Sars-cov-2 protein epitopes and use thereof in prevention and diagnosis of coronavirus infections

Abstract

The subject of the invention are novel peptides derived from SARS-CoV-2 coronavirus proteins, the peptides being immunoreactive epitopes that interact with convalescent serum, use thereof in prevention and diagnosis of SARS-CoV-2 infections, and an innovative SARS-CoV-2 vaccine, comprising immunoreactive peptides and a thermostable nanoadjuvant that enables effective intranasal administration.

Description

[0001] SARS-CoV-2 protein epitopes and use thereof in prevention and diagnosis of coronavirus infections

[0002] The subject of the invention are novel peptides derived from SARS-CoV-2 coronavirus proteins, said peptides being immunoreactive epitopes intereacting with convalescent serum, use thereof in the prevention and diagnosis of SARS-CoV-2 infections, and an innovative SARS-CoV-2 vaccine, comprising immunoreactive peptides and a thermostable nanoadjuvant enabling effective intranasal administration.

[0003] State of the art

[0004] According to the data collected by the World Health Organization (WHO) in 2022, the COVID-19 pandemic resulted in more than 450 million cases worldwide and more than 6 million deaths. COVID-19 is a disease caused by the SARS-CoV-2 coronavirus, which is characterized by rapid spread and a high mutation rate. SARS-CoV-2 is a typical mucosal pathogen that is transmitted from person to person via droplet route. It infects human respiratory epithelial cells, binding to angiotensin-converting enzyme 2 (ACE2) receptors via the viral receptor-binding domain (RBD). The development of highly effective COVID-19 vaccines played a pivotal role in controlling and preventing the pandemic. The vaccines are based on 5 main platforms: messenger RNA (mRNA), viral vector, inactivated virus, subunit (consisting of proteins or peptides), and DNA [WHO R&D Blueprint Team. COVID-19 Vaccine Tracker and Landscape. World Health Organisation 2021. https: / / www.who. int / covid-19 / vaccinesl.

[0005] Spike protein (S protein), located outside the virion, is the primary antigen in vaccines against COVID-19. It is an effective target for neutralizing antibodies that block and prevent S binding to ACE2 receptors on host cells or S rearrangement, thus preventing the virus entry. Different S protein domains are used as vaccine antigens, e.g. N-terminal domain (NTD) or RBD [Buchholz UJ, Bukreyev A, Yang L et al. Contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity. Proc Natl Acad Sci USA 2004;101 :9804-9], For example, the BNT162b2 vaccine (developed by Pfizer and BioNTech) and the mRNA-1273 vaccine (developed by Moderna) consist of mRNA surrounded by lipid nanoparticles. mRNA is injected intramuscularly, where it is taken up by host cells and translated. Due to the relative fragility of mRNA, it needs to be stored in very low temperatures. Such requirements impede the manufacturing, storage and distribution of vaccines. mRNA is also less stable in vivo, which often requiring the development of stabilizing elements, e.g. base modification to incorporate N1 -methyl-pseudouridine (ml ^P). Viral vector vaccines use genetically modified virus which infects human cells. S protein gene of SARS-CoV-2 is inserted into a non-human adenoviral vector. For instance, Vaxzevria vaccine (developed by AstraZeneca and University of Oxford) comprises ChAdOxI chimpanzee adenovirus, with a replication defect caused by E1 and E3 deletion. The S protein gene embedded in the vector has optimized codons with tissue plasminogen activator (tPA) leader sequence [Voysey M, Clemens SAC, Madhi SA et al. Safety and efficacy of the ChAdOxI nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK. Lancet 2021 ;397:99-111 ], Next, NVX-CoV2373 vaccine (Nuvaxovid) developed by Novavax is a subunit vaccine comprising a full-length recombinant trimeric perfusion S protein (SARS- CoV-2-3Q-2P) and a saponin-based Matrix-M nanoadjuvant. The protein was produced by recombination using protein expression in insect cells with the baculovirus system [Paul T. Heath et al. Safety and Efficacy of NVX-CoV2373 Covid-19 Vaccine. N Engl J Med 2021 ; 385:1172-1183 DOI:

[0006] 10.1056 / NEJMoa2107659],

[0007] Mucosal immunity is essential for adequate and long-lasting protection against a viral infection. All licensed COVID-19 vaccines are administered via intramuscular injection, being ineffective in inducing mucosal immunity. They do not prevent upper respiratory track viral infections, which in part due to a failure to activate mucosal immunity. Furthermore, emerging novel SARS-CoV-2 variants highlights the need for the next generation of vaccines against COVID- 19, and intranasal vaccination method is highly desirable for effective induction of both mucosal and systemic immune response. As many as 7 different intranasal vaccines are currently being tested in clinical studies [Carl Zimmer, Jonathan Corum, Sui-Lee Wee, Coronavirus Vaccine Tracker, https: / / www.nytimes.com / interactive / 2020 / science / coronavirus-vaccine-t, Yury Valdes-Balbin, Darielys Santana-Mederos, Lauren Quintero, Sonsire Fernandez, Laura Rodriguez, Belinda Sanchez Ramirez, Rocmira Perez-Nicado, Claudia Acosta, Yanira Mendez, Manuel G. Ricardo, Tays Hernandez, Gretchen Bergado, Franciscary Pi, Annet Valdes, Tania Carmenate, Ubel Ramirez, Reinaldo Oliva, Jean-Pierre Soubal, Raine Garrido, Felix Cardoso, Mario Landys, Humberto Gonzalez, Mildrey Farinas, Juliet Enriquez, Enrique Noa, Anamary Suarez, Cheng Fang, Luis A. Espinosa, Yassel Ramos, Luis Javier Gonzalez, Yanet Climent, Gertrudis Rojas, Ernesto Relova-Hernandez, Yanelys Cabrera Infante, Sum Lai Losada, Tammy Boggiano, Eduardo Ojito, Kalet Leon, Fabrizio Chiodo, Frangoise Paquet, Guang-Wu Chen, Daniel G. Rivera, Dagmar Garcia-Rivera, and Vicente Verez Bencomo, SARS-CoV-2 RBD-Tetanus Toxoid Conjugate Vaccine Induces a Strong Neutralizing Immunity in Preclinical Studies. ACS Chem. Biol. 2021 , 16, 1223-1233. https: / / doi.Org / 10.1021 / acschembio.1 c00272, Xingyue An, Melisa Martinez-Paniagua, Ali Rezvan, Samiur Rahman Sefat, Mohsen Fathi, Shailbala Singh, Sujit Biswas, Melissa Pourpak, Cassian Yee, Xinli Liu, Navin Varadarajan. Single-dose intranasal vaccination elicits systemic and mucosal immunity against SARS-CoV-2. iScience 24, 103037, September 24, 2021. https: / / doi.Org / 10.1016 / j.isci.2O21 .103037, Xuan-Yi Wang, Bin Wang and Yu-Mei Wen, From therapeutic antibodies to immune complex vaccines. Vaccines (2019) 4:2; https: / / doi.Org / 10.1038 / s41541 -018-0095-z, Amanda Przedpelski, William H. Tepp, Sabine Pellett, Eric A. Johnson, Joseph T. Barbieri. 2020. A novel high-potency tetanus vaccine. mBio 11 :e01668-20. https: / / doi.Org / 10.1128 / mBio.01668-20, Aqu Alu, Li Chen, Hong Lei Yuquan Wei Xiaohe Tian, and Xiawei Wei. Intranasal COVID-19 vaccines: From bench to bed. eBioMedicine 2022;76:103841 . Published online 24 January 2022 https: / / doi.Org / 10.1016 / i.ebiom.2022.103841 , Sonia Jangra, Jeffrey J. Landers, Raveen Rathnasinghe, Jessica J. O’Konek, Katarzyna W. Janczak, Marilia Cascalho, Andrew A. Kennedy, Andrew W. Tai, James R. BakerJr., Michael Schotsaert and Pamela T. Wong. A Combination Adjuvant for the Induction of Potent Antiviral Immune Responses for a Recombinant SARS-CoV-2 Protein Vaccine. Front. Immunol. 12:729189. doi: 10.3389 / fimmu.2021 .729189, Zezhong Liu, Wei Xu, Shuai Xia, Chenjian Gu, Xinling Wang, Qian Wang, Jie Zhou, Yanling Wu, Xia Cai, Di Qu, Tianlei Ying, Youhua Xie, Lu Lu, Zhenghong Yuan, Shibo Jiang. RBD-Fc-based COVID-19 vaccine candidate induces highly potent SARS-CoV-2 neutralizing antibody response. Signal Transduction and Targeted Therapy (2020) 5:282; https: / / doi.Org / 10.1038 / s41392-020-00402-5]. These include products from e.g. Indian, Chinese and British manufacturers. Six of them use viral vectors, and the seventh vaccine comprises an inactive fragment of an immunity-stimulating protein. Intranasal vaccination is not new - a flu vaccine administered in the form of an aerosol already exists. However, in contrast to the Covid-19 vaccine, it contains attenuated, but still active viruses. Consequently, it is not recommended for individuals with compromised immune system, including individuals with diabetes, HIV or undergoing antineoplastic therapy. Protein S, among others, that enables the virus to infect cells, was used to produce aerosol vaccine against coronavirus on the market. However, it was highly attenuated and stabilized, and then incorporated into adenovirus. This makes it a type of vector vaccine, similar to products from Astra Zeneca and Janssen (Johnson & Johnson).

[0008] Authors of Aqu Alu et al., Intranasal COVID-19 vaccines: From bench to bed, eBioMedicine 2022;76: 103841 present a compilation of existing knowledge regarding optimization of COVID-19 vaccines manufacturing. Intramuscular vaccines are currently not effective enough to prevent upper respiratory viral infection, partly due to failure to activate mucosal immunity. Intranasal vaccines have the potential to induce both mucosal and systemic immunity. In particular, the authors emphasize the need to provide improved intranasal adjuvants that will ensure high effectiveness and safety, while forming a simple and stable composition, easy to manufacture and affordable.

[0009] The object of the invention is to provide a safe and effective SARS-CoV-2 vaccine formulation.

[0010] The subject of the invention is a SARS-CoV-2 protein peptide having sequence selected from: SEQ NO 1 , SEQ NO 2, SEQ NO 3, SEQ NO 4 or SEQ NO 5.

[0011] Preferably, the peptide is conjugated to a carrier protein.

[0012] Preferably, the carrier protein is KLH or TT or equivalent. Preferably, the peptides are produced synthetically or by recombination.

[0013] Another subject of the invention is the said peptide, optionally conjugated to a carrier protein, such as KLH, TT or identical, for use in inducing immunological response against SAR-CoV-2 virus in a patient, prevention and treatment of COVID-19 disease caused by SAR-CoV-2 coronavirus infection.

[0014] Preferably, the peptide is administered intranasally.

[0015] Another subject of the invention is a vaccine composition comprising the SARS-CoV2 protein peptide having sequence selected from: SEQ NO 1 , SEQ NO 2, SEQ NO 3, SEQ NO 4 or SEQ NO 5, optionally conjugated to a carrier protein, such as KLH, TT or identical, and a pharmaceutically acceptable carrier and / or adjuvant or nanoadjuvant.

[0016] Preferably, the nanoadjuvant comprises 65% emulsified soybean oil, 8% ethanol, 21 % water, 5% Tyloxapol, 1 % benzalkonium chloride.

[0017] Another subject of the invention is the vaccine composition as defined above for use in inducing immunological response against SAR-CoV-2 virus in a patient, prevention and treatment of COVID-19 disease caused by SAR-CoV-2 coronavirus infection.

[0018] Preferably, the vaccine composition is administered intranasally.

[0019] Another subject of the invention is a method for detecting a coronavirus infection, in particular SARS-CoV2 infection, characterized in that

[0020] (a) the peptide as defined in claim 1 is provided,

[0021] (b) the peptide as defined in claim 1 is contacted with a biological sample from a patient, e.g. blood and saliva, presence of complexes of the peptide as defined in claim 1 with antibodies specific to the peptide as defined in claim 1 is detected, wherein the presence of such complexes is indicative of the patient being infected with the virus.

[0022] Detailed description of the invention Selection of vaccine antigens

[0023] The vaccine antigens were selected based on the reverse vaccinology method, i.e. the immunoreactivity profile of convalescent sera with viral proteins was investigated. Reactivity of SARS-CoV-2 lysate protein mixture with sera from convalescents, ill individuals and pre-pandemic healthy individuals was analyzed for the presence of IgM, IgG and IgA classes of antibodies (immunoblotting). As it had been demonstrated that the antibody classes of interest are reactive against S and M proteins, they were selected as the focus of investigation.

[0024] Bioinformatic prediction analyses of B, T and IFN epitopes were performed, and the results obtained were compared to available results from literature. The natural process of acquired immunity to the pathogen was considered while selecting antigens for the vaccine, by means of analysis with immunoreactive viral structures. These results were complemented by genomic sequencing of SARS-CoV-2 viruses present in the Polish population. Bioinformatics data on known potential epitopes that do not cross-react with human tissues were also considered in the selection process to ensure vaccine safety.

[0025] Determination of peptide sequences recognized by antibodies using Pepscan epitope mapping method

[0026] The smallest reactive peptide sequences were selected. Application of the epitope mapping technology using synthetic peptides on rods allowed also for determination of relevant amino acids in the epitopes for interactions with sera. Immunoreactivity with sera from convalescents and healthy individuals was tested to verify that correct peptides / epitopes were selected for the vaccine.

