초록 ▼

○ 연구결과
1. 연구개발 결과(성과)
가. 특허출원
◦ 현재 진행중인 특허(2002년 12월중 출원 완료 가능)
◦ 대상국 : 일차 국내 출원후 자료 보완하여 국외도 출원할 계획임.
◦ 제목 : 경구용 면역증강제의 활성을 가지는 한국산 겨우살이 렉틴성분
◦ 내용 : 본 과제 연구결과중 한국산 겨우살이 추출물(KM-110)과 lectin성분 (KML-C)가 점막면역을 자극하여 면역기능을 증강시키는 효과에 근거하여 특허청구범위를 정하고 이를 이용할 경우 제약을 비롯한 다양한 제품을 개발할 수 있어 산업

○ 연구결과
1. 연구개발 결과(성과)
가. 특허출원
◦ 현재 진행중인 특허(2002년 12월중 출원 완료 가능)
◦ 대상국 : 일차 국내 출원후 자료 보완하여 국외도 출원할 계획임.
◦ 제목 : 경구용 면역증강제의 활성을 가지는 한국산 겨우살이 렉틴성분
◦ 내용 : 본 과제 연구결과중 한국산 겨우살이 추출물(KM-110)과 lectin성분 (KML-C)가 점막면역을 자극하여 면역기능을 증강시키는 효과에 근거하여 특허청구범위를 정하고 이를 이용할 경우 제약을 비롯한 다양한 제품을 개발할 수 있어 산업적인 가치가 매우 클 것으로 기대됨.
나. 특허출원 준비중
◦ 3개월 이내 출원 가능할 것으로 예상됨
◦ 대상국 : 한국 및 중국
◦ 제목 : 한국산 겨우살이 lectin성분이 함유된 백신용 면역증강제(adjuvant) 조성물
◦ 내용 : KM-110이나 KML-C가 체액성 및 세포성 면역증강효과를 나타내며 이를 이용하여 동물백신용 adjuvant로 활용할 수 있는 실재적인 조성물에 관한 내용
다. 논문 발표
특허출원 및 사업화를 위한 전략적인 면에서 그간 논문발표는 보류되어 왔지만 이제 그간의 결과들을 토대로 논문을 발표할 것임. 예상되는 주요 논문의 제목을 정리하면 다음과 같다.
1) 한국산 겨우살이에서 분획된 viscotoxin의 in-vitro 세포독성효과와 in-vivo 독성효과 비교
2) Humoral immunoadjuvant activity of KML-C
3) Cellular immunoadjuvant activity of KML-C
4) 한국산 겨우살이의 잎, 줄기 및 열매 내에 존재하는 lecin의 함량조사
5) Effects of mistletoe preparations in the mixture with other known adjuvants on the production of antibodies for the development of new adjuvant system
6) Mucosal adjuvant effect of Korean mistletoe in mice
7) Involvement of caspase and serine protease in cell death by mistletoe lectin
8) Immune enhancement effect of lectin for genetically engineered proteins
9) Enhancement of immunogenicity of inactivated vaccines of NDV and IBV
10) 돼지 오제스키병에 있어서 렉틴의 독성이 미치는 영향

Abstract ▼

It has been well known that mistletoe has various biological activities such as anti-cancer and immunological activities including adjuvant activity. The ultimate purpose of this project is to develop a new adjuvant for animal vaccine.
For this, we have investigated the following studies and the

