WO2019108155A1

WO2019108155A1 - Treatment (therapeutic) and protective immunization in papillomavirus infected animals by autologous and homologous mucosal pulverized vaccine

Info

Publication number: WO2019108155A1
Application number: PCT/TR2018/050577
Authority: WO
Inventors: Bilge Kaan TEKELIOGLU; Kasim BERBER; Cagla PARKAN YARAMIS
Original assignee: Tekelioglu Bilge Kaan; Berber Kasim; Parkan Yaramis Cagla
Priority date: 2017-11-30
Filing date: 2018-10-10
Publication date: 2019-06-06
Also published as: TR201719260A2

Abstract

The invention relates to an autologous and homologous mucosal pulverized vaccine that provides treatment against the disease caused by papillomaviruses in animals and to the production method of this vaccine.

Description

TREATMENT (THERAPEUTIC) AND PROTECTIVE IMMUNIZATION IN PAPILLOMAVIRUS INFECTED ANIMALS BY AUTOLOGOUS AND HOMOLOGOUS MUCOSAL PULVERIZED VACCINE

TECHNICAL FIELD

PRIOR ART

Papillomaviruses are DNA viruses and are highly species-specific. They infect mainly the squamous cells of the epithelium. Papillomaviruses are divided into different genotypes depending on the degree of sequence difference. More than 170 human papillomavirus (HPV) species have been identified. Papillomavirus infection commonly causes benign skin tumours called warts, which are usually small, cauliflower or solid-shaped and formed with coarse and irregular growths. Warts contain large amounts of infectious non-enveloped papillomaviruses that are relatively stable. Contamination between animals occurs by direct or indirect contact with infected animals, by virus-infected pastures and areas, dirty fence posts or barriers. Contamination by tattooing or other equipment containing viruses is another common source of infection. Warts due to papillomavirus infection are usually seen on the lips, mouth and skin of the animals and less on the eyelids, even on the eye surface or on the toes. Warts are usually present as wart groups, not as focal foci. The duration of infection varies from one month to one year and reoccurence is possible. In the treatment and prophylaxis of papillomavirus infection, various therapeutic and immunologic methods of protection have been tried to date. Until today, as confirmed by reference information, a permanent treatment that prevents the recurrence of the disease could not be found and also no vaccine has been produced that protects against disease.

BRIEF DESCRIPTION OF THE INVENTION

The invention provides an autologous and homologous pulverized mucosal vaccine obtained by inactivation of the samples taken from papillomas (warts) of the mouth, lips, eyelids and skin of naturally infected animals, which provide treatment against disease caused by papillomaviruses in animals and which provides protective immunity, relates to the production method.

