WO2022191742A1 - A method for assessment of the cellular immune - Google Patents

Abstract

An invention relates to medicine, molecular biology, pharmaceuticals and can be used for the diagnostics of immunity to coronavirus infection. A method for assessment of the cellular immune response against coronavirus infection is proposed, consisting in intradermal administration of coronavirus antigen preparation and visual assessment of the skin reaction at the injection site after 24-96 hours. The advantages are a simplicity of implementation – the analysis can be carried out on an outpatient basis, for example, in a state-funded health center by a nurse, as well as a high specificity and sensitivity of the method. The method can be used for a massive, fast and cheap assessment of specific cellular immunity against SARS-CoV-2 coronavirus without a special equipment.

Description

[Title established by the ISA under Rule 37.2] A METHOD FOR ASSESSMENT OF THE CELLULAR IMMUNE

An invention relates to medicine, molecular biology, pharmaceuticals and can be used for the diagnostics of immunity to coronavirus infection.

Infection with SARS-CoV-2 coronavirus leads to the development of respiratory viral infection (COVID-19). Mild clinical manifestations or moderate symptoms are in the most of patients with COVID-19, but severe pneumonia develops in about 15% of patients, and about 5% of patients have acute respiratory distress syndrome (RDS) and multiple organ failure. COVID-19 mortality ranges from 1 to 5%, the most severe forms develop in patients with concomitant comorbid conditions, such as cardiovascular diseases, diabetes, chronic diseases of kidneys, liver etc. [Chen e.a., 2020 (c); Huang e.a., 2020; Xu e.a., 2020].

Coronaviruses (Coronaviridae) are a family of RNA-containing viruses with a single-stranded positively charged RNA molecule capable of infecting humans and some animals. In humans, previously known coronaviruses can cause both mild forms of acute respiratory infection and severe acute respiratory syndrome (SARS).

SARS-CoV-2 coronavirus, the representative of Betacoronavirus genus, belongs to the second group of pathogenicity, because it can lead to a very rapid formation of acute inflammation with the development of severe bilateral pneumonia, hemorrhagic fever and organ dysfunction.

The main method of etiological diagnosis of COVID-19 is the study of biological material from the upper and lower respiratory tract using nucleic acid amplification methods, usually PCR. It allows to detect the virus or its fragments directly in the selected samples.

However, the assessment of an immune response to SARS-CoV-2 is currently an unresolved problem. The currently massively used IgM and IgG antibody test provides only an understanding of whether a person has been exposed to SARS-CoV-2 virus. However, the results of this test, even if positive, do not allow to make an unambiguous conclusion about whether a person has formed an immunity which is sufficient to protect against re-infection and/or from a severe form of infection and/or from complications on various systems and organs.

The protective role of antibodies in COVID-19 has been questioned. 2 studies with the results of the analysis of 222 and 173 patients are published, where it is shown that patients with more severe manifestations of COVID-19 had higher total antibody titers and IgG class antibody titers against SARS-CoV-2, and this was associated with more severe disease outcomes [Zhao e.a., 2020]. It is possible that in some cases there is an antibody-dependent coronavirus infection enhancement, especially if the antibodies are directed against the sites of interaction of viral proteins with specific cellular receptors, or if the virus infects cells by antibody-dependent phagocytosis. It is also possible that antibody titers just correlate with the viral load, not performing protective functions in the early stages of infection.

Analysis of antibody titers in recovered patients with asymptomatic, mild, moderate and severe forms of the disease has shown a positive correlation between the severity of symptoms and the neutralizing activity of antibodies, moreover, neutralizing antibodies were almost not produced in asymptomatic patients [Chen e.a., 2020 (b)]. The titers of antibodies to the virus, the titers of neutralizing antibodies, as well as their dynamics, can vary significantly in the blood serum of different patients.

Thus, also taking into account that the level of antibodies decreases after infection, the antibody test does not always give a truthful and particularly unambiguous answer about whether a person is protected - whether he has an immune response profile that provides sufficient protection.

We propose a solution of this problem.

The aim of a present invention is the creation of a test that allows to evaluate cellular immunity against coronavirus in a human and can be implemented massively and quickly.

A method for assessment of the cellular immune response against coronavirus infection is proposed, which consists in intradermal administration of coronavirus antigen preparation and a visual assessment of the skin reaction at the injection site after 24–96 hours, optimally after 72 hours. The presence and size of redness, as well as the formation of an inflammatory papule at the injection site are detected.

