WO2011093594A2

WO2011093594A2 - Vaccine vehicle including mycobacterial cell-wall skeleton and method for preparing vaccine using the same

Info

Publication number: WO2011093594A2
Application number: PCT/KR2010/009240
Authority: WO
Inventors: Tae-Hyun Paik
Original assignee: Tae-Hyun Paik
Priority date: 2010-01-27
Filing date: 2010-12-23
Publication date: 2011-08-04
Also published as: WO2011093594A3; KR20110087727A; KR101125764B1

Abstract

The present invention relates to a vaccine vehicle capable of greatly enhancing the immunogenicity of a protein or peptide antigen that is a subunit vaccine having low immunogenicity, and a method for preparing a vaccine using the vaccine vehicle. The vaccine vehicle comprises a peptidoglycan containing a carboxyl group and is the cell-wall skeleton of mycobacteria, which is covalently coupled to a peptide or protein antigen by a peptide bond between the carboxyl group and the amine group of the peptide or protein antigen. The vaccine vehicle can form a covalent bond with a protein or peptide antigen at high coupling efficiency, thereby preparing a highly immunogenic and safe vaccine.

Description

VACCINE VEHICLE INCLUDING MYCOBACTERIAL CELL-WALL SKELETON AND METHOD FOR PREPARING VACCINE USING THE SAME

The present invention relates to a vaccine vehicle capable of greatly enhancing the immunogenicity of a protein or peptide antigen that is a subunit vaccine having low immunogenicity, and a method for preparing a vaccine using the vaccine vehicle. More particularly, it relates to a vaccine vehicle including the mycobacterial cell-wall skeleton and a method for preparing a vaccine using the vaccine vehicle.

Vaccines include killed or attenuated pathogens or subunit vaccines, such as proteins, synthetic peptides and polysaccharide-peptide conjugates, and are widely used to prevent or treat microbial infections as well as malignant tumors (cancers) and allergic diseases.

Vaccines need to provide or induce two types of signals in order to elicit a strong protective immune response. First, vaccines need to deliver the antigen, which triggers antigen-specific receptors on T and B lymphocytes. Second, effective vaccines need to induce the expression of co-stimulatory molecules by antigen presenting cells (APCs), which then strongly activate the antigen-triggered lymphocytes. When using vaccines containing live pathogens, the second signal can be effectively activated, but when using inactivated vaccines, particularly highly purified subunit vaccines or peptide antigens, this signal is not provided, resulting in a great decrease in the immunogenicity of the vaccines. Either the addition of an adjuvant that can activate this second signal or the use of a vaccine vehicle will enhance the effectiveness of the vaccine to further enhance an immune response.

Adjuvants (or immune stimulators) refer to substances that stimulate the immune system to enhance the immune response to antigens. Such adjuvants stimulate a local inflammatory response resulting from the recruitment and activation of antigen-presenting cells, such as dendritic cells (DCs) or macrophages, at the site of infection, and then result in the activation of T cells, thereby increasing the immunogenicity of the antigen. Adjuvants stimulate the expression of costimulatory molecules and cytokines such as IL-12, which activate T cells, in antigen-presenting cells that recruited to the site of infection, and these adjuvants also increase the expression of peptide-HMC complexes on the surface of antigen-presenting cells. Thus, administration of an antigen together with an adjuvant can promote cell-mediated immunity and T cell-dependent antibody production to induce effective immune responses. Most strong adjuvants are bacteria or bacterial products, typical of which are mycobacterial cell-wall components.

Vaccine vehicles refer to substances that effectively enhance immunogenicity either by expressing an antigen gene in microorganisms exhibiting adjuvant effects or by covalently coupling an antigen to a microorganism, a microbial component or a carrier. Such vaccine vehicles can effectively induce humoral immunity as well as cell-mediated immunity.