It has been well known that mistletoe has various biological activities such as anti-cancer and immunological activities including adjuvant activity. The ultimate purpose of this project is to develop a new adjuvant for animal vaccine.
For this, we have investigated the following studies and the results are summarized as followed:
1. Investigation of the optimal conditions for the preparation of extract(KM-llO) and purication of lectins from Korean mistletoe.
1) We have established the optimal conditions for the preparation of
extmct(KM-110), which contains lectins with immunological activities and the highest yield.
2) The conditions for the isolation and purification of lectins from Korean mistletoe were also established: fraction containing two different kinds of lectins namely KML-C by hydrolyzed Sepharose-4B colum; purification of lectins in a single component by itmnunoaffinity chromatography using monoclonal antibody.
3) Isolation of viscotoxin by various methods
2. Production and characterization of monoclonal antibodies specific to KML-IIU for the development of ELISA for the assay of KML-IIU and for the making of immunoaffinity colum.
1) We have got several hybridoma secreting monoclonal antibodies to KML-IIU
The antibodies were characterized in terms of titer and specificity. One of them was very specific to KML-llL. which did not cross react with other lectins.
2)Two-sites sandwich ELISA method for the measurement of KML-llU was development using monoclona and polyclonal antibodies, which can be used as a Q.C method.
3)Immunoaffinity chromatography was established for the isolation of KML-llU with monoclonal antibody
3. Preliminary toxicity and general pharmacology of KML-C were investigated ill terms of determination of LD50 general behavors hexobarbital-induced sleeping time, pentylenetetrazole-induced convulsion etc : no significant effects
1) Acute toxicities of toxin fraction and heparine-binding protein fraction were also invesligated.
2) Detennination of the effective dose of KML-C : 250ng/kg was the minimum dose showing immunological activity wheras 12.5-25ug/kg might be the highest dose which did not show any serious side effects.
4. Immunologicla activities
1) Effect of cytotoxcity in vitro on the normal cell(splcen cells)
2) Mitogenic activity with and without other mitogens such as con-A and LPS
3) Induction of cytokine from macrophage with KML-IIU and IIL, viscotoxin HBP.
5. Non-antigen specific resistance effects in experimental animal challenge test with several bactterial strains including Hordetella bronchiseptica, Pasteurella multocida etc.
6. Development of a new adjuvant for the production of antibody
I) Effect of KM-IIO on the adjuvant activity of aluminium hydroxide and ASA25
: KM -110 showed synergistic effects on lhe tiler of antibody produced
2) Development of new oil adjuvant containg KM-110 : when KM-110 was
administered with conventional and the most typical oil adjuvanl FIA and FCA, it showed significant synergistic effect on the rasing of the antibody titer
7. Effects of KM-110 and KML-C on the humoral immuno-activity
1) Determination of the optimal amounts showing adjuvant activities through comparison studies with FIA and FCA
2) Antigen-specific cytokine induction ability and subclass of antibodies produced were investigated to see whether their responses are Thl or Th2
3) KML-C changed a population and surface marker of spleen lymphocytes by flowcytometer, CD69 was increased while CD3 was decreased, decreased, indicating that it has lymphocyte activation activity.
8. KVIL-C showed also cellular-immuno activity such as DTH. which was further confirmed by showing that it induced IL-2 which IS associated with the proliferation of T-cell.
9. Proliferation of spleen cells stimulated by antigen with KML-C previously was increased when stimulated with antigen in-vitro.
10. In order to develop a new formulation containing mistletoc preparation, several components of adjuvant such as alum, squalene, PCA and FIA were mixed with KM-110 or KML-C und used for the immunization with various kinds of antigen and vaccines. We believe that mistletoe preparation has potentiality to be one of active components used for the adjuvant.
11. Mucosal routes for vaccine delivery have advantages, however for this a powerful mucosal adjuvant is required. We have demonstrated that mistletoe preparation such as KML-C and KM-110 have a strong mucosal adjuvant activity.
12. 12. To determine general toxicity m chickens, the 1.1 ng, 2.2 ng and 22.2 ng of the lectin per 1 g of body weight, respectively, were injected into legs of chicks or l-week-old, intramuscularly. The chicks were observed ror changes in blood and histopathology at 4-days' intervals during 16 days, and the results arc summarized as follows.
: During the experimental period, no significant changes were observed in values of RBC, WBC, Hb, PCV, MCV, MCHC, GOT, GPT, BUN, creatinine and total protein in the chicks injected with lectin of 1.1-22.2/g of body weight, which mean the lectin has no toxic effects on blood circulation and functions of liver and kidney. In hisopathological observation, no changes were found in the brain, liver, lung, spleen, kidney, thymus, bursa of Fabricius and muscle of leg in which the lectin was injected with does of 2.2ng/g of body weight.
13. In comparison of immune-enhancing effects of lectin (4.4ng/g of body weight) and Al(OH)₃ on inactivation vaccines of NDV in chicks of 1-week old, HI antibody titers by lectin-adjuvanted B1, LaSota and Ulster 2C were log₂3.9, log₂4.8, and log₂5.3, respectively, at 4 weeks after after vaccination. Althoug the HI titers were lower than those in the same strains adjuvanted with Al(OH)₃ (log₂7.3, log₂8.3, log₂9.3, respectively) the chicks withstood challenge by the Kyojeongwon strain, a very virulent NDV, and the HI titers reached to high level as in the Al(OH)₃-adjuvanted vaccines at 6 weeks after vaccination. The antibody induced by lectin-adjuvanted vaccines revealed reactivity to the NDV antigens as in the Al(OH)₃-adjuvanted vaccines. It was, therefore, recognized that lectin increased immunogenicity of inactivated NDV vaccines.
