EP3212664A1 - Manufacture and use of hyperimmune egg pc2 - Google Patents

Abstract

This invention refers to the manufacturing method of the hyperimmune egg-PC2 consisting of preparation of antigens and immunization of laying chickens with a mixture of antigens prepared from human- and animal-pathogenic germs. This invention refers to the composition of antigens prepared from a mixture of antibiotic-resistant strains from a single bacterial species and from a mixture of various species of bacteria, viruses and fungi, all representing a stimulus administered in each chicken.

Description

MANUFACTURE AND USE OF HYPERIMMUNE EGG PC2

Inventors:

Patra§cu Ionel Victor, Chiurciu Viorica, Chiurciu Constantin,

Topilescu Georgiana and Mihai Iuliana

Technical field

Medicine, immunology, polyclonal antibodies, preventive and curative treatments by passive immunization, immunological health support of over 60 years old patients and immunosuppressive patients.

This invention describes a manufacturing method of the polyvalent hyperimmune egg IMU OINSTANT. This invention is directly related to the manufacturing method of the complex, highly purified antigen from the mixture of various antibiotic-resistant bacterial strains or standard from various bacterial species. This invention is directly related to the manufacturing method of the antigen by mixture of inactivated bacterial strains, yeasts and viruses with a special adjuvant QS21.

Description of invention:

Description of the stage of technique including references

During 1996 Prof. Dr. Roy Walford (1) determined that the biological decline of the adult is based on the immunological aging of the organism, called immunosensescence. Dr. Walford explains at length that the immune system decline is the major cause of systemic aging, including the increased risk of cancer, infections and swelling¹.

Researchers' concern for finding various solutions to cure diseases often starts with the study of the human immune system. Without this defense "invisible wall" provided by a strong immune system, the man is susceptible of almost any possible disease, from pneumonia to arthritis and diabetes^2"4. Even cancer or cardiovascular di seases take advantage of the weakness or overactivity of normal immune functions ⁵·⁶.

Infections and cancer occur when the immune system loses its normal surveillance or intensive control capacity⁷. Cancer patients are extremely vulnerable to infections because both the disease and treatments keep on attenuating the immune system. On the other side, autoimmune diseases occur when the natural restrictions of the immune system reactivity decrease, allowing instead an uncontrolled inflammatory response which, in its turn, may increase the risk of cancer⁸'⁹.

The good news is that by protecting or even stopping the aging of the immune system we can substantially delay the pathological impact of aging itself¹⁰'^{1 1}. Scientists are constantly looking for new immunity enhancement methods via food supplements, diets, physical exercise, rest and of course through expensive medication applied by the pharmaceutical industry.

Scientists have discovered a very interesting way of providing a natural immunological support using immune-protective proteins from chicken eggs. This discovery engages to substantially increase immunity for just a small part of the price of medication¹². This is good news for each of us as we get older and great news at the same time for all people whose immune system is weak, such as cancer or HIV/AIDS patients.

Recent studies on the biological support of human body during aging using the hyperimmune egg have demonstrated that immunoglobulins react particularly with the pathogenic germs they were produced for in the organism of immunized chickens. This activity reflects upon the intestinal mucosa where it improves cell membrane functions also generating a biological comfort by neutralizing the toxins in the digestive contents. Hyperimmune egg consumption protects and even stops immune system aging and substantially delays the pathological impact of aging itself ^{10;1 !} . Hyperimmune egg consumption allows the substantial enhancement of immunity for just a small part of the price of medication ¹².

In the last 25 years, chickens have been more and more used instead of mammals for antibody production. The biggest advantage is the fact that antibodies are collected from the egg and not from the serum. At the same time, the amount of antibodies produced by a laying chicken is larger than that produced by a mammal of the same size. Mammalian immunoglobulin purification is a time-consuming and expensive procedure. Nowadays chickens are considered a cheap and convenient source of antibodies. The amount of immunoglobulin obtained from one egg is equal to that prepared from 300 ml rabbit blood.

Eggs today can be customized so as to provide various types of immunity by consuming them. 24 distinct organisms have been used for the immunization of a single chicken which can further produce eggs for the passive immunization for all these organisms¹⁴.

Cholesterol control. Atherosclerosis is another symptom of immunosenescence. The increasing lack of control of swelling facilitates the formation of clogged arteries within a process the Americans call "inflamm-aging"^{18 19}. The US Army Research Institute of Environmental Medicine has become interested in this process due to the extremely large number of soldiers with a high cholesterol level and decided to study the potential of hyperimmune egg extract in decreasing the cardiovascular risk²⁰.

It was discovered that the cholesterol of soldiers who had drunk a solution of hyperimmune egg powder was stable compared to the placebo-treated control group, whose cholesterol level kept increasing²⁰. This finding contradicts the general preconceptions that the egg is a cause of cholesterol increase.