A positive test - a skin reaction formed at the injection site – is if in a person a cellular immunity against SARS-CoV-2 has formed as a result of the following events: a person is sick with COVID-19 (active or asymptomatic infection), or has had a coronavirus infection, including without obvious clinical manifestations, or has been vaccinated against a coronavirus infection. The test will be negative if a person does not have a cellular immunity against coronavirus.

We have proposed just such a solution, because a cellular immunity associated with the formation of specific T-lymphocytes recognizing viral antigens should be considered the most important component of protective immunological reactions against coronavirus.

T-lymphocytes are the main cells of the acquired antiviral immunity. Among them, CD8+ T lymphocytes recognize heterogenous viral antigens in association with class I histocompatibility molecules and kill virus-infected cells. Helper CD4+ T-lymphocytes recognize viral antigens processed and presented on antigen-presenting cells (mainly professional antigen-presenting dendritic cells) in association with class II histocompatibility molecules and act as assistants in the synthesis of specific antiviral antibodies by B-lymphocytes.

Both types of T-lymphocytes are important for the fight against coronaviruses, but in SARS-CoV infection models it has been shown that the degree of protection depends more on CD4+ T cells. Experimental removal of them led to blocking the release of all types of lymphocytes into the lung tissue, a decrease of neutralizing antibodies and cytokines synthesis, and ended with a significant delay in the purification of the lungs from coronavirus [Chen e.a., 2020 (a)]. In convalescent patients infected with SARS-CoV, the formation occurs of virus-specific CD4+ T-lymphocytes synthesizing IL-2, IFNγ, TNF, apparently related to type 1 Th clones, and CD8+ T-lymphocytes synthesizing IFNγ and TNF. A strong T-cell response correlated with high titers of neutralizing antibodies [Li e.a., 2020].

All known methods for the assessing of the cellular immunity against SARS-CoV-2 are ex vivo, in vitro methods - the methods for the evaluation of the cytokine synthesis by stimulated cells using ELISPOT and cytofluorimetry. These methods are not widely used due to the high cost of laboratory equipment and reagents, the complexity of sample preparation, the speed of research and data analysis. Accordingly, these methods are very expensive, require special equipment, reagents, work of highly qualified personnel and are associated with invasive methods of taking venous blood from the proband individuals. All this limits the ability to assess the cellular immunity against coronaviruses. We consider mentioning these methods as analogues impossible.

Thus, the proposed method is unique and has no analogues.

The method can be used for a massive, fast and cheap assessment of specific cellular antiviral immunity against SARS-CoV-2 coronavirus without a special equipment. The advantages of this method are also a simplicity of implementation – the analysis can be carried out on an outpatient basis, for example, in a state-funded health center by a nurse, as well as a high specificity and sensitivity of the method. This can be signified as technical results.

Also, this method is safe. It is based on the formation of a delayed-type hypersensitivity reaction (DTH), which manifests itself in the form of redness (and induration - papule) at the injection site in the presence of a cellular immune response against SARS-CoV-2 in the body. The test is performed by a nurse licensed to perform intradermal tests.

The technical result also is in the possibility of the assessing of the immune response after vaccination, including by those vaccines that do not contain or do not encode fragments of coronavirus proteins on which antibodies are formed that can be assessed by existing diagnosticums for IgG and IgM antibodies. For example, these are vaccines that do not contain a full-length S-protein or RBD-domain. When using the proposed method in this case, it is necessary to use an antigen containing a fragment available for recognition by the immune system and which is present or encoded in the vaccine. A positive response in this case will be observed in vaccinated and recovered patients who have formed an immune response to this fragment.

The technical result also consists in expanding the range of tests to coronavirus and immunity to coronavirus. In the course of unwillingness to use other tests due to their shortcomings described above (insufficient information content, difficult accessibility), or if there are contraindications, for example, if there are difficulties with blood sampling, or if there is not access to them, this solution will allow to diagnose coronavirus and immunity to it. The specified technical result is achieved by using the method according to the present invention.

