WO2016032352A1 - Manufacture and use of modern ovotransferrin (otf-m) - Google Patents

Abstract

This invention refers to the method of manufacture and purification of specific ovotransferrin also called modern ovotransferrin (OTF-M) from the egg white, consisting of preparation of antigens, immunization of laying chickens with a certain antigen, purification and assessment of egg white-extracted OTF-M. The specific ovotransferrin is extracted by a two-stage precipitation with various concentrations of ammonium sulfate and citric acid, followed by dialysis and freeze-drying. This invention also refers to the composition of antigens prepared from a mixture of antibiotic-resistant strains from a single species of bacteria or of an antigen prepared from a mixture of various antibiotic-resistant species of strains, isolated from patients in Romania. This invention is susceptible for industrial use.

Description

DESCRIPTION OF INVENTION

Title

MANUFACTURE AND USE OF MODERN OVOTRANSFERRIN (OTF-M)

TECHNICAL FIELD

Immunology, medicine, preventive treatments, biotechnolog applications

This invention describes a method of manufacture of specific ovotransferrin (OTF-M) for a target antigen. The eggs the specific ovotransferrin is extracted from are provided by poultry (Gallusdomesiicus) immunized with antigens prepared from bacteria, viruses, fungi, parasites, venom, allergens. This invention refers to the preparation of specific ovotransfemns using antibiotic-resistant bacteria isolated from patients with clinical symptoms hospitalized in Romania. This invention refers to the preparation of specific monovalent or polyvalent ovotransferrins prepared from eggs of poultry (Gallusdomesiicus) immunized with a monovalent antigen or with an antigen prepared from various species of bacteria. This invention refers to the preparation of specific ovotransferrin against the antigens prepared from β-factame bacteria and from other antibiotic-resistant gram positive bacterial strains.

DESCRIPTION OF THE STAGE OF TECHNIQUE INCLUDING

REFERENCES

The chicken (Gallusdomesiicus) egg has been used as food since long time ago. It contains various nutrients which promote both living and growth. Egg proteins are essential nutrients with a perfect amino acid balance necessary for cell construction. Egg proteins are found 50% in egg white and 40% in the yolk. The rest of proteins are found in the egg shell and membranes. Besides the nutritive value, egg proteins have unique biological activities. Hyperimmunized chickens provide an economical and specific source of egg yolk-extracted immunoglobulin (IgY), important for preventing bacterial, viral, mycotic infections etc. Egg white proteins, such as ovotransferrin (OTF), lysozyme, avidin perform a series of biological activities [1].

More than 60 years ago, various researchers proved that ovotransferrin was an iron-binding protein with bacteriostatic effect [26]. Bacteria need iron for their growth and due to the binding properties, OTF is able to prevent bacteria from using iron [17]. OTF is synergic with other bacterial proteins like lysozyme, found in large amounts in the egg white.

Even the saturated form of iron is active, and inhibition is the result of agglutination by OTF of the bacteria having its cell walls split by lysozyme (11).

1. Characteristics of ovotransferrin

Ovotransferrin (OTF) or conalbumin is the major glycoprotein in the egg white (12-15%) consisting of a single chain with 686 amino acids, with a molecular weight of about 78 kDa. It is synthesized in the oviduct of the chicken and stored in egg white. OTF belonging to the family of transferrins which are divided into four categories:

- serum transferrin (isoelectric point (Ip) = 7.4), present in plasma, with main role in iron transport;

- lactoferrin (Ip = 8.8) present in milk and secretions of mammals, with main role in natural immune defense;

- melanotransferrin (Ip= 6.8-7.1), a protein located on the surface of melanoma cells;

- ovotransferrin (Ip - 6.0) present in egg white.

OTF comprises two domains (N- and C- terminal end) and each domain contains binding sites. Each domain is divided into two parts, containing 160 amino acids. OTF contains 15 disulfide bridges which stabilize the protein tertiary structure. OTF transports iron and is able to bind Fe (3+) ions in combination with an anion, usually bicarbonate. Each OTF molecule can closely but reversibly bind two iron atoms. Both iron binding sites are alike but not identical.

There are two types of ovotransferrin: apo- and holo-. Apo-transferrin does not contain iron and can be destroyed by physical and chemical treatment. Holo-transferrin is iron-saturated, the i ron-ovotransferri n complex, being stable under proteolytic hydrolysis and thermal denaturation [21]. Apo-OTF is colorless and easily destroyed under physical and chemical treatment, while Fe- binding ovotransferrin (holo-) is pink and resistant under proteolytic hydrolysis and thermal denaturation. Since most chemical reagents and denaturation conditions decrease Fe affinity to OTF, a particular OTF space configuration is necessary. Fe binding to OTF requires a CO3^2" or HCO3 molecule on the Fe³⁺ atom. Reiter and co. (1975) have demonstrated that bicarbonate rather than pH is an essential factor for the bacteriostatic capacity of OTF. A single anion (pyrophosphate, sulfate or chlorine) is necessary for in vitro Fe³⁺ release from iron ovotransferrin. Iron might be released from the transporting protein at low pH (pH = 4.5) in the presence of excessive sodium citrate (17). The OTF structure and function are similar to those of milk lactoferrin.

A fraction of faaman blood has the same iron-binding effect like ©votransfe s. This serum protein was called siderophilin, and nowadays it is called human transferrin. Ovotransferrin and blood transferrin have the same amino acid and carbohydrates composition [20].

Ovotransferrin is glycosylated and contains a single glycan chain (consisting of mannose and TSl-acetyl glucosamine residues) in the C-terminal domain. Ovotransfemn is a neutral glycoprotein synthesized in the oviduct of chickens and stored in egg white. It has two similar domains in the H and C terminal regions, each of them binding a Fe⁺⁺⁺, Cu^, Af^""^" atom very closely and specifically. The recognition of transferring molecules is mediated by membrane receptors.

