KR20230094973A - Vaccine composition comprising inactivated bordetella pertussis and method for preparing the same - Google Patents

Abstract

The present application relates to a vaccine composition comprising inactivated pertussis strains. The problem that the present application seeks to solve is to provide the vaccine composition containing inactivated pertussis strains. The inactivated pertussis vaccine composition according to an example of the present application has no toxicity or proliferative power and is effective in maintaining storage stability at room temperature while inducing an excellent immune response.

Description

Vaccine composition containing an inactivated pertussis strain and method for preparing the vaccine composition

The present application relates to a vaccine composition against diseases caused by pertussis.

Recently, the importance of vaccines has been emphasized due to the epidemic of infectious diseases including coronavirus, and in particular, interest in reducing vaccine production costs and safety of vaccines during transportation and storage is growing due to climate warming.

For this medical need, vaccine formulations stable at room temperature are being developed, but most are viral vaccines, and in some virus vaccine manufacturing processes, even if the strain itself is heat-resistant, the final vaccine formulation requires refrigeration (Osman et al., 2021). Bacteria cannot survive again if they are inactivated, and it is difficult for bacteria involving many proteins to achieve stability at room temperature, so even bacterial vaccines cannot achieve long-term stability at room temperature.

Therefore, when manufacturing vaccines using bacteria, bacteria are inactivated and used as whole cell vaccines (Pace et al., 1998), but when these dead cell vaccine products are inactivated, proteins are denatured and cell membranes collapse, resulting in pathogenic bacteria. There is a problem that all of them must be refrigerated or frozen because they will die.

On the other hand, when microorganisms such as viruses or bacteria invade, ingest microbial pathogens through phagocytosis and degrade microorganisms while presenting epitopes to immune cells to maintain innate immune defense and homeostasis (Lee et al., 2020). When bacteria are captured by phagocytosis, phagosomes (endosomes) are formed, and phagosomes go through a series of 'maturation' stages and are gradually acidified. effectively remove Through this mechanism, phagocytes, including macrophages, neutrophils and dendritic cells, initiate adaptive immune responses by presenting antigens to T cells as well as dealing with microbial pathogens. Thus, phagocytosis serves as a bridge between innate and acquired immunity (Lee et al., 2020).

Regardless of antigen-presenting cell type, antigen-presenting cells (APCs), such as dendritic cells and macrophages, interact with a specific T-cell subset that expresses specific antigen-specific T-cell receptors on their surface. MHC class II binds to antigenic peptides produced by degradation of self and non-self proteins in endosomes and lysosomes, and presents the peptides to antigen-specific CD4+ T cells. Recognition of peptide-MHC class II by CD4+ T cells promotes activation and differentiation into a subset of T helper cells, mediating interactions between antigen-specific B cells and T helper cells. When the class I complex on dendritic cells recognizes the peptide-MHC class I complex by CD8+ T cells, it promotes the differentiation of CD8+ T cells into cytotoxic T cells (Ewanchuk and Yates, 2018)

Viruses can grow if nutrients are replenished after crystallization, but pathogens cannot survive again once inactivated. Subunit vaccines using viruses or antigenic proteins involve several proteins, but it is difficult to achieve room temperature stability in bacteria involving many proteins. In other words, since the growth temperature of pathogens is constant, they die when the temperature exceeds a certain temperature. Thus, in contrast to viral vaccines, none of the bacterial vaccines achieved long-term stability at room temperature. Therefore, when producing dead cell vaccines using bacteria, they are used as whole cell vaccines by heating, treating with solvents such as alcohol or acetone, or inactivating bacteria using glutaraldehyde (Pace et al., 1998), but these dead cell vaccine products All are refrigerated or frozen.

Therefore, it was necessary to develop a bacterial vaccine capable of achieving long-term stability at room temperature without refrigerating or freezing storage.

Osman N, Goovaerts D, Sultan S, Salt J, Grund C. Vaccine Quality Is a Key Factor to Determine Thermal Stability of Commercial Newcastle Disease (ND)Vaccines. Vaccines (Basel). 2021 Apr 9;9(4):363. Pace JL, Rossi HA, Esposito VM, Frey SM, Tucker KD, Walker RI. Inactivated whole-cell bacterial vaccines: current status and novel strategies. Vaccine. 1998 Oct; 16(16):1563-74. Lee HJ, Woo Y, Hahn TW, Jung YM, Jung YJ. Formation and Maturation of the Phagosome: A Key Mechanism in Innate Immunity against Intracellular Bacterial Infection. Microorganisms. 2020 Aug 25;8(9):1298. Ewanchuk BW, Yates RM. The phagosome and redox control of antigen processing. Free Radic Biol Med. 2018 Sep; 125:53-61.