A large number of adjuvants have been developed and studied from the beginning of the 20^th century, and among them, alum is the only adjuvant approved for human use, but it has a limitation in that it mainly increases humoral immunity. Thus, it is required to develop a novel adjuvant or vaccine vehicle which can effectively stimulate humoral immunity and cell-mediated immunity. In order to use the newly developed adjuvant or vaccine vehicle in practice, the safety, and effectiveness thereof need to be sufficiently verified.

The absence of a highly effective adjuvant as described above constitutes a significant obstacle to the successful development of vaccines, particularly those directed against intracellular pathogens, requiring cellular immunity. Particularly, in the case of subunit vaccines, such as protein or peptide antigens, which require the addition of potent adjuvants, their weak immunogenicity for vaccination still remains as a major problem. Thus, it is urgently required to develop a novel adjuvant and vaccine vehicle, which improve the immunogenicity of an antigen, can constantly stimulate a potent immune response and have guaranteed safety and reliability.

Generally, the mycobacterial cell-wall components strongly stimulate host inflammatory responses, leading to granuloma formation, the up-regulation of antigen presentation and inflammatory cytokines, and subsequent increases immune responses. Mycobacterium bovis bacillus Calmette-Guerin (BCG), the current live attenuated vaccine for tuberculosis, has been administered to approximately three billion people since 1921 and is one of the safest and most widely used vaccines. BCG has been much investigated as a tuberculosis vaccine as well as anticancer immunotherapeutic agents for various malignant tumors, including bladder cancer. In recent years, as vaccine vehicle systems for the expression of foreign genes in mycobacteria have advanced, recombinant BCG has been examined as delivery vehicles for various vaccine antigens.

Mycobacterial cell wall consists of peptidoglycan, arabinogalactan, mycolic acid, lipoarabinomanan (LAM), superficial lipids and mycoside, which are stacked to a thickness of about 20 nm (FIG. 1). Mycobacterial cell wall contains very high lipid content (60 wt % on a dry weight basis); many of which have been shown to have immune modulatory activity. Killed mycobacterial cells as well as mycobacterial cell-wall components, including cell-wall skeleton (CWS), muramyl dipeptide (MDP), trehalose dimyclolate (TDM), methanol extractable residue (MER) and the like are known as strong adjuvants that stimulate innate immunity. Among them, the CWS is a strong immunoactive microparticle consisting of peptidoglycan, arabinogalactan and mycolic acid, which promotes immune responses and also induces the proliferation of CTL and the activation of NK cells to exhibit anticancer immune effects. The clinical efficacy of the BCG-CWS as an anticancer immunotherapeutic agent has been much studied from 1970s by Yamamura et al. (Ann N Y Acad Sci 277: 209-227). The mechanism of BCG-CWS-mediated host immune activation partially contributes to the maturation of dendritic cells that is induced by Toll-like receptor (TLR) 2 and TLR4. Recently, it was reported that purified BCG-CWS activates the nuclear factor-kB promoter in the TLR2-dependent manner. Korean Patent Laid-Open Publication No. 2006-126963 discloses that a cell-wall fragment derived from the Mycobacterium tuberculosis-complex may be used as an adjuvant for immunotherapy.

Among the above-described mycobacterial CWS components, the peptidoglycan complex consists of alternating units of disaccharides including N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), which are cross-linked by tetrapeptide (L-alanyl-D-glutaminyl-meso-diaminopimelyl-d-alanine) side chains (FIG. 2). The glutamate and meso-diaminopimelic acid in the murein tetrapeptides have free carboxyl groups which can be coupled to the free amino groups of various protein or peptide antigens. Based on this fact, the present invention has been completed.

The present invention has been made in order to solve the above-described problems occurring in the prior art, and it is an object of the present invention to provide a vaccine vehicle that can greatly enhance the immunogenicity of a protein or peptide antigen.

Another object of the present invention is to provide a vaccine vehicle that inhibits an allergic immune response.