This 26-week study included army staff with cholesterol over 180 mg/dl²⁰. All subjects had the same physical characteristics and participated during the study in a food educational program to reduce fat and cholesterol intake. Until the end of the study the total cholesterol level of the control group increased while the subjects receiving the supplement revealed no change. Similarly, placebo subjects had an increased total cholesterol-to-HDL ratio, while no change was noticed in the supplemented group. These findings proved that the hyperimmune egg could be beneficial in changing the serous lipoprotein level, reducing cardiovascular disease risk²⁰

Fight against swelling and arthritis. During the immune decline, the human body can face increased immune system reactivity as excessive swelling. This paradox appears due to the fact that the immune system includes strong defense mechanisms which normally control swelling once the real threat disappeared. If the immunological functions are damaged, the organism becomes vulnerable to excessive swelling¹⁸.

Once with aging, the immune-regulatory function decreases, increasing at the same time the risk of chronic autoimmune diseases such as lupus or rheumatoid arthritis. Normally, these diseases are difficult to cure but the hyperimmune egg proved to be considerably successful in reducing this type of swelling, on animals^14,21"23. Research conducted on human subjects at Weill Cornell Graduate School of Medical Sciences demonstrated the efficacy of hyperimmune egg therapy in patients with various kinds of arthritis²⁴. There was a significant improvement of patients after starting supplementation including rheumatoid arthritis patients. This improvement lasted 30 days after the end of treatment. Researchers reached to the conclusion that hyperimmune egg products can reduce the intestinal load with organisms tending to cross-react with the human tissue during the autoimmune response²⁴.

Oral cavity health. Immunosenescence brings along many negative changes inside the oral cavity particularly in teeth and gums which can hide pathogenic germs and swellings resulting eventually in chronic conditions such as cardiovascular diseases^25"26. The dental plaque is a real bacteria and inflammatory molecule biofilm increasing the risk of cavity or gum swelling (gingivitis).

The hyperimmune egg extract can reduce the volume of microorganisms in the dental plaque decreasing mouth swelling²⁷. A group of animals receiving hyperimmune egg against tooth decay bacteria had a decreased number of tooth decays than the control group ^28"29. Hyperimmune egg throat wash was also successful in people to reduce bacterial diseases; it seems that the extract remains active and present in the mouth at least overnight, providing long-term protection^30"32. Fighting against teeth and gum diseases also involved tooth pastes and chewing gums containing hyperimmune egg extracts³³.

Gastrointestinal infections. Immunosenescence is involved in the gastrointestinal tract (Gl), which is considered one of the biggest organs creating the bond between human body and environment. During normal immunological protection, the GI tract allows the selective passage of nutrients and beneficial fluids excluding millions of harmful organisms. The normal GI tract produces beneficial IgA antibodies which cover and isolate bacteria. The GI tract also contains a series of immune system-specific guard cells which recognize and act instantly against threats.

Once with aging, all these GI functions decrease, making us vulnerable not only to digestive infections or diarrhea, but also to other malign diseases such as colon cancer, one of the most severe diseases of elders^34"36. The bacterial superinfection extending to the small intestine is a major concern, often misdiagnosed in elders³⁷.

The most common viral infections of the GI tract are unresponsive to antibiotics and even exacerbated by them^{38 39}. A more efficient method is needed which does not require chemical killing of organisms, but inactivating and preventing them from spreading. The hyperimmune egg specifically provides this approach. The most common toxic microorganisms in people are Salmonella and E. coli bacteria and rotavirus. Studies on animals revealed almost 100% efficacy of hyperimmune egg preparations against these bacteria, even if treatment is carried out post infection^{40 44}. For instance, mice were protected against rotavirus and Salmonella infections by oral administration of hyperimmune egg^44"46. The animals used in the trial also received a partial protection against strains other than those chickens had been immunized against at the beginning^42"44! Of equal importance for public health specialists, the subjects receiving hyperimmune egg have almost zero excretion of viruses or bacteria compared to the extremely high level of transmission of these microorganisms in control animals^47"50. The hyperimmune egg is also effective against already-onset infections, with up to 84% healing rate and reducing germ discharge in the stool.

Another interesting study was conducted on human subjects by worldwide expert S.A. Sarker from the International Diarrheal Disease Research Center. He proved that the hyperimmune egg shown similar efficacy with milk-based products concerning rotavirus treatment in children ^17"51.

It is obvious that the hyperimmune egg gas a prevention and treatment potential against extremely dangerous infections of not only the digestive tube, as we shall further demonstrate.

Other antibacterial effects. Of course, the immunosenescence-induced poor immunity makes us vulnerable to a great variety of bacterial infections outside the gastrointestinal tract as well. The hyperimmune egg extracts have a high potential against such infections.

Cystic fibrosis (CF) patients are particularly exposed to Pseudomonas aeruginosa (PA) colonization in the lungs, an organism which grows in its own secretion. PA infections are in fact the major cause of death and paralysis in CF patients⁵². Among the cystic fibrosis patients who performed throat wash with a hyperimmune egg obtained from PA-immunized chickens, none was chronically colonized with this organism while 24% of the control subjects were colonized⁵².

The hyperimmune egg proved its efficacy against other respiratory infections as well. A group of patients with bacteria-induced throat aches used a throat spray containing hyperimmune egg and experienced a dramatic diminution of symptoms, compared to placebo patients⁵³.

