise to the P.SC2.ORF8.50 peptide. Residue number 1 corresponds to the N-terminus and 97 corresponds to the C-terminus.

[061]. Figure 3 - Absorbance values obtained in the standardization of the indirect ELISA assay using the peptides P.SC2.ORF9b.27 and P.SC2.ORF8.50 as antigen. The absorbance values obtained for a pool of positive and negative sera for each antigen are shown.

[062]. Figure 4 - Receiver operator characteristic curve (ROC CURVE) of the ELISA test using the P.SC2.ORF9b.27 peptide for immunodetection of total antibodies (IgA, IgM and IgG) anti-ORF9b.

[063]. Figure 5 - Distribution of reactivity of positive and negative individuals in the indirect ELISA test using the P.SC2.ORF9b.27 peptide for immunodetection of total antibodies (IgA, IgM and IgG) anti-ORF9b. 66 positive sera and 30 negative sera were used.

[064]. Figure 6 - Receiver operator characteristic curve (ROC CURVE) of the ELISA test using the P.SC2.ORF8.50 peptide for immunodetection of total antibodies (IgA, IgM and IgG) anti-ORF8.

[065]. Figure 7 - Distribution of reactivity of positive and negative individuals in the indirect ELISA test using the P.SC2.ORF8.50 peptide for immunodetection of total antibodies (IgA, IgM and IgG) anti-ORF8. 66 positive sera and 30 negative sera were used.

Definitions

[066]. Antibodies or immunoglobulins (Ig) are blood proteins produced in response to a specific antigen. Antibodies chemically combine with substances that the body recognizes as foreign, such as bacteria, viruses, and foreign substances in the blood.

[067]. An antigen is a molecule capable of stimulating an immune response. Each antigen has distinct surface characteristics, or epitopes, resulting in specific responses. An epitope is a small portion of this molecule where the interaction with antibodies actually occurs. (ABBAS, A. K.; LICHTMAN, A. H. H.; PILLAI, S. Cellular and Molecular Immunology. ed. 9. Philadelphia: Elsevier Saunders, 2017).

[068]. Sensitivity refers to the test's ability to recognize infection (positive result) when the individual has actually been infected.

[069]. Specificity refers to the ability of the test to provide a negative result when the individual has not been infected.

[070]. Yonden's J index summarizes the performance of a diagnostic test, being calculated by the sum of sensitivity and specificity minus one unit. The AUC index demonstrates the peptide's ability to differentiate infected from uninfected individuals.

[071]. The cut off is the absorbance value used to determine a positive or negative result. It will be positive above the cut off and negative below.

Claims (10)

1. Peptide characterized by the fact that it comprises at least one sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2. 2. Peptide according to claim 1, characterized in that it is produced synthetically or recombinantly. 3. Peptide according to claim 1, characterized in that it is presented as an isolated sequence or is linked to a carrier such as, for example, bovine serum albumin (BSA) or hemocyanin (KLH). 4. Peptide according to claim 1, characterized in that it is for use as an antigen in the detection of binding molecules in immunological assays for serological diagnosis, detection of viral infection or epidemiological surveillance for COVID-19. 5. Use of the peptide according to claim 1, characterized in that it is for use in immunoenzymatic assay tests, Western Blot, immunofluorescence, immunohistochemistry, EIA, MEIA, immunoagglutination tests, electrochemical sensors, for detection of circulating antibodies or other binding molecules related directly or indirectly to COVID19. 6. Use of the peptide according to claim 1, characterized in that it is for use in the detection of immunoglobulins in samples of body fluids, such as serum, saliva, urine, blood, plasma, to detect the presence of circulating antibodies or other binding molecules against COVID-19 . 7. Method for using the peptides according to any of the preceding/preceding claims for immunodiagnosis of COVID-19 characterized by comprising the steps of: a) binding the antigenic peptide alone or any combination thereof to a solid support, more specifically, nitrocellulose, nylon, latex, polypropylene or polystyrene; b) expose the antigenic peptides bound to the solid support in: (a) a biological sample (serum, plasma, tear, urine, blood, containing patient antibodies, in conditions conducive to binding of the antibody to the antigen peptide; c) detect the presence of immunoglobulins (IgT, IgA, IgM, IgG, IgE) bound to the peptide, bound to the solid support in (b). 8. ELISA METHOD according to claim 7 and any of the previous claims, characterized by carrying out step (a) using the peptide in the concentration range 10 to 300 ng/well, which may be in its original form, with modification at their ends and in the form of polymers made up of repetitions of a peptide sequence, which can be separated by spacers or linkers. 9. ELISA METHOD according to claim 7 and any one of the previous claims, characterized by carrying out step (a) using a bovine serum albumin (BSA) blocking solution ranging from 1 to 10%, carrying out the step (b) by diluting the biological sample in incubation buffer (casein at a concentration between 1.6 and 4.4% (w/v) in phosphate-saline buffer solution pH 7.4) in the range of 1:50 to 1:400 , carrying out step (c) by diluting the secondary antibody at a concentration of 1:5000 to 1:30000 in incubation buffer. 10. USE OF THE PEPTIDE as defined according to any of the preceding/preceding claims, characterized by containing at least one physiologically acceptable adjuvant to be used in the preparation of immunogenic compositions.