Still another object of the present invention is to provide a method of preparing a vaccine, having excellent immunogenicity, by forming a covalent bond between a protein or peptide antigen and the vaccine vehicle at high coupling efficiency.

To achieve the above objects, the present invention provides a vaccine vehicle comprising a peptidoglycan containing a carboxyl group, wherein the vaccine vehicle is the cell-wall skeleton of mycobacteria, which is covalently coupled to a peptide or protein antigen by a peptide bond between the carboxyl group and the amine group of the peptide or protein antigen.

The mycobacterial cell-wall skeleton is a complex of peptidoglycan-arabinogalactan-mycolic acid having high immune activity, wherein the disaccharide chains of the peptidoglycan are cross-linked by tetrapeptides. The glutamic acid and meso-diaminopimelic acid in the tetrapeptides have free carboxyl groups, and thus can be covalently coupled to the free amino groups of a peptide or protein antigen. Based on this fact, the present inventor has prepared a vaccine by covalently coupling a peptide or protein antigen to the mycobacterial cell-wall skeleton serving as a vaccine vehicle, and have confirmed the immunogenicity of the vaccine, thereby completing the present invention.

Examples of the present invention describe experimental results for the use of the cell-wall skeleton of BCG as a vaccine vehicle, but it is to be understood that mycobacteria other than the BCG can show effects which are equal or similar to the effects described in the Examples, because they have the same structure as that of the cell-wall skeleton of Mycobacterium bovis.

The cell-wall skeleton (CWS) can be isolated by conventional methods, including the methods described in J. Natl. Cancer. Inst. 52: 95-101(1974) and USP 6,593,096, and an isolation method which will be newly developed in the future may also be used in the present invention, because the method for preparing the mycobacterial cell-wall skeleton of the present invention is not limited.

Since the amino group in the antigen molecules is coupled to the carboxyl group of the cell-wall skeleton, any peptide, protein and glycoprotein antigen may be coupled to the vaccine vehicle of the present invention.

Examples of the present invention illustrate only OVA (ovalbumin), KLH (keyhole limpet hemocyanin) and BSA (bovine serum albumin) as protein antigens that are typically used for the verification of immunogenicity, as well as the peptide antigen Mart1 (a melanoma-associated antigen that is recognized by T cells), but the scope of the present invention is not limited thereto, and it is to be understood that the CSW may be coupled to all peptide, protein and glycoprotein antigens having a free amino group.

The present invention also provides a method for preparing a vaccine using the above-described vaccine vehicle, the method comprising the steps of: (A) preparing a suspension of a mycobacterial cell-wall skeleton (hereinafter referred to as CWS); (B) activating the free carboxyl group of peptidoglycan of the mycobacterial CWS; (C) allowing the CWS (activated precursor), having the activated carboxyl group, to react with a peptide or protein antigen, thereby covalently coupling the antigen to the CWS by a peptide bond; and (D) isolating and purifying the antigen-coupled CWS from the reaction solution of step (C).

In order to increase the reproducibility and coupling efficiency of the coupling reaction of the antigen with the CWS, it is preferable that the CWS in the reaction solution is uniformly suspended as single particles. However, since the CWS is hydrophobic in nature, it tends to form aggregates in an aqueous solution or a hydrophilic solution, thus making it significantly difficult to prepare a uniform suspension. In order to solve this problem, the CWS suspension of step (A) is preferably prepared by suspending the CWS in a C_1-C₄ alcohol, such as methanol, ethanol, propanol or butanol, or benzyl alcohol. Although Examples of the present invention do not suggest specific data, the results of suspending the CWS in the above-described alcohol indicated that the CWS was maintained in a stable state at -20 ℃ for at least 2 years without forming an aggregate even when the concentration of the CWS reached 10% (dry weight: 100 mg/ml). Also, although Examples of the present invention describe only the use of 2-propanol, the use of other alcohols could provide the same results as the use of 2-propanol.

