WO2022103351A1

WO2022103351A1 - Laboratory and clinical test method for the follow-up of t cell response in the diagnosis and monitoring of covid 19 disease

Info

Publication number: WO2022103351A1
Application number: PCT/TR2020/051311
Authority: WO
Inventors: Ercüment OVALI; Zeynep Emel DEMİRALP; Derya Dilek KANÇAĞI; Cihan TAŞTAN
Original assignee: Acibadem Labmed Sağlik Hi̇zmetleri̇ Anoni̇m Şi̇rketi̇; Celltek Sağlik Ve Danişmanlik Hi̇zmetleri̇ Li̇mi̇ted Şi̇rketi̇
Priority date: 2020-11-12
Filing date: 2020-12-16
Publication date: 2022-05-19
Also published as: TR202018115A2

Abstract

The present invention relates to laboratory and clinical test method in terms of response to diagnosis or treatment that provides to show whether the person has come upon the virus and/or has developed immunity against the virus in viral infections, including SARS-CoV-2, where the antibody response is insufficient.

Description

LABORATORY AND CLINICAL TEST METHOD FOR THE FOLLOW-UP OF T CELL RESPONSE IN THE DIAGNOSIS AND MONITORING OF COVID 19 DISEASE

Technical Field

The invention, relates to a laboratory and clinical test method for the follow-up of T cell response in the diagnosis and monitoring of COVID 19 disease.

Particularly, the invention relates to response test regarding diagnosis or treatment that provides to show whether the person has come upon the virus and/or has developed immunity against the virus in viral infections, including SARS-CoV-2, where the antibody response is insufficient.

State of the Art

Today, there are three basic methods for the diagnosis of SARS-CoV-2. The first one is the presence of the PCR test, the second one is the detection of the antigen, and the third one is the antibody response. Although the specificity of all three tests is good, their sensitivity is low. Particularly, on one hand the false negativity rates of PCR tests are >30%, on the other hand the results of the antibody test are very problematic in SARS- CoV-2. Herein, there is no such a test that shows explicitly whether immunization is formed or not after the illness or whether patients who do not have PCR positivity previously have come upon this virus or not even if they have not developed an antibody response. Due to these problems, there are important problems in detecting the correct immune mass. Moreover, it is not known that patients have this disease will experience re-infection subsequently.

One of the applications found in the literature review as an example of PCR tests, is the national application number 2020/06162 and is related to the semi-multiplex coronavirus real-time polymerase chain reaction detection kit that uses primer and probe sets. The national application numbered 2020/04448 relates to the multiplex coronavirus qrt-pcr test kit that uses primer and probe sets. Patent numbered KR102018201 B1 relates to the method and kit for detecting batcoronavirus by using real-time PCR. It is mentioned that the tests for various coronaviruses passed by people are performed using real-time PCR using minimal primers according to the present invention. However, these tests will not be able to show whether the patients whose PCR is not positive come across this virus or not.

As a result, due to the abovementioned disadvantages and the insufficiency of the current solutions regarding the subject matter, a development is required to be made in the relevant technical field.

Brief Description of the Invention

The invention, relates to a laboratory and clinical test method for the follow-up of T cell response in the diagnosis and monitoring of COVID 19 disease, which fulfills the abovementioned requirements, eliminates all disadvantages and brings some additional advantages.

Studies performed regarding SARS-CoV-2 show that antibody response is highly variable and antibody stability period is short in PCR positive patients. In fact, it is known that T cell responses occur in all patients without question. The present invention aims to provide a solution to this problem by showing the clinical and in vitro T Cell response of inactive virus and/or virus mRNA, spike proteins and/or other proteins considering this information.

Thus, the present invention aims to offer a solution to the problem wherein inactive virus and/or virus mRNA, spike proteins and/or other proteins a. Causing T cell proliferation and expression of activation markers under in vitro conditions in incubation, using ELISPOT (Enzyme-linked Immunospot Assay), ELISA (Enzyme-linked Immunosorbent Assay) tests that analyze the secretions of gamma interferon or different cytokines from T cells for this aim; b. Showing delayed-type hypersensitivity after intradermal application with the logic of Mantoux method (DTH: delayed-type hypersensitivity).