The problem to be solved by the present application is to provide a vaccine composition comprising an inactivated pertussis strain.

The problem to be solved by the present application is to provide a method for preparing a vaccine composition capable of maintaining a stable storage state at room temperature and room temperature.

The problem to be solved by the present application is to provide a method for preparing an inactivated pertussis strain capable of maintaining a stable storage state at room temperature and room temperature.

The problem to be solved by the present application is to provide an inactivated pertussis strain capable of maintaining a stable storage state at room temperature and room temperature.

In order to solve the above problems, one embodiment of the present application provides a vaccine composition including a pertussis strain inactivated to satisfy Equation 1 below.

[Equation 1]

(In Equation 1, A is the hydrophobic fluorescence value measured in the strain before inactivation, and B is the hydrophobic fluorescence value measured in the inactivated strain.)

In one embodiment of the present application, the degree of intracellular protein aggregation of the strain may satisfy Equation 2 below.

[Equation 2]

(In Equation 2 above, PA is the intensity of aggregated protein in the cell cross-section, and PT is the total area of the cell cross-section.)

In one embodiment of the present application, the strain may be inactivated by treatment at 55 ° C to 70 ° C for 30 minutes to 90 minutes.

In one embodiment of the present application, the vaccine composition can be maintained in a stable storage state for at least 24 months at a temperature of 1 ° C to 65 ° C.

In one embodiment of the present application, the composition may further include one or more selected from the group consisting of a pharmaceutically acceptable carrier, diluent, preservative, stabilizer, buffer, and immune adjuvant.

In one embodiment of the present application, the stabilizer may be one or more amino acid stabilizers selected from the group consisting of alanine, histidine, arginine, lysine, isoleucine, valine, methionine, glycine, and proline.

In one embodiment of the present application, the preservative is trehalose, skim milk, sorbitol, dextran, ascorbic acid, and red ginseng extract. It may be one or more selected from the group consisting of

In one embodiment of the present application, the vaccine composition is for intraperitoneal, intramucosal, intramuscular, intradermal, subcutaneous, intravenous, intrarectal, intrapulmonary, intrathecal, oral, transdermal or intranasal administration can

One embodiment of the present application, in the inactivated strain, 5 to 10% by weight trehalose, 5 to 15% by weight skim milk, 2 to 5% by weight sorbitol and 3 to 5% by weight dex based on the total weight of the composition at least one selected from the group consisting of tran; and 0.1 to 10% by weight of ascorbic acid; adding a preservative further comprising; Freezing at -80 ° C to -70 ° C for 24 hours to 48 hours; Freeze-drying for 48 hours to 60 hours under conditions of -50 ° C to -40 ° C and 15 Pa to 30 Pa;

One embodiment of the present application, culturing the pertussis strain; and inactivating the strain to satisfy Equation 1 below; It provides a method for producing an inactivated pertussis strain comprising a.

[Equation 1]

(In Equation 1, A is the hydrophobic fluorescence value measured in the strain before inactivation, and B is the hydrophobic fluorescence value measured in the inactivated strain.)

In one embodiment of the present application, the inactivating step may be inactivated by treating the strain at 55 ° C to 70 ° C for 30 minutes to 90 minutes.

One embodiment of the present application provides an inactivated pertussis strain prepared by the above production method and satisfying Equation 1 above.

One embodiment of the present application provides a method for preventing or treating pertussis disease caused by pertussis infection by a vaccine composition comprising a pertussis strain inactivated to satisfy Equation 1 above.

One embodiment of the present application provides a use of a pertussis strain inactivated to satisfy Equation 1 or a vaccine composition containing the pertussis strain for preventing or treating pertussis disease.

One embodiment of the present application provides a method of inducing an immune response against the pertussis strain in an administered subject by administering a pertussis strain inactivated to satisfy Equation 1 or a vaccine composition containing the pertussis strain.

In one embodiment of the present application, the strain may satisfy Equation 2 above.

The inactivated pertussis vaccine composition according to one embodiment of the present application has no toxicity and proliferative ability, and maintains storage stability at room temperature while inducing an excellent immune response.

Figure 1 shows the hydrophobic fluorescence values of live cells and inactivated pertussis strains measured using a fluorescence spectrophotometer.
Figure 2 is a photograph of the degree of protein inactivation of live cells and inactivated pertussis strains measured by Transmission Electron Microscope (TEM).
Figure 3 is a TEM photograph of Figure 2 showing the degree of protein inactivation as intracellular protein aggregation.
Figure 4 shows the specific IgG antibody titer efficacy by inoculation with inactivated pertussis strain.
Figure 5 shows the level of TNF-a induction by inoculation with an inactivated pertussis strain.

Hereinafter, the present application will be described in more detail.