The activating step (B) is a step of activating the free carboxyl group of the CWS to make highly reactive with primary amine groups of antigen. This may be carried out using any conventional organic method for forming a peptide bond between carboxylic acid and primary amine. In Examples of the present invention, the carboxyl group was activated by allowing the CWS to react with EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride) / NHS (N-hydroxysuccinimide) so as to prepare NHS-ester, but the scope of the present invention is not limited thereto.

FIG. 3 is a sequence diagram showing chemical reactions that occur in a process for constructing a vaccine using the CWS as a vaccine vehicle. As shown in FIG. 3, free carboxyl groups in the CWS react with EDC/NHS to form NHS-ester, and then react with the free amino groups of an antigen to form a peptide bond.

In order to quantitatively evaluate the coupling efficiency of the antigen in the preparation of the vaccine, and to induce an effective immune response by the vaccine, the antigen-coupled mycobacterial CWS is preferably uniformly dispersed as single particles other than aggregates. However, the antigen-coupled CWS tends to form aggregates in an aqueous solution or a hydrophilic solution, like the CWS. Thus, the antigen-coupled CWS prepared in step (D) is preferably suspended in a suspension buffer containing 1-10% (v/v) of ethanol, isopropanol or benzyl alcohol. As the suspension buffer, any buffer may be used as long as it is generally used for the suspension of vaccines. In Examples of the present invention, 0.02% Tween 80-1% ethanol-PBS was used as suspension buffer, but the scope of the present invention is not limited thereto. The use of said solution as suspension buffer allowed the antigen-coupled CWS to be prepared into a uniform suspension containing single particles.

Examples of the present invention illustrate only OVA (ovalbumin), KLH (keyhole limpet hemocyanin) and BSA (bovine serum albumin) as protein antigens and Mart1 as a peptide antigen, but the scope of the present invention is not limited thereto. Also, as can be seen in Examples of the present invention, the antigens could be covalently coupled to the CWS at high efficiency, and as the concentration of the antigen was increased, the amount of antigen coupled to the CWS was also gradually increased. Coupling efficiency in Examples of the present invention was similar or superior to the results of other studies on the coupling of proteins to other various vehicles [Bioconjug. Chem. 19(7): 2485-1490, Immunology 107(4): 523-529]. Furthermore, it is to be understood that the antigen can be coupled to the vaccine vehicle of the present invention at higher coupling efficiency by optimizing reaction conditions while changing reaction solution concentration, reaction time, reaction temperature during the reaction of the peptide or antigen with the CWS.

The present invention also provides a activated CWS precursor for preparing the vaccine according to the above-described method, wherein the activated CWS precursor is produced by the reaction of the mycobactrial CWS with EDC 〔1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride)/NHS (N-hydroxysuccinimide)]. The activated CWS precursor of the present invention is very stable and has little or no effect on antigen coupling efficiency even when it is stored for 1 year or more. Thus, it can be advantageously used as a activated CWS precursor for the preparation of the vaccine.

Animal tests for confirming the immunogenicity of the OVA-CWS, KLH-CWS and BSA-CWS vaccines prepared according to the present invention were performed and, as a result, all the vaccines showed significantly excellent immunogenicity even when the antigens were administered in a very small antigen amount of 1-2 ㎍, compared to when the antigens were administered alone or administered together with alum as an adjuvant. Also, when compared with the case in which 20 ㎍ of the antigen was used together with the same amount of the CWS as an adjuvant, the antigen-CWS vaccine according to the present invention showed equal immunogenicity in an antigen dose corresponding to 1/8~1/2 of said case, suggesting that the vaccine vehicle of the present invention greatly enhance the immunogenicity of the antigen. As described above, it is expected that the vaccine vehicle of the present invention can greatly reduce the dose of the antigen, and thus can greatly increase the safety of the vaccine.