Human transferring has two N-glycan compounds in the N-terrninal end, while OTF has a single N-glycan chain. The significant structural similarities between lactoferrin and ovotransferrin account for their similar biological role. [24]

OTF belongs to the group of proteins called metalloproteinase which induce the formation of thermal shock-responsible compounds. Due to this characteristic, OTF can be used in protective lotions and ointments against cold or other types of environmental stress.

OTF - an ideal immunomodulator

OTF activity demonstrates that the role of the molecule is closely related to the importance of iron. Iron deficiency, the anemia, is a common concern both for people, particularly in developing countries, and for animals during the first weeks of life. In its ionic form, excessive iron can become harmful for the organism. It is an essential element necessary for the development of pathogenic bacteria and tumor cells. The immune system cells protect tissues against toxicity, playing a primordial role in controlling the concentration of this metal. The presence of iron-binding proteins, such as transferrin and ovotransferrin which are able to neutralize the iron, results in the elimination of metal poisoning by binding it [12].

Cell-mediated immunity and intracellular bacterial damage induced by polymorphonuclear leukocytes are reduced in iron-deficient subjects. This fact is due to the lack of iron necessary for cell proliferation, DNA synthesis and iron-dependent enzyme activity [20].

Bullens (1983) demonstrated that lactoferrin and ovotransferrin limit the amount of ionic iron available in body fluids up to 10^*18 M. This amount is not enough for the normal bacterial growth, and pathogenic bacteria obtain iron by using hem components. The iron-binding proteins together with antibodies have a strong in vitro bacteriostatic effect and are essential for protection against various infections. Lactoferrin is important for the bactericide function of leukocytes against Pseudomonasaeruginosa.

Brock [22] showed that there is a connection between metal and immunity. Immune system cells protect tissues against toxicity, playing a primordial role in controlling the concentration of this metal. The mechanism proves that the synthesis of this type of cells and the presence of iron-binding proteins such as lactoferrin, ovotransferrin and ferritin, able to neutralize the iron, results in the elimination of metal poisoning by binding it.

2. Biological functions of ovotransferrin

Antimicrobial activity

Many researchers have demonstrated not only the bacteriostatic effect but also the bactericide effect in vitro of lactoferrin and OTF against a wide range of microorganisms including gram- positive and gram-negative aerobic and anaerobic bacteria, fungi and viruses such as the human cytomegalovirus (HCMV), herpes simplex virus (HSV1) or human immunodeficiency virus (HIV).

In vivo, milk lactoferrin can perform its inhibitor effect on microbial growth in the intestine of the newborn. The antimicrobial activity of lactoferrin has a role in the selection of newborn's intestinal flora and prevents enter opathogenic organism colonization. Elttson (1989) demonstrated that transferrins destroy the outer membrane of gram negative enter obacteriaceae. Tests have been conducted on lactoferrin and transferrin property of releasing radioactively labeled lipopolysaccharides (LPS) from Escherichia coli and Salmonellatyphimurium ceil wall. Moreover, transferrin enhances the antibacterial effect of rifampicin concentration, a substance which does not penetrate the outer membrane of the bacterium. This trial proves that iron-binding proteins destroy gram-negative bacteria membrane and impair its permeability [22].

Zagulskisi and co. (1995) have conducted trials to demonstrate the protective effect of bovine lactoferrin (BL) when intravenously administered in guinea pigs 24 h before a lethal dose of Escherichia coli. About 70% of BL-treated guinea pigs survived the trial. The survival rate of guinea pigs treated only with E. coli was 4-8%. Human lactoferrin has almost the same protective effect as BL.

At low concentration, lactoferrin determines a markedly inhibition of E. coli_i S.typhi and Shigelladysenteriae growth. The addition of 0.2 mg/ml lactoferrin in the culture medium resulted in a gradual decrease of bacteria a different incubation times between 0 and 12 hours. A maximum inhibition rate was recorded for E. coli (94%), followed by Salmonella typhi (78%) and Shigelladysenteriae (75%) [13].

The bacteriostatic effect of transferrins was associated with the iron-binding capacity. Almost all bacteria need iron and due to the capture properties, transferrins are able to prevent the iron from being used by bacteria. The also contribute to the blocking of bacteria carbohydrate metabolism or to cell wall damage by calcium and magnesium binding. Lactoferrifl is in synergy with other antibacterial proteins such as lysozyme present in secretions [25].

The property of certain transferrins to donate iron to the duodenal mucosa was studies using an in vitro incubation technique.

Bovine, human and goat iactoferrins were shown to have bacteriostatic effect against BacHhsstearothermophilus and BaciUiissubtilis, both in the presence and absence of insignificant amounts of metal. Bovine lactoferrin inhibited spore germination and growth of Bacillusstereothermophilus vegetative forms [10, 14],

OTF acts as growth inhibitor of a wide variety of microorganisms, the antibacterial mechanism of action being attributed to its property of binding and retaining Fe which is essential for bacterial growth. The authors proved that apo-OTF, Cu-OTF and Zn-OTF considerably inhibited E. coli and Staphylococcus aureus culture growth. Studies have been done on the O I F growth effect on the development of bifidobacteria. Their growth is stimulated by addition of apo-transferrin, Cu-ovotransferrin or Fe-OTF in the environment. Among the studied species, B. breve and B. hifidum were the most sensitive.

Soukka and co. (1990) studied the synergic inhibitor effect of lactoferrin and lactoperoxidase system on the viability of Streptococcusmutans, serotype C, in vitro. Both had significant antibacterial effect against S. mutans at low pH [23].

