KR102643430B1 - Vaccine delivery system using pH-responsive nanocomplex - Google Patents
Vaccine delivery system using pH-responsive nanocomplex Download PDFInfo
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- KR102643430B1 KR102643430B1 KR1020230026615A KR20230026615A KR102643430B1 KR 102643430 B1 KR102643430 B1 KR 102643430B1 KR 1020230026615 A KR1020230026615 A KR 1020230026615A KR 20230026615 A KR20230026615 A KR 20230026615A KR 102643430 B1 KR102643430 B1 KR 102643430B1
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Abstract
본 발명은 (a) 양이온성 폴리 아미노산 고분자 및 음이온성 고분자를 포함하는 나노 복합체; (b) 항원; 및 (c) 면역증강제를 포함하고, 상기 폴리 아미노산 고분자는 이미다졸기 또는 히스티딘으로 개질된, 백신 전달체에 관한 것으로, 본 발명에 따른 pH 감응형 나노 복합체를 포함하는 백신 전달체는 항원의 면역세포 전달 및 인식을 향상시켜 항체생성 및 사이토카인 발현을 증가시킬 수 있으므로, 백신의 면역원성을 향상시켜 특정 항원에 대한 예방을 가능하게 한다.The present invention provides (a) a nanocomposite containing a cationic poly amino acid polymer and an anionic polymer; (b) antigen; and (c) an immune adjuvant, wherein the poly amino acid polymer is modified with an imidazole group or histidine. The vaccine carrier comprising the pH-sensitive nanocomposite according to the present invention delivers antigen to immune cells. And recognition can be improved to increase antibody production and cytokine expression, thereby improving the immunogenicity of the vaccine and enabling prevention against specific antigens.
Description
본 발명은 pH 감응형 폴리 아미노산 고분자, 항원 및 면역증강제를 포함하는 백신 전달체, 및 상기 백신 전달체를 포함하는 백신 조성물에 관한 것이다. The present invention relates to a vaccine carrier comprising a pH-responsive poly amino acid polymer, an antigen, and an adjuvant, and a vaccine composition comprising the vaccine carrier.
최근 인플루엔자 또는 COVID-19 등 다양한 바이러스의 유행으로 인해 전세계적으로 매 해 사회적, 경제적 손실이 발생하고 있다. 이러한 팬데믹 상황에서 바이러스 예방을 위한 백신기술 개발이 매우 중요하다. Recently, social and economic losses are occurring around the world every year due to the spread of various viruses such as influenza or COVID-19. In this pandemic situation, the development of vaccine technology to prevent viruses is very important.
일반적으로 백신은 생백신, 약독화백신, 사백신, 재조합 백신이 있다. 생백신 및 약독화 백신은 바이러스의 제조 시일이 오래 걸리고 생산 단가가 높을 뿐 아니라 안정성 문제를 수반하고 있다. 그러므로 안정성이 뛰어나고 생산 효율이 좋은 재조합 백신의 연구 개발이 활발히 진행되고 있으나 재조합 백신의 경우 기존 바이러스 유래의 백신에 비해 면역원성이 낮은 단점이 있다. In general, vaccines include live vaccines, attenuated vaccines, killed vaccines, and recombinant vaccines. Live and attenuated vaccines not only take a long time to manufacture the virus and are expensive to produce, but also have stability problems. Therefore, research and development of recombinant vaccines with excellent stability and high production efficiency are actively underway, but recombinant vaccines have the disadvantage of lower immunogenicity compared to vaccines derived from existing viruses.
따라서, 면역원성 증강을 위해 면역세포에의 항원전달능이 향상된 백신전달체의 개발이 요구되는 실정이다. 이에 본 발명자들은 항원을 효과적으로 세포에 전달하고 자극하여 면역 반응을 활성화시키고, 특정 항원에 대한 예방 및 면역력을 강화시킬 수 있는 백신 전달체에 관한 발명을 완성하였다. Therefore, there is a need to develop a vaccine delivery system with improved antigen delivery ability to immune cells to enhance immunogenicity. Accordingly, the present inventors have completed the invention of a vaccine delivery system that can effectively deliver and stimulate antigens to cells, activating immune responses, and strengthening prevention and immunity against specific antigens.
본 발명의 목적은 pH 감응형 고분자를 이용하여 면역원성이 증가된 백신 전달체를 제공하는 것이다. The purpose of the present invention is to provide a vaccine delivery system with increased immunogenicity using a pH-sensitive polymer.