[0027] Conjugation of five selected peptides to carrier protein; immunoreactivity and cytotoxicity tests

[0028] As a part of the efficacy and safety testing, the immunoreactivity of the conjugates with sera from convalescents and healthy individuals was analyzed to confirm their suitability for the vaccine. The conjugates were tested for cytotoxicity on three human lines of nasal and pulmonary epithelium and cerebral microvascular endothelium, demonstrating no cytotoxicity and no cytokine induction. The research task regarding the influence of investigated preparations on blood-brain barrier integrity and cytokine secretion was conducted on lines of human astrocytes and human cerebral capillary endothelium. It was demonstrated that the antigen-carrier protein conjugates are not neurotoxic in in vitro tests on cell lines and do not induce NO. The conjugates were demonstrated to have no effect on tight junction protein expression, in the human blood-brain barrier model, no toxic influence on cells in this barrier, as well as no effect on blood-brain barrier integrity and cytokine secretion. Better selection of antigens for the vaccine was facilitated by surface plasmon resonance (SPR) analysis (performed using BiaCore) of the strength of interaction between the conjugates and convalescent antibodies.

[0029] Formulation of nanoadjuvant with conjugates

[0030] Adjuvant research, conducted in parallel, revealed the phenomenon of selective response to antigen in the conjugate following intranasal administration, while intramuscular administration induces a response to the conjugate carrier. Based on these results, it is possible to provide systemic protection against the administered epitope bound in the conjugate following its intranasal administration. Nanoadjuvant formulations based on Pharmacopeia-listed ingredients, used in pharmacological preparations as intranasal suspensions, were developed.

[0031] In vivo experiments

[0032] Nanoconjugate is prepared by mixing the ingredients and mechanical emulsification.

[0033] Mice were subjected to intranasal immunization with K1 -K5 conjugates in the presence of the developed nanoadjuvant. Functionality of the nanoadjuvant was determined as compared to a model adjuvant. Intranasal administration of the conjugates mixed with the nanoadjuvant induces specific IgG and IgA antibodies (with adjuvant, K1 -K3), regardless of adjuvant type (K2), and with no adjuvant added (K2 and K3). Results of clinical and pathomorphological studies demonstrated that no harmful effects of the vaccine were indicated by the animals’ clinical condition. The conjugates induce systemic humoral and cellular response (analyzed in serum, BAL, and spleen cells). Analysis of intracellular cytokine secretion by stimulated splenocytes following intranasal vaccination in mice demonstrated that the vaccine induces cellular response after intranasal administration. Bioinformatic analysis indicated that selected peptides were not mutated in Omicron variant, therefore all selected epitopes remain protective in the vaccine, including protection against the Omicron variant. Also, Omicron mutations BA.2, BA.4, BA.5 do not affect peptide sequences used for the vaccine. Safety of the vaccine is further supported by the fact that its peptide ingredients do not comprise any sequence fully (100%) overlapping with and identical to human proteins.

[0034] The invention provides a safe and effective vaccine formulation against SARS-CoV-2 virus at the level of in vitro tests on cell lines and in vivo tests on the murine animal model, confirmed in GLP studies. The vaccine according to the invention comprises exclusively immunoreactive, protective antigens which immunize in the natural immunity process, not cross-reacting with human tissues nor crossing the blood-brain barrier. Intranasal formulation does not require the use of needles, reduces mass vaccination cost, facilitates repeated immunization, is less invasive, does not cause stress related to vaccination, extends indications for immunization, is more convenient to use, provides good systemic response, demonstrates potential to protect against Omicron variants, ensures better protection of mucous membranes and portals of entry, simultaneously providing effective IgG, IgA and cellular response. The peptide vaccine according to the invention does not depend on carbohydrate coat of glycoproteins having unknown structure, composition and function as a result of their biosynthesis following vaccination with other vaccines. Such a solution offers full control over immunization and - based on the platform developed - enables rapid adjustment of the vaccine to a new viral variant.

[0035] Description of figures

[0036] Fig. 1 Immunoreactivity of sera from convalescents and pre-pandemic healthy individuals with SARS-CoV-2 lysate. Electrophoretic analysis of SARS-CoV-2 lysate proteins. Fig. 2 Reactivity of viral proteins with IgA in individual convalescent sera.

[0037] Fig. 3 Immunoreactivity of viral proteins with serum - immunoblotting method.

[0038] Fig. 4 Study of immunoreactivity profile of convalescent sera with viral proteins. Reactivity of serum samples from the same patient, collected on day 0 and after 3 weeks.

[0039] Fig. 5 Identification of immunoreactive proteins. Analysis of reactivity of IgG antibodies in serum with selected peptides (5 most reactive and one control). Peptides were synthesized on polyethylene rods. A higher response in convalescents than in ill individuals may be indicative of strong protective properties. In vaccinated individuals, a high response to membrane protein-derived sequences GAVILRGHLRIAGHHLGR and ATSRTLSYYK was reported.

[0040] Fig. 6 Identification of immunoreactive proteins. Analysis of reactivity of IgA antibodies in serum with selected peptides (5 most reactive and one control). Peptides were synthesized on polyethylene rods.

[0041] Fig. 7a and 7b In vitro studies of conjugates. Cytotoxicity of conjugates and peptides (24 h induction). Test conducted on murine TC-1 cell line.

[0042] Fig. 8 In vitro studies of conjugates. Cytotoxicity of conjugates and peptides (24 h induction). Test conducted on primary human HSAEC, human nasal epithelial RPMI2650 and human lung carcinoma A549 cell lines.

[0043] Fig. 9 In vitro studies of conjugates. Cytotoxicity of conjugates and peptides (6 h and 24 h induction) in a test conducted on human cerebral microvascular endothelial HBEC-5i line.

[0044] Fig. 10 In vitro studies of conjugates. Analysis of activation of TLR2, TLR4, TLR5 and N0D2 receptors by peptides and conjugates.

[0045] Fig. 11 In vitro studies of conjugates. Induction of cytokines by conjugates and peptides - murine TC-1 line (24 h culture).

[0046] Fig. 12 In vitro studies of conjugates. Induction of cytokines by conjugates and peptides - human HSAEC line (24 h culture). Fig. 13 Induction of cytokines by conjugates and peptides - human RPMI 2650 line (24 h culture).

[0047] Fig. 14 Induction of cytokines by conjugates and peptides - human A549 line (24 h culture).

[0048] Fig. 15 IgG antibody level determination in murine serum. Conditions: conjugate 50 pg / ml; sera 1 :50; anti-IgG 1 :10000 (with AP). Legend: red bar - plate coated with conjugate, green bar - plate coated with KLH.

[0049] Fig. 16 IgA antibody level determination in serum. Conditions: conjugate 50 pg / ml; sera 1 :10; anti-lgA 1 :10000 (with AP). Legend: red bar - plate coated with conjugate.