Bovine laetoferrin, the acute phase protein, present in bovine uterus discharge, as shown to bind to 6 species of coagulase negative staphylococci isolated from intramarrrmar bovine infections. The study results determined the presence of specific receptors of lactoferrin responsible for its binding to bovine-isolated staphylococci (Satyanarayan, 1990),

References

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6) A WADE A. C. (1996) -On hen egg fractionation: Application of liquid chromatography to the isolation and the purification of hen egg white and egg yolk proteins; Eur. Food Res. Technol., 202: 1-14

7) AZARI P.R. and R.F. BAUGH (1967). A simple and rapid procedure for preparation of large quantities of pure ovotransferrin. Arch. Biochem. Biophys. 118 : 138-144

8) BATTISTUZZI G„ M. SOLA (1992) -Fe3+ binding to ovotransferrin in the presence of a-amino-acids, BiochJBioph. Acta 1 118, 313-317

9) BULLENS J. J. ( 1982)-Semnificatia fierului in infectie - Bioch. Bioph. Acta 718, 42-48 ) CHUNG M.C., S.L. CHAN si s. SHEVUZU, 1991 0 Purification of transferrin and lactoferrin using DEAE Afii- Gel Blue, Int. J. Biochem. 23: 605 - 616.

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) GUO M., I. HARVER, W. YANG, L COGHDLL, D.J. CAMPOPIANO J., A. PARKINSON, R R MAC GBLLIVRARY, W.R. MARRIS, P.J. SADLER (203) - Sinergic anion and metal binding to the ferric ion - binding protein from Neisseria gonorrhoeae. J. Biol. Chem. 278: 2490 - 2502.

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) RE ITER B., J.H. BROCK and E D. STEEL (1975) - Inhibition of Escherichia coli by bovine colostrum and post-col ostral milk. U The bacteriostatic effect of lactoferrin on a serum susceptible and serum resistant strain of E. coli. Immunology, 28: 83-95

) SONKKA T„ M. LUMIKARI, J. TENOVUO (1990) - Efectul inhibitor combinat al lactoferinei si sistemului lactoperoxidazelor asupra viabilitatii Streptococcus mutans, Serotip C- J. Clin. Microbiol. 98, 2312-2319 ) TRANSSON G.B., KERSTIN THOREN TOLLING, BERN JONES (1983) - Absorb†ia fierulu! la purcelusji de lapte, J. of PedrGastr. Nutr. 2, 693-700

) VACHEER M.C., M. PIOT, O.J. AWADE (1995) -Isolation of hen egg white lyzozyme, ovotransferrin and ovoalbumin, using a quarternary ammonium bound to a highly crosslinked agarose matrix. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 664 : 201-210

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) CHIURCR7 VIORICA, I V. PATRASCU, C G CHIURCIU, GEORGIAN A TOPILESCU, B. FRUNZAREANU (2014)- IMB-PaChi Assay for Growth Inhibition of Bacteria by Neutralizing IgY Antibodies. European Biotechnology Congress Lecce 2014) CHIURCRJ VIORICA, I.V.PATRASCU,C.G. CHIURCIU, GEORGIANA TOPILESCU, R.T. CRISTINA(2014)- Growth Inhibition of antibiotic resistant bacteria by neutralizing IgY antibodies. European Biotechnology Congress Lecce 2014

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SHORT DESCRIPTION OF THE INVENTION

Brief formulation of technical solutions

The objective of this invention is to design a method of production for specific ovotransferrin against antibiotic-resistant or sensitive bacterial antigens, viral antigens. Bacterial or viral strains are provided by patients with clinical symptoms hospitalized in Romania.

The specific ovotransferrin has antibacterial and antiviral effect, helping the norma! growth of organisms. An intensive research program was developed in order to obtain specific OTF, with the following purpose: achievement of polyclonal antibodies in egg yolk (IgY) and GTF- M with specific effect on various epitopes, use of methods of production which preserve the highest level of specific activities and application of some control methods for the accurate assessment of their effect on the target antigen. The monovalent or polyvalent antigen was used in mixture with an adjuvant QS21 according to the methodology described in this invention patent. This technology is easy to use and specific OTF can be sold at an accessible price and exploited in the medical institutions of Romania. By using the ELISA assessment kit and IMB-PaChi kit which assess the strains isolated from each patient, a specific treatment program can be designed using OTF which has a direct effect and blocks the in vitro bacterial development.

This invention refers to the production of OTF from egg white on the following stages:

(a) preparation of a complex antigen using antibiotic-resistant bacterial strains isolated from patients in Romania. The antigen is a mixture of antibiotic-resistant strains From a single species of bacteria or an antigen prepared from a mixture of various species of strains from various species of bacteria;

(b) mixture of the antigen with adjuvant QS21;

(c) immunization of poultry with the antigen prepared as per section (a) and b) generating specific antibodies in the chickens, which are then transferred and built up in the egg yolk;

(d) egg white isolation from the eggs of chickens immunized with the target antigen and extraction of the water-soluble OTF-containing fraction, by separation from the water- insoluble fraction; (e) passing of the water-soluble fraction through 0.45 μιη prefilters and 0.22μπι filters;

(f) OTF concentration by tangential filtration through 30kDa cassettes.

Another objective of this invention is the use of specific OTF in clinics and laboratories. The specific OTF manufactured by this procedure is easy to prepare and has an accessible price. These OTF-M are specific for the often antibiotic-resistant bacterial strains present in the environment of Romania. The specific OTF can be marketed as monovalent product consisting of a single specific OTF against a single pathogenic germ or as mortiple OTF-M which is a mixture of specific OTF against various species of human-isolated pathogenic bacteria.

A number of methods of separations of specific ovotrartsferrins were tested.

Ovotransfenin can be separated from the egg white by ethanol fractionation procedures (Bain and Deutsch, 1948; Warner and Weber, 1951), ammonium sulfate precipitation or egg albumin coagulation (Warner, 1954; Azari and Baugh, 1967). Nevertheless, the disadvantages of these techniques of ovotransferrin purification are protein denaturation, and the achieved purity is relatively poor (Vachier and col., 1995).

Some researchers used, for ovotransferrin purification, besides ethanol and ammonium sulfate precipitation, ion exchange chromatography with DEAE-cellulose, Q-Sepharose columns (KO and Ahn, 2008). Chung and co. (1991) applied DEAE-Affi Gel Blue chromatography as a first fractionation stage of egg white proteins. The ovotransferrin-containing fraction obtained on DEAE Affi Gel Blue was further purified using Fast Flow Liquid Chromatography.

