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Title:
BIOLOGICAL PRODUCT WITH ANTIVIRAL ACTIVITY AGAINST COVID-19 VIRUS
Document Type and Number:
WIPO Patent Application WO/2022/111782
Kind Code:
A1
Abstract:
The coronavirus disease 2019 (COVID‑19) is a pandemic outbreak caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2). According to official data from the World Health Organization, by Dec 28th, 2020, >81,746,833 people have been affected by the SARS-CoV-2, and > 1,783,597 patients had died due to the disease. Camels are host and reservoir for different strains of the coronaviruses such as SARS and MERS-SARS and SARS-Cov-2 viruses and they produce effective neutralizing antibodies to these viruses [3]. Therefore, they can be used as an important source to generate strong immune responses against different strains of the coronaviruses [4]. Therefore, immunizing camels with an inactivated form of the current coronavirus strain (SARS-CoV-2) the causative agent of the current COVID-19 pandemic disease will produce a strong immune response against the virus components and the generation of specialized antibodies. Interestingly, these antibodies are present in serum and milk at the same concentration. This invention aimed to immunize camel against COVID-19 virus and used the generated antibodies in milk or serum as a treatment for COVID-19.

Inventors:
TAMAN MOHAMED (EG)
ABDOUN AHMED (EG)
Application Number:
PCT/EG2020/000033
Publication Date:
June 02, 2022
Filing Date:
November 26, 2020
Export Citation:
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Assignee:
TAMAN MOHAMED AHMED ELHADY (EG)
ABDOUN AHMED SABRY SALAH ELDIN (EG)
International Classes:
C07K16/08; A23C9/00; C07K16/04
Foreign References:
US10336817B22019-07-02
Other References:
HASSON SIDGIS, AL-JABRI ALIA: "Immunized camels and COVID-19", ASIAN PACIFIC JOURNAL OF TROPICAL MEDICINE, vol. 13, no. 6, 14 July 2020 (2020-07-14), Singapore , pages 239 - 137, XP055942479, ISSN: 1995-7645, DOI: 10.4103/1995-7645.282561
ZHAO JINCUN, PERERA RANAWAKA A. P. M., KAYALI GHAZI, MEYERHOLZ DAVID, PERLMAN STANLEY, PEIRIS MALIK: "Passive Immunotherapy with Dromedary Immune Serum in an Experimental Animal Model for Middle East Respiratory Syndrome Coronavirus Infection", JOURNAL OF VIROLOGY, vol. 89, no. 11, 1 June 2015 (2015-06-01), US , pages 6117 - 6120, XP055942481, ISSN: 0022-538X, DOI: 10.1128/JVI.00446-15
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Claims:
Claims

1 -Producing antibodies of Covid- 19 derived from camels, through immunizing camels against the virus.

2-Using the serum derived from camels immunized against Covid- 19 treating those affected by the virus.

3 -Using milk of camels immunized against Covid- 19 for treating those affected by the virus.

Description:
biological product with antiviral activity against covid-19 virus

Technical Field :

Product against covid-19 virus .

Background Art :

The coronavirus disease 2019 (COVID-19) is a pandemic outbreak caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). According to official data from the World Health Organization, by Dec 28 th , 2020, >81,746,833 people have been affected by the SARS-CoV-2, and > 1,783,597 patients had died due to the disease [1],

The clinical outcomes in elder patients or those with underlying diseases such as cardiovascular and lung affections, cancer or renal dysfunction may suffer from more severe symptoms and mortality [2], Infected individuals usually exhibit nonspecific symptoms such as a cough and fever. Clinically, antiviral drugs are usually used for treatment, but there are no specific drugs and vaccines that are used. Treatments for COVID-19 mostly involve symptom alleviation measures, non-specific broad spectrum antiviral drugs, as highly effective antiviral drugs and there is currently no cure or vaccine for COVID-19. However, transfusion of critically ill patients with serum from convalesced individuals, which contain human antibodies against the virus, has been shown to greatly improve clinical outcome. This process is called the passive immunization, but it is hard to identify the right individuals with the right antibodies and to give such a blood product safely.

Camels are well-known as a host and reservoir for different strains of the coronaviruses such as SARS and MERS-SARS and SARS-Cov-2 viruses and they produce effective neutralizing antibodies to these viruses [3]. Therefore, they can be used as an important source to generate strong immune responses against different strains of the coronaviruses [4]. Therefore, immunizing camels with an inactivated form of the current coronavirus strain (SARS-CoV-2) the causative agent of the current COVID-19 pandemic disease will produce a strong immune response against the virus components and the generation of specialized antibodies.