본 발명의 또 다른 목적은 면역원성이 증가된 백신 전달체를 포함하는 백신 조성물을 제공하는 것이다. Another object of the present invention is to provide a vaccine composition comprising a vaccine carrier with increased immunogenicity.
이에 본 발명자들은 pH 감응형 고분자에 항원 및 면역증강제를 혼합하여 복합체를 형성하는 경우, 항원 및 면역증강제의 전달능이 향상되고 항체생성 및 사이토카인 발현율이 향상됨을 확인함으로써 본 발명을 완성하였다. Accordingly, the present inventors completed the present invention by confirming that when a pH-sensitive polymer is mixed with an antigen and an adjuvant to form a complex, the delivery ability of the antigen and the adjuvant is improved and the antibody production and cytokine expression rates are improved.
따라서, 본 발명은 (a) 양이온성 폴리 아미노산 고분자 및 약제학적으로 허용가능한 음이온성 고분자를 포함하는 나노 복합체; (b) 항원; 및 (c) 면역증강제를 포함하고, 상기 폴리 아미노산 고분자는 이미다졸기 또는 히스티딘으로 개질된, 백신 전달체를 제공한다. Therefore, the present invention provides (a) a nanocomposite comprising a cationic poly amino acid polymer and a pharmaceutically acceptable anionic polymer; (b) antigen; and (c) an immune adjuvant, wherein the poly amino acid polymer is modified with an imidazole group or histidine.
또한, 본 발명은 상기 백신 전달체 및 약학적으로 허용가능한 담체를 포함하는 백신 조성물을 제공한다. Additionally, the present invention provides a vaccine composition comprising the vaccine delivery vehicle and a pharmaceutically acceptable carrier.
본 발명에 따른 pH 감응형 나노 복합체를 포함하는 백신 전달체는 항원의 면역세포 전달 및 인식을 향상시켜 항체생성 및 사이토카인 발현을 증가시킬 수 있으므로, 백신의 면역원성을 향상시켜 특정 항원에 대한 예방을 가능하게 한다. The vaccine carrier containing the pH-sensitive nanocomposite according to the present invention can increase antibody production and cytokine expression by improving immune cell delivery and recognition of antigens, thereby improving the immunogenicity of the vaccine to prevent specific antigens. Make it possible.
도 1은 양이온성 폴리 아미노산 고분자 (PLI)의 합성 과정을 나타낸 것이다.
도 2는 PLI 합성 결과를 FT-IR 분석을 통해 확인한 것이다.
도 3은 PLI 합성 결과 NMR 분석을 통해 확인한 것이다.
도 4는 PLI 고분자와 CpG-ODN 의 몰비율에 따른 나노입자의 입자크기 및 표면 전하를 확인한 것이다.
도 5는 PLI 고분자와 CpG-ODN 의 몰비율에 따른 CpG-ODN retardation을 확인한 것이다.
도 6은 HA/PLI/CpG-ODN 입자의 입자 크기를 확인한 것이다.
도 7은 HA/PLI/CpG-ODN/OVA의 제조에서 항원의 질량 및 투입 시기를 나타낸 것이다.
도 8은 다양한 HA/PLI/CpG-ODN/OVA 복합체의 입도 분석 결과를 나타낸 것이다.
도 9는 PLI 입자의 pH5.5~7.4 구간에서 proton sponge capacity를 확인한 것이다.
도 10은 OVA-FITC의 세포내 전달능을 확인한 것이다.
도 11은 CpG-ODN-FITC의 세포내 전달능을 확인한 것이다.
도 12는 각 샘플에서 사이토카인(IL-6, TNF-alpha) 발현량을 비교한 것이다.
도 13은 마우스에 각 샘플을 처리한 후 항체 생성능을 확인한 것이다. Figure 1 shows the synthesis process of cationic poly amino acid polymer (PLI).
Figure 2 shows the PLI synthesis results confirmed through FT-IR analysis.
Figure 3 shows the results of PLI synthesis confirmed through NMR analysis.
Figure 4 confirms the particle size and surface charge of nanoparticles according to the molar ratio of PLI polymer and CpG-ODN.
Figure 5 confirms CpG-ODN retardation according to the molar ratio of PLI polymer and CpG-ODN.
Figure 6 confirms the particle size of HA/PLI/CpG-ODN particles.
Figure 7 shows the mass and introduction time of antigen in the production of HA/PLI/CpG-ODN/OVA.
Figure 8 shows the particle size analysis results of various HA/PLI/CpG-ODN/OVA complexes.
Figure 9 confirms the proton sponge capacity of PLI particles in the pH range of 5.5 to 7.4.