A two-stage chromatography was also used: on Superose-6 Prep column and on Q-s*sperose Fast Flow column (Awade and co. 1 94).

The large scale application of these techniques is challenging because they are difficult and very expensive (Awade, 1996).

Ko and Ahn (2008) applied a simple and economical purification method for the large scale production of egg ovotransferrin. It was separated from the yolk and diluted with the same volume of distilled water. In order to prevent denaturation during separation, ovotransferrin was transformed in holo- by adding 20 mM iron chloride solution (FeCb.6H20). It was iron-bound and separated from the egg white using various concentrations of ethanol (30% up to 50%). Using the final ethanol concentration of 43% and pH 9.0 were the best conditions for separation of ovotransferrin from the egg white.

The precipitated holo-transferrm was dissolved in distilled water arad subjected to chromatograph on ton exchange column (AGI-X2) to remove the ovotransferrin-bound iro after pH adjustment to 4.7. Apo-ovotransferrin which was obtained through this protocol had more than 80% purity degree. This method is simple, economical and fit for the large scale production of egg white ovotransferrin. The isolated ovotransferrin can be used in human food because the single solvent used in this process is ethanol. The AG1-X2 ion exchange agent and the ethanol used in this process can be regenerated as well.

Abeyrathxie (2013) described a method for ovotransferrin isolation from the egg white without using organic solvents, with more than 85% purity, by combination of ammenium sulfate precipitation and citric acid.

One of the objectives of this invention is to describe a simple and easy method to obtain ovotransferrin without using organic solvents.

Ovotransferrin is prepared by the following steps:

(a) Prepare antigens using antibiotic-resistant bacterial strains isolated from patients in Romania.

(b) Prepare antigens by mixture with an adjuvant QS21.

(c) immunize chickens with antigens prepared as per sections (a) and (b) generating specific antibodies in chickens, which are transported via the oviduct and built up in egg white and yolk.

(d) Separate the egg white from the yolk and dilute 1 : 1 with deionized water.

(e) Remove ovomucin by centrifugation or tangential filtration, adjusting the pH at 4.5-5.0.

(f) Add ammonium sulfate and citric acid to the obtained supernatant and store at 4°C overnight.

(g) Centrifuge or filter the suspension and dissolve in deionized water the obtained precipitate which contains ovotransferrin.

(h) Perform the second precipitation using distinct ratios of ammonium sulfate and citric acid.

(i) Dissolve in deionized water the precipitate obtained after centrifugation or filtration and subject it to dialysis to remove salts.

(j) Identify the protein by agar immunodiffusion assay (ID) using anti- ovotransferrin serum. (k) Assess ovotransferrin specificity by immunoenzymatic assay (ELISA) and antibacterial activity by bacterial growth inhibition assay (IMBPaChi).

This method of OTP prepar tion is easier and Jess expensive than those prev ously d scribed; ovotransferrin can be used in clinics and laboratories since it is specific for some strains present in Romania which are often antibiotic-resistant. Ovotransferrin can be marketed as a monovalent product because it is specific against a single pathogenic germ or as natural complex protein with antibacterial properties against various human-isolated acterid species.

The specific ovotransferrin can be used in antimicrobial, antiviral treatments and as iron supplement in human medicine.

BRIEF DESCRIPTION OF PROCEDURES

The objectives of this patent are revealed by the description below:

Annex # 1. It presents the preparation of specific ovotransferrin by egg white dilution and two-stage precipitation with various concentrations of ammonium sulfate and citric acid and describes these processes by biochemical and immunological methods:

a) Identification and assessment of the total protein content by Bradford method

b) Identification of OTF-M by agar get immunodiffusion assay against anti-avian rabbit serum

c) Identification of OTF-M by radial agarose gel immunodiffusion assay against anti-avian rabbit serum

d) Identification of specific OTF-M by ELISA using anti-avian conjugate

e) Identification of specific OTF-M by bacterial growth inhibition assay (IMB-PaChi) using standard ATCC bacterial cultures.

DETAILED DESCRIPTION OF INVENTION

The extraction of ovotransferrin from the egg white of chickens immunized with a target antigen is an economical cost-effective process. According to this invention, the preparation of specific ovotransferrin consists of various stages: preparation of antigen (1), immunization of conventional or SPF laying chickens (2), extraction and partial purification of ovotransferrin (3), qualitative and quantitative antibody assessment (4).

1, Preparation of antigen

According to this invention, antibiotic-resistant bacterial strains were isolated from patients. The bacterial cells cultivated on selective media are harvested and washed three times wit phosphate buffer (PBS) and centiifuged at 4000 rpm for 15 minutes.

2. Immunization of conventional or SPF laying chickens

The antigen is administered by intramuscular inoculation of 0.5 ml in four distinct sites on the chest muscles of conventional or SPF laying chickens. The antigen is inoculated three times every 14 days. The presence of specific antibodies in biood and eggs is tested after the second administration, by ELISA, ID and BVlB-speciftc PaChi. Eggs are harvested 14 days after the third antigen inoculation, when antibody titer is assessed on regular basis from the eggs of chickens immunized with the target antigen.

Large amounts o ovotransferrin can be obtained from the eg white of chi¾k¾n¾ immun zed with the target antigen, without significant capital.

The immunization of laying chickens with a target antigen is a well known technique. This invention can undertake any method of chicken immunization which consists of the administration of the target antigen by any route: subcutaneous, intracutaneous, intramuscular, intravenous.

Adjuvant QS21 was used for this invention. Other types of adjuvants can also be used, such as complete or incomplete Freund adjuvant or a mixture of them. Using antigen QS21 mixed with the target antigen enhances the immune response, induces no local reactions and has been demonstrated to be efficient for the production and maintenance of a high titer for a long period of time.

3. Preparation of ovotransferrin from the egg white

A simple procedure was used in this patent, allowing the quantitative and qualitative storage of ovotransferrin obtained from the eggs of chickens immunized with a target antigen. The egg white is extracted after chicken immunization.