These antibodies are able to gain access to tissues and neutralize SARS-CoV-2 viruses outside and inside infected cells [5,6], These antibodies possess an antibacterial and antiviral activity [7,8]. Also, they were demonstrated to be stable at high temperatures, resist stomach enzymes digestion and can pass into the bloodstream as intact antibody molecules [9], They are less immunogenic than most mammalian IgGs, and when administered intravenously they are less inclined to prompt serum sickness and anaphylactic adverse reactions [10]. This has already been demonstrated in a similar strategy that have invented and implemented in phase I clinical trial for the treatment of HIV patients, with a patent being granted by the US Patent Office [6]. Interestingly, these antibodies are present in serum and milk at the same concentration [11].

The neutralizing antibodies in serum or milk of immunized camels will give passive immunity to COVID-19 infected patients as a treatment or as a means of prophylaxis to prevent those at risk of SARS-CoV-2 infection. Sera obtained from MERS-immune camels augment the kinetics of MERS-CoV clearance and reduced the severity of pathological changes in infected lungs, with efficacy proportional to the titer of MERS-CoV-neutralizing serum antibody [12], Once confirmed its safety, the purified antisera can be transfused directly into the bloodstream of the infected patient.

This is in the same manner where current anti-snake venoms are generated and used.

References

[1] World Health Organization (WHO): WHO Coronavirus Disease (COVID-19) Dashboard. WHO, Geneva,

2020. urihttps://covid 19. who.int/. Accessedsimplehttps ://covid 19. who. int/. Accessed

Dec 27, 2020.

[2] Chen N, Zhou M, Dong X, Qu J, Gong F, Han Y, Qiu Y, Wang J, Liu Y, Wei Y, et al: Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: A descriptive study. Lancet. 395:507- 513. 2020.

[3] Lau SKP, Chan JEW. Coronaviruses: emerging and re-emerging pathogens in humans and animals. Virol J (2015) 12. https://doi.Org/10.1186/s 12985-015-0432-z

[4] Killerby ME, Biggs HM, Midgley CM, Gerber SI, Watson JT. Middle East Respiratory Syndrome Coronavirus Transmission. Emerg Infect Dis. (2020)

26(2): 191-198. https://dx.doi.org/10.3201/eid2602.190697 [5] Cortez-Retamozo V, Lauwereys M, Hassanzadeh Gh G, Gobert M, Conrath K, Muyldermans S, De Baetselier P, Revets H. Efficient tumor targeting by singledomain antibody fragments of camels. Int J Cancer. (2002) 98(3):456-62.

[6] Hasson SS & Al-Jabri AA. Therapeutic composition. US 10336817B2, filed by SULTAN QABOOS UNIVERSITY. Being granted by the US Patent Office in 2016 (Priority to ZA16/7084 2016-10-14).

[7] EL-Fakharany EM, Abedelbaky N, Haroun BM et al. Anti-infectivity of camel polyclonal antibodies against hepatitis C virus in Huh7.5 hepatoma. Virol J (2012)

9, 201. https://doi.org/10.1 186/1743-422X-9-201 .

[8] El Agamy El, Ruppanner R, Ismail A, Champagne CP, Assaf R. Antibacterial and antiviral activity of camel milk protective proteins. J Dairy Res. (1992)

59(2).T 69-75.

[9] Jason VS & Burnett BP. Survival and digestibility of orally-administered immunoglobulin preparations containing IgG through the gastrointestinal tract in humans. Nutr. J. (2015) 14, 22.

[10] Herrera MG, Leon A, Segura F, Meneses B, Lomonte J, Philippe JP, Chppaux P, Gutierrez JM. Factors associated with adverse reactions induced by caprylic acid- fractionated whole IgG preparations: comparison between horse, sheep and camel IgGs. Toxicon. (2005) 46:775-781.

[11] Hamers-Casterman C, Atarouch T, Muyldermans S, Bendolman N, Flamers R. Naturally occurring antibodies devoid of light chains. Nature. (1993) 363: 446-448.

[12] Zhao J, Perera RAPM, Kayali G, Meyerholz D, Perlman S, Peirisb M (2015). Passive immunotherapy with dromedary immune serum in an experimental animal model for Middle East Respiratory Syndrome coronavirus infection. Journal of Virology 89(11): 6117-6120.