A coronavirus antigen and a preparation based on it are proposed for use in the method. A recombinant protein that repeats the structure of the coronavirus can be used as the antigen. The antigen is either isolated from the destroyed or inactivated coronavirus and purified, and it contains a protein fraction. Hybrid proteins based on at least two proteins from the M, S, N or E proteins of the coronavirus, or their fragments, for example, set forth as the amino acid sequence of SEQ ID NO.:1,2 or 3; M, S, N or E protein of the coronavirus, or its fragment, for example, set forth as the amino acid sequence of SEQ ID NO.:4 or 5 are examples of recombinant proteins. It can also be other full-length proteins of the new coronavirus, their fragments, for example, amino acid sequences LTRPLLESELVIGAV of the M protein, TRFASVYAWNRKRISN of the S protein, RLNQLESKMSGKGQQ, GMEVTPSGTWLTYTG, DKDPNFKDQVILLNK of the N protein, SEETGTLIVNSVLLF, ALRLCAYCCNIVNVS of the E protein, but not limited to, combinations of these or other fragments, their parts. The coronavirus antigen containing a protein fraction can be isolated from the destroyed coronavirus, for example, with temperature, ultraviolet and other methods, or it can be an inactivated virus, for example, a vaccine strain; with further purification.

The use of the coronavirus antigen described above is proposed for using in the proposed method. Analogs have also not been revealed by us.

A preparation of the coronavirus antigen described above is also proposed – for use in the proposed method. It contains such an antigen in an amount from 0.01 to 50 micrograms, optimally - 10 micrograms, and a target additive. Analogs have also not been identified by us. The target additive includes pharmaceutically acceptable carriers and buffer solutions known from the prior art - described in various texts, such as, for example, Remington's Pharmaceutical Sciences.

The use of the coronavirus antigen and a preparation based on it for a new purpose - for the implementation of the proposed method - allows to implement the development, even if it has not shown the effectiveness in a study for vaccination. It will also allow to increase the production of domestic preparations and substances.

It is important to note that the antigen used in the proposed method for the assessment of vaccine action must contain a fragment of the coronavirus contained in the vaccine or encoded by the nucleic acid at the base of the vaccine.

In the context of the ongoing pandemic, it is extremely important to carry out coronavirus diagnostics quickly and on a large scale, especially the assessment of immunity against coronavirus. This will make it possible to understand who is at risk and who needs to be unequivocally vaccinated, and possibly once again with another vaccine, for example, in the absence of immunity after vaccination, or after coronavirus in the absence of a positive test result, or with a weak test result.

Due to the fact that the problem of the new coronavirus continues to be very acute, and not many succeed in bringing diagnostic methods and preparations to the market, this group of inventions will increase the chances of fighting this infection.

The invention is illustrated by the following drawings.

Fig.1

A photo of a hand after administration of coronavirus antigen – of a hybrid protein set forth as SEQ ID NO.:1, to a healthy person who has not been ill with COVID-19 and has not been vaccinated (injection reaction).

Fig.2

A photo of a hand after administration of various doses of coronavirus antigen – of a hybrid protein set forth as SEQ ID NO.:1, to persons who have had COVID-19, when assessed after 72 hours.

Fig.3

A photo of a hand after administration of various doses of coronavirus antigen – of a hybrid protein set forth as SEQ ID NO.:1, to vaccinated persons when assessed after 72 hours.

Examples

The authors of the present invention have conducted laboratory studies confirming the possibility of described invention implementation. The obtained research results are illustrated by examples (1,2).

Example 1. Obtaining an antigen and a preparation based on it

Different variants of the antigen were prepared to evaluate the effectiveness of the proposed method for determining cellular antiviral immunity in vivo by assessing the skin reaction to the administration of a viral antigen.

The N protein of the new coronavirus, set forth as SEQ ID NO.:4, was obtained using genetic engineering and biotechnology methods. The coding polynucleotide optimized in codon composition for expression in E.coli, set forth as SEQ ID NO.: 9, was cloned in pET22b (+) vector - for protein production in E.coli, competent cells of E.coli BL21 Star (DE3) and Lemo21 (DE3) were transformed. Further, the protein was produced using the obtained producer strain and purified by chromatography, in one of the variants with the removal of methionine at the N-terminus.

The peptide set forth as SEQ ID NO.:5 – a fragment of the S protein of the new coronavirus – was synthesized chemically.

A hybrid protein set forth as SEQ ID NO.:3 and containing fragments of S and N proteins of the new coronavirus was synthesized chemically. The protein has a good solubility. Such a hybrid protein was also obtained with the addition of methionine at the N-terminus.

Hybrid proteins set forth as the amino acid sequences SEQ ID NO:1 and 2 were developed and presented for the first time in the application for the invention of the Russian Federation №2020112937 dated 05.04.2020 and international application PCT/RU2020/000257, filing date 02.06.2020, priority date 05.04.2020. Hybrid proteins consist of 424 and 422 amino acid residues, with methionine at the N-terminus - 425 and 423 amino acid residues.

Purified hybrid protein set forth as SEQ ID NO.:1 and fusion protein set forth as SEQ ID NO.:2 were obtained using genetic engineering and biotechnology methods.