[0050] Fig. 17 Intracellular cytokine secretion by stimulated splenocytes following intranasal vaccination in mice (intracellular cytokine staining, ICS). In groups of mice vaccinated intranasally with A1 , A1+NAC and A1 +MF59, an increased percentage of population of CD4+ and CD8+ cells secreting IFN-y and TNF-a was observed when compared to control groups: PBS, NAC adjuvant and MF59 control adjuvant. Expression of CD69 marker was not changed. The results indicate cellular response after intranasal administration of the vaccine.

[0051] Fig. 18 Splenocyte (CD4+ and CD8+) response to A1 and A3 stimulation with regard to IL-2 and IL-4 production. A1 antigen induced IFN-y and IL-2 cellular response in CD4+ lymphocytes both alone and in combinations with NAC and MF59 adjuvants. A1 antigen induced cellular response by IFN-y induction in combination with MF59 adjuvant. A3 antigen induced IFN-y and IL-2 in CD8+ lymphocytes in combination with MF59 adjuvant. A3 antigen induced IL-2 also in combination with MF59 adjuvant.

[0052] Description of reference symbols:

[0053] P1 - KVGGNYNYLYRLFRKSNLKPC (SEQ ID NO: 1 )

[0054] K1 is identical to A1 - conjugate KVGGNYNYLYRLFRKSNLKPC - KLH

[0055] P2 - LPDPSKPSKRSFIEDLLFNKC (SEQ ID NO: 2) K2 is identical to A2 - conjugate LPDPSKPSKRSFIEDLLFNKC - KLH

[0056] P3 - GAVILRGHLRIAGHHLGRC (SEQ ID NO: 3)

[0057] K3 is identical to A3 - conjugate GAVILRGHLRIAGHHLGRC - KLH

[0058] P4 - ATSRTLSYYKC (SEQ ID NO: 4)

[0059] K4 - conjugate ATSRTLSYYKC - KLH

[0060] P5 - TESNKKFLPFQQFGRDIAC (SEQ ID NO: 5)

[0061] K5 - conjugate TESNKKFLPFQQFGRDIAC - KLH

[0062] Example 1

[0063] Preparation of sample set of convalescent and control serum

[0064] Biological material for testing was collected from COVID-19 patients admitted to the J. Gromkowski Regional Specialist Hospital (pol. Wojewodzki Szpital Specjalistyczny im. J. Gromkowskiego). Hospital staff collected blood (venous, 5 ml) and nasopharyngeal swabs from convalescents, patients in acute phase of the disease upon hospital admission and controls, and secured the samples for further testing. In hospital laboratory serum was obtained and concentration of IgG and IgM class specific antibodies was determined, and then the biological material was transferred to IITD (pol. Instytut Immunologii i Terapii Doswiadczalnej, Institute of Immunology and Experimental Therapy) for testing. Swab and blood samples were collected from 89 patients (first collection); second blood collection included 19 samples. Patient age ranged from 32 to 82 years. Among the patients there were 36 females and 53 males, 85% were undergoing antibiotic therapy, 1 person had undergone SARS-CoV-2 infection and 1 person had received the first dose of Covid-19 vaccine. Due to plasma therapy applied, the number of patients who did not receive plasma was significantly lower, which extended the time required to collect serum samples from such individuals. Apart from that, pre-pandemic serum and umbilical blood serum samples, leftover from other projects, and sera from individuals who received 2 vaccine doses were collected for comparison purposes. The broad panel of serum samples allowed for an in-depth evaluation of their reactivity with SARS-CoV-2 proteins, which in turn facilitated selection of immunoreactive proteins as vaccine antigens for further testing.

[0065] Example 2

[0066] Immunoreactivity analysis, establishing epitope structure, conjugate preparation

[0067] First research step included selection of antigens for the vaccine, thus firstly immunoreactivity of SARS-CoV-2 antigens (commercially available SARS-CoV-2 lysate) was analyzed using patient sera (obtained in example 1 ), recombinant viral proteins (commercially available SARS-CoV-2 proteins: S, M, N and E protein), and peptides selected by means of bioinformatic in silico methods (obtained by PEPSCAN method) [J.M. Carter, Epitope Mapping of a Protein Using the Geysen (PEPSCAN) Procedure, in: Peptide Analysis Protocols, Humana Press, New Jersey, 2014: pp. 207-224. http: / / link.springer.eom / 10.1385 / 0-89603-274-4:207 (accessed February 25, 2014)]. The peptides were synthesized on NCP-type polyethylene pins (Mimotopes) by addition of individual amino acids via coupling reaction on a 96-well plate. F-moc amino acid derivatives with blocked side groups (Mimotopes) were used for the synthesis. In the first step, the pins were deprotected using 20% piperidine solution in DMF for 1 hour in a sealed container. Then, following 2 min DMF wash and 4 x 1 min methanol wash, the pins were dried for 1 hour under fume hood. Subseguently, the coupling reaction was performed, by immersing the pins in 100 pl of 60 mM amino acid, 60 mM DIC and 65 mM HOAt solution in DMF. Reaction progress was monitored by addition of 0.5 mM bromophenol - discoloration of the solution indicated that the coupling reaction was completed (typically after approximately 4 h). The reaction was carried out at room temperature, in a sealed container, in order to prevent the solution from evaporating. After washing off unbound amino acids with methanol (5 min), drying and 5 min incubation in DMF, the pins were subjected to another deprotection and coupling reaction, until a peptide having seguence of set length was obtained. Then, the peptides were deprotected to remove chemical compounds that block side groups, using 2.5% anisole and 2.5% dithioethane solution in trifluoroacetic acid. Following final washing (methanol 10 min, solution of 0.5% acetic acid in 50% methanol in water 1 x 1 h, methanol 2 x 2 min) and drying, pins with bound peptides were stored at -20°C. After full-length peptide synthesis, and after each immunoenzymatic analysis, the pins were disrupted in order to purify them. Disruption buffer consisted of 1 % SDS, 0.1 % [3-mercaptoethanol, 0.1 M sodium phosphate, pH 7.2. Sonicator was filled with the disruption buffer heated up to 55-65°C. Polyethylene blocks were immersed in the disruption solution, with pins facing down, and sonicated for 10 min (7 kW / 25 kHz).

[0068] Analysis of reactivity with sera was performed by immunoblotting methods (Fig. 1 to Fig. 4) and ELISA test.