In addition, all groups, either immunized with either OVA antigen (a typical allergy-causing antigen) alone, immunized with OVA in combination with alum or BCG-CWS as an adjuvant, showed a strong Th2-biased immune response, whereas, in a group immunized with the vaccine of the present invention, the immune response of the Th2 antigen OVA was strongly switched to a Th1 immune response. This suggests that the inventive vaccine, obtained by covalently coupling Th2 antigen (that can cause allergy) to the CWS can be used for the prevention and treatment of allergic diseases.

As described above, according to the inventive method for preparing the vaccine vehicle and the vaccine, a specific antigen is covalently coupled to the cell-wall skeleton. Thus, the inventive method can be advantageously used to prepare a highly immunogenic and safe vaccine.

FIG. 1 is a schematic diagram showing the cell-wall structure of mycobacteria.

FIG. 2 is a schematic diagram showing the detailed structure of peptidoglycan of the cell-wall skeleton (CWS).

FIG. 3 is a sequence diagram showing chemical reactions that occur during a process of constructing a vaccine using the CWS as a vaccine vehicle.

FIG. 4 is a graphic diagram showing the amount of OVA-specific IgG antibody in mouse serum after immunization.

FIG. 5 is a graphic diagram showing the effects of OVA antigens on the proliferation of lymphocytes.

FIG. 6 is a graphic diagram showing the effects of immune responses by OVA antigens on IL-2 production.

FIG. 7 is a graphic diagram showing the effects of immune responses by OVA antigens on IL-12p40 production.

FIG. 8 is a graphic diagram showing the effects of immune responses by OVA antigens on IFN-γ production.

FIG. 9 is a graphic diagram showing the amount of OVA-specific IgG antibody in mouse serum after immunization.

FIG. 10 is a graphic diagram showing the effect of KLH antigen on the proliferation of lymphocytes.

FIG. 11 is a graphic diagram showing the effects of immune responses by KLH antigens on IL-2 production.

FIG. 12 is a graphic diagram showing the effects of immune responses by KLH antigens on IL-12p40 production.

FIG. 13 is a graphic diagram showing the effects of immune responses by KLH antigens on IFN-γ production.

Hereinafter, the present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are illustrative purposes only and are not to be construed to limit the scope of the present invention.

Examples

Example 1: Preparation of BCG-CWS and conjugation of protein or peptide antigen

The BCG-CWS was prepared according to the conventional method described in the following literature: J. Natl. Cancer. Inst. 52: 95-101(1974) or USP 6,593,096. The prepared BCG-CWS was suspended in 2-propanol at a concentration of 50 mg/ml (on a dry weight basis) and then stored at -20 ℃ until use.

As antigens, the protein antigens, OVA (ovalbumin, Pierce, Rockford, Ill., USA), KLH (keyhole limpet hemocyanin, Pierce) and BSA (bovine serum albumin, Pierce), and the peptide antigen Mart 1 (Peptron, Daejeon, Korea), were used. The protein antigens were dissolved in 0.1M PBS, and the peptide antigen was dissolved in DMSO. Also, the antigens were dissolved at a concentration of 1, 2, 4 or 8 mg/ml, thereby preparing antigen solutions.

0.5 ml of the BCG-CWS suspension (dry weight: 50 mg/ml), stored -20℃, was transferred to a 2.0-ml microtube, and 1 ml of 2-propanol was added thereto, followed by shaking in a bead beater. The mixture was centrifuged at 14,000g for 5 minutes, and the BCG-CWS pellets were collected. To the BCG-CWS pellets, 1 ml of an activation solution of 20mM EDC/50mM NHS in 2-propanol was added, followed by shaking in a bead beater and then allowed to react by mixing end-to-end for 30 minutes at room temperature. The resulting EDC/NHS-activated CWS pellet was washed with 1.5 ml of 2-propanol.