The structural and characteristic features of the present invention will be understood clearly by the following drawings and the detailed description made with reference to these drawings and therefore the evaluation shall be made by taking these figures and the detailed description into consideration. Figures Clarifying the Invention

Figure 1 , is a schematic view of the inventive method, which is based on showing T cell response.

Figure 2, is a schematic view of the inventive method based on showing the delayed-type hypersensitivity with the Mantoux method (DTH) logic.

Figure 3 is the cytokine release profile which shows the serum cytokine amount change after stimulation with the Sars-CoV-2 viral antigenic suspension.

Figure 4 is the cytokine release profile which shows the serum cytokine percentage % change after stimulation with the Sars-CoV-2 viral antigenic suspension.

Figure 5 is the interferon gamma spot number and it is the T cell response profile activated after stimulation with the Sars-CoV-2 viral antigenic suspension.

Detailed Description of the Invention

In this detailed description, the inventive method is described only for clarifying the subject matter in a manner such that no limiting effect is created.

The invention is a laboratory and clinical test method for the follow-up of T cell response in the diagnosis and monitoring of COVID 19 disease, comprising the following processes,

- Showing T Cell response or

- Showing delayed type hypersensitivity with the Mantoux method approach.

The laboratory test method comprising the step of showing T cell response, that is shown in Figure 1 schematically, comprises the following process steps: i. Preparing SARS-CoV-2 viral antigenic suspension

Intradermal injection fluid for T Cell stimulation is prepared as follows:

Stimulant antigenic structures are liquid or lyophilized product prepared in appropriate carrier, balancing stabilizer liquids in the range of 0.1 pg - 5 pg. ii. Peripheral blood mononuclear cell (PBMC) isolation: It is the T Cell to be tested by peripheral smear. iii. Incubation

It is the 24-48 hour incubation of viral antigenic structures in PBMC cell culture. iv. Determination of T Cell activation and proliferation after incubation Determination of the proliferation amount of T cells and activation markers by flow cytometric analysis (CD25, CD107a, CD3, CD4, CD8, CD16, CD19, CD56) v. Determination of IFN-gamma release in SARS-CoV-2 specific T cells after incubation

It is the detection of gamma interferon or other cells secreting cytokine by ELISPOT method for SARS-CoV-2 specific T cell response follow up. vi. Determination of cytokine release in SARS-CoV-2 specific T cells after incubation For SARS-CoV-2 specific T cell response follow up, it is to monitor the cytokines secreted by Elisa method or CBA (difference from control group).

According to an embodiment of the invention; the clinical test method comprising the step of showing delayed type hypersensitivity with the Mantoux method approach, that is shown in Figure 2 schematically: i. Preparing SARS-CoV-2 viral antigenic suspension

Intradermal injection fluid for T Cell stimulation is prepared as follows:

Stimulant antigenic structures are liquid or lyophilized product prepared in appropriate carrier, balancing stabilizer liquids in the range of 0.1 pg - 5 pg. ii. Intradermal injection of SARS-CoV-2 viral antigenic suspension

50-400 pl of the suspension is administered intradermally to form bulla. Simultaneously, the same amount of SF is administered to the other arm intradermally. iii. Incubation

It is exposed to incubation for 48-72 hours. iv. SARS-CoV-2 clinical response monitoring 48-72 hours after the injection; o Redness of 0-5 mm is negative o Redness of 6-9 mm is encountering the virus o Redness of >10 mm is active disease symptom.

Diagnostic tests (including nasopharyngeal swab) may give false negative results. A false negative result leads to the asymptomatic non-isolated person to infect a large number of people. In a research made in Wuhan when 205 throat swabs, 490 nasal swabs, and 142 sputum samples were collected and were analyzed by RT-PCR test, 11% of sputum, 27% of nasal and 40% of throat samples were detected at 1 to 7 days after the onset of the disease was informed as false negative results. In another study including 957 patients with suspected Covid-19 "or" confirmed, a false-negative diagnosis rate was noticed between 2% and 29%. Though the evidence is limited as a whole, in the studies carried out, negative RT-PCR results that are obtained falsely in a frequent manner draws attention. For this reason, there are important problems in detecting the correct immune mass. Herein, a test method showing whether the patients have come across this virus or not is particularly important in the vaccination step.