[0069] Immunoblotting

[0070] Immunoreactivity of viral proteins (viral lysate) separated in polyacrylamide gel was tested using the Western blot method. Electrophoresed SDS-PAGE gel (according to Laemmli et al.) was incubated in transfer buffer (10 mM Tris-HCI, 150 mM glycine, 20% methanol, pH 8.3) for 30 min, and then was transferred under semi-dry conditions onto a PVDF Immobilon P membrane (Merck Millipore) using Trans Blot® SD Semi-Dry Transfer Cell system (Bio-Rad). The transfer was carried out at 25 V for 60 min. Free sites on the PVDF membrane were blocked with 1 % BSA solution in TBS (Tris buffered saline) buffer comprising 20 mM Tris, pH 7.5, 150 mM NaCI, pH 7.5, with the addition of 0.1 % Tween®20 (TBS-T) for 1 hour at room temperature with mixing. Primary antibodies (blood serum from a selected group of patients), diluted 1 :100 in TBS-T buffer with the addition of 0.1 % BSA solution, were added and the membrane was incubated overnight in 4°C. Next, the membrane was washed

[0071] 3 times in TBS-T, each time for 10 min, to remove unbound antibodies. In the next step, anti-human IgG or IgA secondary antibodies, conjugated to alkaline phosphatase (Sigma-Aldrich), diluted 1 :10000 in TBS-T buffer, were added and incubated for 1 hour at room temperature with mixing. Another washing was performed as described above, followed by induction of color reaction by adding NBT and BCIP mixture as alkaline phosphatase substrate. The reaction was stopped by washing in MiliQ water. Membrane documentation was prepared using Gel Doc™.

[0072] Immunoenzymatic test Immunoreactivity of the peptides obtained was tested using immunoenzymatic ELISA method [A. Jarzqb, D. Witkowska, E. Ziomek, A. Dqbrowska, Z. Szewczuk, A. Gamian, Shigella flexneri 3a Outer Membrane Protein C Epitope Is Recognized by Human Umbilical Cord Sera and Associated with Protective Activity, PLoS ONE. 8 (2013) e70539. https: / / doi.org / 10.1371 / journal.pone.0070539]. Sera from COVID-19 patients, convalescents, pre-pandem ic healthy individuals and individuals who received two COVID-19 vaccine doses were used in the test. In the first step, pins were thawed and equilibrated in TBS-T solution for 10 min at room temperature. Free sites on the pins were blocked by incubating them in 1 % BSA solution in TBS-T for 1 h at room temperature. Sera from study groups were diluted 1 :1000 in TBS-T before use. Pins were incubated in serum solution for 2 hours at room temperature. Anti-human IgG, IgA secondary antibodies conjugated to alkaline phosphatase were diluted 1 :10000 in TBS-T solution before use; pins were incubated in secondary antibody solution for 1 hour at room temperature. Color reaction was induced for 30 min using phosphatase substrate - Alkaline Phosphatase Yellow Liquid Substrate System (Sigma-Aldrich). Following absorbance determination at 405 nm (PowerWave HT, BioTek Instruments, Winooski, USA), the antibodies were washed off from the pins by 10 min sonication in disruption solution. Following pin washing in water at 60°C (1 x 10 min) and methanol at 60°C (1 x 10 min), the pins were dried and frozen at -20°C for long-term storage or stored at 4°C for use within the next few days. Each test was repeated at least four times. Reactivity of sera with synthetic peptides as epitopes was carried out on polyethylene rods (pins, pegs) using PEPSCAN technology. This analysis provided a representation of reactivity of IgG and IgA classes of antibodies for selection of the vaccine antigen (Fig. 5 and Fig. 6).

[0073] Search for functional epitopes was enabled by bioinformatic analysis of immunoreactive viral epitopes. Thus, by means of the PEPSCAN (pin) method, several hundreds of peptides were synthesized on polyethylene rods and their reactivity with sera was determined, which enabled selection of vaccine peptides. Sequence analysis of epitopes from databases was complemented by the epitope prediction method for B lymphocytes, for T lymphocytes and epitope analysis for IFNy based on BEPIPred, BCPred, TepiTool and IFNepitope tools. This analysis enabled selection of shared sequences, comparison of these sequences with literature data, and subsequent synthesis of selected epitopes and analysis of their immunoreactivity with sera. One of immunoreactive peptide sequences was identified as KVGGNYNYLYRLFRKSNLKP - Spike444-463 (a sequence recognized by neutralizing antibodies). Owing to such approach, several epitopes of spike (S) and membrane (M) protein, apart from the S1 subunit of spike protein, could be picked as potential vaccine antigens.

[0074] Selection of peptides / epitopes, synthesized on rods, for the vaccine was confirmed by testing immunoreactivity with sera from convalescents and healthy individuals. Serum IgA antibody response is weaker than in the case of IgG, but strongest in convalescents. Peptides best recognized by convalescent antibodies were selected. The original spike protein peptide from RBD region (KVGGNYNYLYRLFRKSNLKP) eventually remained unchanged, as its recognition by tested sera was not significantly affected by implemented mutations. That also means that in the case of a natural mutation, the vaccine should remain effective.

[0075] Eventually, 5 epitopes were picked:

[0076] KVGGNYNYLYRLFRKSNLKPC spike proteins Preparation 1

[0077] LPDPSKPSKRSFIEDLLFNKC spike proteins Preparation 2

[0078] GAVILRGHLRIAGHHLGRC membrane protein Preparation 3

[0079] ATSRTLSYYKC membrane protein Preparation 4

[0080] TESNKKFLPFQQFGRDIAC spike protein Preparation 5

[0081] Selected peptide epitopes were conjugated to KLH carrier protein (commercially prepared by GenScript).

[0082] Selected epitopes after conjugation to KLH carrier protein:

[0083] (1 ) KVGGNYNYLYRLFRKSNLKPC - KLH - the most important sequence from RBD region of spike protein: preparation 1

[0084] (2) LPDPSKPSKRSFIEDLLFNKC - KLH - spike protein: preparation 2

[0085] (3) GAVILRGHLRIAGHHLGRC - KLH - membrane protein: preparation 3

[0086] (4) ATSRTLSYYKC - KLH - membrane protein: preparation 4

[0087] (5) TESNKKFLPFQQFGRDIAC - KLH - spike protein: preparation 5 Carrier protein conjugates were first used for cytotoxicity study on cell lines.

