MYCOPLASMA BOVIS VACCINE CONTAINING BORON NITRIDE NANOPARTICLES

Abstract

The invention relates to a vaccine formulation used in the field of veterinary vaccine technologies and developed to create immunity against Mycoplasma bovis. This vaccine formulation contains boron nitride nanoparticles prepared in nanosuspension form using nanoprecipitation techniques, along with an antigen containing Mycoplasma bovis. The boron nitride nanoparticles in the formulation are used together with the antigen as part of the vaccine component. Within the scope of this invention, boron nitride nanoparticles are converted into nanoparticle form using surfactants, and the resulting nanoparticle system is combined with the Mycoplasma bovis antigen to form the vaccine formulation. The invention relates to a Mycoplasma bovis vaccine formulation containing boron nitride nanoparticles.

Description

MYCOPLASMA BOVIS VACCINE CONTAINING BORON NITRIDE NANOPARTICLES Technical Area The invention involves boron nitride nanoparticles in the field of veterinary vaccine technologies. Prepared in nanosuspension form using nanoprecipitation technique and It provides both bacterial inactivation and adjuvant properties against Mycoplasma bovis. It relates to the vaccine formulation that shows effectiveness. State of the Art In inactivated virus and bacteria vaccines, the causative microorganisms cause disease. formaldehyde or formaldehyde, for example, are used to eliminate the forming properties. Chemical inactivating agents are used. This inactivation process... As a result, the microorganism loses its ability to multiply, while Antigenic regions are preserved, thus enabling the formation of an immune response in the host. The mechanisms of action of the inactivating agents used vary, and some These agents bind to DNA, some disrupt basic protein structures, and some This causes damage to the cell membrane. In the known state of the art, aluminum is among the vaccine adjuvants used. These include salts, montanide, saponin, protein, and virus-like particles. However, these adjuvants did not exhibit any inhibitory properties towards bacteria. Therefore, bacteria are also inhibited during the preparation of vaccine formulations. The use of chemical substances is required. This situation, the use of more than one chemical component in the formulation It makes it possible. Aluminum is a concern due to the use of aluminum-containing vaccine adjuvants. toxicity, reduction of iron binding capacity due to binding to transferrin, inhibition of magnesium transfer, disruption of calcium metabolism, and Negative consequences such as oxidative stress can occur. Furthermore... 2 Aluminum can cross the blood-brain barrier, leading to accumulation in the brain. it can show, it can pass into bone tissue and during pregnancy it can affect the fetus It is stated that it can have toxic effects on the embryo. In addition, Aluminum salts stimulate only humoral immunity, not cellular immunity. It is stated that it has no effect. Application in saponin-containing vaccines used in the known state of the art swelling in the region, lameness, loss of appetite, weakness, problems with organ function, and Side effects such as dizziness may occur. In oil-based adjuvants, however, the antigen... Problems may occur during homogenization, and also during application. Inflammatory reactions, granuloma and ulcer formation occur in the region. It is possible. Current vaccine formulations contain, in addition to adjuvant components: At least one chemical substance that provides inactivation must be used. Some formulations contain more than one adjuvant and preservative. This situation is causing vaccine costs to increase. Furthermore... Cost and raw material supply due to importing existing vaccine adjuvants Additional burdens arise in these processes. Purpose of the Invention The invention introduces a new approach to this field, different from the structures currently used in the technology. the creation of a structure with different technical features that brings about innovation It aims to... The primary aim of the invention is to provide effective immunity against Mycoplasma bovis infections. The goal is to develop a new vaccine formulation that can generate a response. Another aim of the invention is to utilize boron nitride nanoparticles as both an adjuvant and a functional agent. a dual-function vaccine system in which it acts as a bacterial inactivation agent to provide. 