The following specific structural or functional descriptions are merely exemplified to explain embodiments according to the concept of the present application, and embodiments according to the concept of the present application may be implemented in various forms, and the embodiments described herein should not be construed as limiting.

Embodiments according to the concept of the present application may be applied with various changes and may have various forms, so specific embodiments are intended to be described in detail herein. However, this is not intended to limit the embodiments according to the concept of the present application to a specific disclosure form, and should be understood to include all changes, equivalents, and substitutes included in the spirit and technical scope of the present application.

Terms used in this specification are only used to describe specific embodiments and are not intended to limit the present application. Singular expressions include plural expressions unless the context clearly dictates otherwise.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and are not interpreted in an ideal or excessively formal sense unless explicitly defined herein. .

Throughout the present specification, when a part "includes" a certain component, it means that it may further include other components without excluding other components unless otherwise stated.

As used throughout this specification, the terms "about," "substantially," and the like are used at or approximating that value when manufacturing and material tolerances inherent in the stated meaning are given, and do not convey the understanding of this application. Accurate or absolute figures are used to help prevent exploitation by unscrupulous infringers of the disclosed disclosure. The term "step of (doing)" or "step of" as used throughout the present specification does not mean "step for".

Throughout this specification, reference to "A and/or B" means "A, B, or A and B".

As used herein, the term "prevention" refers to any activity that suppresses or delays the onset of a disease by administering a vaccine composition.

As used herein, the term "treatment" refers to all activities that improve or benefit symptoms of a disease already caused by administration of a vaccine composition.

As used herein, the term "vaccine" is an antigen used for immunity to prevent bacterial infection or bacterial disease, and is prepared from an inactivated bacterial strain, and is weakened or artificially weakened to obtain immunogenicity against a specific infection. It means administering the killed pathogenic microorganisms into the subject. When stimulated by a vaccine, the immune system in the individual operates to produce antibodies, and when re-infection occurs while maintaining the sensitivity, the disease can be overcome by effectively producing antibodies in a short time. However, the vaccine containing the inactivated pertussis strain according to the present application differs from conventional vaccines in accordance with the principle of immunotolerance, not only protecting against a specific antigen component, but also covering a wide range of diseases including secondary bacterial infectious diseases that can cause the bacterial disease. It can have preventive and curative effects on diseases.

As used herein, the term "inactivation" refers to a condition in which a virulent strain loses its ability to proliferate before transformation, and includes dead bacteria. The inactivated strain refers to a state in which replication division is impossible in the host.

The inactivated pertussis strain may be inactivated to satisfy Equation 1 below, and the inactivation method is not limited.

[Equation 1]

(In Equation 1, A is the hydrophobic fluorescence value measured in the strain before inactivation, and B is the hydrophobic fluorescence value measured in the inactivated strain.)

As used herein, the term "hydrophobic fluorescence value" is a fluorescence value measured by anisotropy for the degree of hydrophobicity according to inactivation conditions of the pertussis strain with a fluorescence probe for the non-polar portion of the protein. When the hydrophobic fluorescence value of the fluorinated pertussis strain satisfies the range of Equation 1 above, the immune effect is excellent.

The degree of intracellular protein aggregation of the inactivated strain may satisfy Equation 2 below.

As used herein, the term “intracellular protein aggregation” refers to the ratio of intensity to area of protein aggregated within a cell with respect to the total intracellular area, and is represented by Equation 2 below.

[Equation 2]

(In Equation 2, PA is the strength of the aggregated protein based on the cross-section of the cell, and PT is the total area based on the cross-section of the cell.)

The term "intracellular protein" in the present application refers to all proteins present in strain cells. A cross section of a cell may refer to a cross section having the largest diameter. The immune effect is excellent when the intracellular protein aggregation degree satisfies the range of Equation 2 above.

The inactivated pertussis strain having an aggregation degree within the range of Equation 2 has the advantage of eliminating toxicity and proliferative ability, exhibiting excellent antibody titer like the strain before inactivation, excellent immune effect, and improving vaccine efficacy.

The inactivated pertussis strain satisfying all of Equation 1 above has an excellent immune effect. In addition, an inactivated strain that satisfies both the values of Equation 1 and Equation 2 above has an excellent immune effect.

The inactivated strain may be inactivated by treating the strain at a temperature of 55 ° C or higher, 70 ° C or lower, or 60 ° C or lower for 30 to 90 minutes, but is not limited thereto. If the strain is treated at a temperature of less than 55 ° C. or treated for less than 30 minutes, some mutant viable cells may not be inactivated, resulting in viable cells being detected. When the strain is treated at a temperature of greater than 70 ° C, greater than 65 ° C, or greater than 60 ° C, or treated for more than 90 minutes, there is a problem that the hydrophobicity value of the cells increases due to protein denaturation and the degree of protein aggregation in the cells also increases. So, there is a problem that the immune effect of the vaccine is not good because the antibody titer is lowered.