In this invention, the yolk is separated from the egg white, diluted 1 : 1 with desonized water, mixed and pH is adjusted to 4.5-5.0; ovomucin is removed by keeping the egg white diluted for 24 h at 4°C. Ovotransferrin was obtained by 5% (w/v) ammonium sulfate precipitation followed by 2% (w/v) citric acid precipitation. The precipitate harvested after centrifugation was dissolved in deionized water and precipitated with 2% (w/v) ammonium sulfate and 1.5% (w/v) citric acid. The sediment obtained after centrifugation is subjected to dialysis with O.lSMNaCI soiutioB.

Ovotransferrin is subjected to qualitative and quantitative assays. The quantitative determinations consist of testing the total protein content by Bradford method, radial immunodiffusion, ELISA and IMB-specific PaChi. The qualitative determinations are performed by agar gel immunodiffusion assay and ELISA.

This method preserves the natural structure and stability of ovotransferrin. The method is simple and the extraction cycle is short.

RECOMMENDED METHODS OF USING THIS INVENTION

The examples below are for illustration only and have no intention of restricting the purpose of this invention.

Example 1

Preparation of OTF-M a) Attach a quality certificate issued by a competent authority to each bacterial strain, b) Store each strain at -75°C as original product. Bacterial growth for preparation of antigen should be performed in the recommended culture medium;

c) From the 24 hours bacterial culture, prepare a bacterial sediment equal to SxlO^CFU, by centriftiga sefj! for 15 minutes at 4000 rpm at +2 ° C;

d) Re-suspend the obtained sediment in PBS at the initial volume and freeze-dry 2 ml in 4 ml vials. After freeze-drying, store the bacterial culture at -2Q°C;

e) Re-suspend the sediment obtained as per section d) at the initial volume in PBS with 0.4% formalin and incubate overnight at +37°C. Remove formalin by centrifugation for 15 minutes at 4000 rpm at +20°C. Re-suspend the inactivated bacteria in PBS and freeze-dry. After freeze-drying, store the vials at -20°C;

f) Prepare the antigen using 500 μg inactivated cells/ml or 3-4.5 μg protein/ml reconstituted in 0.5 ml PBS and mix with 0.5 ml adjuvant QS21. For chicken immunization, dilute equal parts of the mixture and 5% Tween 20 emuf sifier,

g) The antigen is a mixture of strains from the same species of bacteria or a mixture of distinct species of bacterial strains;

h) 140-180 days old, clinically healthy, conventional or SPF laying chickens are reared in groups of 10, in cages provided with specially designed battery cage and automatic feeding and watering systems. Each chicken is labeled on both wings;

i) Inoculate the chickens intramuscularly in four distinct sites with 2 ml of target antigen prepared as per section 1.f)

j) After the first administration, inoculate chickens twice with the same antigen, by the same administration route, at 14 days interval; k) Take blood samples during the second and third administration and perform the immune response control against the target antigen;

1) Treat the blood samples taken from chickens after the second and third administration of antigen in order to express the serum extracted from the clot and store at +4°C or -20°C. m) Assess the immune response by qualitative and quantitative ELISA, agar gel immunodiffusion assay (SPGA), single radial immunodiffusion assay (ID S) and EvLB- specific PaChi assay (28). Assess the activity of antibodies extracted from the yolk using each assay kit. Check OTF-M by:

- Agar gel immunodiffusion assay (SPGA), using anti-avian rabbit serum compared to standard OTF.

- SPGA using binary OTF-M dilutions tested against the target antigen. Assess the higest dilution providing a specific response and compare with the in-house standard.

- Qualitative ELISA

- Quantitative ELISA

- Bradford method for protein determination

- IMB-specific PaChi assay

n) Chickens with positive response to the target antigen are approved for OTF-M production, o) The best time for egg harvesting is when 100-200 mg of IgY are extracted from the yolk and 10-20 mg OTF-M are extracted from the egg white.

Example 2

OTF-M filling

After testing OTF-M by SPGA, IDRS and bacterial growth inhibition asd after total protein determination, the prepared batches are filled as per the following steps: a) Perform OTF-M purification by tangential filtration using 30kDa cassettes, by successive change of the wash solution (PBS) to pH7 from the filter device;

b) Concentrate OTF-M by tangential filtration removing the excessive saline (PBS);

c) Filter OTF-M prepared as per section 2b) by 0.45 μπι filters in specially designed places with sterile air filtered by 99.997 yield cassettes for 0.1 μπι particles into a GMP-certified working area;

d) Freeze-dry OTF-M prepared as per section 2b): 20 ml OTF-M solution in 50ml vials. e) Mix OTF-M prepared as per section 2b) in proportion of 20 mg OTF per gram gel or Vaseline for skin applications, and 20 mg specific OTF in 80 ml phosphate buffer for oro- pharyngeal-gastrointestinal treatment.

f) Fill the prepared OTF-M by sterile distribution into vials, ampoules or tubes according to dosage or administration route.

Example 3

Final control of OTF-M a) The below described methods of eoatrol are vised for t¾e filial forisuiatksR of OTF-M solution for skin, intranasal, oro-pharyngeal-gastrointestinal applications:

- Microbiological sterility;

- Determination of specific OTF activity by ELISA and SPGA;

- Determination of OTF content by ELISA;

- Determination of bacterial growth inhibition by IMB-specific PaChi assay b) Only the control-appropriate batches of specific OTF are approved for human consumption.