Encoding polynucleotides codon-optimized for expression in E.coli, for example, set forth as SEQ ID NO.:9, were cloned in a vector for protein production in E.coli, for example, pET22b (+), competent E.coli cells, for example, BL21 Star (DE3), were transformed. Next, the protein was produced using the obtained producer strain and purified by chromatography, in one of the variants with the removal of methionine at the N-terminus.

The coding polynucleotides set forth as SEQ ID NO.: 6 or 7 were obtained based on viral sequences or optimized for expression in mammalian cells, with restriction sites flanking the target gene, and also with the addition of the Kozak sequence before the start codon to initiate translation, after the start codon – a signal sequence, for example, IGF, HGH, respectively, for the secretion of a synthesized protein from an eukaryotic cell. Cloning was performed in the pcDNA3.1(+) vector according to the instruction for the vector. Mammalian cells (CHO) were transfected with the created plasmids. Further, the proteins were produced using the obtained producers and purified by chromatography.

"CoviVak" registered vaccine preparation was also used for the study, which is a purified concentrated suspension of SARS-CoV-2 coronavirus "AYDAR-1" strain, obtained by reproduction in a transferable culture of Vero cells and inactivated with beta-propiolactone (from the Instruction for "CoviVak" vaccine).

The obtained antigens were mixed with the saline or PBS and used in the study.

Example 2. Demonstration of the developed method effectiveness

Preparations obtained according to Example 1 were used to perform a skin test.

Intradermal administration of the antigen in 0.2 ml of saline solution was carried out in the forearm area of both hands in the amount of 0,01; 0,1; 0,5; 5,0; 10,0; 50,0 micrograms at a distance of 5-10 cm from each other. As a control, a saline solution in a volume of 0.2 ml was taken.

Five groups of volunteers participated in the study:

1) Healthy persons who have not had COVID-19 and have not been vaccinated (control)

2) Persons who have had COVID-19

3) Persons immunized with the EpiVacCorona vaccine (Vector SSC (state scientific center) of virology and biotechnology of Rospotrebnadzor Federal Budgetary Institution of Science - FBIS)

4) Persons immunized with the Gam-COVID-Vac vaccine (Federal State Budget Institution (FSBI) N.F. Gamaleya National Research Center for Epidemiology and Microbiology, Ministry of Health of the Russian Federation) when assessing after 72 hours

5) I.V. Dukhovlinov – the developer and creator of a vaccine against coronavirus infection based on a genetic construct encoding a hybrid protein set forth as SEQ ID NO.: 1, presented in the application for the invention of the Russian Federation №2020112937 of 05.04.2020 and international application PCT/RU2020/000257, having immunized himself with this vaccine.

As a result of assessing the development of a skin reaction within 6 days, it was noted that the first signs of a reaction in the form of redness appear after 24 hours, and the maximum size of redness of the skin at the injection site, as well as the formation of an induration - papule – was noted after 72 hours, this is the optimal time to assess the reaction. Later the reaction fades and passes without a trace. The result can be estimated up to 96 hours after the administration of the antigen, afterwards the picture is not informative.

As a result of the analysis, it was revealed that in different people of the same group (except for the control) the administration of doses as described above causes the formation of a reaction, "spots" are formed according to a gradient – the more is the dose, the more is the area and brightness of the redness, the more papule is formed. Having found this out, a few more people of the groups indicated above were administered a dose of 0.1; 0.5; 5 or 10 micrograms of antigen. The results were captured by photography. Thus, Figures 1-3 show photos which demonstrate a skin reaction in individuals of groups 1-5 who were injected with an antigen – the hybrid protein set forth as SEQ ID NO.:1. Only a part of the photos is presented in this application since the type of reaction is similar, and the photos provided allow to confirm the absence or development of the reaction and demonstrate its appearance.

In the first group of healthy individuals at all periods of study, when all types of antigen were administered in all doses, only an injection reaction was noted without redness or other manifestations – as shown on . Such reaction was observed after the administration of the hybrid protein preparation set forth as SEQ ID NO.:1, as well as of preparations of other antigens described in Example 1, including those obtained in prokaryotic and eukaryotic producers.

A dose-dependent skin reaction was noted in the form of a redness spot around the injection site with a size of 10-60 mm ( , 3), or redness with the formation of a papule in the center of induration ( , 3) in the most of experiments in 2-5 groups of individuals after administration of the hybrid protein preparation set forth as SEQ ID NO.:1. The control administration of saline solution in these individuals ended only with the formation of an injection reaction without redness, as it is shown on . A similar picture was obtained after administration of other antigens preparations described in Example 1.