The inactivated strain may be completely inactivated when the strain is heat-treated at 55 ° C for 90 minutes, at 56 ° C for 30 minutes, at 60 ° C for 30 minutes, and at 70 ° C for 30 minutes, but is not limited thereto. Temperature and time may be appropriately adjusted within a range that satisfies Equation 1 or Equation 2.

The vaccine composition of the present application can maintain a storage stability at room temperature, room temperature, lukewarm temperature, refrigeration or freezing. In the present specification, room temperature means 15 ~ 25 ℃, room temperature means 1 ~ 35 ℃. “Stability at room temperature” means stability in a temperature range higher than normal room temperature in hot regions, for example, in the range of 28° C. to 32° C. in Italy or Texas.

Specifically, the vaccine composition has a freezing temperature, 0 ° C or higher, 1 ° C or higher, 5 ° C or higher, 15 ° C or higher, 25 ° C or higher, 35 ° C or higher, 40 ° C or higher, 50 ° C or higher, 60 ° C or higher, 65 ° C or higher It can maintain stable storage condition even at temperature. The vaccine composition can maintain a storage stability at the temperature for a period of 1 month or more, 3 months or more, 6 months or more, 12 months or more, 24 months or more, or 36 months or more.

Specifically, the vaccine composition can maintain a stable storage state for 24 months or longer at a temperature of 0 ° C or higher, and the vaccine composition can maintain a stable storage state for 24 months or longer at a temperature of 1 ° C or higher. The vaccine composition may maintain a storage stability for 24 months or longer at a temperature of 5°C or higher, and the vaccine composition may maintain a storage stability for 24 months or longer at a temperature of 15°C or higher. The vaccine composition can maintain a stable storage state for 24 months or longer at a temperature of 25 ° C or higher, and the vaccine composition can maintain a stable storage state for 24 months or longer at a temperature of 35 ° C or higher. The vaccine composition can maintain a storage stability for 24 months or more at a temperature of 40 ° C or higher, the vaccine composition can maintain a storage stability for 24 months or more at a temperature of 50 ° C or higher, and the vaccine composition can maintain a storage stability for 24 months or more at a temperature of 60 ° C or higher. It can be stored in a stable state for more than 24 months.

In one embodiment of the present application, the vaccine composition may further include one or more selected from the group consisting of a pharmaceutically acceptable carrier, diluent, preservative, stabilizer, buffer, and adjuvant.

The stabilizer may contain an amino acid stabilizer contributing to the stability of the formulation, and the stabilizer may be any one or more of the group consisting of non-ionic and basic amino acids.

The stabilizer may be at least one amino acid stabilizer selected from the group consisting of alanine, histidine, arginine, lysine, isoleucine, valine, methionine, glycine, and proline.

Examples of non-polar and basic amino acids include, but are not limited to, alanine, histidine, arginine, lysine, ormitine, isoleucine, valine, methionine, glycine, and proline. For example, the amino acid stabilizer can be glycine or proline, typically glycine. In addition, the stabilizer may be a single amino acid or a combination of two or more amino acids. Amino acid stabilizers can be natural amino acids, amino acid analogs, modified amino acids or amino acid equivalents. Generally, amino acids may be L-amino acids. For example, when proline is used as a stabilizer, it will typically be L-proline, although it is possible to use amino acid equivalents, such as proline analogs.

The amino acid stabilizer is present in an amount of at least 100 mM. Specifically, the amino acid stabilizer is present in an amount of 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM or at least more. In addition, the amino acid purity of the stabilizer should be at least 98%, at least 99%, or at least 99.5% or higher.

The preservative may be at least one selected from the group consisting of trehalose, skim milk, sorbitol, dextran, and ascorbic acid.

The preservative may serve as a cryopreservative when the vaccine composition is freeze-dried.

As used herein, the term "lyophilization" refers to biological materials such as cells, tissues, organs, and organisms at a temperature below 0°C, specifically below -80°C, which is a temperature at which solid carbon dioxide is used to freeze them, and more specifically, when frozen using liquid nitrogen. It refers to a method of drying in an environment having a temperature of less than -130 ° C.

Biological materials are typically susceptible to damage caused by unregulated biochemical kinetics. During the lyophilization process, the biological material being dried by cooling or freezing typically comes into contact with one or more cryopreservatives. Lyophilization may be obtained by any lyophilization procedure known in the field of this application, including processes such as slow freezing and multi-step vitrification and one-step vitrification as described elsewhere in this application. do.