Figure 10 confirms the intracellular delivery ability of OVA-FITC.
Figure 11 confirms the intracellular delivery ability of CpG-ODN-FITC.
Figure 12 compares the expression levels of cytokines (IL-6, TNF-alpha) in each sample.
Figure 13 shows the antibody production ability after treating each sample in mice.
이하, 본 발명을 상세하게 설명한다. Hereinafter, the present invention will be described in detail.
본 발명은 (a) 양이온성 폴리 아미노산 고분자 및 약제학적으로 허용가능한 음이온성 고분자를 포함하는 나노 복합체; (b) 항원; 및 (c) 면역증강제를 포함하고, 상기 폴리 아미노산 고분자는 이미다졸기 또는 히스티딘으로 개질된, 백신 전달체를 제공한다. The present invention provides (a) a nanocomposite comprising a cationic poly amino acid polymer and a pharmaceutically acceptable anionic polymer; (b) antigen; and (c) an immune adjuvant, wherein the poly amino acid polymer is modified with an imidazole group or histidine.
본 발명에서, 상기 양이온성 폴리 아미노산 고분자는 하기 일반식 1로 표현될 수 있다:In the present invention, the cationic poly amino acid polymer can be expressed by the following general formula 1:
[일반식 1][General Formula 1]
XmYn X m Y n
(상기 일반식 1에서, X는 리신, 히스티딘, 및 아르기닌으로 이루어진 군에서 선택된 어느 하나이고, Y는 이미다졸기 및 히스티딘으로 이루어진 군에서 선택된 어느 하나가 개질된 리신이며, m, n은 각각 1 이상의 정수이다.) (In the above general formula 1, It is an integer above.)
본 발명의 일 구현예에서, 상기 일반식 1의 X는 리신일 수 있으며, 상기 일반식 1의 Y는 이미다졸기로 개질된 리신일 수 있다. In one embodiment of the present invention, X in Formula 1 may be lysine, and Y in Formula 1 may be lysine modified with an imidazole group.
본 발명에서, 상기 m, n은 각각 1 이상 10 이하의 정수일 수 있다. 또한, 본 발명에서, 상기 일반식 1의 m와 n은 10:1 내지 1:10, 예컨대 7:1 내지 1:7, 5:1 내지 1:5, 3:1 내지 1:3, 또는 1:1 내지 1:3일 수 있으나 이에 제한되는 것은 아니다. 상기와 같이 m와 n의 비율을 조정함으로써, 상기 폴리 아미노산 고분자의 pH 감응성을 부여 및 강화할 수 있다.In the present invention, m and n may each be integers from 1 to 10. In addition, in the present invention, m and n in General Formula 1 are 10:1 to 1:10, such as 7:1 to 1:7, 5:1 to 1:5, 3:1 to 1:3, or 1 :1 to 1:3, but is not limited thereto. By adjusting the ratio of m and n as described above, pH sensitivity of the poly amino acid polymer can be imparted and strengthened.
본 발명에서, 상기 양이온성 폴리 아미노산 고분자는 음이온성 고분자와 혼합되어 나노 복합체를 형성할 수 있다. 예컨대, 상기 나노 복합체는 양이온성 고분자와 음이온성 고분자의 정전기적 인력에 의해 결합된 복합체(폴리플렉스, polyplex)일 수 있다. In the present invention, the cationic poly amino acid polymer can be mixed with an anionic polymer to form a nanocomposite. For example, the nanocomposite may be a composite (polyplex) in which cationic polymers and anionic polymers are bonded by electrostatic attraction.
본 발명에서, 상기 음이온성 고분자는 약제학적으로 허용가능하며 음전하를 띄는 고분자라면 특별히 한정되지 않는다. 구체적으로는, 카르복시메틸셀룰로오스, 덱스트란, 잔탄검, 알긴산, 카라기난, 아라비아검, 젤란검, 옥시키틴 및 옥시풀루란, 콘드로이틴, 히알루론산, 카보머, 폴리갈락투론산, 폴리L-글루탐산, 폴리아스파르트산, 폴리아크릴산 등의 수용성 고분자, 및 이들의 유도체 등을 들 수 있고, 이들 중 1종 또는 2종 이상을 사용할 수 있다. 또한, 이들은 천연이나 합성 등의 기원에 의해서 특별히 한정되지 않는다. 예를 들어, 상기 음이온성 고분자는 히알루론산일 수 있다. In the present invention, the anionic polymer is not particularly limited as long as it is a pharmaceutically acceptable polymer and has a negative charge. Specifically, carboxymethyl cellulose, dextran, xanthan gum, alginic acid, carrageenan, gum arabic, gellan gum, oxykitin and oxypullulan, chondroitin, hyaluronic acid, carbomer, polygalacturonic acid, poly L-glutamic acid, poly Examples include water-soluble polymers such as aspartic acid and polyacrylic acid, and derivatives thereof, and one or two or more types of these may be used. Additionally, they are not particularly limited by origin, such as natural or synthetic. For example, the anionic polymer may be hyaluronic acid.