3 Another aim of the invention is to effectively activate the immune system while preserving antigenic structures. The goal is to obtain a stimulating vaccine formulation. Another purpose of the invention is to provide the additional inactivation needed in traditional vaccines. The goal is to offer a vaccine technology that eliminates the use of chemicals. Another aim of the invention is to simplify the vaccine production process, thereby reducing production time and The goal is to develop a formulation that reduces costs. Another purpose of the invention is to prevent toxic effects, abscesses, and granulomas at the vaccine administration site. a vaccine formulation that prevents inflammation and other pathological changes from occurring to provide. Another objective of the invention is that it can be used safely in pregnant animals. It is the fact that. Another objective of the invention is to demonstrate that boron nitride can be used as a vaccine adjuvant, domestically. a system that enables the utilization of resources and offers economic advantages The goal is to develop vaccine formulations. The structural and characteristic features and all the advantages of the invention are given below. a detailed explanation written with figures and references to these figures This will make it clearer, and therefore the evaluation will also be based on this. This should be done taking into account the figures and detailed explanations. Technical Effects Provided by the Invention Thanks to the invention; • Boron nitride nanoparticles inhibit bacteria while simultaneously boosting immunity. It can stimulate the immune system and generate a protective immune response. • It exerts an inhibitory effect by preserving antigenic determinants and inhibits immunity. It enables the formation of a response. • Boron nitride derivatives inhibit mononuclear cell proliferation in the blood and It increases cytokine stimulation. 4 • A single product provides both bacterial inactivation and adjuvant effects. It can be provided. • Eliminating the inactivation phase facilitates vaccine production. This saves time and costs during the process. • It positively contributes to the protection of the vaccine against contaminants. • Toxic effects, pathological changes, abscess, granuloma at the vaccination site. and it does not cause inflammation. • Higher level of immune response compared to control groups It constitutes. • IgG, IgM, and IFN-Σ levels were higher in the vaccinated group compared to the control group. This ensures that it is higher. • No scattering observed after intramuscular administration It provides. Detailed Description of the Invention In this detailed description, the preferred configurations of the invention are presented, only in relation to the subject matter. for a better understanding and without creating any limiting effects. This is explained as follows. The invention is a Mycoplasma bovis vaccine formulation containing boron nitride nanoparticles. It is related to. The formulation in question provides bacterial inactivation and the same prepared using nanoprecipitation technique, which also exhibits vaccine adjuvant properties. It contains boron nitride nanoparticles in nanosuspension form. Formulation; boron nitride (1), Captex 355 (2), Gelucire 48 / 16 (3), acetone (4), ethanol (5), dimethyl sulfoxide (6), ultrapure water (7) and M. bovis containing antigen (8) It consists of its components. The vaccine formulation subject to the invention contains 0.5% boron nitride (1), 2.5% by weight. Captex 355 (2), Gelucire 48 / 16 (3) at 2.5%, ultra at 44.5% It contains pure water (7) and antigen (8) containing 50% M. bovis. Alternative Boron nitride (1) 0.1-25%, Captex 355 (2) 0.1-25%, Gelucire in applications 48 / 16 (3) 0.1-25%, ultra-pure water (7) 1-50% and M. bovis containing antigen (8) 25- It can be used in the range of 75%. Acetone (4), ethanol (5) and dimethyl sulfoxide (6) solvents used in nanoparticle production and removed at the end of the process and are not among the final formulation components. Boron nitride (1) is the main component and adjuvant of the formulation and is used to treat bacteria. Captex 355 (2) and Gelucire are responsible for inactivation and immune stimulation. 48 / 16 (3), surface used in converting boron nitride into nanoparticle form are the active ingredients. Acetone (4), ethanol (5) and dimethyl sulfoxide (6), nanoparticles Organic solvents used in the production phase. Ultra-pure water (7), It is one of the basic components used in the preparation of the formulation. It plays a role in the creation of a nanoparticle system containing M. bovis. Antigen (8) constitutes the antigenic component of the vaccine formulation. The invention includes boron nitride (1), Captex 355 (2), Gelucire 48 / 16 (3), acetone (4), Nanoparticle form using ethanol (5) and dimethyl sulfoxide (6) It is converted. For this purpose, boron nitride (1), in an amount of 10 mg, in a 1:1 ratio 10 mL organic phase consisting of a mixture of acetone (4) and ethanol (5) and 1 mL dimethyl It is dissolved in a medium containing sulfoxide (6). In a separate phase, 50 mg Captex 355 (2) and 5 mL aqueous phase containing 50 mg Gelucire 48 / 16 (3) is prepared and organic The phase is added dropwise into the aqueous phase, which is stirred at a speed of 750 rpm. The mixing process should be carried out at a speed of 500-1000 rpm for 15-60 minutes. This can be achieved at 750 rpm for 30 minutes in the preferred application. is applied. After mixing, the mixture is given a power rating of 25-75%, between 1-15. Sonication can be applied for a duration of 1-4 minutes, in 1-4 cycles. In the