HIV/AIDS people. Many ways, the human immunodeficiency virus (HIV) infection resembles the accelerated immunosenescence by low immunity and characteristic excessive swelling⁵⁹. A study on HIV/AIDS people demonstrated the remarkable benefits associated with hyperimmune egg intake⁶⁰. The supplemented subjects experienced overall improvement of living, including less anorexia, abdominal pains, diarrhea, short-term memory loss, dizziness, headaches and insomnia. Breathing pains and difficulties were also reduced and all subjects significantly gained weight until the end of the 8 weeks study. The swelling markers decreased significantly and the number of the most important lymphocyte CD4, an immune function indicator, improved in 20% of patients.

This is a major finding, considering the immune system suppression degree usually seen in HIV/AIDS patients and the outrageous expenses of the standard treatment. It is a real progress to be able to prevent so much sufferance through a cheap and easy to administer egg. Immune support in cancer patients. Immunosenescence is an important factor for cancer occurrence, because in this case, the high level of normal immune system surveillance against potential malign diseases is reduced. At the same time, cancer as much as HIV/AIDS, brings along some kind of accelerated immunosenescence, with cancerous calls invading and weakening their unlucky hosts. Chemotherapy aggravates the situation, making cancer patients even more vulnerable to infections. The Gl is extremely impaired both by the disease and by chemotherapies, both increasing the intestinal permeability which eventually allows the occurrence of infections in the body⁶¹.

Surprisingly, various types of orally administered antibodies had a promising effect in improving cancer and chemotherapy symptoms in the GI tract⁶². A mixture of human IgA and IgG antibodies orally administered in patients with bone marrow transplant prevented the harmful increase of intestinal permeability noticed in placebo patients⁶³.

Animal immunoglobulin (similar with the biologically active proteins from the hyperimmune egg extract) was able to reduce fungi colonization during oral administration in patients with bone marrow transplant⁶⁴. These studies validated the use of hyperimmune egg to support digestive system immunity in immunosuppressive cancer patients and opened the door to new studies with even more dramatic results.

We live in an unfriendly world, surrounded by an environment full of microbes and toxins which can intensely impair our health or can cause us chronic pains and ailment. As we grow old, immunosenescence weakens our immune system making us vulnerable not only to infections but also to cancer or other autoimmune or inflammatory diseases. The medical biochemistry or pharmaceutical industry approaches not always succeed in healing, and they even may increase the risk of such diseases (as in the case of excessive antibiotic prescription resulting in the development of new organisms harmful for human health).

Nevertheless, the hyperimmune egg laid by the chicken is a natural reservoir of protective elements which can improve human health. Chickens immunized against disease-causing organisms lay eggs enriched with substances able to improve human health, both in the GI tract and other parts of the body. Gastrointestinal diseases and also infections and excessive swelling are real targets for hyperimmune egg supplementation. Moreover, hyperimmune egg supplements can improve the immunity of HIV/AIDS people and even proved to be able to support the immunity of cancer patients. There is no doubt: hyperimmune eggs are a unique approach for the protection of health and longevity.

The number of old people is in unprecedentedly permanent increase at present. Due to the recent state-of-the-art technology and better feed, people live longer than before, especially in the developed countries where lifespan doubled from 45 to 80, in 2000. A good health for old people requires medical scientific studies on aging. The immune system is one of the body functions deeply impaired by aging. Research on aging must focus on the immunological aging phenomena in order for us to understand aging as a whole.

Many changes in old people are related to the innate immunity of those people as well as the immunity acquired during lifetime. The immune system impairment in elders is generally regarded as immunity deterioration called immunosenescence. Various immunological parameters are often reduced in elders and the ratio between innate and acquired immunity remains relatively similar throughout lifetime. Lately, the acknowledgment of the immune system role in elders has increased the number of publications in geriatric and gerontology science magazines. The magazine Immunity & Ageing was issued in 2004, being an "on-line" magazine with the purpose of publishing the large number of papers that did not have enough room in the printed publications.

Immunogerontology, the study of the immune system of elders, is a relatively new field, therefore no key information is yet available on this topic. The conclusions of studies on animals or tissue cultures and on humans were inconsistent, but these discrepancies shall probably be solved once more information is available. This situation is due to the fact that gerontologists did not use universally accepted definitions regarding the beginning of aging or when a person is considered old. Studies on aging involve a comparison between a young population and old population without trying to define who is old and who in young. Without a clear definition of these terms it is difficult to determine whether the immunological difference noticed in elders are the result of aging or age-related diseases. Many studies were exclusively focused on the fact that in order to live for 100 years, the old person should be exceptionally healthy. Even if this population is very limited, the conclusion of these studies helped deciding the basic paperwork for broader documentation on aged youth.

In 1984, a team of researchers developed the SENIEUR protocol as an attempt to define the selection criteria for truly healthy old people for immune-gerontology studies. The SENIEUR protocol described four health stages based on clinical and laboratory data. In their effort to reduce their involvement in other studies, they compared the phenomena noticed in the natural healthy aging processes with the phenomena attributable to age-related diseases. Using the SENIEUR protocol, a few recent studies have contradicted many hypotheses on the immune system aging process. The following paragraphs present some of these recent immune- gerontology findings.

It is well-documented that immunocompetence decline occurs once with aging or better said it is related to the age of people. In this case, the immune system starts losing some of its functions and can no longer respond fast or efficiently to stimuli. Age-related changes of the host:

^ in the immune system;

^ chemical changes and modification of proteins found on cells' surface; ^ impairment of the entire organism.