All studied antigens caused the formation of a reaction in group 2 – of recovered from coronavirus and in group 5, the picture was similar to that shown on and 3, respectively.

In group 3 – in immunized with the EpiVacCorona vaccine - the reaction as shown on was observed after the administration of hybrid protein preparations set forth as SEQ ID NO.:1,2,3, of the N protein set forth as SEQ ID NO.:4, of the CoviVac vaccine preparation. No reaction was observed to the preparation of the antigen – a fragment of the S-protein – set forth as SEQ ID NO.:5, the picture was as on .

In group 4 – in persons immunized with the Gam-COVID-Vac vaccine - the reaction as shown on was observed after the administration of preparations of hybrid protein set forth as SEQ ID NO.:1,2,3, S-protein fragment set forth as SEQ ID NO.:5, the CoviVac vaccine preparation. No reaction was observed to the preparation of the antigen – the N protein set forth as SEQ ID NO.:4, the picture was as on .

The reliability of the obtained results was confirmed after the analysis of the sequences on which the vaccines are based. Thus, the reaction is observed after the administration of those antigens that contain the fragments contained in a vaccine. It can be concluded that this principle must be followed when using antigens in the proposed method.

The administration of the antigen preparation (variants described in Example 1) in an amount from 0.01 to 50 micrograms demonstrated that the optimal amount for use in the proposed method is 5-10 micrograms. We consider a dose of 10 micrograms be the optimal universal dose of the antigen, at which the redness (and the induration – papule) is clearly formed, and it is possible to evaluate the result of the research.

Thus, a skin reaction for the administration of a coronavirus antigen occurs in those who have been ill, for the administration of a relevant coronavirus antigen - in vaccinated individuals. The method can be used for a massive, fast, cheap and without the need for special equipment evaluation of a specific cellular immune response against coronavirus infection, against SARS-CoV-2. The antigen according to the invention and the preparation based on it are applicable in the proposed method.

<110> Dukhovlinov I.V., Simbirtsev A.S., Totolyan A.A.

<120> A method for assessment of the cellular immune response against coronavirus infection

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<223> S domain, from 306 to 380 amino acid residue of S protein of the new coronavirus

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<210> SEQ ID NO : 2

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Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Gly Gly Gly Gly Asp

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Lys Met Ser Gly Lys Gly Gln Gln Gln Gln Gly Gln Thr Val Thr Lys

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Asp Tyr Lys His Trp Pro Gln Ile Ala Gln Phe Ala Pro Ser Ala Ser

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Ile Leu Thr Ala Leu Arg Leu Cys Ala Tyr Cys Cys Asn Ile Val Asn

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Val Ser Leu Val Lys Pro Ser Phe Tyr Val Tyr Ser Arg Val Lys Asn

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Leu Asn Ser Ser Arg Val

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<210> SEQ ID NO : 3

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<213> Artificial sequence

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<221> domain

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<223> S domain, from 345 to 360 amino acid residue of S protein of the new coronavirus

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Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu

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Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro

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Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly

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Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr

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Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp

115 120 125

Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp

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His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln

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Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser

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Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn

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Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala

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Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu

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Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln

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Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys

245 250 255

Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln

260 265 270

Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp

275 280 285

Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile

290 295 300

Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile

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Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala

325 330 335

Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu

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Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro

355 360 365

Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln

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Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu

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Thr Gln Ala

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<223> a polynucleotide based on viral sequences encoding a hybrid protein of 424 a.a. with the addition of the IGF secretory seq.