preferred application, the sonication process is performed at 60% power for 5 minutes and 2 It is performed in a cyclical manner. Following the sonication process, organic Evaporation is used to remove solvents. Evaporation process is carried out at a temperature range of 30-75°C and for a duration of 1-15 minutes under vacuum. This can be done below this level, and the preferred method is 5 minutes at 45°C. It is applied for a limited period of time. The system obtained after evaporation removes residual solvents. For this purpose, it is subjected to ultracentrifugation. The ultracentrifugation process is performed at 10,000- 6 At a speed range of 15,000 rpm, for a duration of 10-60 minutes, and at a temperature range of 5-25°C. This can be achieved at a speed of 12,500 rpm for 30 minutes in the preferred application. It is applied for a period of time at 15°C. As a result of this process, residual solvents are removed. Dimethyl sulfoxide (6) is removed from the system. Then 1 mL is added to the system. ultra pure water (7) is added and the second time at 60% power, for 5 minutes and 2 Sonication is applied in a cyclical manner. Thus, boron nitride nanoparticles are processed. is obtained. The resulting boron nitride nanoparticles were subjected to sterilization using a 250-280 nm wavelength. The neck can be exposed to UV light for 1-4 hours, which is the preferred method. In this procedure, sterilization is performed for 2 hours at a wavelength of 260 nm. Samples taken from the formulation after sterilization were liquid Thioglycollate. It is inoculated onto culture medium and cultured under aerobic, anaerobic and microaerobic conditions at 37°C. It is left to incubate for 14 days. With the nanoparticle system that has completed sterilization, an antigen containing M. bovis (8) was detected. By combining these components, a vaccine formulation is created. In this context, the antigen is 2×10⁹ It is prepared in the form of a buffer solution containing CFU / mL M. bovis and boron nitride. The nanoparticle system is mixed with the nanoparticle system at a 1 / 1 (v / v) ratio. The resulting mixture... It is incubated in a shaking incubator at room temperature for 48 hours. Following the incubation process, samples taken from the formulation were subjected to sterility testing. For this purpose, they are seeded onto suitable culture media and incubated for 14 days. The formulation is left to pass sterility tests and is ready for use. is being brought in. Instead of Captex 355 (2) used in the invention, Compritol 888 ATO, Gelot 64, isopropyl myristate, sodium lauryl sulfate, Poloxamer 188, Tween species, Span species, Lecithin, Gelucire species, Caprylol 90, Lauroglycol 90, Labrasol, Labrafil species, Captex species, Brij 72, Capmul species, Poloxamer 407 or any of these. Surfactants containing combinations of these can be used. Similar In this way, instead of Gelucire 48 / 16 (3), surfactants in the same group can be used. or combinations thereof may be preferred. Also, instead of acetone (4) ethanol, acetonitrile, dichloromethane, ethyl acetate or methanol; instead of ethanol (5) Acetone, acetonitrile, dichloromethane, ethyl acetate, or methanol can be used. 7 In the vaccine formulation that is the subject of the invention, boron nitride (1) has both bacterial inhibition. It also acts as a component that stimulates the immune system. The formulation exerts an inhibitory effect while preserving antigenic determinants, and It facilitates the formation of an immune response. Boron derivatives in mononuclear cells by increasing the proliferation and cytokine stimulation of the immune system This contributes to their stimulation. Furthermore, within the same system, both... due to both inactivation and the provision of adjuvant effect in the production process The inactivation phase is eliminated, saving time and costs. This is ensured. The formulation contributes to protection against contaminants. and toxic effects, pathological changes, abscess, granuloma and inflammation at the application site It has been determined that it does not create.

Claims

1. The invention relates to a vaccine used against Mycoplasma bovis in the field of veterinary vaccine technologies. It is a vaccine formulation, characterized by its use in nanoprecipitation techniques. with boron nitride nanoparticles prepared in nanosuspension form It contains Mycoplasma bovis antigen.

2. Complies with Claim 1, and its characteristic is the formulation of boron nitride nanoparticles. It is present as an adjuvant.

3. Complies with Claim 1, its characteristic being; boron nitride nanoparticles to bacteria. It is present as the component that enables inactivation.

4. Complies with Claim 1, and its characteristic is that boron nitride nanoparticles conform to Captex 355 and It was obtained using Gelucire 48 / 16.

5. Compliant with Claim 1, its characteristics include; 0.1%-25% boron nitride by weight, 0.1%-25% Captex 355, 0.1%-25% Gelucire 48 / 16, 1%-50% ultrapure water and 25%-75% It contains M. bovis antigen in certain proportions.