Studied separately, some of these changes seem strange, but when studied together they reveal to radically impair the overall health of the person. A major change encountered in the aged body is the involution of thymus. The thymus is an organ where T-cells maturate. T-cells are very important since they are a specialized lymphocyte population with various functions:

^ they kill bacteria;

^ they help other immune system cells.

The thymus is naturally atrophied once with aging. At the age of 60, the thymus is less than 5% compared to birth if we consider a lifespan of 120 years, when the thymus will be completely disappeared. T-cells are permanently produced throughout lifetime and thymus atrophy results in permanent decrease of T-cells during life. It is unknown why thymus deteriorates. It seems that it is an extremely low energetic organ required by the organism during the first stages of life. It does not have time to develop its own resistance against extraneous antigens. Once with the full development of the immune system, to protect the host from billions of antigens, keeping the thymus at energetic level is very difficult. For the organism it is better regarding evolution to decrease the amount of thymic tissue and use the energy that would support the thymus for other purposes. Since T-cells are very important for immunity, people who live longer always need a fresh T-cell reservoir that would protect the organism against new antigens and this slow but certain loss of the thymic tissue deeply impairs the immune system during aging. T-cell aging effects have various symptoms.

Many T-cell subpopulations are found in thymus and bloodstream, among which naive T- cells and memory T-cells. Naive T-cells are those T-cells that have never been exposed to any kind of extraneous antigen. These are activated and stimulated to identify extraneous antigens. Using memory T-cells, the immune system eliminates extraneous antigens from the body and stores the information in T-cell memory. T-cell memory enters a state of dormancy and is reactivated only by another exposure to the same antigen. A difference regarding naive and memory T-cell subpopulations was noticed between young and old persons. Newborns have more naive T-cells than memory T-cells. The ratio is backwards in adults because most naive T-cells transformed into memory T-cells by exposure to antigens. Old people no longer have naive T- cells due to the progressive deterioration of thymus once with aging and the population of naive T-cells does not regenerate. Therefore the naive T-cell stock decreases and the aged immune system can no longer respond as well as in young people to new antigens.

Once with the decline of T-cell subpopulation, there are important changes on cell surface of all T-cells. The T-cell uses the T receptor protein found in the cell surface to bind to an antigen, communicating the stimulus inside the cell. Many molecules are involved in signal transduction. The antigen coupling and identification signal transmission processes pass through the cell membrane. The transduction signal is a chemical tandem reaction, each reaction depending on the case. Aged T-cells no longer recognize antigen CD 28, an important molecule for transduction signal and T-cell activation on the cell surface. In the absence of this protein, T-cells remain active without response to extraneous pathogenic stimuli. A T-cell transduction signal dysfunction clue is that cells have no response to extraneous pathogenic stimuli. Another clue is that the presence of antigen CD69 on cell surface is less in elders. T-cells produce the antigen CD69 only after binding to an antigen at the cell receptor. If the antigen binding signal is not transmitted inside the T-cell, the antigen CD69 will not appear on cell surface and this is a clue that no transduction signal is found in elders.

Another flaw in T-cell activity in elders is related to calcium decrease. Calcium is a vital element for many biochemical reactions including transduction signal. A calcium deficiency in T- cells stops the transduction signal by absence of enzymatic stimulation including of protein kinase C, MAPK and MEK, which need calcium for their own functions. Calcium decrease can inhibit the production of cytokines, proteins responsible for the coordination of antigen interaction and enhancement of the immune response.

A much studied cytokine is interleukin 2 (IL-2), a cytokine produced and secreted by T- cells, inducing cell proliferation and supporting the long-term increase of T-cells. Aged T-cells lose their capacity to produce and respond to IL-2. When exposed to an antigen, memory T-cells divide rapidly and proliferate in order to produce more cloned T-cells which, in their turn fight the antigen. This proliferation takes place with IL-2. If not enough cytokine IL-2 is produced or if T- cells cannot efficiently respond to IL-2, T-cell functions are much reduced. Changes were recorded for other cytokines such as cytokine 4, tumor necrosis Alfa factor and gamma-interferon, but it is yet unknown the impact of these changes on the aged immune system.

The age-related genetic impairment is found in T-cells. The in vitro study on human T- cells cultivated for a longer time have shown that division cycles become slower and then stop, right when cells grow in the presence of IL-2; the conclusion is that T-cells stop dividing and become too old to function. Many T-cell engineering-related issues are noticed once with cell growth decrease. The protein transcription factors (NF-kB and AP-1) which use DNA to create RNA during protein production processes seem to become unfit or inactive. Aging also contributes to this process decreasing T-cell response capacity by lack of activation of the genes required for T-cell stimulation. Aged T-cells are more sensitive to apoptosis or are programmed to die, by gradual loss of telomeres which are a problem for DNA damage-preventing chromosomes. T-cell receptor gene rearrangement issues contribute to thymus regression by formation of less apoptosis-resistant cells. Therefore, T-cells seem to have a limited life and immunosensescence and are genetically programmed.