<400>

gccgccacca tgggcaagat cagcagcctg cccacccagc tgttcaagtg ctgcttctgc 60

gacttcctga aggtaacttt agcttgtttt gtgcttgctg ctgtttacag aataaattgg 120

atcaccggtg gaattgctat cgcaatggct tgtcttgtag gcttgatgtg gctcagctac 180

ttcattgctt ctttcagact gtttgcgcgt acgcgttcca tgtggtcatt caatccagaa 240

actaacattc ttctcaacgt gccactccat ggcactattc tgaccagacc gcttctagaa 300

agtgaactcg taatcggagc tgtgatcctt cgtggacatc ttcgtattgc tggacaccat 360

ctaggacgct gtgacatcaa ggacctgcct aaagaaatca ctgttgctac atcacgaacg 420

ctttcttatt acaaaggagg aggaggagga ggattcactg tagaaaaagg aatctatcaa 480

acttctaact ttagagtcca accaacagaa tctattgtta gatttcctaa tattacaaac 540

ttgtgccctt ttggtgaagt ttttaacgcc accagatttg catctgttta tgcttggaac 600

aggaagagaa tcagcaactg tgttgctgat tattctgtcc tatataattc cgcatcattt 660

tccactttta agtgttatgg aggaggagga ggaggagatg ctgctcttgc tttgctgctg 720

cttgacagat tgaaccagct tgagagcaaa atgtctggta aaggccaaca acaacaaggc 780

caaactgtca ctaagaaatc tgctgctgag gcttctaaga agcctcggca aaaacgtact 840

gccactaaag catacaatgt aacacaagct ttcggcagac gtggtccaga acaaacccaa 900

ggaaattttg gggaccagga actaatcaga caaggaactg attacaaaca ttggccgcaa 960

attgcacaat ttgcccccag cgcttcagcg ttcttcggaa tgtcgcgcat tggcatggaa 1020

gtcacacctt cgggaacgtg gttgacctac acaggtgcca tcaaattgga tgacaaagat 1080

ccaaatttca aagatcaagt cattttgctg aataagcata ttgacgcata cggaggagga 1140

ggaggaggat cggaagagac aggtacgtta atagttaata gcgtacttct ttttcttgct 1200

ttcgtggtat tcttgctagt tacactagcc atccttactg cgcttcgatt gtgtgcgtac 1260

tgctgcaata ttgttaacgt gagtcttgta aaaccttctt tttacgttta ctctcgtgtt 1320

aaaaatctga attcttctag agtttaatga

<210> SEQ ID NO : 7

<211> 1356

<212> DNA

<213> Artificial sequence

<220>

<223> a polynucl. codon-opt. for mammals encoding a hybrid prot. of 422 a.a. with the addition of the HGH secretory seq.

<400>

gccgccacca tggccaccgg ctcccggacc tccctgctgc tggccttcgg cctgctgtgc 60

ctgccctggc tgcaggaggg ctccgccgtg accctggcct gcttcgtgct ggccgccgtg 120

taccggatca actggatcac cggcggcatc gccatcgcca tggcctgcct ggtgggcctg 180

atgtggctgt cctacttcat cgcctccttc cggctgttcg cccggacccg gtccatgtgg 240

tccttcaacc ccgagaccaa catcctgctg aacgtgcccc tgcacggcac catcctgacc 300

cggcccctgc tggagtccga gctggtgatc ggcgccgtga tcctgcgggg ccacctgcgg 360

atcgccggcc accacctggg ccggtgcgac atcaaggacc tgcccaagga gatcaccgtg 420

gccacctccc ggaccctgtc ctactacaag ggcggcggcg gcggcggctt caccgtggag 480

aagggcatct accagacctc caacttccgg gtgcagccca ccgagtccat cgtgcggttc 540

cccaacatca ccaacctgtg ccccttcggc gaggtgttca acgccacccg gttcgcctcc 600

gtgtacgcct ggaaccggaa gcggatctcc aactgcgtgg ccgactactc cgtgctgtac 660

aactccgcct ccttctccac cttcaagtgc tacggcggcg gcggcggcga cgccgccctg 720

gccctgctgc tgctggaccg gctgaaccag ctggagtcca agatgtccgg caagggccag 780

cagcagcagg gccagaccgt gaccaagaag tccgccgccg aggcctccaa gaagccccgg 840

cagaagcgga ccgccaccaa ggcctacaac gtgacccagg ccttcggccg gcggggcccc 900

gagcagaccc agggcaactt cggcgaccag gagctgatcc ggcagggcac cgactacaag 960

cactggcccc agatcgccca gttcgccccc tccgcctccg ccttcttcgg catgtcccgg 1020

atcggcatgg aggtgacccc ctccggcacc tggctgacct acaccggcgc catcaagctg 1080

gacgacaagg accccaactt caaggaccag gtgatcctgc tgaacaagca catcgacgcc 1140

tacggcggcg gcggcggctc cgaggagacc ggcaccctga tcgtgaactc cgtgctgctg 1200

ttcctggcct tcgtggtgtt cctgctggtg accctggcca tcctgaccgc cctgcggctg 1260

tgcgcctact gctgcaacat cgtgaacgtg tccctggtga agccctcctt ctacgtgtac 1320

tcccgggtga agaacctgaa ctcctcccgg gtgtga 1356

<210> SEQ ID NO : 8

<211> 1272

<212> DNA

<213> Artificial sequence

<220>

<223> a polynucleotide codon-optimized for E.coli, encoding a hybrid protein of 422 а.a.