Most people infected with SARS-CoV-2 exhibit an antibody response between 10th day and 21 st day after the infection. Detection may take a long time (four weeks or more) in mild cases and antibodies (ie IgM, IgG) are not detected in any way in a small number of cases. IgM and IgG antibodies against SARS-CoV-2 develop between 6-15 days after the onset of the disease based on the available data. In the coronavirus cases encountered previously (1985-2020), it attracts the attention that the protective immune response time is substantially short. Moreover, antibody titer reduces to the initial levels in a short period of time in patients with low neutralizing antibody levels. The studies performed not only show an average of 12 months between the first infection and the second infection, but also show the decrease of the antibody levels (50%) substantially within in a period of 6 months. At the end of a year, the antibody levels that reduce 75%, reach the initial level after 4 years. What matter is that it is not known whether the presence of antibodies indicates that the person is immune to the disease later on or not or how long it will have a protective effect. Lymphocytes and their subgroups play critical role so as to maintain immune system function. As in the case of immune diseases and other infectious diseases, virus infection can cause disorder in the levels of lymphocyte subsets. In the light of data obtained from SARS patients in the years 2003-2004, protective immunity was formed after most acute viral infections. For this reason, development of CD4 + T cell, CD8 + T cell and neutralizing antibody responses against SARS-CoV-2 that play a critical role in removing all acute infection becomes a possibility (humoral and cytotoxic immunity). Moreover, naturally some of the T and B cells are kept within the body for a long time as immunological memory and protective immunity against SARS-CoV-2 infection. SARS- CoV2 specific T cell response can be protective or pathogenic. This situation may be a scenario to be contemplated separately or simultaneously, depending on the timing, composition or size of the response. The definition and evaluation of human antigenspecific SARS-CoV-2 T cell responses can be made in the best manner directly by using broad-based epitope pools and ex vivo analysis such as flow cytometric, ELISA, ELISPOT that can be able to detect T-Cell derived cytokines. For example, in an ex-vivo study carried out with COVID 19 patients, the SARS-CoV-2 specific T lymphocyte ratio was observed as 100% for CD4 and 80% for CD8. Similarly, 20% SARS-CoV-2 specific T lymphocyte level drew the attention in control individuals due to cross reaction based on other coronavirus types. It was shown that a strong T-cell response was formed against spike protein and Th1 , Th2 and cytokine releases. Such information can act as a guide in selecting the vaccine strategies that have the highest probability to provide protective immunity against SARS-CoV-2. In addition to this, information regarding T cell responses to COVID-19 can act as a guide in selecting appropriate immunological endpoints for COVID-19 candidate vaccine clinical trials that have already been initiated. However, this information is still not used so as to evaluate the immune response of patients, shaping the treatment, and following up the population immunity with population screening. Therefore, the present invention suggests the use of these tests as diagnostic and followup tests. However, more importantly, these in vitro tests are costly and require high technology. For this reason, they are tests which are difficult to generalize socially. Thus, the skin test proposed by the present invention can answer all these questions and also it is easy, cheap and accessible.

The antibodies (B cell-derived humoral immune response) created by the body against the receptor binding site, nucleocapsid protein and other antigenic structures of the spike protein can be determined by enzyme-linked immunosorbent assay (ELISA) 14 days after the start of the symptoms in most of the patients. However, the T cell-based response has a more complex structure. The recent studies have set forth that, type I IFN plays a critical role in the passage from natural to adaptive immunity during the acute phase of SARS and patients with bad results exhibited type I IFN-mediated immunopathological events and insufficient adaptive immune responses. The definition and evaluation of virus antigen-specific SARS-CoV-2 T cell responses can be performed directly by ELISPOT analysis of IFN-gamma secretion that belongs to the fresh peripheral mononuclear cell. Originally, the ELISPOT test was developed so as to detect antigen-specific antibody secreting B cells and subsequently was used so as to count T cells secreting antigenspecific cytokines From that time, ELISPOT test has been used so as to measure antigenspecific T cells by detecting cytokines secreted by T cells after stimulation with synthetic peptides (used as pools or single peptides) or complete antigens (such as proteins or lysates). The ELISPOT test not only allows determining the amount of T cells responding to a specific antigen, but also allows the detection of functionally relevant molecules upon specific stimulation of effector T cells. However, this test was used only in studies until now; it was not used in diagnosis and monitoring. However, more importantly, these in vitro tests are costly and require high technology. For this reason, they are tests which are difficult to generalize socially.