T-cell function decrease in elders impairs type B cell function since T-cells act together with B-cells to regulate the antibody production. T-cells induce B-cells to activate immunoglobulin genes, which in exchange creates the antibody range which is more limited in elders than T-cells range in youth. The production of immunoglobulin M, one of the five classes of antibodies, is particularly impaired. During infection, IgM is the first class of antibiotics to respond. The incapacity of elders to fight infections compared to young people can be related to the reduced IgM immune response. B cells aging rate decreases with age. B cells are produced in the bone marrow throughout life but their number decreases with age. With few aged B cells, the amount of antibodies produced as immune response to infection decreases.

Autoantibodies, the antibodies reacting against self antigens, are usually the distinctive symptoms of autoimmune disease. Still, the presence of autoantibodies is often correlated with age, even in healthy old people without autoimmune disease. Interleukin 10 (11-10) is a molecule which stimulates autoantibody production in autoimmune disease patients. 11-10 seems not to impair the health of old people. In exchange, autoantibody production in elders can be related to the above-mentioned functional modifications in T-cells. The production of small amounts of autoantibodies is normal during aging, the activity of which is unknown. A possibility would be that T-cell gene mutations which occur once with aging should create a T-cell subpopulation which recognizes the antigen of the host. Normally, these T-cells are eliminated from the thymus before full aging, but thymic regression may keep T-cell population. These T-cells can induce B cells to produce autoantibodies against own antigens. This theory seems to be supported by studies on mice proving that transplanting a thymus from a fetus to elders with autoimmune disease may recover functions and treat the autoimmune disease.

Other immune system cells are also impaired by aging. The activity of leukocytes, including macrophages, monocytes, neutrophils and eosinophils is reduced in elders. There is little information on the effects of aging on these types of cells.

Natural killer (NK) cells. These cells are created to secrete cytokines and kill other cells, and they have been studied very much. According to some data, NK cells decrease with age but this theory was based on studies on mice and was changed because it had been found out that their number in the human body was very little changed. The SENIEUR protocol was not taken into account in the beginning. The studies conducted with the SENIEUR protocol revealed that NK cell activation is very little changed in time, and a cross-study suggested that even if cytotoxic (cell-killing) functions of NK cells were maintained, the secretory activity of cytokines would be uncertain. While cytokines play an important role in tumor development and removal of infections, NK cell immunosenesecence may have an extensive effect on the immune system. Further studies on NK cells are necessary to settle these discrepancies. What are the implications of all these changes in the aged immune system regarding elders' health? Two topics were studied to ensure health: vaccination and nutrition.

Old people are known for their bad tolerance to vaccination, unlike young people. Vaccination is done to educate the immune system against an infectious agent. Vaccine provide an non infectious substance containing the same antigen as pathogenic agents for the development of memory T-cell population and antibody-producing B cells so as to prevent further infections. Some vaccines like the avian smallpox vaccine need to be administered only once to provide lifetime immunity. Other vaccines, such as flu vaccine, need to be administered each year due to the existence of various virus strains and prevalence of a virus strain against others. Flu and pneumonia are two diseases which affect elders mostly.

For this reason, vaccines are a priority for medical assistance during aging. Nevertheless, the elders face particular vaccine-related modifications. The antibody response in elders is slower and weaker than in young people and T-cell subpopulations respond poorly to vaccination T-cell memory in elders does not react so quickly and for a long time like in young subjects. Many studies attempted to improve vaccines administered in old people. Studies are now conducted on a new type of vaccine which can release liposome or DNA (naked DNA) to develop a stronger immune response. There is an attempt of using new methods of vaccination among which the intranasal or oral spray to stimulate mucosal immunity.

Nutrition is particularly important for the immune response of seniors. At the same time, old people often face malnutrition. Less calories in food means slow aging and help maintaining a large number of naive T-cells and IL-2 level. Vitamin E and zinc are important nutrients for the appropriate functioning of the immune system. Long-term zinc deficiency in old people results in decreased cytokine production and therefore, low activity of T-cells. Vitamin E is a potential medicine in treating Alzheimer (CSA's Discovery Guide Series: Alzheimer's Disease de Fred Spangler). It was discovered at the same time that vitamin E supplements can enhance the immune system. In mice and people, a daily dose of vitamin E higher than the one recommended by the USA methodology, improves T-cell functions in the cell-mediated immunity. Vitamin E is also an antioxidant which can protect lymphocytes, brain and other tissues from the destructive free radicals.

We have seen that aging affects many components of the immune system which interacts with every organ in the body. A clear understanding of the immunological modifications resulting in aging is necessary for quality medical assistance of old people (65, 66). Glossary

1. Adjuvant: a vaccine-incorporated non-reactive substance increasing the immune response to a vaccine. Adjuvants cannot stimulate the immune response without antigens.

2. Antibodies: proteins produced by B cells as response of an antigen's stimuli. Once recognized, B cell antigen produces the same type of antibodies which will bind only this antigen which has stimulated B cells.

3. Antigen: an extraneous substance binding with the antibodies or T-cell receptors which produce an immune response. Antigens can be proteins, carbohydrates, nucleic acids, hormones or other substances. The immune system usually responds against an extraneous antigen, but in case of autoimmune disease the immune system reacts against self-antigens released by the organism as if they were its own but extraneous antigens.