As used herein, the term "biological material" refers to cells, cell aggregates, tissue samples, organs, biological fluids, (multi)cellular organisms and any other membranes (eg, liposomes), nucleosides (DNA or RNA), nucleosides Means a cleoside (virus), lipid or proteinaceous entity.

Therefore, when the cryopreservative is used, there is an effect of having high efficiency without changing the optical / physical aspect in cryopreserving or drying the strain, and there is an effect that standardization and quality control thereof are possible.

Carriers that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline quality cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

As the immune adjuvant, immune adjuvants commonly used in the art may be used without limitation, cholera toxin binding unit, aluminum salts, lipid emulsion (MF-59), synthetic detergent (Tween), microspheres, liposomes, mucoadhesive polymers, etc. It may include, but may not be limited thereto.

The vaccine composition may further include a buffer. The buffer is not limited to any specific component, and any one or more of various buffers that are pharmaceutically acceptable may be used as needed. Specifically, the buffer may be saline, purification buffer (eg, 0.01M PBS), buffered saline, dextrose, water, glycerin, isotonic aqueous buffer, and combinations thereof.

The vaccine composition may further include a pH adjusting agent, a surfactant, and the like.

The dosage form of the vaccine composition may be, for example, a gel (jelly), cream, ointment, semi-solid formulation such as a warning agent, liquid formulation, powder, fine granule, granule, film, tablet, suppository, patch, microneedle It may be selected from solid preparations such as solvent preparations, orally disintegrating tablets, mucosal sprays such as aerosol preparations, and inhalants, but may not be limited thereto. In addition, when the pharmaceutical composition is administered to the mucous membrane of a human or animal, the semi-solid preparation and the solid preparation may be dissolved by body fluids and/or body temperature.

The formulation of the vaccine composition may be a formulation for oral administration or a formulation for parenteral administration.

The oral preparation may be used in formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols. A solid preparation for oral administration may be prepared by mixing at least one or more excipients, for example, starch, calcium carbonate, sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Non-solid preparations for oral administration may include, but are not limited to, various excipients such as wetting agents, sweeteners, aromatics, and preservatives in addition to simple diluents such as water and liquid paraffin.

The parenteral preparation may include a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, a suppository, a sterilized injection, a patch preparation, and a spray preparation, and non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, and olive oil. Vegetable oils such as, injectable esters such as ethyl oleate, etc. may be used, but may not be limited thereto.

In one embodiment of the present application, the vaccine composition is for intraperitoneal, intramucosal, intramuscular, intradermal, subcutaneous, intravenous, intrarectal, intrapulmonary, intrathecal, oral, transdermal or intranasal administration can

The location of the mucosa is not particularly limited, and body locations used for mucosal administration in the art. For example from nasal mucosa, nasal mucosa, oral mucosa, ocular mucosa, ear mucosa, genital mucosa, throat mucosa, pharyngeal mucosa, airway mucosa, bronchial mucosa, lung mucosa, gastric mucosa, intestinal mucosa, rectal mucosa, etc. can be selected appropriately.

Intranasal administration of the vaccine has an advantageous effect of enhancing immunity against a specific bacterial infection in which bacteria infect the host from the mucosal surface of the upper and/or lower respiratory tract.

Transdermal administration of the vaccine may be administered via a patch having a plurality of microprotrusions, a microneedle patch or a needle-free patch.

The vaccine composition may be in the form of a freeze-dried powder that can be reconstituted immediately before use, and has an effect of increasing stability and inhibiting the growth of microorganisms.

Subjects to which the vaccine composition can be administered are not limited, and may include mammals such as rats, mice, livestock, and humans, but may not be limited thereto. The effective dosage range of the above vaccine composition is gender, body surface area, body weight, type and severity of disease, age, drug sensitivity, administration route and excretion rate, administration time, treatment period, target cell, expression level, Whether the vaccine composition is used as a single agent or as an adjuvant agent, it may vary depending on various factors well-known in the medical field, including the degree of development according to pathological conditions, and can be appropriately determined by experts in the field. can

The vaccine composition may be administered several times or multiple times. For example, the vaccine composition of the present application may be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more. Administration may be at intervals of about 1 week to about 16 weeks, and in certain specific embodiments, at intervals of about 1 week to about 4 weeks. In the case of repeated infection or secondary infection by a specific bacterium targeted by the vaccine of the present invention, it can be re-administered on a regular basis.

In one embodiment of the present application, inactivating the bacterial strain to satisfy Equation 1 above; It provides a method for producing an inactivated strain comprising a. Equation 1 and the description of the inactivated strain are as described above.

The inactivating step in the manufacturing method may be inactivated by treating at a temperature of 55 ° C to 70 ° C for 30 minutes to 90 minutes.