Example 4

A. Quantitative determination of OTF-M by indirect ELISA ELISA is "in house" prepared for each and every assay.

The minimum detected amount of OTF-M is 10 nanograms in the tested material. Due to the specificity and reproducibility of immunoenzymatic reaction, ELISA is assd in the manufacturing process of OTF, on production stages and during qualitative and quantitative control.

a) Perform the ELISA to be used in the quantitative control of OTF by comparing with an international OTF standard (USA) or with an OTF substandard prepared atRomvac.

b) Cover the wells with 150 μΐ anti -avian rabbit serum IgG at 3.75 μηι/ml concentration in carbonate-bicarbonate buffer;

c) Incubate the 96-ELISA well plates for 90 minutes at +37°C;

d) Wash four times in 300 μΐ PBT-Tween using an automatic plate washer;

e) Add 200 μΐ of 1% BSA in PBS-Tween in each well and incubate for 45 minutes at 37°C; f) Wash the reaction plate four times in PBS-Tween as per section d);

g) Add in triplicate 150 μΐ specific OTF-M or OTF SPF (25, 12,5, 6,25 μg ml) in PBS;

h) Add In parallel in triplicate 150 ul SIGMA standard OTF (25, 12,5, 6,25 μg/ml);

i) Incubate plates at +37°C for 90 minutes;

j) Wash plates four times in PBS-Tween;

k) Add 150 μΐ IgG anti IgY conjugated with peroxidase to 1:5000 dilution;

I) Add 150 μί TMB and incubate for 5-15 minutes at room temperature in the dark m)Freeze the reaction with 150 μΐ HC1;

n) Read the reaction under DO450 filter spectrophotometer;

o) Perform comparative standard curves and consider the highest positive dilution to 0.200 DO. B. Quantitative determination of OTF-M by indirect ELISA a) Cover the plate overnight at +4°C or for 2 hours at room temperature with OTF to be tested and standard OTF each in three replicates, in binary dilutions starting with 1:1000 dilution in carbonate-bicarbonate solution;

b) Keep wells Al and HI as OTF controls;

c) Wash 3 times with wash solution;

d) Add 100 μΐ anti-avian 1 :5000 diluted conjugate;

e) Incubate the plate for 2 hours at +37°€;

f) Wash 4 times with wash solution;

g) Add 100 μ! TMB and leave at room temperature for 5-15 min;

h) Add 100 ul stop solution;

i) Read the reaction absorbance under 450 nm filter spectrophotometer.

j) Assay the reaction by blank wells control where DO450 should be maximum 0.060. The reacti on is not valid if other values are obtained,

k) The dilution for which DO450 is 0.200 than the international standard OTF is considered positive reaction for OTF presence.

1) Perform the μg/ml OTF content in comparison with reference OTF taking into account that

ELISA detects minimum 10 ng ml and it is performed in comparison with standard OTF.

Example 5

C Determination of specific OTF-M content by ELISA

The specific OTF activity is determined by a quantitative method against the antigen represented by the whole bacterial cells inactivated and freeze-dried as per section 1). The reaction plate is covered with an antigen and specific OTF is tested in successive binary dilutions starting from 1 : 1000 dilution in triplicate. The highest positive dilution is when the reaction is equal or higher than 0.200 OD or the numerical value for the dilution higher than 0.200 OD. The positive reaction to this dilution is given by 5-10 ng of specific OTF per well, per 150 μΐ.

a) Cover an ELISA plate with 150 μΐ from the suspension of bacteria freeze-dried at 1.67- 1.70 g cells per ml or 10 g bacterial protein per ml in carbonate-bicarbonate buffer (0.05 M, pH 9.6);

b) Store the covered plate for 12 hours (overnight) at +4°C;

c) After removal of the liquid, wash the plate 3 times in PBS-Tween 20 (2%) wash solution; d) Stop the reaction with fixation buffer, 300 μΐ/ well and incubate the plate for 30 minutes at room temperature;

e) Remove the stop liquid;

f) Dry the plate for 30 minutes in desiccator;

g) Distribute in each well 100 μΐ from the OTF suspension diluted 1: 1000 in binary dilutions up to 1 :24 as per the plate configuration. Test OTF to be assessed in triplicate;

h) Keep wells Al and HI as antigen controls, wells Bl, CI and Dl as negative reaction controls using OTF-SPF and wells El, Fl and G3 as positive reaction controls using reference specific OTF;

i) Incubate the plate for 2 hours at + 7°C;

j) Wash 3 times in wash solution;

k) Add 100 μΐ of 1 :5000 diluted anti-avian conjugate using the dilution buffer as diluent;

1) Incubate the plate for 2 hours at +37°C ,

m) Wash the plate 4 times in wash solution;

n) Add 100 ul TMB and leave at room temperature for 5-15 min.

o) Add 100 ul stop solution;

p) Read the reaction under 450 nm absorbance spectrophotometer;

q) The reaction is valid when the reaction in the control blank wells Al and HI reveal values lower than 0.060 DO, when the reaction in OTF-SPF (negative) control wells Bl, CI and Dl reveal values of 0.060-0.090 DO and positive control wells El, Fl, Gl reveal values of 1.400-1.800 DO.

EXPERIMENT: ELISA EC Oil

Control of reaction:

Al, A8, HI and H8 (blank): OD 0.045

OTF-SPF (negative) Bl, CI, Dl and B8, C8 and D8: OD-0.065

Positive reactions for various specific OTF-M:

a Seriel

a Serie2

S Serie3

1 2 3 4 5 6 7 8

Anti S.enteritidis OTF-M x 3 replicates

—Φ—Seriel -^«5lr"Serie3

1 2 3 4 5 6 7 8

Assessment of ELISA plate:

Series 1 - blue line, anti S.enteritidis specific OTF-M

Series 2 - red line, anti E. coli specific OTF-M batch #05

Series 3 - green line, anti R coli specific OTF-M batch #04

Example 6

Determination of OTF-M contents by single radial immunodiffusion assay (Mancini Method)

Single radial immunodiffusion (IDRS) has been accepted as safe method of quantitative determination of the antigen and/or serum using a standard reagent. IDRS can very accurately confirm the protein content in OTF against anti-avian serum. The agarose- or agar-prepared gel is recommended in this case and the test consists of the following steps:

a) Prepare the 1% agarose mixture in PBS containing sodium azide 0.05%;

b) Heat agarose at 80-90°C;

b. l) Mix the agarose gel at 56°C with anti-avian serum at 56°C and pour into disposable plates with 5 cm diameter;

c) Allow the agarose to solidify in the plate at room temperature;

d) Perform 4 mm diameter wells at minimum 20 mm distance;

e) Pour a warm agarose drop in each well;

f) Distribute 30 μΐ of whole OTF in successive dilutions 1/2, 1/4, 1/8 etc. in PBS in gelose wells;

g) Store reaction plates at room temperature in liquid medium and read the reaction at 24, 48 and 72 hours;

h) Agar staining with Coomassie Brilliant Blue.