4. Apoptosis: programmed cell death, consisting of chromosome condensation, degraded DNA and destruction of killer cells.

5. Autoimmune disease: the immune system attacks its own organism and destroys tissues. The host's immune system makes errors on its self antigens against extraneous antigens and induces an immune response of tissues that have self antigens. Lupus, rheumatoid arthritis and multiple sclerosis are examples of autoimmune diseases in people.

6. B cells: a lymphocyte population maturing in bone marrow and producing antibodies. B cells can be found in bone marrow and bloodstream.

7. Antigens CD: proteins on the surface of lymphocytes serving as biochemical markers characteristic for a type of cells. CD can be used for differentiation (cluster differentiation) indicating the connection or maturation stage of lymphocytes. Molecules are approved to be called CD followed by a number (CD 28 or CD 69). Some antigens CD have known functions while others don't.

8. Cytokines: proteins synthesized by immune system cells reacting with the immune, nervous and endocrine systems. Cytokines can be immunostimulators or immunosuppressive. Cytokines include interleukins, interferon and some growth factors.

9. Immunocompetence: a quality or state of the immune system with appropriate functions.

10. Immunogerontology: study of the aging process of the immune system. 11. Immunoglobulin: another word for antibodies. Immunoglobulins are subdivided into 5 classes: IgM, IgA, IgE, IgD and IgG.

12. Immunosenescence: immune system aging.

13. Leukocytes: white blood cells. The leukocyte population consists of B cells, T-cells, monocytes, macrophages, basophiles, eosinophils, neutrophils and natural killer cells.

14. Liposome: fat vesicles containing a substance (drug or vaccine) intended for a special type of cell

15. Lymphocytes: a population of white blood cells, among which a cell subpopulation, B cells and natural killer cells.

16. Mucosal immunity: a part of the immune system associated with the mucosal tissues of the respiratory, gastrointestinal and urogenital tracts.

17. Natural killer cells: cells which attack and destroy virus-infected and tumor cells.

18. Signal transduction: a process in which a cell reacts to a stimulus from the environment by transmitting the information from the membrane surface inside the cell. The cell membrane has proteins/receptors for specific molecules. When molecules bind to a receptor, this produces a cascade of chemical reactions in the cell, involving enzymes, proteins and ions (especially calcium). Each reaction depends on the situation and if it is not part of it, it is lost and the transduction signal is interrupted.

19. T-cell receptor: a protein complex on the surface of T-cells binding with the antigen. Like antibodies, T-cell receptors are specific for certain antigens.

20. T-cells: a population of lymphocytes in the blood, maturing in the thymus. T- cells can be found in the thymus, lymph nodes and bloodstream. T-cells face many infections including the assistance of other cells of the immune system, killing extraneous pathogens or the infected cells even suppressing the immune response.

21. Telomere: special proteins to repeat DNA sequences at the end of the chromosome protecting chromosomes from degradation.

22. Thymus: an organ above the heart, where T-cells mature

23. Transcription factors: proteins helping RNA synthesis using a DNA template. Protein NF-kB and protein AP-1 are two very well known transcription factors. References:

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BRIEF DESCRIPTION OF INVENTION

Short description of technical solutions

Studies during 201 1-2014 were conducted to obtain an identified hyperimmune egg: hyperimmune egg PC2, containing antibodies for 10-30 pathogenic agents and/or toxic substances. This whole egg can be consumed raw, boiled at 65°C for 25 minutes, boiled at 100°C for 4.5 minutes. Under these conditions, the specific activities of all immunoglobulins are maintained. The hyperimmune egg PC2 can be preserved by drying using low temperature atomization or freeze-drying. The powder can be consumed as such or in mixture with cacao, water-dissolved vitamins, mineral water, soda or milk.

The immunization consists of the inoculation of minimum 10 bacterial antigens or mixture of 10-30 bacterial, viral and micotic antigens, dried by freeze-drying and mixed with an adjuvant QS21. This technology described in this patent is easy to use, and specific IgY can be sold at accessible price and exploited in the medical units of Romania.

This invention was to produce a hyperimmune egg which contains a group of 10-30 IgY of the yolk, consisting of the following steps:

(a) preparation of a complex antigen using antibiotic-resistant bacterial strains isolated from patients from 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, with viral and micotic antigens;

(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) hyperimmune egg PC2 is used in the feed of over 65 years old people or as per recommendations in immunosuppressive patients, with oral, digestive diseases or mucosal, tissue or organ impairments.

BRIEF DESCRIPTION OF PROCEDURES

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

Annex # 1. The hyperimmune egg PC2 is prepared accordingly:

(a) IgY-specific identified by ELISA using IgG anti rabbit IgY (photo);

(b) IgY-specific quantitatively determined by ELISA (photo)

(c) IgY-specific qualitatively determined by ELISA (photo) DETAILED DESCRIPTION OF INVENTION

According to this invention, the preparation of hyperimmune egg PC2 consists of various stages: preparation of antigen (1), immunization of conventional or SPF laying chickens (2) qualitative and quantitative antibody assessment (3).