본 발명에서, 용어 "항원"은 살아있는 동물의 면역계가 그것을 인식하도록 하기 위해 면역 반응을 유도하고자 하는 분자 또는 분자의 조합을 지칭한다. In the present invention, the term “antigen” refers to a molecule or combination of molecules intended to induce an immune response so that the immune system of a living animal recognizes it.
본 발명에서, 상기 항원은 펩타이드, 단백질, 핵산, 당(sugar), 병원균, 약독화된 병원균, 비활성화된 병원균, 바이러스, 바이러스-유사 입자(VLP), 세포 및 세포 조각으로 구성된 군으로부터 선택되는 것일 수 있다. 상기 단백질은, 예컨대 재조합 단백질, 서브유닛(subunit), 스플릿(split) 단백질 항원을 포함할 수 있으며, 상기 세포는, 예컨대 수지상세포를 포함할 수 있다.In the present invention, the antigen is selected from the group consisting of peptides, proteins, nucleic acids, sugars, pathogens, attenuated pathogens, inactivated pathogens, viruses, virus-like particles (VLPs), cells and cell fragments. You can. The protein may include, for example, a recombinant protein, a subunit, or a split protein antigen, and the cell may include, for example, dendritic cells.
본 발명에서, 상기 항원은 코로나바이러스(Coronavirus)의 항원, 일본뇌염바이러스(Japanese encephalitis virus)의 항원, HIB(Heamophilus influenzae type B)의 항원, 지카바이러스의 항원, 녹농균(Pseudomonas aeruginosa)의 항원, 백일해 항원, 결핵균 항원, 탄저균 항원, HAV(Hepatitis A virus)의 항원, HBV(Hepatitis B virus)의 항원, HCV(Hepatitis C virus)의 항원, HIV(human immunodeficiency virus)의 항원, HSV(Herpes simplex virus)의 항원, 나이세리아 메닝기티디스(Neisseria meningitidis)의 항원, 코리네박테리움 디프테리아(Corynebacterium diphtheria)의 항원, 보르데텔라 퍼투시스(Bordetella pertussis)의 항원, 클로스트리듐 테타니(Clostridium tetani)의 항원, HPV(human papilloma virus)의 항원, 바리셀라 바이러스(Varicella virus), 엔테로코키(Enterococci)의 항원, 포도상구균(Staphylococcus aureus)의 항원, 폐렴간균(Klebsiella pneumonia)의 항원, 아시네토박터바우마니(Acinetobacter baumannii)의 항원, 엔테로박터(Enterobacter)의 항원, 헬리코박터 파일로리(Helicobacter pylori)의 항원, 말라리아의 항원, 뎅기바이러스의 항원, 쯔쯔가무시(Orientia tsutsugamushi)의 항원, 중증열성혈소판감소증후군(severe fever with thrombocytopenia syndrome Bunyavirus; SFTS Bunyavirus)의 항원, 인플루엔자 바이러스의 항원, 에볼라바이러스의 항원 및 폐렴구균의 항원으로 구성된 군으로부터 선택되는 것일 수 있다.In the present invention, the antigen is a coronavirus antigen, a Japanese encephalitis virus antigen, a HIB (Heamophilus influenzae type B) antigen, a Zika virus antigen, a Pseudomonas aeruginosa antigen, and pertussis. Antigen, Mycobacterium tuberculosis antigen, Anthrax antigen, HAV (Hepatitis A virus) antigen, HBV (Hepatitis B virus) antigen, HCV (Hepatitis C virus) antigen, HIV (human immunodeficiency virus) antigen, HSV (Herpes simplex virus) Antigen of Neisseria meningitidis, Antigen of Corynebacterium diphtheria, Antigen of Bordetella pertussis, Antigen of Clostridium tetani, Antigen of HPV (human papilloma virus), Varicella virus, Enterococci antigen, Staphylococcus aureus antigen, Klebsiella pneumonia antigen, Acinetobacter baumannii baumannii antigen, Enterobacter antigen, Helicobacter pylori antigen, malaria antigen, dengue virus antigen, Orientia tsutsugamushi antigen, severe fever with thrombocytopenia syndrome It may be selected from the group consisting of Bunyavirus (SFTS Bunyavirus) antigen, influenza virus antigen, Ebola virus antigen, and pneumococcal antigen.