Hereinafter, the present invention will be described in detail by Examples, Comparative Examples, and Experimental Examples. However, the following Examples and Comparative Examples are only to illustrate the present invention, and the content of the present invention is not limited to the following Examples.

<Example> Preparation of inactivated pertussis strain

Inactivated pertussis strains of Examples 1 to 4 were prepared by treating live pertussis strains (Comparative Example 1) in a constant temperature water bath at the following temperatures for each time period. Each temperature and treatment time are shown in Table 1 below.

temperature Processing time (minutes) Example 1 55℃ 90 Example 2 56℃ 30 Example 3 60℃ 30 Example 4 70℃ 30

<Experimental Example 1> Confirmation of anisotropy of inactivated pertussis strains

In the above experiment, the hydrophobic fluorescence value was measured for the degree of fluorescence of the viable cells of Comparative Example 1, the control group, and the pertussis strains of Examples 1 to 4, the experimental group, reacted with the Bis-ANS reagent using a fluorescence spectrophotometer.

Specifically, 1 × 10 ⁸ to 1 × 10 ⁹ pertussis strains inactivated under each condition and 10 μM Bis-ANS (4,4′-Dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt, Sigma Chemical Co., USA) was reacted by adding PBS to a final volume of 2 ml and stirring at room temperature for 1 hour.

In order to measure the fluorescence intensity after reacting each strain with Bis-ANS for 1 hour, the reacted solution was transferred to a quartz cell/cuvette for a fluorescence photometer, followed by a fluorescence spectrophotometer (Cary eclipse flouorescence spectrophotometer, Agilent Technologies Co., Ltd.). USA) was used to measure fluorescence values. At this time, among the spectrophotometer scan conditions, the excitation wavelength was set to 350 nm and the emission wavelength range was set to 400 nm to 690 nm. After scanning using a fluorescence spectrophotometer, the maximum value appearing between 485 nm and 505 nm was measured, and the hydrophobicity intensity fluorescence values are shown in FIG. 1 and Table 2.

As a result, the hydrophobic fluorescence value of the viable cells of Comparative Example 1 was 25. The hydrophobic fluorescence values of the pertussis strains inactivated as in Examples 1 to 4 were 201, 146, 179, and 247, respectively, and it was confirmed that they satisfied the range of Equation 1.

Referring to Figure 1, B / A (A is the hydrophobic fluorescence value measured in the strain before inactivation, B is the hydrophobic fluorescence value measured in the inactivated strain) according to Equation 1 is in the range of 5.5 to 10 It can be confirmed that it is in

<Experimental Example 2> Transmission Electron Microscope (TEM) analysis of inactivated pertussis strain and confirmation of protein aggregation

In the above preparations, the inactivated pertussis strain was polymerized into resin at a concentration of 1 x 10 ⁸ to 1 x 10 ^{9 ,} and the transmission electron microscope (TEM) picture and protein aggregation were digitized and shown in FIGS. 2 and 3 was

Specifically, in order to understand the characteristics and structure of the inactivated pertussis strain using a transmission electron microscope (Transmission Electron Microscope, TEM), the outer surface of the bead and the cut cross section were imaged, and the inactivated pertussis strain was imaged using Image J The composition ratio of the inactivated protein inside the strain was measured. At this time, the inactivated pertussis strain was ultra-thin sectioned through primary fixation, secondary fixation, dehydration, and polymerization with resin to analyze cross-sectional characteristics and measure the degree of protein inactivation.

In order to measure the intensity of protein aggregation within the cells of each cell, the PA value, which is the degree of intracellular protein aggregation based on the cell cross section, was measured, and this was divided by the PT value, which is the total area of the cell cross section. The PA value, which is the intensity of protein aggregation of each inactivated cell, means the intensity of aggregated protein in the entire area of the cell cross section. However, at this time, the background value that appears when measuring the live cell densitometer was removed from the area value of the inactivated protein. In the case of viable cells, intracellular protein aggregation did not occur, so the intensity of the aggregated protein was set to 0. The average value of the area between groups was compared and expressed as density, and based on this, the intensity of protein aggregation was quantified and expressed. When measuring the protein aggregation of each cell, the n number was set to 20 or more.