After completion of the washing process, 0.5 ml of the above-prepared antigen solution was added thereto, followed by shaking in a bead beater, and then allowed to react in a microtube rotator (5 rpm) at 4℃ for 18 hours. Then, the reaction solution was centrifuged at 14,000 g for 5 minutes to collect BCG-CWS (antigen-CWS) pellets to which the antigen had been conjugated by covalent coupling. The unbound protein antigen in antigen-CWS pellets was removed by washing with 1.5ml of 0.01M PBS(pH 7.2) containing 0.02% Tween 80 and 1% ethanol(suspension buffer). The resulting antigen-conjugated CWS pellets was resuspended in suspension buffer(0.02% Tween 80/1% ethanol/PBS) and shaken in a bead beater, thereby preparing an antigen-CWS suspension. Then, the concentration of the protein or peptide antigen conjugated to the BCG-CWS in the suspension was measured using a bicinchoninic acid (BCA) protein assay kit (Pierce) and calculated after substracting the protein content of unconjugated CWS. The coupling efficiency of the protein or peptide antigen bound to the BCG-CWS was calculated by dividing the total amount of antigen bound to the BCG-CWS by the amount of antigen used in the coupling reaction, and the results of the calculation are shown in Table 1 below.

Table 1

As can be seen in Table 1 above, the protein antigens showed a high coupling efficiency of more than 90% to the BCG-CWS when used at a concentration of 1 mg/ml, and the peptide antigen Mart1 showed a coupling efficiency of 54%. As the concentration of the antigen solution was increased, the coupling efficiency was somewhat reduced, but the amount of antigen conjugated to the BCG-CWS was gradually increased. Particularly, the OVA showed the highest coupling efficiency and the largest amount of conjugated antigen.

Example 2: Confirmation of immunogenicity of OVA-CWS

In order to confirm the immunogenicity of the protein antigen conjugated to the BCG-CWS, mice were immunized with the OVA antigen-bound BCG cell wall skeleton (OVA-CWS) vaccine prepared using 8.0 mg/ml of the OVA antigen solution, and the immunogenicity of the vaccine was examine.

Specifically, as shown in Table 2 below, 6-week-old BALB/c mice were divided into 10 groups, each consisting of 5 animals. The mice received three immunizations at two-week intervals subcutaneously.

At 2-week-inrtervals after each immunization, serum was collected and the amount of OVA-specific IgG was quantified by an ELISA assay. The results are shown in Table 2 and FIG. 4. As can be seen in Table 2 and FIG. 4, group 9, immunized with the OVA-CWS vaccine according to the present invention, showed an IgG antibody titer which was substantially similar to that of groups 1 and 2 immunized with 20 ㎍ OVA alone or together with alum as an adjuvant, even though the OVA antigen was administered in an amount of only 2.6 ㎍. In addition, groups 7 to 10 showed IgG antibody titers which were substantially similar to those of groups 3 to 6 immunized with 20 ㎍ OVA together with BCG-CWS as an adjuvant, even though the amounts of antigen administered were only about 1/10~1/2 of those in groups 3 to 6. This suggests that the inventive vaccine prepared using the BCG-CWS shows very strong immunogenicity.

Table 2

Meanwhile, in order to determine the characteristic of the immune response of each group to OVA antigen (a typical allergy-causing antigen), each of the Th2 antibody OVA-specific IgG1 and the Th1 antibody OVA-specific IgG2a was quantified by an ELISA assay, and the results are shown in Table 3 below.

Table 3

As can be seen in Table 3 above, as expected from the characteristics of OVA antigen, the Th2 antibody IgG1 in all of groups 1 to 6 immunized with OVA alone together with alum or BCG-CWS as an adjuvant was highly induced compared to the Th1 antibody IgG2a. On the other hand, groups 7 to 10 immunized with the OVA-CWS according to the present invention were switched to a Th1 antibody response, and thus the production of IgG2a antibody therein was highly induced compared to those in groups 1 to 6.