- Fix the gelose with a mixture of ethanol and acetic acid for 30 minutes at room temperature Remove the fixing solution.

- Stain the gel with 10 volumes of Coomassie Brilliant Blue, for one hour or overnight at room temperature (the dye solution can be reused);

- Decolorize the gel in 10 volumes of decolorizing solution at room temperature for 30 minutes;

- Repeat decolorizing;

- Dabble the gel for 15 minutes in 1-2% glycerol in deionized water;

- The gel is ready to be photographed.

- At the same time, measure the diameter of each precipitation ring;

Calculate the equivalent in milligrams protein (OTF) per ml solution accordingly:

reaction ring diameter minus diameter of the well, divided by 2 (thickness on both sides) and multiplied by 3.3 correction coefficient for the amount of tested OTF-M per well against the reference serum. The result can be represented graphically, depending on the dilutions used for OTF-M (Annex #1DRS).

Total protein:

- OTF AIE before filtration = 58.20 mg/m!

- OTF ATE after filtration through 0.45 μηι = 31.39 mg / ml

- OTF BLV before filtrati on =33 mg/ml

- OTF BLV after filtration through 0.45 μπι = 29.40 mg/ml

Central well, wells 1 and 4 : anti IgY rabbit serum

In wells 2 and 6 : Whole OTF ATE + Carre S.03/20.02.2014

In wells 3 and 5 : IgY K.p. + S t. 5.04/12.02.2014 dilution 1/32

Example 7

Determination of protein content by Bradford method

Bradford method is used for determination of protein content in various biological products among which there are OTF-M suspensions.

Bradford method is based on the Coomassie Briliant Blue G-250 stain binding to the protein, resulting in a protein-stain complex. It has a high extinction coefficient resulting in great sensitivity of protein assessment. The protein binding of the stain is a very fast process (about 2 min.), and the protein-stain complex remains dispersed in the solution for about 1 hour. The assay is performed in acid pH, with maximum absorption at about 595 ran. Protein concentration is determined by comparison with the response of the standard depending on the nature of tested protein: serum albumin (BSA), bovine gamma globulin (BGG) or avian immunoglobulin (standard OTF).

(a) Reagents

- Coomassie Briliant Blue G-250 (Sigma-Aldrich);

- Orthophosphoric acid 85% (Merck);

- Ethyl alcohol 95 % (Chimopar);

- Bovine serum albumin (Sigma);

- Standard OTF (Sigma).

(b) Materials and equipment

- ELISA plate reader

- ELISA plates

(c) Preparation of Bradford reagent. Stock solution: Dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 ml ethyl alcohol 95%, add 100 ml orthophosphoric add and dilute with distilled water to a fixed volume of 200 ml. Filter the solution through filter paper (Whatman #1 or equal). Store the so-prepared stain in brown vial at +4°C, being stable for at least 6 months. Prepare the working Bradford reagent by diluting the stock solution with distilled water in 1 :5 ratio. It can be stored at +4°C being stable for maximum 7 days.

(d) Preparation of standard protein solutions. Standard stock protein solution: dissolve 0.2g of the reference material (BSA) in the same buffer used for the preparation of test solution and bring to fixed volume of 100 ml. The solution has a concentration of 2 mg/ml. Prepare the standard OTF solution as per section 9(d).

(e) Standard working solutions: dilute parts of the stock solution with the same buffer solution to obtain five to seven standard dilutions with concentrations between 1 and 1500 ug protein per ml.

φ Preparation of test solution. Dissolve an appropriate amount of test protein in buffer so as to obtain a solution with concentration within the limits of standard working solution concentration range.

(g) Distribute on the ELISA plate 25 μΐ sample or standard in separated wells and in replicates.

(h) Add 300μ1 Bradford reagent - working solution and easily stir for 30 seconds. Avoid frothing, which causes errors when reading the absorbance. Incubate for 10 minutes at room temperature in the dark.

(i) Measure the absorbance at 595 nm against a control (buffer); read within 60 min.

(j) Calibration curve testing. In order to draw the standard curve, prepare seven working solutions with concentrations of: 62.5; 125; 250; 500; 750; 1000; 1500 g BSA or OTF/ml. Test each point on the curve in quadruplicate. The optical density variation of the Coomassie Brilliant Blue G-250-protein comple depending on th protein standard concentration is shown in Fig. 1 and in Photo 1. The regression line equation for the standard curve is y = 1.0017x + 0.0083.

(h) Method validation. Testing of standard in quadruplicate should yield reproducible results.

Test sensitivity is assessed as appropriate if 62.5 g protein/ml in the sample provides 0.095 absorbance units.

Fig.l. Calibration curve

Photo 1. Plate - calibration curve

(I) Bradford method for protein determination is a sensitive, reproducible method providing a convenient signal linearity range: 1-750 μg/ml. Bradford method has the advantage of good stability of staining reagent (Commassie Brilliant Blue G-250). The assays conducted using Bradford method, require very small amounts of samples (25 μΐ). Results can be read 10 minutes after the start of staining reaction.