[0088] Example 3

[0089] SARS-CoV-2 genome sequencing and bioinformatic analysis

[0090] Identification of vaccine antigens included sequencing of viral genomes and bioinformatic analysis of epitopes. 91 materials were collected from COVID-19 patients. Patient nasopharyngeal swabs were used to isolate total RNA. Next, viremia level was examined by means of real-time PCR technique. Samples with Ct<30 were subjected to further sequencing. Full-length SARS-CoV-2 genome sequences were obtained. A library for high-throughput sequencing was prepared using EasySeq kit, REF RC-COV096v2, by NIMGEN. Sequencing reaction was performed on MiSEQ instrument using Sequencing Kit v3 (300 cycles). Sequences were assembled using spades software and Wuhan-Hu-1 (NC_045512.2) reference sequence. Quality of the sequences obtained was verified in fastqc software. Average coverage of the analyzed samples was 610. Full-length viral sequences were assembled and genes were annotated. No concomitant viruses were detected. Testing was also performed on material from patients exhibiting SARS-CoV-2 symptoms despite previous infection with the virus (two infections within 1 -2 months) and patients who developed the infection after 2-3 weeks of receiving the second dose of vaccination. The following mutations were identified in the samples: 69 / 70 del, 145Y del, N501Y, A570D, D614G, T716I, S982A, D11 18H within the spike gene. Based on the identification, it was deemed that the tested samples comprise B.1.1.7 variant of SARS-CoV-2. Next, S1 protein sequences, as expressed by prof. Czerwihski’s team, were analyzed. Clustalomega was used to map the sequences, and Jalview was used for visualisation and mutation analysis. These sequences were then compared to NCBI Reference Sequence: NC_045512.2 (as reference sequence) using clustalomega server. One mutation, i.e. wro1 , 1840 A-G, was found. The following results were obtained: S1 protein comprised all mutations of the Delta variant. Furthermore: del69 / 70, del144, N501Y, A570D - found in the British variant (B.1.1.7); K417N, E484K, N501Y (present also in B.1 .1 .7) - for the South African variant (B.1 .351 ). In total, 97 SARS-CoV-2 genomes were collected and sequenced, including the following variants: original Wuhan, British, South African, and Delta. Moreover, the secured swab material comprising total (including derived from respiratory epithelium) RNA was used to prepare RNA for analysis of epithelial cells expression profile during SARS-CoV-2 infection. Nasopharyngeal swabs from healthy individuals (constituting control group) were matched to the analyzed samples derived from infected patients. As a result of the experiment, genes whose expression was potentially correlated with infection and seventy of symptoms relating to SARS-CoV-2 infection could be distinguished. Also, mutations from the period of 2021 -06-29 to 2021 -07-29 for Europe (approximately 80 thousand sequences) were analyzed. Bioinformatic analysis of S1 gene nucleotide sequence agreement and distribution of different S1 protein variants in European population revealed marked predominance of the Delta variant. Based on the analysis of mutations in the Omicron variant and comparison of peptide sequences used for immunization, comprising sequences of different variants found in the population, with the Delta variant for S protein (including glycosylation sites) and N, M, E proteins, it has been demonstrated that peptides selected for the vaccine remained unmutated, also in the Omicron BA.2 variant.

[0091] Mutations in COVID-19 non-structural proteins (nsp1 -nsp16) for Delta and Omega (BA.2) variants were analyzed. The analysis was carried out using covidcg.org server and GISAID database. Statistical data on prevalence of individual mutations for both versions was compared. Structures and functions of nsp proteins were analyzed based on pdb database and available literature. Mutations were mapped onto protein structures, and their influence on viral function and operation of translation-replication mechanism was analyzed.

[0092] A model of response to individual vaccine preparations was prepared using artificial intelligence techniques, with a view to employing it for the present vaccine. Linear regression algorithm was used to predict the difference in antibody levels between the first and second dose. The model was built based on multiple linear regression, according to an appropriate formula.

[0093] Bioinformatic studies and genome sequencing of viruses that infect post-vaccination support the vaccine development strategy, as such approach enables rapid reaction to emerging threats. Example 4

[0094] In vitro studies of conjugates - cytotoxicity and induction of cytokines Cytotoxicity of conjugates (K; pol. koniugat) and peptides (P) (6 h and 24 h induction) was studied on primary human HSAEC, human nasal epithelial RPMI2650 and human lung carcinoma A549 cell lines, on murine TC-1 cell line, and on human cerebral microvascular endothelial HBEC-5i line (Fig. 7 to Fig. 9). The conjugates and peptides did not exhibit toxicity. Conjugates do not induce cytokines or NO; only K2 induces a low level of IL6 and IL8. No cytotoxicity against A549 and RPMI lines was observed. K1 and K3 conjugates induce minor HBAC-5i cell proliferation, while K5 exhibits slight toxicity in 6-hour incubation, however, such effect was not observed after 25 hours of incubation (Fig. 9). Proliferative activity was lower than the one observed for 6 h. For HSAEC line, K3 and K4 conjugates exhibited low toxicity, and K5 induced HSAEC cell proliferation (Fig. 8).

[0095] Analysis of activation of TLR2, TLR4, TLR5 and NOD2 receptors by peptides and conjugates demonstrated that none of the peptides and conjugates is recognized by TLR5 and NOD2. K2, K4 and K5 conjugates are recognized by TLR2, while K2 and K4 also induce TLR4 receptor activation (Fig. 10).

[0096] Induction of cytokines by conjugates and peptides in murine TC-1 line (24 h culture). A panel of Th1 / Th2 / Th17 cytokines: IL-17E, IFNy, IL-1 B, IL-2, IL-4, IL-6, IL-22, IL-10, IL-12p70, IL-17A and TNFa was analyzed. Only IL-6 and IL-17E in culture supernatants were detected. IL-17E levels were comparable in all tested samples. P2 peptide exhibited a higher level than other samples, but it was not statistically significant. In the case of IL-6, a considerable variation in cytokine production was observed. K1 and K2 conjugates induced slightly higher levels of IL-6 than in the case of K3, K4 or K5 (Fig. 11 ).

[0097] Cytokine and chemokine induction by conjugates and peptides in human HSAEC line (24 h culture) was investigated. A panel of Th1 / Th2 / Th17 cytokines: IL-17E, IFNy, IL-1 B, IL-2, IL-4, IL-6, IL-22, IL-10, IL-12p70, IL-17A and TNFa, and chemokines: eotaxin, IP-10, MCP-1 was analyzed. Levels of IL-4, IL-10, IL-17A, IL-17E, IL-22, IL-12, and eotaxin, IP-10 and MCP-1 were comparable in all tested samples; no statistically significant differences between tested groups (peptides, conjugates) and a carrier or medium were observed. For IL-6, IL-8, IFNy and IL-1 b, a stimulant-dependent variability in cytokine level was observed. K2 conjugate induced a higher level of IL-6, IL-1 b, IFNy and IL-8 than the other samples (fig. 12).

[0098] Results regarding induction of cytokines by conjugates and peptides examined using human RPMI 2650 line (24 h culture) are presented in Fig. 13. A panel of Th1 / Th2 / Th17 cytokines: IL-17E, IFNy, IL-1 B, IL-2, IL-4, IL-6, IL-22, IL-10, IL-12p70, IL-17A and TNFa, and chemokines: eotaxin, IP-10, MCP-1 was analyzed. No statistically significant differences between the samples were observed. For IL-10, a very low level - 0.8 pg / ml - was observed, which corresponds to the test's limit of sensitivity.