Also, 2 weeks after the final immunization, splenocytes were collected and cultured in RPMI complete medium supplemented with fetal bovine serum (Gibco-BRL, Rockville, NY, USA), after which 5 ㎍/㎖ Concanavalin A (Con-A; Fluka, Milwaukee, WI, USA), 5 ㎍/㎖ lipopolysaccharide (LPS; Fluka) or 20 ㎍/㎖ OVA (Pierce) was added. Then, the cells were cultured in a CO₂ incubator for 48 hours. Cell proliferation was analyzed using a cell counting kit (Dojindo Laboratories, Kumamoto, Japan), and the results of the analysis are shown in FIG. 5. Also, the culture supernatants were collected, and the production of each of IL-2, IL-4, IL-10, IL-12p40 and IFN-γ in the supernatants was measured using a BD OptEIA set (BD Biosciences, San Diego, CA). The results of measurement for IL-2, IL-12p40 and IFN-γ are shown in FIGS. 6 to 8, respectively. Because the levels of expression of IL-4 and IL-10 in response to OVA stimulation were relatively low, it was difficult to comparatively analyze the expression levels. In the figures, ** and *** indicate significant differences from the OVA + alum group (group 2; P < 0.01 and P < 0.001, respectively).

As can be seen in FIG. 5, only groups 7 and 8 immunized with the OVA-CWS according to the present invention showed a very significant increase in lymphocytes in response to OVA stimulation, compared to that in group 2 immunized with OVA in combination with alum as an adjuvant, suggesting that the OVA-CWS showed strong lymphoproliferative ability.

As can be seen in FIGS. 6 to 8, the expression levels of IL-2, IL-12p40 and IFN-γ in response to OVA stimulation were similar to each other, and thus in the groups immunized with either OVA plus BCG-CWS or OVA-CWS, the expression levels of IL-2, IL-12p40 and IFN-γ were significantly increased compared to those in other groups, including group 2. Particularly, the OVA-CWS greatly increased the expressions of IL-2, IL-12p40 and IFN-γ and the ability of the OVA-CWS to produce IFN-γ was more than 240 times higher than that of the OVA-alum (group 2).

Such results revealed that, when OVA antigen is used for immunization in a state in which it was conjugated to the BCG-CWS according to the present invention, it more effectively proliferates lymphocytes in response to antigen stimulation and also stimulate the expression of cytokines such as IL-2 and IL-12p40. In addition, it is believed that, only when OVA antigen (Th2 antigen) is used for immunization in a state in which it was conjugated to the BCG-CWS, it effectively stimulates innate immunity to strongly induce the expression of IFN-γ thereby switching the immune response to a Th1 response.

Example 3: Conformation of immunogenicity of KLH-CWS

Mice were immunized with a KLH-CWS vaccine prepared using 8.0 mg/ml of antigen solution of Example 1, in order to confirm the immunogenicity of the KLH-CWS vaccine.

Specifically, as shown in Table 4 below, 6-week-old BALB/c mice were divided into 8 groups, each consisting of 5 animals. The mice received three immunizations at two-week intervals subcutaneously. After the immunization, the KLH-specific expression of IgG, the proliferation of lymphocytes in response to KLH stimulation, and the production of IL-2, IL-12p40 and IFN-γ were measured in the same manner as Example 2, and the results of the measurement are shown in Table 4 and FIGS. 9 to 13, respectively.

Table 4

As can be seen in Table 4 above and FIG. 9, the KLH-CWS vaccine according to the present invention showed strong immunogenicity compared to when the antigen was administered alone, even in the case of group 8 in which the dose of antigen used was 1.34 ㎍ which was only 6% of the dose of the KLH antigen of groups 1 to 5. Also, the IgG antibody titer of group 8 in the first immunization was lower than those of groups 2 to 5 immunized with 20 ㎍ antigen together with alum or CWS as an adjuvant, but as immunization was repeated, the IgG antibody titer was rapidly increased, and after the third immunization, groups 3 to 8 showed higher IgG antibody titers than that of group 2 administered with alum as an adjuvant. Even when the same amount of CWS was used as an adjuvant, a similar IgG antibody titer was measured even in group 8 immunized with 1.34 ㎍ of the antigen. This suggests that, when not only OVA, but also KLH antigen, are prepared into vaccines using the vaccine vehicle of the present invention, the immunogenicity of the prepared vaccines is greatly enhanced.