Experiment no. 30/25.06.2014

Total protein:

OTF-M AIE before filtration = 58.20 mg ml

OTF-M AIE after filtration through 0.45 μιη = 31.39 mg / ml

OTF-M BLV before filtration =33 mg/ml

OTF-M BLV after filtration through 0.45 μιη = 29.40 mg/ml

The results reveal difference in the protein content of specific OTF-M depending on the degree of purification by filtration (Millipore 0.45μη filters). Example §

Determination of bacterial growth inhibition using IMB-specific PaChi assay

(1) . 1MB PaChi is the standard kit used for the assessment of in vitro specific bacterial growth infeibitkin of immunoglobulin (OTF-M). This test is based on the capacity of specific antibodies to inhibit and neutralize bacterial growth, IMBPaChi assay is monovalent and effective against a single group of epitopes found in a single bacterial species. 1MB PaChi assay reveals the presence of these epitopes (15-50%) in other bacterial species as well. The assay reveals the specific OTF inhibition capacity against other antibiotic-sensitive or resistant bacterial strains.

(2) . 1MB PaChi assay kit contains

(a) bacterial growth culture medium

(b) culture medium with OTF SPF negative control

(c) culture medium with specific OTF-M for the test bacterial strain

(3) . Working protocol

(a) Grow the bacterial suspension to be tested in the medium of the black-labeled tube #1.

To this extent, place 0.1 ml of the bacterial culture in 9.9 ml culture medium. Incubate the culture at 37 °C for 4, 6 or 24 hours.

(b) Take 0.1 ml from the din bacterial suspension removed from thermostat after 4, 6 or 24 hours and mix it with 9.9 ml culture medium, mix well and read optical density under 600 nm filter spectrophotometer. The optical density should be about 0.05 DO<soo.

(c) from the dilution tube, aseptically distribute 2 ml bacterial suspension in each of the two tubes left in the kit. Incubate samples at 37 °C and stir continually. Read the results 4 and 8 hours after incubation. Perform final reading 24 hours after incubation. Read with the naked eye and/or under 600 nm filter spectrophotometer.

(d) the specific bacterial growth inhibition can be noticed 4 and 8 hours after incubation at 37°C. Perform final reading 24 hours after incubation at 37 °C. The specific inhibitory effect of OTF-M can be seen with the naked eye when the culture medium remains transparent in the OTF-M sample, and the medium from the positive control sample has a stronger and stronger turbidity after 4, 8 and 24 hours.

(e) a sample is positive when there is a visible difference between control sample turbidity and specific OTF-containing sample turbidity. When optical density is assessed under spectrophotometer, the sample is positive if the difference between control and specific OTF-containing sample is higher than 0.1 DO 600 nm.

(f) the best bacterial growth inhibition effect occurs if bacterial growth is stopped 24 hours after incubation at 37 °C and optical density is 0.060 DO$oo.

(g) the efficacy of specific OTF can be assessed depending on the inhibited amount of germs. Treatment can be formulated under these conditions using a single or various doses per day.

Experiment no. 51/07.05.2014

1. Purpose of experiment: Testing of bacterial growth inhibition under the influence of specific OTF-M against E. coli Kp bacteria cultures

2. Reagents:

2.1. Bacterial s rains

E. coli 56637 ^passage performed on 06.05.2014, from the collection of strains of VB Hospital, mixed with 20% glycerin, stored at -75°C, on 12.12.2013)

> Klebsiella pneumoniae 5S826 (passage performed on 06.05.2014, front the collection of strains of VB Hospital, mixed with 20% glycerin, stored at -75°C, on 12.12.2013 )

2.2. Specific OTF-M

OTF-M Ec, prepared on 31.03.2014, freeze-dried, reconstituted with TSB medium (200 mg 2 ml)

> OTF-M Kp, prepared on 17.01.2014, freeze-dried, reconstituted with TSB medium (200 mg 2 mi).

2.3. IgY SPF batch 04 (non sterile), freeze-dried, prepared on 21.01.2014, reconstituted on

25.03.2014 with TSB medium (1 :1 ratio) Results:

Reading under DOsoo spectrophotometer

OTF-M K. pneumoniae QTF-SPF 24 hours after incubation

B! Seriel m Serie2

Red line x 3 replicates - control OTF-SPF

Blue line x 3 replicates - K. pneumonia-specific OTF-M

A total inhibition is noticed within 4 hours with slight increase after 24 hours, which confirms the specific inhibition capacity of K. pneumonia-specific OTF-M

OTF-SPF compared toE. Colt- specific OTF-M

4 h of incubation 24 h of incubation

0,549 1,130

0,552 1,109

0J55 0,951 a Seriel

■ Serie2

Column 2: control OTF SPF

Column 1: E. Coli- specific OTF-M

The specific inhibition of anti E. coli OTF-M against OTF-SPF is noticed after 4 and 24 hours.

Claims

CLAIMS Title MANUFACTURE AND USE OF MODERN OVOTRANSFERRIN (OTF-M)

1. Method of manufacture of OTF-M, which refers to the achievement of this protein (OTF- M) by immunization of laying chickens with an antigen prepared from a mixture of antibiotic-resistant strains from a single species of bacteria or of an antigen prepared from a mixture of various antibiotic-resistant species of strains, isolated from patients from Romania. It is extracted from the egg white of chickens immunized with a target antigen. Dilute the egg white with deionized water in proportion of 1/1, adjust to pH 5 and perform a two-stage precipitation with ammonium sulfate and citric acid. Preserve by freeze- drying.

2. Claims for the method in section 1 refer to the fact that the antigen is a mixture of antibiotic-resistant strains from a single species of bacteria.

3. Claims for the method in section 1 refer to the fact that the antigen is a mixture of bacterial strains froni various antibiotic-resistant species of bacteria.

4. Claims for the method in section 1 refer to the fact that the antigen contains a mixture of inactivated bacteria mixed with an adjuvant QS21 which is very well tolerated by chickens inoculated intramuscularly.

5. Claims for the method in section 1 refer to the fact that the antigen contains a mixture of inactivated bacteria mixed with an adjuvant QS21 which provides a very good antigenic stimulus in the organism of immunized chickens, achieving a high OTF-M titer.

6. Claims for the method of manufacture of specific ovotransferrin (OTF-M) extracted from eggs of chickens immunized with antigens prepared from antibiotic-resistant bacterial strains isolated from hospitalized patients with clinical symptoms.