DETAILED DESCRIPTION OF THE INVENTION

The first step of the invention which describes the method of vaccine production for the treatment of infections caused by papillomaviruses of animals is the use of naturally occurring papillomas (warts) of the mouth, lips, eyelids and skin of infected animals. For this purpose, papillomas (warts) of the naturally infected animals which formed on the mouth, lips, eyelids and skin should be removed without bleeding with a size of 0.1 -0.5 cm, preferably with an electrocautery or by the help of a scissors or a scalpel. Removed papilloma tissue then should be place in a glass tube containing 5 ml 96% ethanol. Then the vibrations emitted by the high frequency ultrasonic sound waves and the capsid structure of the papillomavirus are separated. High frequency sound waves have a disruptive effect on microorganisms. Thus, both inactivation with 96% ethanol and ultrasonic effect the virus becomes adequate for the preparation of the stock solution of the mucosal pulverized vaccine. For this purpose, viruses are inactivated at +4°C using high-frequency ultrasonic sound waves at 25 kHz for 5 minutes. When preparing a papillomavirus mucosal vaccine, infected wart tissues were used for virus isolation. High frequency sound waves are used to expose the virus particles in the dense tissue. High-frequency sound waves not only expose the virus particles inside the tissues, but also provide inactivation of the viruses in the tissues where the 96% ethanol cannot effect. The samples in the glass tube are then continuously vigorously shaking for 3000 rpm with a vortex mixer to ensure complete homogenization. The obtained product is stored at +4°C and vigorously shakes with vortex for 5 minutes once every hour during the day as described in the virus inactivation procedure and maintained at the same temperature overnight. Liquid (stock solution) containing 96% ethanol and inactivated virus is used for the preparation of mucosal pulverized autologous and homologous vaccines to treat papillomavirus infection and generate protective immunity against disease. The stock solution stored in the refrigerator must be shaken with vortex for 5 minutes before each use. For dilution, 1 ml of stock solution should be taken and transferred into a glass tube containing 9 ml (20%) ethanol + 80% distilled water in order to obtain a 1/10 dilute solution in the first step (1/10). It is thoroughly mixed and homogenized at 3000 rpm with vortex for 5 minutes. The same procedure is repeated 8 more times to obtain 9 times dilution. After taking 1 ml of the final dilution, add 9 ml of 96% ethanol and mix by vortexing for 5 minutes at 3000 rpm. From the prepared liquid mixture, 7.5 ml of the solution is transferred to a brown colour glass vial with pulverization apparatus, and 2.5 ml of chitosan solution (0.5 mg / ml) with 40 ml of distilled water is added to the mixture. The final solution is vortexes at 3000 rpm for a further 5 minutes and as long as it is used to generate protective immunity by autologous and homologous treatment against infection it is kept at +4°C. The final solution com prim ises of the inactivated virus, 7.5 ml of ethanol (15% ethanol/ volume), up to 1.5 mg chitosan (maximum dosage for 50 ml) and 40 ml distilled water. The above-mentioned method and its contents are used as a pulverized mucosal vaccine to generate autologous treatment and protective immunity to infection in animals in which papillomavirus vaccine strain was isolated. The produced vaccine can be used to provide for homologous treatment and protective immunity against infection in other animals in the same stable whether sick or healthy. In any case, the produced vaccine is used twice a day in the morning and in the evening. Dose rate is 1 ml in adults and 0.6 ml in young animals for 4-12 weeks by oral or nasal pulverization or by direct mixing to the food or water. At a therapeutic dose, the symptoms of the disease generally regress within 7-10 days and after recovery from the infection to generate a permanent and long-term immunity the vaccine should be used for at least 4 more weeks. When used for 12 weeks, it provides a longer immunity and prevents recurrent infections in animals that are recovering from the infection. The mechanism of autologous vaccination is a holistic therapeutic treatment that is based on the stimulation of the immune system. The main technique is isolating and inactivating the pathogenic virus that causes infection in the infected animal. It is based on the principle that the vaccine mixture contains both the inactivated viral nucleocapsid complex and the diluted antigens with the appropriate adjuvant material. It can be used safely in both sick and non-sick animals and when used can treat infection or create protective immunity. In homologous vaccination, the inactivated viral nucleocapsid-adjuvant complex can also be administered to other sick animals of the same species after proper dilution. For example, the vaccine prepared with the isolated virus from a papillomavirus infected dog can be given to other sick dogs. Similarly the vaccine prepared with isolated virus from a papillomavirus infected cattle or horse can also be given to other sick cattle or horses. Autologous and homologous treatment and the pathways to create treatment and the protective immunity against the infection are based on the nucleocapsid complex which is the basic structural units of the virus. 96% ethanol affects the nucleocapsid complex by populating proteins and lipids. 