As a result, in Comparative Example 1, which is a live cell, there was no protein denaturation when the cell internal structure was confirmed, so the intracellular protein aggregation density (intracellular protein aggregation density) PA / PT of Equation 2 (PA is the protein aggregated in the cross section of the cell). Intensity, PT, total area of cell cross-section) value was 0. In the case of the inactivated pertussis strains of Examples 1 to 4, it was confirmed that the PA/PT values were 0.05, 0.047, 0.032, and 0.017, respectively.

hydrophobic fluorescence value protein aggregation Comparative Example 1 25 0 Example 1 201 0.05 Example 2 146 0.047 Example 3 179 0.032 Example 4 247 0.017

<Experimental Example 3> Confirmation of antibody titer increase effect by inoculation of inactivated pertussis strain

1. Preparation

3 x 10 ⁷ viable cells of Comparative Example 1 or 3 x 10 ⁷ inactivated pertussis strains as in Examples 1 to 4 were inoculated into the abdominal cavity of mice twice at one week intervals. Seven days after the final inoculation, the experimental animals were sacrificed by asphyxiation with CO ₂ , and blood was collected from the abdominal vena cava through laparotomy, and serum was obtained and stored at -80° C. until ELISA. After PBS washing, 1 ml of pertussis live bacteria (OD ₆₀₀ =1.0) is diluted 1/10, and 100 ul each is added to all wells of a 96-well immunoplate (SPL), and cultured at 4℃ overnignt. After washing the plate 3 times with wash buffer, it was incubated for 1 hour at room temperature using blocking buffer. After washing twice, 125 ul of serum sample prepared by diluting to 1/100 was dispensed to the top row A of the plate, and 100 ul of blocking buffer was dispensed to the remaining rows. After 1/5 serial dilution from row A to row H, the cells were incubated for two hours at room temperature. After washing 3 times, 100 ul of secondary antibody (Goat anti mouse IgG HRP, BIO-RAD) was added to all wells and incubated for 1 hour at room temperature. After washing three times, 100 ul of TMB substrate solution (R&D System) was added and incubated for 15 minutes. After adding 50 ul of stop solution (2N H ₂ SO ₄ , Duksan) to all wells, the plate was put into a micro plate spectrophotometer and OD ₄₅₀ was measured.

2. Results

IgG antibody titers against the pertussis strains in the serum of mice immunized twice with 3 x 10 ⁷ live cells of Comparative Example 1 and 3 x 10 ⁷ inactivated pertussis strains of Examples 1 to 4 were measured and shown in FIG. 4 . .

As a result, the antibody titer of the inactivated pertussis strain inoculated group of Examples 1 to 4 was significantly increased compared to the control group at a level equivalent to that of the live cell inoculated group.

Looking at Examples 1 to 4, B / A (A is the hydrophobic fluorescence value measured from the pertussis strain before inactivation, B is the hydrophobic fluorescence value measured from the inactivated mutant strain) according to Equation 1 is 5.5 to 10 It can be confirmed that it is in the range of , and PA / PT according to Equation 2 (PA is the intensity of the aggregated protein in the cross section of the cell, PT is the total area of the cross section of the cell) is also in the range of 0.02 to 0.07. can

<Experimental Example 4> Confirmation of TNF-a induction by inoculation of inactivated pertussis strain

1. Inoculation of live and inactivated pertussis strains

A total of 3 x 10 ⁷ CFU/100 ul of the viable cells of Comparative Example 1 and the inactivated pertussis strains of Examples 1 to 4 were intraperitoneally inoculated twice at weekly intervals.

2. Splenocyte Isolation

One day before the spleen was harvested, anti-CD3e (1 μg/ml; BioLegend) and anti-CD28 (3 μg/ml; BioLegend) antibodies were used to stimulate T lymphocytes, and the plates to which the spleen cells were to be seeded were coated. One week after the final inoculation, the spleen was removed and washed with cold PBS until the reagent became clear. The washed spleen tissue was crushed using a filter (70 μm cell strainer), and red blood cells (RBC) in the tissue were removed with ACK buffer (Gibco, A10492-01). After washing with culture medium, splenocytes were diluted to 5 x 10 ⁵ /ml and each well of a 12-well plate coated with anti-CD3e (1 μg/ml; BioLegend) and anti-CD28 (3 μg/ml; BioLegend) antibodies. 2 ml was dispensed into each. 1.25 x 10 ⁷ each of pertussis bacteria were treated in wells in which splenocytes were dispensed. Harvested after 6 hours of culture, the level of cytokines in the culture medium was measured.

3. Cytokine measurement

Tumor necrosis factor-alpha (TNF-a) was measured in spleen cells, and the concentration of TNF-a was measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Invitrogen, mouse). TNF-a ELISA KIT) was measured according to the manufacturer's instructions.

In order to support the immunotolerance response, changes in TNF-a cytokine levels in the splenocytes of mice were measured over time and are shown in FIG. 5 . Splenocytes were infected with the pertussis strain for 24 hours, and the amount of TNF-a increased by about 14 times or more in the splenocytes of the live cells of Comparative Example 1 and the inactivated pertussis strains of Examples 1 to 4 compared to the negative control group.