The human skin represents a tissue appropriate for the study of human immune responses in terms of its relative availability. The skin represents the largest and mostly exposed interface of the body with the environment. Besides its acting as a passive, mechanical barrier, it plays an important role in the host defense. The Mantoux Test (MT) is a classic delayed-type hypersensitivity (DTH) response to intradermal injection of tuberculin purified protein derivative (PPD). Typically the test determines immunity to tuberculosis in people, and positive reactions that develop in people who have previously been exposed to Mycobacterium tuberculosis and in people who were vaccinated with the Bacillus of Calmette and Guerin (BCG) vaccine. The cutaneous T cell mediated memory recall represents the immune response. Most of the infiltrated cells are neutrophils at very early time points (4-6 hours) in DTH response. About 12 hours after the thread, T cells begin to be seen around the dermal blood vessels. The maximum number of migrating active macrophages is observed at 24 hours, but most of the cells that migrate at 48 hours are T cells. Most of T cells accumulate perivascularly, but more diffuse T cells are found in the epidermis and interstitium. CD4 + T cells are more in number than CD8 + T cells. It is observed that, so early after the injection, the pro-inflammatory cytokines IFN, TNF, and lymphotoxin seem to stimulate the expression of adhesion molecules on the endothelium and increase the permeability of the local blood vessel. For this reason, the T-Cell response, which becomes clear after 48 hours when antigens are administered intradermally, becomes visible with the findings of skin redness and swelling (induration). For this purpose, the DTH test is used for many years in mycoplasma. Moreover, it has been considered that Mycobacterium tuberculosis infection progresses with an immune response which is very similar to that of SARS-CoV-2. Likewise, we observe the fact that the antibodies in Mycobacterium tuberculosis are not used for diagnostic purposes in follow-up and treatment depending on their unclarity. Whereas the DTH test successfully shows whether the person comes across the bacteria and forms a sufficient immune response for Mycobacterium tuberculosis or not. Therefore, the ideal method to be used for diagnosis and monitoring in SARS-CoV2 where antibody responses and PCR tests are controversial and re-infection such as tuberculosis is contradictive, is DTH test.

References:

1. Gabriella D, et al. SARS-Cov-2 infection: Response of human immune system and possible implications for the rapid test and treatment. International Immunopharmacology. 2020; 84: 106519.

2. Le Bert N, et al. SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls.

3. Shurin MR, et al. Assessing Immune Response to SARS-CoV-2 Infection. Immuno Targets and Therapy. 2020;9:111-114.

4. Azkur AK, et al. Immune response to SARS-CoV-2 and mechanisms of immunopathological changes in CO VID- 19. Allergy. 2020;75:1564-1581.

5. La Marca A, et al. Testing for SARS-CoV-2 (COVID-19): a systematic review and clinical guide to molecular and serological in-vitro diagnostic assays. RBMO. 2020;41 (3):483-499.

6. Caruana G. Diagnostic strategies for SARS-CoV-2 infection and interpretation of microbiological results. Clinical Microbiology and Infection. 2020 ;26: 1178-1182.

7. Woloshin S, et al. False Negative Tests for SARS-CoV-2 Infection — Challenges and Implications. N Engl J Med. 2020;383(6):e38(1)- e38(3).

8. Bastos MY, et al. Diagnostic accuracy of serological tests for covid- 19: systematic review and meta-analysis. BMJ2020;370:m2516.

9. Deeks JJ, et al. Antibody tests for identification of current and past infection with SARS- CoV-2. Cochrane Database of Systematic Reviews. 2020, Issue 6.

10. Watson J, et al. Interpreting a covid- 19 test result. BMJ 2020;369:m1808.

11. Antibody (Serology) Testing for COVID-19: Information for Patients and Consumers

12. https ://www. fda. gov/medical-devices/coronavirus-covid- 19-and-medical-devices/antibodv- serok)av-testino-covid-19-information-patients-and-consumers. (Last access date: 07.09.2020)

13. Roy S. COVID-19 Reinfection: Myth or Truth? SN Compr Clin Med. 2020:1-4.

14. Kirkcaldy Rd, et al. COVID- 19 and Postinfection Immunity Limited Evidence, Many Remaining Questions. JAMA. 2020;323(22):2245-46.