[0099] Results regarding induction of cytokines by conjugates and peptides examined using human A549 line (24 h culture) are compiled in Fig. 14. A panel of Th1 / Th2 / Th17 cytokines: IL-17E, IFNy, IL-1 B, IL-2, IL-4, IL-6, IL-22, IL-10, IL-12p70, IL-17A and TNFa, and chemokines: eotaxin, IP-10, MCP-1 was analyzed. No statistically significant differences between the samples were observed. Only K2 induces an elevated level of IL6 and IL8.

[0100] All cytokines and chemokines were tested using MILLIPLEX MAP Human or Mouse Cytokine / Chemokine Assay kits with Luminex xMAp technology on Luminex 200 System.

[0101] Example 5

[0102] Analysis of influence of tested preparations on blood-brain barrier integrity and cytokine secretion

[0103] To examine the influence of tested preparations on blood-brain barrier integrity and cytokine secretion, human cerebral capillary endothelium HBEC-5i (ATCC CRL-3254), i.e. a model of human blood-brain barrier, and human astrocytes of U373 (Upsala) line were used as cell models. Cytotoxicity of tested samples was assayed by means of SRB test. The results indicated no toxic effect on the tested cells, no significant influence of the tested samples on NO production / secretion, and also no significant influence of the conjugates on tight junction protein expression in human blood-brain barrier model (occludin, E-cadherin, claudin 5, ZO-1 ). ELISA test results demonstrated that the tested conjugates do not induce TNFa, IL-10, IL-6. In human blood-brain barrier model, the conjugates do not affect the barrier integrity, NO production, cytokine secretion, nor tight junction protein expression, i.e. the conjugates do not have a toxic effect on blood-brain barrier cells. Viral protein immunoreactivity was determined, epitope structure was selected and established, conjugates were prepared and their safety was demonstrated by means of in vitro testing. Next, with a view to establishing effectiveness of the vaccine, they were - in combination with nanoadjuvant - administered intranasally to mice.

[0104] Example 6

[0105] Preparation of nanoadjuvants, physicochemical studies thereof, and cell induction studies

[0106] Adjuvant for the vaccine was selected from a panel of 13 previously prepared and characterized formulations. The selected nanoadjuvant is characterized by long-term stability and induces a high level of serum IgA and IgG antibodies, specific to the antigen included in the formulation, following intranasal administration in mice. The nanoadjuvant was manufactured in quantities required for the planned in vitro testing, and its physicochemical parameters, such as droplet size (412 nm ± 50 nm) and zeta potential (35.49 mV ± 2.65 mV) were established. Polydispersity parameter was determined to be 0.262 ± 0.04, which indicates moderate polydispersity. The adjuvant is not cytotoxic in mice. The nanoadjuvant (NAC) developed is based on well-defined ingredients, known from Pharmacopeia, as an emulsion comprising ingredients in appropriate proportions, that determine its droplet size and zeta potential.

[0107] NAC nanoadjuvant ingredients (mechanical emulsification):

[0108] Soybean oil 65%

[0109] Ethanol 8%

[0110] Water 21 %

[0111] Tyloxapol 5%

[0112] Benzalkonium chloride 1 % Pharmacopeia: Benzalkonii chloridum NAC cytotoxicity, tumor necrosis factor a (TNF-a), induction and their influence on antigen absorption by immune cells were studied by means of in vitro testing.

[0113] Complementary properties of NAC were verified by intranasal vaccination in mice in combination with model protein, ovalbumin (OVA).

[0114] NAC adjuvants exhibit extraordinary stability (3 years and it is still being analyzed), are stable in pH 5.7-8.2, preserve 100% of the antigen, and do not require any special storage conditions. Sub-cytotoxic dose, a parameter required for an adjuvant, was established on RPMI2650 (human, nasal septum) and FaDu (human, pharyngeal epithelium) lines. NACs induce TNFa ejection when administered with very low doses of antigen, i.e. they enhance response to antigen. We did not observe a similar effect for other cytokines. NACs alone do not produce such effect; the adjuvant needs to be administered in combination with the antigen. NACs enhance TLR receptor expression in respiratory epithelial cells; this effect is also dependent on the presence of antigen. In tests with model protein, the nanoadjuvant exhibits extraordinary adjuvant properties, and may be used as adjuvants for intranasal vaccinations. In the case of IgA antibodies response - induction of antibodies occurs when administered intranasally, mainly as a response to epitopes. When administered intramuscularly, basically no IgAs are induced. A completely different response was obtained than in the case of intranasal administration. NAC immunizes more strongly than alum. Following intranasal administration, IgA antibodies are induced, mainly in response to epitopes. Following intramuscular administration, no IgA induction occurs.

[0115] Example 7

[0116] Determination of vaccine effectiveness in vivo - humoral response in mice

[0117] Immunization of mice was performed, and vaccine effectiveness and safety were analyzed at the animal model level. After completion of cell line studies and obtaining appropriate approvals for animal experiments from Ethics Committees, murine model studies were conducted at the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences (pol. Polska Akademia Nauk, PAN) and at ICP PAN Lukasiewicz in Pszczyna. The studies were conducted in Pszczyna in line with Good Laboratory Practice (GLP) standards. Humoral response was analyzed in peripheral blood serum samples and BAL preparations. The results are presented in Fig. 15 to 17.

[0118] Following third intranasal vaccination with individual conjugates and S1 protein, the level of specific class IgG and IgA antibodies was analyzed using immunoenzymatic ELISA method. Reactivity of class IgG antibodies was observed for all antigens; it was most specific in the case of conjugates 1 and 3, while for conjugates 2, 4 and 5 reactivity with the carrier was observed as well. Response to conjugates 2-5 was also significant without the adjuvant. Regarding systemic response in class IgA, a considerable level of antibodies was observed for conjugates 1 , 2, 4 and 5. In the case of BAL lavage analysis, a lower antibody titer was exhibited for conjugates 1 , 2 and 3. Thus, immunogenicity of conjugates and S1 protein was demonstrated.

[0119] Next, testing of intracellular cytokine secretion by stimulated splenocytes following intranasal vaccination in mice (intracellular cytokine staining, ICS) was performed. Results indicate cellular response following intranasal administration of the vaccine, as in groups of mice vaccinated intranasally with conjugates, an increased percentage of population of CD4+ and CD8+ cells secreting IFN-y i TNF-a was observed when compared to control groups: PBS, NAC and MF-59. Expression of CD69 marker was not changed. Testing result is presented in Fig. 18 and 19.