<400>

atggtgaccc tggcgtgctt tgtgctggcg gcggtgtatc gtattaattg gattaccggc 60

ggcattgcga ttgcgatggc gtgcctggtg ggcctgatgt ggctgtccta ttttattgcg 120

tcctttcgtc tgtttgcgcg tacccgttcc atgtggtcct ttaatccgga aaccaatatt 180

ctgctgaatg tgccgctgca tggcaccatt ctgacccgtc cgctgctgga atccgaactg 240

gtgattggcg cggtgattct gcgtggccat ctgcgtattg cgggccatca tctgggccgt 300

tgcgatatta aagatctgcc gaaagaaatt accgtggcga cctcccgtac cctgtcctat 360

tataaaggcg gcggcggcgg cggctttacc gtggaaaaag gcatttatca gacctccaat 420

tttcgtgtgc agccgaccga atccattgtg cgttttccga atattaccaa tctgtgcccg 480

tttggcgaag tgtttaatgc gacccgtttt gcgtccgtgt atgcgtggaa tcgtaaacgt 540

atttccaatt gcgtggcgga ttattccgtg ctgtataatt ccgcgtcctt ttccaccttt 600

aaatgctatg gcggcggcgg cggcgatgcg gcgctggcgc tgctgctgct ggatcgtctg 660

aatcagctgg aatccaaaat gtccggcaaa ggccagcagc agcagggcca gaccgtgacc 720

aaaaaatccg cggcggaagc gtccaaaaaa ccgcgtcaga aacgtaccgc gaccaaagcg 780

tataatgtga cccaggcgtt tggccgtcgt ggcccggaac agacccaggg caattttggc 840

gatcaggaac tgattcgtca gggcaccgat tataaacatt ggccgcagat tgcgcagttt 900

gcgccgtccg cgtccgcgtt ttttggcatg tcccgtattg gcatggaagt gaccccgtcc 960

ggcacctggc tgacctatac cggcgcgatt aaactggatg ataaagatcc gaattttaaa 1020

gatcaggtga ttctgctgaa taaacatatt gatgcgtatg gcggcggcgg cggctccgaa 1080

gaaaccggca ccctgattgt gaattccgtg ctgctgtttc tggcgtttgt ggtgtttctg 1140

ctggtgaccc tggcgattct gaccgcgctg cgtctgtgcg cgtattgctg caatattgtg 1200

aatgtgtccc tggtgaaacc gtccttttat gtgtattccc gtgtgaaaaa tctgaattcc 1260

tcccgtgtgt aa 1272

<210> SEQ ID NO : 9

<211> 1260

<212> DNA

<213> Artificial sequence

<220>

<223> a polynucleotide cod.-opt. for E.coli, encoding N prot. of the new coronavirus