15. Wikramaratna P, et al. Estimating false-negative detection rate of SARS-CoV-2 by RT- PCR. MedRxiv. 2020. doi: https://doi.orci/10. 1101/2020.04.05.20053355.

16. Gudbjartsson DF, et al. Humoral Immune Response to SARS-CoV-2 in Iceland. N Engl J Med. 2020. DOI: 10.1056/NEJMoa2026116.

17. Chen Z and Wherry EJ. T cell responses in patients with COVID-19. Nature Reviews Immunology 2020;20:529-536.

18. Chowdhury MA, et al. Immune response in COVID-19: A review. Journal of Infection and Public Health xxx (2020) xxx-xx. (Article in press)

19. Nayak S and Acharjya B. Mantoux test and its interpretation. Indian Dermatol Online J. 2012;3(1): 2-6. 20. Liu W, Liu L, Kou G, Zheng Y, Ding Y, Ni W, et al. Evaluation of Nucleocapsid and Spike Protein-based ELISAs for detecting antibodies against SARS-CoV-2. medRxiv. 2020:2020.03.16.20035014.

21. Thijsen S, et al. Elevated nucleoprotein-induced interferon-y release in CO VID-19 patients detected in a SARS-CoV-2 enzyme-linked immunosorbent spot assay. The Lancet Preprints. 2020.

22. Li CK, et al. T Cell Responses to Whole SARS Coronavirus in Humans. J Immunol. 2008;181(8): 5490-5500.

23. Calarota SA and Baldanti F. Enumeration and Characterization of Human Memory T Cells by Enzyme-Linked Immunospot Assays. Clinical and Developmental Immunology. 2013; https://doi.org/10. 1155/2013/637649

24. De Blasi S, et al. Marked T cell activation, senescence, exhaustion and skewing towards TH17 in patients with COVID-19 pneumonia. NATURE COMMUNICATIONS. 2020. https://doi.org/10. 1038 /s41467-020- 17292-4.

25. Zhou R, et al. Acute SARS-CoV-2 Infection Impairs Dendritic Cell and T Cell Responses. 2020, Immunity 53, 1-14.

26. Vukmanovic-Stejic M, et al. Mantoux Test as a model for a secondary immune response in humans. Immunology Letters. 2006;107:93-101.

27. Nayak S and Acharjya B. Mantoux test and its interpretation. Indian Dermatol Online J. 2012;3(1): 2-6.

28. Woloshin S, et al. False Negative Tests for SARS-CoV-2 Infection — Challenges and Implications. N Engl J Med. 2020;383(6):e38(1)- e38(3).

29. Woelfel R, Corman VM, Guggemos W, Seilmaier M, Zange S, Mueller MA, et al. Clinical presentation and virological assessment of hospitalized cases of coronavirus disease 2019 in a travelassociated transmission cluster. medRxiv. 2020:2020.03.05.20030502.

30. Zhao J, Yuan Q, Wang H, Liu W, Liao X, Su Y, et al. Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019. medRxiv. 2020:2020.03.02.20030189.

31. OKBA NMA, Muller MA, Li W, Wang C, GeurtsvanKessel CH, Corman VM, et al. SARS- CoV-2 specific antibody responses in COVID- 19 patients. medRxiv. 2020:2020.03.18.20038059.

32. Liu W, Liu L, Kou G, Zheng Y, Ding Y, Ni W, et al. Evaluation of Nucleocapsid and Spike Protein-based ELISAs for detecting antibodies against SARS-CoV-2. medRxiv. 2020:2020.03.16.20035014.

33. Long Q-x, Deng H-j, Chen J, Hu J, Liu B-z, Liao P, et al. Antibody responses to SARS- CoV-2 in COVID-19 patients: the perspective application of serological tests in clinical practice. medRxiv. 2020:2020.03.18.20038018.

34. Wan WY, Lim SH, Seng EH. Cross-reaction of sera from COVID-19 patients with SARS- CoV assays. medRxiv. 2020:2020.03.17.20034454.