The problem in previous art:

1. Treatments for COVID-19 mostly involve symptom alleviation measures, non-specific broad -spectrum antiviral drugs.

2. There is currently no cure or vaccine for COVID-19.

3. Currently, there are three COVID-19 vaccines released to the market of which Pfizer, Modema and AstraZeneca vaccines and the SinoVac (Chinese vaccine). However, there is no evidence that any existing vaccines will protect against

COVID-19.

4. This invention is the first trial to confirm the antiviral activity of camel serum,

IgG and milk.

Disclosure Of The invention ;

Detailed description

This invention is based on the following:

1. Immunization of dromedary camel against SAR-Cov2 virus (Egyptian strain) isolated from Egyptian patients. Dromedary camel was immunized with a total of 2 mg of COVID-19 virus suspended in 2 ml of complete Freund's adjuvant, and followed by of 6 booster doses of COVID-19 virus in 1 ml of incomplete Freund's adjuvant. Five days after the last booster dose, 100 ml of blood was collected and serum was separated and stored at -80C.

2. Milk samples were also collected to test against COVID-19 virus.

3. Test of camel milk against SAR-Cov-2 virus,

4. Checking the antiviral acti vity of immunized camel serum against COVID-19 virus.

Methodology:

> MTT cytotoxicity assay (TC50)

Samples were diluted with Dulbecco's Modified Eagle's Medium (DMEM). Stock solutions of the test compounds were prepared in 10 % DMSO in ddH 2 O. The cytotoxic activity of the extracts were tested in Vero E6 cells by using the 3 -(4, 5- dimethylthiazol -2-yl)-2, 5-diphenyltetrazolium bromide (MTT) method (Mossman, 1983) with minor modification. Briefly, the cells were seeded in 96 well-plates (100 mΐ/well at a density of 3* 105 cells/ml) and incubated for 24 hrs at 37°C in 5%CO 2 . After 24 hrs, cells were treated with various concentrations of the tested compounds in triplicates. After further 24 hrs, the supernatant was discarded and cell monolayers were washed with sterile phosphate buffer saline (PBS) 3 times and MTT solution (20 mΐ of 5 mg/ml stock solution) was added to each well and incubated at 37°C for 4 hrs followed by medium aspiration. In each well, the formed formazan crystals were dissolved with 200 mΐ of acidified isopropanol (0.04 M HC1 in absolute isopropanol = 0.073 ml HCL in 50 ml isopropanol). Absorbance of formazan solutions were measured at λmax 540 nm with 620 nm as a reference wavelength using a multi-well plate reader. The percentage of cytotoxicity compared to the untreated cells was determined with the following equation.

The plot of % cytotoxicity versus sample concentration was used to calculate the concentration which exhibited 50% cytotoxicity (TC50).

% cytotoxicity

= ( absorbance of cells without treatment

— absorbance of cells with treatment)X 100 /( absorbance of cells without treatment )

Reference:

√ Mossman T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods; 65(l-2):55-63.

> Plaque reduction assay

Assay was earned out according to the method of (Hayden et al, 1980) in a six well plate where Vero E6 cells (10 5 cells / ml) were cultivated for 24 hrs at 37°C. Middle East respiratory syndrome-related corona virus isolate NRCE-HKU270 (Accession Number: KJ477103.2) virus was diluted to give 10 PFU/ well and mixed with the safe concentration of the tested compounds and incubated for 1 hour at 37°C before being added to the cells. Growth medium was removed from the cell culture plates and the cells were inoculated with (100 μl / well) virus with the tested compounds, After 1 hour contact time for virus adsorption, 3 ml of DMEM supplemented with 2% agarose and the tested compouds was added onto the cell monolayer, plates were left to solidify and incubated at 37°C till formation of viral plaques (3 to 4 days). Formalin (10%) was added for two hours then plates were stained with 0.1 %crystal violet in distilled water. Control wells were included where untreated virus was incubated with Vero E6 cells and finally plaques were counted and percentage reduction in plaques formation in comparison to control wells was recorded as following

% inhibition= viral count (untreated) - viral count (treated)/viral count (untreated) x 100

Reference:

√ Hayden, F.G., Cote, K.M., Douglas, R.G., (1980). Plaque inhibition assay for drug susceptibility testing of influenza viruses. Antimicrob. Agents Chemother; 17, 865-870.

Method of utility

The present invention relates to the ability of camels to produce antibodies of Covid-19, whether in blood serum of milk, in order to use it for treating those affected by the virus.

Antiviral activity of immunized camel serum against COVlD-19 Antiviral activity of immunized camel milk against COVID-19