96% ethanol acts on proteins and lipids by creating structural changes in the nucleocapsid complex. With the destruction of the nucleocapsid complex, the virulence is reduced. Inactivation of the virus with ethanol was previously reported by different researchers. The use of ethanol for inactivation is a convenient and safe method; it is edible, tolerable and safe for environment and animals. When the final diluted solution is obtained, the containing ethanol in the total volume reaches up to 15% max. The therapeutic daily dose delivered to the animal reaches a maximum of 0.8 ml, which is safe for animals. The diluted pulverized mucosal vaccine solution induces the first immune response in epithelial cells after inoculation by nasal and oral mucosa, including the inactivated viral nucleocapsid-adjuvant complex. Stimulation of secretory immunoglobulin A (S-lgA) production is one of the main targets in order to prevent the adhesion of virus which causes papillomavirus infection to epithelial tissue cells. The development of cellular Th1 (cytotoxic and helper T cells) response is induced by stimulating mucosal immune response to generate protective immunity against papillomavirus infection. By using a mucosal vaccine containing a pulverized inactive virus, CD8, cytotoxic and helper T cells secretions against papillomavirus infection are stimulated. This leads to the development of the cellular immune response of the animals against papillomavirus infection and the subsequent formation of increased T cell activity. Autologous and homologous vaccination against papillomavirus infection stimulates cellular immune response. Subsequently, an enhanced humoral response involving B and plasma cells activity is achieved and an increase in antibody production is achieved. The use of chitosan in this vaccine technique, which is our invention, is also a novelty, and when chitosan is used as a biological adjuvant, it increases protein absorption through mucosal surfaces and is therefore suitable for use in the delivery of systemic and mucosal-acting drugs and vaccines. In addition to its carrier role during the delivery of drugs, chitosan acts as an adjuvant used to increase the effect of the active substance or to reduce its side effects, as it has the effect of activating the immune system. Additionally by its bio- adhesive and bio-carrier properties, chitosan is effectively taken up by phagocytic cells to provide a strong stimulation of systemic and mucosal immune responses. Although this activity of chitosan is not fully understood, the stimuli it generates on the immune system can be explained by well known immune response mechanisms. It is thought that chitosan stimulates the immune system by activating NLRP3 infiltration and thus leads to the production of potentiated lnterLeukin-I b. In the experimental vaccine models tested, chitosan forms a balanced Th1 / Th2 response. No toxic or other side effects were observed in the mice treated with subcutaneous injection twice a day at 7 days intervals with chitosan at a daily dose of 1 .5 mg/mouse. In addition, an effective immune response of a dose of 50 micrograms in immunization studies by subcutaneous (SC-subcutaneous) injection using normal- particle chitosan has been identified. It has also been found that chitosan has a strong potential to increase both cellular and humoral immune responses and induces a balanced Th1 / Th2 response and is also safe and has effective adjuvant potential and also suitable for a wide range of prophylactic and therapeutic vaccine spectrum. Chitosan was applied at the upper limit dose of 1 .5 mg in our trails based on reference information. It has been observed that unwanted reflexes such as sneezing have occurred in the vaccinated animals. These unwanted reflexes reduce the adherence of the target vaccine dose to the mucosal surface and inhibits the desired immune response effect. For these reasons, it has been found that Chitosan does not cause undesirable effects and provides optimum results when used with a dose of not more than 1 .5 mg for 50 ml. Another reason for the use of chitosan in the indicated dose is that the wart tissues due to papillomavirus contain intense amounts of antigenic virus particles and infected tissue fragments. The increase in the number of virus particles in the resulting stock solution naturally increases the amount of antigenic particles. The adjuvant effect of chitosan at lower concentrations was found to be sufficient due to both undesirable side effects and high concentration of virus particles and antigen. When applied at 300 micrograms/ml (1 .5 mg / 50 ml) of dosage, the absence of side effects and the desired therapeutic and prophylactic effects of the pulverized mucosal vaccine were observed. The virus particle rate at the vaccine concentration is 3 microliters/ml (150 microliter/50 ml). Using the vaccines obtained by the method of our invention, it was observed that the symptomatic and clinical symptoms of papillomavirus infection were treated (therapeutic effect) and successful prophylactic results were obtained in the prevention of infection (prophylactic effect). Our method is to produce autologous and homologous pulverized mucosal vaccines prepared from papillomaviruses isolated from infected tissue samples obtained from infected animals which were not protected by an effective vaccine to date. The produced vaccine is administered to the sick animals by a special bio based adjuvant substance which also developed by us. After administration, the clinical symptoms begin to regress within 7-10 days and completely recover within 4 weeks. Vaccine administration can be done at the same time to the sick animals and clinically healthy animals, providing recovery in sick animals and stopping the spread of the disease. Sick animals are treated and prophylactic immunity is generated in infected, healthy or non infected animals.

Claims

1 . An autologous and homologous mucosal pulverized vaccine for the treatment of papillomavirus in animals characterized in that comprising ethanol-inactivated virus, 7.5 ml ethanol (15% ethanol / volume), at most 1 .5 mg chitosan and 40 ml distilled water in 50 ml, which is the most suitable dose, in order to not to show any toxic effect and to minimize the antigenic effect.