35. Edridge, A. et al. Coronavirus protective immunity is short-lasting. medRxiv, doi:10. 1101/2020.05. 11.20086439 (2020). 36. Seow J, et al. Longitudinal evaluation and decline of antibody responses in SARS-CoV-2 infection. medRxiv preprint. 2020. doi: 10.1101/2020.07.09. 2014 8429

Antibody (Serology) Testing for COVID- 19: Information for Patients and Consumers https://www.fda.gov/medical-devices/coronavirus-covid-19-and-medical-devices/antibody- serology-testing-covid-19-information-patients-and-consumers (Last access date: 09.09.2020)

37. Wang F, Nie J, Wang H, Zhao Q, Xiong Y, Deng L, et al. Characteristics of Peripheral Lymphocyte Subset Alteration in COVID- 19 Pneumonia. The Journal of Infectious Diseases. 2020;221(11):1762-9.

38. Grifoni A, Weiskopf D, Ramirez SI, Mateus J, Dan JM, Moderbacher CR, et al. Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals. Cell. 20202020/05/20/

39. Weiskopf D, Schmitz KS, Raadsen MP, Grifoni A, Okba NMA, Endeman H, et al. Phenotype of SARS-CoV-2-specific T-cells in COVID-19 patients with acute respiratory distress syndrome. medRxiv. 2020:2020.04. 11.20062349.

40. Immune responses and immunity to SARS-CoV-2 https://www.ecdc.europa.eu/en/ covid- 19/latest-evidence/immune-responses (Last access date: 09.09.2020)

41. Zhang F, et al. Adaptive immune responses to SARS-CoV-2 infection in severe versus mild individuals. Signal Transduction and Targeted Therapy (2020) 5:156.

42. Le Bert N, et al. SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls. Nature. 2020; 584: 457-462.

43. Altmann DM and Boyton RJ. SARS-CoV-2 T cell immunity: Specificity, function, durability, and role in protection. Sci. Immunol. 2020 ;5, eabd6160.

44. Vukmanovic-Stejic M, et al. Mantoux Test as a model for a secondary immune response in humans. Immunology Letters. 2006;107:93-101.

45. Thijsen S, et al. Elevated nucleoprotein-induced interferon-y release in CO VID- 19 patients detected in a SARS-CoV-2 enzyme-linked immunosorbent spot assay. The Lancet Preprints. 2020.

46. Li CK, et al. T Cell Responses to Whole SARS Coronavirus in Humans. J Immunol. 2008;181(8): 5490-5500.

47. Calarota SA and Baldanti F. Enumeration and Characterization of Human Memory T Cells by Enzyme-Linked Immunospot Assays. Clinical and Developmental Immunology. 2013; https://doi.org/10. 1155/2013/637649

Claims

1. A laboratory or clinical test method for the follow-up of T cell response in the diagnosis and monitoring of COVID 19 disease, characterized in that, the method comprises the process steps of;

- Showing T Cell response or

- Showing delayed type hypersensitivity with the Mantoux method approach.

2. The method according to claim 1 , characterized in that; the laboratory test method comprising showing T Cell response, comprises the process steps of; i. Preparing SARS-CoV-2 viral antigenic suspension, ii. Peripheral blood mononuclear cell (PBMC) isolation, iii. Incubation, iv. Determination of T Cell activation and proliferation after incubation, v. Determination of IFN-gamma release in SARS-CoV-2 specific T cells after incubation, vi. Determination of cytokine release in SARS-CoV-2 specific T cells after incubation.

3. The method according to claim 2, characterized in that; the incubation time in the process step (iii) is 24-48 hours.

4. The method according to claim 2, characterized in that; the determination in the process step (v) is made by ELISPOT method.

5. The method according to claim 2, characterized in that; the determination is made by ELISA method or cytokine bead analysis by flow cytometry in the process step (vi).

6. The method according to claim 1 , characterized in that; the clinical test method comprising showing delayed type hypersensitivity with the Mantoux method approach, comprises the process steps of; i. Preparing SARS-CoV-2 viral antigenic suspension, ii. Intradermal injection of SARS-CoV-2 viral antigenic suspension iii. Incubation, iv. SARS-CoV-2 clinical response monitoring. . The method according to claim 6, characterized in that; the incubation time in the process step (iii) is 48-72 hours.