[0120] Example 8

[0121] Veterinary examination of animals

[0122] Veterinary observations of animals after the first (I) and second vaccination (II) of mice with the conjugates during the experiment involved evaluation of general condition and weight of the animals. Vaccinations had no major implications for weight gain in individual groups and the animals did not experience increased discomfort during the experiment. In some groups transient muzzle swelling appeared after vaccination. At the same time, in some groups isolated instances of collapsing were observed, likely due to insufficient water intake. After the second vaccination, abrasions in the nose area were noticed in two groups and the PBS control group. In the other groups, no reaction was observed after the second vaccination with a decreased dose. Results of the conducted histopathological examination were evaluated by means of inflammatory infiltration analysis. Inflammatory infiltration of mononuclear cells, i.e. lymphocytes, plasmocytes and histiocytes, in nasal tissue in the group after the second vaccination with conjugate 1 with adjuvant and no infiltration in the no vaccination group. To conclude, histopathological examination results confirmed that intranasal administration of the vaccines (conjugates of peptides with NAC or MF59 adjuvant) induced weak, transient inflammation (infiltration of immune cells) within nasal mucosa and pulmonary tissue in some mice. However, tissue condition essentially did not differ from the condition induced by saline administration or the appearance of tissues in group 0 (no vaccine) mice.

[0123] The results are indicative of effectiveness and safety of intranasally administered vaccines in murine model. The task was completed successfully, since it concludes the experimental preclinical part of research on original Covid-19 vaccine, and indicates its safety and effectiveness at this stage, as confirmed by GLP in vitro and in vivo studies.

[0124] To summarize, when selecting antigens for the vaccine, acquired immunity to the pathogen was considered, by analyzing convalescent sera (IgA, IgG, IgM) with viral proteins. Application of the epitope mapping technology using synthetic peptides on rods allowed for selection of the smallest structures, within full-length pathogen proteins, that are reactive with convalescent serum.

[0125] Antigen-carrier protein conjugates are not cytotoxic when tested on cell lines. Antigen-carrier protein conjugates are not neurotoxic when tested in vitro on cell lines.

[0126] When tested in vitro, the conjugates induce a low and selective level of cytokines.

[0127] The conjugates induce a humoral (IgG, IgA), systemic and cellular (serum, BAL, spleen) response. Intranasal administration of the conjugates induces specific protection against the antigen: IgG and IgA antibodies (with adjuvant, K1 -K3), regardless of adjuvant type (K2), and with no adjuvant added (K2 and K3).

[0128] The results allow for the use of suspension and adjuvant based on Pharmacopeia-listed ingredients, formulations used in pharmacological preparations, as intranasal suspension. The results allow for the use of tetanus or diphtheria toxoid as epitope carrier in conjugate. The bioinformatic data taken into consideration confirm that the selected epitopes do not comprise sequences identical to human tissues. No harmful effects of the vaccine were indicated by the animals’ clinical condition. Omicron and BA.2 variant mutations were considered in vaccine design - antigens selected for the vaccine are conservative and mutation-independent.

[0129] Ease of administration, both in adults afraid of needles and in children, constitutes an invaluable advantage of an aerosol vaccine. Another benefit of a vaccine in the form of nasal drops is also ease of storage, as it does not require temperatures as low as e.g. in the case of the Pfizer vaccine. Moreover, intranasal preparations do not require the use of syringes, which lowers vaccination costs and reduces amount of medical waste generated by vaccination programs. Aerosol vaccine also does not need to be administered by a healthcare professional. As a result, it may be applied in many developing countries by trained volunteers.

[0130] SEQUENCE LIST

[0131] <110> Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences

[0132] <120> SARS-CoV-2 protein epitopes and use thereof in prevention and diagnosis of coronavirus infections

[0133] <130> PK / 9353 / AR

[0134] <160> 5

[0135] <170> Patentin version 3.5

[0136] <210> 1

[0137] <211> 21

[0138] <212> PRT

[0139] <213> SARS-CoV-2

[0140] <400> 1

[0141] Lys Vai Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser 1 5 10 15

[0142] Asn Leu Lys Pro Cys 20

[0143] <210> 2

[0144] <211> 21

[0145] <212> PRT

[0146] <213> SARS-CoV-2

[0147] <400> 2

[0148] Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Phe He Glu Asp Leu 1 5 10 15

[0149] Leu Phe Asn Lys Cys

[0150] 20 <210> 3

[0151] <211 > 19

[0152] <212> PRT

[0153] <213> SARS-CoV-2

[0154] <400> 3

[0155] Gly Ala Vai He Leu Arg Gly His Leu Arg He Ala Gly His His Leu

[0156] 1 5 10 15

[0157] Gly Arg Cys

[0158] <210> 4

[0159] <211 > 11

[0160] <212> PRT

[0161] <213> SARS-CoV-2

[0162] <400> 4

[0163] Ala Thr Ser Arg Thr Leu Ser Tyr Tyr Lys Cys

[0164] 1 5 10

[0165] <210> 5

[0166] <211 > 19

[0167] <212> PRT

[0168] <213> SARS-CoV-2

[0169] <400> 5

[0170] Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gin Gin Phe Gly Arg Asp

[0171] 10 15 lie Ala Cys

Claims

Claims1. A SARS-CoV-2 protein peptide having sequence selected from: SEQ NO 1, SEQ NO 2, SEQ NO 3, SEQ NO 4 or SEQ NO 5.

2. The peptide of claim 1 , characterized in that it is conjugated to a carrier protein.

3. The peptide of claim 2, characterized in that the carrier protein is KLH or TT or identical.

4. The peptide of claim 1 , characterized in that the peptides are produced synthetically or by recombination.

5. The SARS-CoV-2 protein peptide as defined in claims 1-3 for use in inducing immunological response against SAR-CoV-2 virus in a patient, prevention and treatment of COVID-19 disease caused by SAR-CoV-2 coronavirus infection.

6. The peptide for use as defined in claim 5 for intranasal administration.

7. A vaccine composition comprising the peptide as defined in one of claims 1-3 and a pharmaceutically acceptable carrier and / or adjuvant or nanoadjuvant.

8. The vaccine composition of claim 7, characterized in that the nanoadjuvant comprises 65% emulsified soybean oil, 8% ethanol, 21 % water, 5% Tyloxapol, 1 % benzalkonium chloride.

9. The vaccine composition as defined in one of claims 5-6 for use in inducing immunological response against SAR-CoV-2 virus in a patient, prevention and treatment of COVID-19 disease caused by SAR-CoV-2 coronavirus infection.

10. The vaccine composition for use as defined in claim 8, characterized in that it is administered intranasally.

11. A method for detecting a coronavirus infection, in particular SARS-CoV-2 infection, characterized in that(c) the peptide as defined in claim 1 is provided,SUBSTITUTE SHEET (RULE 26)(d) the peptide as defined in claim 1 is contacted with a biological sample from a patient, e.g. blood and saliva,(e) presence of complexes of the peptide as defined in claim 1 with antibodies specific to the peptide as defined in claim 1 is detected, wherein the presence of such complexes is indicative of the patient being infected with the virus.SUBSTITUTE SHEET (RULE 26)

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