Preparation of antigen

According to this invention, identified and multiplied antibiotic-resistant bacterial strains were isolated from patients. The bacterial cells washed 3 times in phosphate buffer (PBS) and centrifuged at 4000 rpm at 20°C, 15 minutes are freeze-dried in 10 ml vials, 4 ml bacterial suspension in each vial. After freeze-drying, the vials are stored at -20°C. 50 mg bacteria are re- suspended in phosphate buffer (PBS) at optical density OD₆oo of 1.00 and mixed with 45μ1 adjuvant QS21. Viral antigens are obtained by cell culture replication of viruses and their inactivation with beta-propiolactone. After cultivation on selective media, fungi are inactivated for 60 minutes at 80°C.

1. 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 blood and eggs is tested after the second administration, by ELISA. Eggs are harvested 14 days after the third antigen inoculation, when antibody titer in serum and yolk is high. The antibody titer is assessed on regular basis from the eggs of chickens immunized with the target antigen.

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. The control methods are detailed in the invention patent no. A/00156 from 25.02.2014

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, 12 months; adjuvant QS21 is important for the preparation of specific antibodies.

Hyperimmune egg PC2 from this invention is used for the preventive or curative treatment of bacterial, viral, micotic, yeast infections, after administration per os against digestive, pulmonary diseases, viral or other infections in the human body. 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

(a) . Immunize each group of chickens with a set of 10-30 bacterial, viral or micotic antigens.

The bacterial and micotic strains can be isolated from patients from the hospitals of Romania. Some viral or micotic strains are represented by a mixture of strains isolated from patients from the hospitals of Romania and certified strains. This set of microorganisms consist of: Shigella dysenteriae, Staphylococcus epidermidis, Staphilococus simulans, Staphilococus aureus, Escherichia coli, Salmonella enteritidis and typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae, Haemophilis influenzae, Stereptococus (6 species), Acinetobacter Baumani, Herpes simplex, Clostridium difficile (spores, vegetative form and exotoxins A and B).

(b) . Store each strain at -85°C as original product. Grow bacteria for antigen preparation in the recommended culture medium;

(c) . From the 24 hours bacterial culture, prepare a bacterial sediment equal to 2xl 0¹⁰CFU, by centrifugation for 15 minutes at 4000 rpm at +20°C;

(d) . Re-suspend the obtained pellet in PBS at the initial volume and freeze-dry 2 ml in 4 ml vials. After freeze-drying, store the bacterial culture at -20°C;

(e) . Re-suspend the sediment obtained as per section c) 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 3-4.5μg 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 emulsifier;

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

(h) . Prepare the viral antigens from cell cultures with beta-propiolactone inactivated virus, purified by filtration through Millipore 0.45μηι filters. (i). Prepare fungi antigens from the fungi cultures in selective media inactivated for 160 minutes at 80°.

(j). 140-180 days old, clinically healthy, conventional or SPF laying chickens are industrially reared, fed with Leghorn or Rhode Island Red laying chicken feed and with automatic watering systems.

(k). Inoculate the chickens intramuscularly in four distinct sites with 2 ml of target antigen;

(I). After the first administration, inoculate chickens twice with the same antigen, by the same administration route, at 14 days interval;

(m). Take blood samples during the second and third administration and perform the immune response control;

(n). 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.

(o). Assess the immune response by qualitative and quantitative ELISA;

(p). Chickens with positive response to the target antigen are approved for hyperimmune egg PC2 production.

(q). The best time for egg harvesting is when 100-200 mg of IgY and 10-20 mg IgY specific are extracted from the yolk.

Example 2

IgY extraction

(a) . Separate the egg white from the yolk and extract after tearing the vitelline membrane from the eggs of immunized chickens 14 days after the last administration of target antigen.

(b) . Measure the volume of yolk and then mix with distilled water at +4°C, pH 4-7 in 1 :7 -1 :10 ratio and stir with a turmix for 5 minutes at room temperature.

(c) . Adjust pH at 4-and add Thimerosal 0.01%

(d) . Freeze the mixture at -20 -40°C directly or gradually, one degree per minute using liquid nitrogen.

(e) . After freezing the entire mixture, thaw at +20°C.

(f) . After separating the liquid and semisolid phases extract the aqueous phase; (g) . Process IgY extracted in aqueous phase by filtration at 20°C using filters which allow the passing of proteins weighing less than 20kDa to retain water-insoluble biomolecules, most lipids and yolk granules.

(h) . Store IgY at +4°C for routine quantitative and qualitative determinations.

Perform quantitative and qualitative determinations using:

qualitative ELIS A;

> quantitative ELISA;

Example 3

Hyperimmune egg PC2 filling

After determining the specific bacterial growth inhibition activity, hyperimmune eggs are filled by:

(a) . Take the eggs from the collecting conveyor and transport them in the frigorific room at

+4- 8°C;

(b) . Select and remove broken and dirty eggs;

(c) . Mark each egg with the date of manufacture and potency;

(d) . Automatically distribute in egg cartons with 6, 12 and 36 eggs;

(e) . On each carton put labels with information on the hyperimmune egg PC2;

(f) . State the storage method, use and expiry date on the label;

(g) . Store and transport the hyperimmune eggs PC2 packed in cartons at +4 - 8°C;

(h) . Store hyperimmune eggs PC2 until use at +4-8°C.