<400>

atgtccgata atggcccgca gaatcagcgt aatgcgccgc gtattacctt tggcggcccg 60

tccgattcca ccggctccaa tcagaatggc gaacgttccg gcgcgcgttc caaacagcgt 120

cgtccgcagg gcctgccgaa taataccgcg tcctggttta ccgcgctgac ccagcatggc 180

aaagaagatc tgaaatttcc gcgtggccag ggcgtgccga ttaataccaa ttcctccccg 240

gatgatcaga ttggctatta tcgtcgtgcg acccgtcgta ttcgtggcgg cgatggcaaa 300

atgaaagatc tgtccccgcg ttggtatttt tattatctgg gcaccggccc ggaagcgggc 360

ctgccgtatg gcgcgaataa agatggcatt atttgggtgg cgaccgaagg cgcgctgaat 420

accccgaaag atcatattgg cacccgtaat ccggcgaata atgcggcgat tgtgctgcag 480

ctgccgcagg gcaccaccct gccgaaaggc ttttatgcgg aaggctcccg tggcggctcc 540

caggcgtcct cccgttcctc ctcccgttcc cgtaattcct cccgtaattc caccccgggc 600

tcctcccgtg gcacctcccc ggcgcgtatg gcgggcaatg gcggcgatgc ggcgctggcg 660

ctgctgctgc tggatcgtct gaatcagctg gaatccaaaa tgtccggcaa aggccagcag 720

cagcagggcc agaccgtgac caaaaaatcc gcggcggaag cgtccaaaaa accgcgtcag 780

aaacgtaccg cgaccaaagc gtataatgtg acccaggcgt ttggccgtcg tggcccggaa 840

cagacccagg gcaattttgg cgatcaggaa ctgattcgtc agggcaccga ttataaacat 900

tggccgcaga ttgcgcagtt tgcgccgtcc gcgtccgcgt tttttggcat gtcccgtatt 960

ggcatggaag tgaccccgtc cggcacctgg ctgacctata ccggcgcgat taaactggat 1020

gataaagatc cgaattttaa agatcaggtg attctgctga ataaacatat tgatgcgtat 1080

aaaacctttc cgccgaccga accgaaaaaa gataaaaaaa aaaaagcgga tgaaacccag 1140

gcgctgccgc agcgtcagaa aaaacagcag accgtgaccc tgctgccggc ggcggatctg 1200

gatgattttt ccaaacagct gcagcagtcc atgtcctccg cggattccac ccaggcgtga 1260

Non Patent Literature

1. Chen N., Zhou M., Dong X. et al. (c) Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study. Lancet. 2020; 395(10223):507-513. doi: 10.1016/S0140-6736(20)30211-7.

2. Huang C., Wang Y., Li X. et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020; 395(10223): 497-506. doi: 10.1016/S0140-6736(20)30183-5.

3. Xu Z., Shi L., Wang Y. et al. Pathological findings of COVID-19 associated with acute respiratory distress syndrome. Lancet Respir. Med. 2020; 8(4): 420–422. doi: 10.1016/S2213-2600(20)30076-X.

4. Chen X., Pan Z., Yue S. (b) Disease severity dictates SARS-CoV-2-specific neutralizing antibody responses in COVID-19. Signal Transduction and Targeted Therapy. 2020; 5:180. doi.org/10.1038/s41392-020-00301-9

5. Zhao J., Yuan Q., Wang H. et al. Antibody Responses to SARS-CoV-2 in Patients With Novel Coronavirus Disease 2019. Clin Infect Dis. 2020 Nov 19;71(16):2027-2034. doi: 10.1093/cid/ciaa344.

6. Chen J., Lau Y., Lamirande E. et al. (а)Cellular immune responses to severe acute respiratory syndrome coronavirus (SARS-CoV) infection in senescent BALB/c mice: CD4+ T cells are important in control of SARS-CoV infection. J.Virol. 2010;84(3):1289-1301. doi: 10.1128/JVI.01281-09.

7. Li C., Wu H., Yan H. et al. T cell responses to whole SARS coronavirus in humans. J.Immunol. 2008;181(8):5490-5500. doi: 10.4049/jimmunol.181.8.5490.

Claims (15)

1. A method for assessment of the cellular immune response against coronavirus infection, consisting in intradermal administration of coronavirus antigen preparation and a visual assessment of the skin reaction at the injection site after 24-96 hours.
2. The method according to claim 1, characterized in that the coronavirus antigen is a recombinant protein that repeats the structure of the coronavirus.
3. The method according to claim 1 or 2, characterized in that the coronavirus antigen is the M, S, N, or E protein of a coronavirus or a fragment thereof.
4. The method according to claim 1 or 2, characterized in that the coronavirus antigen is a hybrid protein based on at least two proteins of the M, S, N or E proteins of the coronavirus or fragments thereof.
5. The method according to claim 4, characterized in that the hybrid protein is set forth as the amino acid sequence of SEQ ID NO.:1,2 or 3.
6. The method according to claim 1, characterized in that the coronavirus antigen contains a protein fraction, it is isolated from the destroyed or inactivated coronavirus and purified.
7. A coronavirus antigen for use in the method according to claim 1.
8. The antigen according to claim 7, characterized in that it is a recombinant protein that repeats the structure of the coronavirus.
9. The antigen according to claim 8, characterized in that it is the S or N protein of the coronavirus.
10. The antigen according to claim 8, characterized in that it is a fragment of the S protein of the coronavirus.
11. The antigen according to claim 10, characterized in that it is set forth as the amino acid sequence of SEQ ID NO.:5.
12. The antigen according to claim 8, characterized in that it is a hybrid protein based on at least two proteins of the M, S, N or E proteins of the coronavirus or fragments thereof.
13. The antigen according to claim 12, characterized in that it is set forth as the amino acid sequence of SEQ ID NO.:1,2 or 3.
14. The antigen according to claim 7, characterized in that it contains a protein fraction, it is isolated from the destroyed or inactivated coronavirus and purified.
15. A coronavirus antigen preparation for use in the method according to claim 1, containing the antigen according to any of claims 7-14 in an amount from 0.01 to 50 micrograms and a target additive.

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