As can be seen in FIG. 10, only group 6 immunized with the KLH-CWS vaccine according to the present invention, and groups 4 and 5 administered with the BCG-CWS as an adjuvant showed a significant increase in lymphocytes in response to KLA stimulation, compared to group 2 administered with alum as an adjuvant.

As can be seen in FIGS. 11 to 13, the expression levels of IL-2, IL-12p40 and IFN-γ in response to KLA stimulation in FIGS. 11 to 13 showed a tendency similar to that of the OVA-CWS vaccine of Example 2. Moreover, the expression levels of IL-4 and IL-10 response to KLH stimulation were low, similar to the expression levels response to OVA antigen in Example 2, and thus it was difficult to comparatively analyze the expression levels.

Mice were immunized with a BSA-CWS vaccine prepared using 8.0 mg/ml of antigen solution of Example 1, and the immunogenicity of the BSA-CWS vaccine was confirmed.

Specifically, as shown in Table 5 below, 6-week-old BALB/c mice were divided into four groups, each consisting of 5 animals. The mice received three immunizations at two-week intervals subcutaneously. After the immunization, the BSA-specific IgG expression levels in the groups were measured in the same manner as Example 2, and the results of the measurement are shown in Table 5 below.

Table 5

As can be seen in Table 5 above, group 4 immunized with the BSA-CWS vaccine according to the present invention showed significantly immunogenicity compared to either group 1 administered with BSA antigen alone or group 2 administered with alum as an adjuvant, even though the dose of the antigen in group 4 was 2.51 ㎍ corresponding to 12.5% of the antigen dose of groups 1 to 3. In addition, group 3 administered with the BCG-CWS as an adjuvant showed an IgG antibody titer similar to that of the vaccine of the present invention.

Claims

A vaccine vehicle comprising a peptidoglycan containing a carboxyl group, wherein the vaccine vehicle is the cell-wall skeleton of mycobacteria, which is covalently coupled to a peptide or protein antigen by a peptide bond between the carboxyl group and the amine group of the peptide or protein antigen.
The vaccine vehicle of claim 1, wherein the mycobacteria are Mycobacterium bovis BCG.
A method of preparing a vaccine using the vaccine vehicle of claim 1 or 2, the method comprising the steps of:

(A) preparing a mycobacterial cell-wall skeleton suspension;

(B) activating the free carboxyl group of peptidoglycan of the mycobacterial cell-wall skeleton;

(C) allowing the cell-wall skeleton, having the activated carboxyl group, to react with a peptide or protein antigen, thereby covalently coupling the antigen to the cell-wall skeleton by a peptide bond; and

(D) isolating and purifying the antigen-coupled cell-wall skeleton from the reaction solution of step (C).
The method of claim 3, wherein the cell-wall skeleton of step (A) is prepared by suspending the cell-wall skeleton in methanol, ethanol, propanol, butanol or benzyl alcohol.
The method of claim 3, wherein the activating in step (B) is carried out by allowing the cell-wall skeleton to react with EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride)/NHS (N-hydroxysuccinimide).
The method of claim 3, wherein the antigen-coupled cell-wall skeleton prepared in step (D) is suspended in buffer containing 1-10%(v/v) of ethanol, isopropanol or benzyl alcohol.
A vaccine precursor obtained by the method of claim 5, wherein the vaccine precursor is produced by reaction of a mycrobacterial cell-wall skeleton with EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride)/NHS (N-hydroxysuccinimide).