Example 4

Preparation of the whole granulated hyperimmune egg PC2 integral by atomization

Wash, break and separate the hyperimmune egg PC2 from its shell by hand or using an automatic device. Mix the contents of the egg into a special mixing machine and transfer at +10C° into a low temperature drying device (+65°C) and then into a stainless steel container and then from a dosage device fill the whole granulated egg into vials of 350g, 170g or aluminum foil sealed vials. Pack the labeled vials into individual and collective boxes.

The individual boxes with 12 and 6 g are distributed in plastic heat-closed bags. The final packaging consists of carton boxes of 50 bags per box.

The granulated hyperimmune egg PC2 can be stored at +20-22°C for one year and at +4- 8°C for at least 2 years. Example 5

Preparation of the whole granulated hyperimmune egg PC2 by vacuum drying (freeze-drying)

Freeze-dry the whole egg mixture according to Ex. 4 using an intensive vacuum drying program, at an eutectic point of -30-56°C, with a special evaporation program at -4°C and final temperature of +30°C.

After freeze-drying fill the granulated hyperimmune egg PC2 according to Ex. 4.

Example 6

Final control of IgY

The below described methods of control are used for the final formulation of hyperimmune egg PC2, for oral administration:

> Microbiological sterility: food standard.

(EC) Regulation no. 2160/2003 of the European Parliament and Council regarding the control of Salmonella infections and other specific food-borne zoonotic agents.

(EC) Regulation no. 852/2004 on food sanitation foresees that eggs are basic food products. All EC legislation provisions on sanitation.

> Determination of specific IgY activity by ELISA;

> Determination of IgY content by ELISA;

Only the control-appropriate batches of specific IgY, according to e.g. 6.a are approved for human consumption.

Example 7

A. Quantitative determination of IgY by ELISA

ELISA is "in house" prepared for each and every assay.

ELISA can detect very high dilutions of immunoglobulins. 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 used in the manufacturing process of IgY, on production stages and during qualitative and quantitative control.

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

(b) . Cover the wells with 150μ1 anti-avian rabbit serum IgG at 3.75 μπι/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 IgY or IgY SPF (25, 12.5, 6.25 μ%/πιΙ) in PBS;

(h) . Add in parallel in triplicate 150 μΐ standard IgY SIGMA ((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;

(1). Add 150 μΐ TMB and incubate for 5-15 minutes at room temperature in the dark

(m). Freeze the reaction with 150μ1 HC1;

(n). Read the reaction under D0₄₅o filter spectrophotometer;

(0) . Perform comparative standard curves and consider the highest positive dilution to 0.200

OD.

B. Quantitative determination of IgY by direct ELISA

(a) . Cover the plate overnight at +4°C or for 2 hours at room temperature with IgY to be tested and standard IgY each in three replicates, in binary dilutions starting with 1 : 1000 dilution in carbonate-bicarbonate solution;

(b) . Keep wells A l and HI as IgY controls;

(c) . Wash 3 times with wash solution;

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

(e) . Incubate the plate for 2 hours at +37°C;

(f) . Wash 4 times with wash solution;

(h). Add 100 μΐ TMB and leave at room temperature for 5-15 min;

(1) . Add 100 μΐ stop solution;

(j). Read the reaction absorbance under 450 nm filter spectrophotometer;

(k). Assay the reaction by blank wells control where D0₄₅o should be maximum 0.060. The reaction is not valid if other values are obtained;

(1). The dilution for which D0₄₅o is 0.200 or higher is considered positive reaction for IgY presence; If the reaction plate is valid, compare positive reactions from test IgY with the international standard IgY;

Perform the g/ml IgY content in comparison with reference IgY taking into account that ELISA detects minimum 10 ng/ml and it is performed in comparison with standard IgY.

Example 8

C. Determination of specific IgY content by ELISA

The specific IgY 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 IgY 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-lOng of specific IgY per well, per 150μ1.

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

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

After removal of the liqu id, wash the plate 3 times in PBS-Tween 20 (2%) wash solution;

Stop the reaction with fixation buffer, 300 μΐ/ well and incubate the plate for 30 minutes at room temperature;

Remove the stop liquid;

Dry the plate for 30 minutes in desiccator;

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

Keep wells Al and HI as antigen controls, wells Bl, CI and Dl as negative reaction controls using IgY-SPF and wells El, Fl and Gl as positive reaction controls using reference specific IgY of Romvac;

Incubate the plate for 2 hours at +37°C;

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 μΐ TMB and leave at room temperature for 5-15 min.

(o). Add 100 μΐ stop solution;

(p). Read the reaction under 450 nm absorbance spectrophotometer;

(r). 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 wells Bl, CI and Dl of control IgY-SPF (negative) reveal values of 0.060-0.090 OD, and positive control wells El, Fl, Gl reveal values of 1.400-1.800 OD.

Claims

1. The method of manufacture of the hyperimmune egg PC2 consists of the immunization of laying chickens with an antigenic mass containing antigens from human-pathogenic bacteria, viruses and fungi.

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

3. Claims for the method in section 1 refer to the fact that the antigen is a mixture of bacterial strains from 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, viruses and fungi 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, viruses and fungi mixed with an adjuvant QS21, which provides an immunological stimulus which generates a uniform immune response in the organism of chickens for each antigen.