However, no significant change was observed between the live bacteria before inactivation and the inactivated pertussis strain inoculated group. It was confirmed that the inoculation of the inactivated pertussis strain of Example 1 also induced a level of TNF-a equivalent to the level of TNF-a induction by the live cell inoculation of Comparative Example 1.

Looking at Example 1, B / A (A is the hydrophobic fluorescence value measured from the mutant strain before inactivation, B is the hydrophobic fluorescence value measured from the inactivated mutant strain) according to Equation 1 is 8.04, ranging from 5.5 to 10 , and PA / PT according to Equation 2 (PA is the intensity of the aggregated protein in the cross-section of the cell, PT is the total area of the cross-section of the cell) is 0.017, which is in the range of 0.02 to 0.07. .

Having described specific parts of the present application in detail above, it is clear that these specific descriptions are only preferred embodiments for those skilled in the art, and the scope of the present application is not limited thereto. Therefore, the embodiments described above are illustrative in all respects, and should be understood as non-limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the present application is indicated by the following claims rather than the detailed description above, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts thereof should be construed as being included in the scope of the present application.

Claims (14)

A vaccine composition comprising a pertussis strain inactivated to satisfy Equation 1 below.
[Equation 1]

(In Equation 1, A is the hydrophobic fluorescence value measured in the strain before inactivation, and B is the hydrophobic fluorescence value measured in the inactivated strain.)
The method of claim 1,
The degree of intracellular protein aggregation of the strain,
A vaccine composition characterized in that it satisfies Equation 2 below.
[Equation 2]

(In Equation 2 above, PA is the intensity of aggregated protein in the cell cross-section, and PT is the total area of the cell cross-section.)
The method of claim 1,
The strain,
Vaccine composition characterized in that inactivated by treatment at 55 ℃ to 70 ℃ for 30 minutes to 90 minutes.
The method of claim 1,
The vaccine composition,
A vaccine composition characterized in that it maintains a stable storage state for at least 24 months at a temperature of 1 ° C to 65 ° C.
The method of claim 1,
The composition,
A vaccine composition further comprising at least one selected from the group consisting of pharmaceutically acceptable carriers, diluents, preservatives, stabilizers, buffers and adjuvants.
The method of claim 6,
The stabilizer,
A vaccine composition, characterized in that it is at least one amino acid stabilizer selected from the group consisting of alanine, histidine, arginine, lysine, isoleucine, valine, methionine, glycine and proline.
The method of claim 6,
The preservative,
A vaccine composition, characterized in that at least one selected from the group consisting of trehalose, skim milk, sorbitol, dextran and ascorbic acid.
The method of claim 1,
The vaccine composition,
A vaccine composition characterized in that it is for intraperitoneal, intramucosal, intramuscular, intradermal, subcutaneous, intravenous, intrarectal, intrapulmonary, intrathecal, oral, transdermal or intranasal administration.
A method for preparing the vaccine composition of any one of claims 1 to 8,
In the inactivated pertussis strain, based on the total weight of the composition
At least one selected from the group consisting of 5 to 10 wt% trehalose, 5 to 15 wt% skim milk, 2 to 5 wt% sorbitol, and 3 to 5 wt% dextran, and
0.1 to 10% by weight of ascorbic acid; adding a preservative further comprising;
Freezing at -80 ° C to -70 ° C for 24 hours to 48 hours; and
Freeze-drying for 48 hours to 60 hours under conditions of -50 ℃ to -40 ℃, 15 Pa to 30 Pa; method for producing an inactivated pertussis vaccine composition comprising.
In the method for producing the inactivated pertussis strain of any one of claims 1 to 8,
culturing the strain; and
Inactivating the strain to satisfy Equation 1 below;
Method for producing an inactivated pertussis strain comprising a.
[Equation 1]

(In Equation 1, A is the hydrophobic fluorescence value measured in the strain before inactivation, and B is the hydrophobic fluorescence value measured in the inactivated strain.)
The method of claim 10,
The degree of intracellular protein aggregation of the strain,
A method for producing an inactivated pertussis strain, characterized in that it satisfies Equation 2 below.
[Equation 2]

(In Equation 2 above, PA is the intensity of protein aggregated within the cell, and PT is the total area of the cell cross-section.)
The method of claim 10,
In the inactivation step,
A method for producing an inactivated pertussis strain, characterized in that the pertussis strain is treated at 55 ° C to 70 ° C for 30 to 90 minutes.
An inactivated pertussis strain, characterized in that produced by the manufacturing method of claim 10.
The method of claim 13,
The degree of intracellular protein aggregation of the strain,
An inactivated pertussis strain, characterized in that it satisfies Equation 2 below.
[Equation 2]

(In Equation 2 above, PA is the intensity of aggregated protein in the cell cross-section, and PT is the total area of the cell cross-section.)

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