14. The lectin (4.4 ng/g of body weight) also increased the immunogenicity of inactivated IBV vaccines of M1 and KM91 strains as in the Al(OH)₃-adjuvanted vaccines of the strains. The HI antibody titers by lectin-adjuvanted vaccines of M41 and KM91 were log₂3.8 and log₂4.4 respectively, at 4 weeks after vaccination. The chicks withstood challenge by a virulent IBV strain, and the HI titers reached to high level as in the Al(OH)₃-adjuvanted vaccines at 6 weeks after vaccination. It was, therefore, clear that lectin increased immunogenicity of inactivated IBV vaccines.
15. To detemine general toxicity of lectin in pigs and reference to its concentration if ti can enhance the immunogenicity of inactivated vaccines, the lectin in various concentrations was injected into post-weanling piglets intramuscularly. The piglets were ovserved for clinical signs and changes in blood and histopathology, and the results are summarized as follows.
: By lectin of 30ng/g of body weight there were signs such as systemic erythema, eutaneous hemorrhage, dypsnea, fever and death. Lesions such as centrilobular congestion fat granules, necrosis, vcuolatin and depletion of nuclei were also observed in liver. In kidney, vacuolation and necrosis in convoluted tubules and hyaline droplets in glomeruli were observed, By lectin of 7ng/g of body weight edema and cutaneous hemorrhage in the injected muscle, lamenes and depression were observed. By lectin of 3-5 ng/g of body weight, signs of depression and inappetence, and congestion in the injected area were shown. Moreover toxic changes were found in liver and kidney. By lectin of 0.5-0.7 ng/g of body weight, no significant changes were observed in values of RBC, WBC, Hb, PCV, BUN, creatinine, AST and ALT while obserbved wre swelling and vacuolation of convoluted tubules by 0.7 ng leci수 and necrosis and fibrosis of muscular fibers by 0.5 ng lectin. Therefore, it was recognized that toxicity of the lecin was marked in pigs, and thus, the concentraiton of lectin for inactivated vaccines which will not exhibit toxicity was considered as less than 0.7ng/g of body weight.
16. In determination of immune-enhancing effects of lectin (0.7ng/g of body weight) on inactivated vaccines of ADV in post-weanling piglets, only population of B lymphocytes (sIgM+) incresed among immunocompetent cells such as MHC class II, CD2+, CD4+, CD8+, B lymphoytes (sIgM+), NK cells and granulocytes, which meant no cellular immune response was activated. In serum neutralizing antibody titers (SN titers) by lectin (0.7ng/g of body weight)-adjuvanted vaccine, tlle SN titer at 4 weeks after vaccination was log₂3.0 as shown by vaccine without adjuvant. However, the piglets with such a low SN titer could withstand challenge by NY,J87-i strain, a virulent ADV, while some of piglets showed slight diarrhea and lever for 1-2 days, The SN titers reached to level of log₂5.0 as in the vaccine without adjuvant at 5 weeks after vaccination. It is, therefore, concluded that lectin has marked toxicity and can not enhance the immunogenicit:y of inactivated ADV vaccine by lo\v dose (0.7 ng/g of body weight) which exhibit no toxicity.
17. To produce HN gene protein of NDV, a recombinant DNA clone named pFBHN was constructed between HN gene (1.7 kb) and pFastBacHTa plasmid, a baculovirus expression vector (4.D kbl. The pFBIN DNA was transformed into compeLent DHlOBac cells, and HN DNA -recombinant bacmid WaS established by transferring the TTN DNA to baemid (baculovirus DNA) which was harbored in the DH10Bac. The HN DNA -recombinant bacmid was extracted and then infected into Sf cells using lipofectin. The protein expressed from HN DNA of the bacmid was determined by occlusion-negative morphology of the infected Sf cells and fluorescent antibody assay using NDV antiserum. The HN protein was extracted and used for the production of vaccine. Lectin-adjuvanted vaccines of HN protein were made in two formulas such as 200 ng of lectin plus 200 ng or 300 ng of HN protein per 0.5 ml injection dose. To compare the immune response, Al(OH)₃-adjuvanted HN protein vaccines was also produced with 200 ng of HN protein and Al(OH)₃ in 34% of the total volume. The vaccines were injected into I-week old, SPF chicks, and the chicks were tested for titers and duration of HI antibody and immune response to challenge for six weeks.
18. In comparison on immune-enhancing effects of lectin and Al(OH)₃ on HN
protein vaccines, HI antibody titers by lectin-adjuvanted and Al(OH)₃-adjuvanted
vaccines at l week after vaccination were almost equal as log₂2.4 and log₂2.6,
respectively. At 4 weeks afterr vaccination, antibody of log₂4.0 was produce by
Iectin-adjuvanted vaccie, which was log₂1.0 lower than the titer of Al(OH)₃-adjuvanted vaccine. After challenge by Kyojeongwon strain, a very virulent NDV, there were no cases of clinically ill and death in both group of the chicks. At 1 week after the challenge, HI titers of lectin-adjuvanted and Al(OH)₃-adjuvanted vaccines reached to log₂6.2 and log₂7.4, respectively, and the titers further reached to log₂7.0 and log₂7.6 respecitely, at 6 weeks after vaccination. On the other hand, HN protein (200ng) without adjuvant induced HI titers of log₂1.6, log₂2.0, log₂2.8 amd log₂3.0 at post weeks, 1, 2, 3, and 4, respectively, from which log₂1.0 difference was found when compared to the titers. by lectin-adjuvanted HN protein at same time course. Moreover, some of the chicks immunized only with HN protein showed signs of depression and mild dyspnca for 2-3 days after challenge. It was, therefore, clearly recognized that the lectin enhanced immunogenicity of HN protein, which enabled chicks resistat to the challenge of virulfent virus.