본 명세서에서의 용어 "코로나바이러스(Coronavirus)"는 코로나바이러스 과(Family Coronaviridae)에 속하는 바이러스로, 외막에 둘러싸인 RNA 게놈을 가진 바이러스를 말한다. 상기 코로나바이러스는 표면 돌기(spike) 단백질의 왕관과 같은 모양을 본떠 붙여진 이름으로, 양성 가닥(positive-strand) RNA를 유전체로 가진다. 이 RNA는 바이러스의 구조를 암호화하는 뉴클레오캡시드(nucleocapsid: N), 외피(envelope: E), 막(membrane: M), 표면 돌기(spike: S) 단백질 등의 정보를 포함하고 있다. 코로나바이러스는 표면 돌기(S) 단백질의 수용체 접합 부위(receptor binding domain: RBD)가 숙주 세포의 수용체와 결합해 융합하면서 숙주 내부로 침투하여 자기 복제를 수행한다.As used herein, the term "Coronavirus" refers to a virus belonging to the Family Coronaviridae and has an RNA genome surrounded by an outer membrane. The coronavirus is named after the crown-like shape of the surface spike protein, and has positive-strand RNA as its genome. This RNA contains information such as nucleocapsid (N), envelope (E), membrane (M), and surface spike (S) proteins that encode the structure of the virus. The coronavirus penetrates into the host and replicates by fusing with the receptor binding domain (RBD) of the surface protrusion (S) protein and the host cell receptor.
상기 코로나바이러스는 알파코로나바이러스, 베타코로나바이러스, 감마코로나바이러스, 및 델타코로나바이러스로 이루어진 군으로부터 선택될 수 있으며, 구체적으로 돼지 유행성 설사 바이러스(porcine epidemic diarrhea virus: PEDV), 개 코로나바이러스(canine coronavirus: CCV), 고양이 전염성 복막염 바이러스(feline infectious peritonitis virus: FIPV), 소 코로나바이러스(bovine coronavirus: BCoV 또는 BCV), 조류 전염성 기관지염 바이러스(avian infectious bronchitis virus: IBV), 전염성 위장염 코로나바이러스(transmissible gastroenteritis coronavirus: TGEV), 중증급성호흡기증후군 코로나바이러스(severe acute respiratory syndrome coronavirus: SARS-CoV), 중증급성호흡기증후군 코로나바이러스(Severe acute respiratory syndrome coronavirus 2: SARS-CoV-2), 중동 호흡기 증후군 코로나바이러스(Middle East respiratory syndrome coronavirus: MERS-CoV), 또는 이들의 조합일 수 있다. SARS-CoV-2는 코로나바이러스감염증-2019(COVID-19)의 주요 원인체일 수 있다.The coronavirus may be selected from the group consisting of alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus, and specifically, porcine epidemic diarrhea virus (PEDV) and canine coronavirus. : CCV), feline infectious peritonitis virus (FIPV), bovine coronavirus (BCoV or BCV), avian infectious bronchitis virus (IBV), transmissible gastroenteritis coronavirus : TGEV), severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (Middle) East respiratory syndrome coronavirus (MERS-CoV), or a combination thereof. SARS-CoV-2 may be the main causative agent of coronavirus disease 2019 (COVID-19).
본 발명에서, "세포내 섭취(endocytosis)"는 세포가 외부 환경의 분자와 입자를 세포질로 내재화하는 세포 과정으로, 외부 유입 물질을 제거하기 위한 대사과정이다. 항원이 세포내 섭취 과정에서 내재화되면 면역 반응을 자극하기 전에 리소좀에서 분해될 수 있으며, 이는 백신의 효능 감소로 이어질 수 있다. 본 발명의 백신 전달체에서, 상기 항원은 고분자 내부에 봉입되어 있는 형태일 수 있다. 이러한 형태로 투입될 경우, 고분자에 의해 고분자의 양성자화(protonation)에 의해 항원이 보호되므로 항원의 제거를 회피할 수 있다. 따라서 본 발명의 백신 전달체를 활용할 경우, 항원 전달률이 극대화될 수 있다는 이점이 있다. In the present invention, “endocytosis” is a cellular process by which cells internalize molecules and particles from the external environment into the cytoplasm, and is a metabolic process to remove external substances. If antigens are internalized during endocytosis, they may be degraded in lysosomes before stimulating an immune response, which may lead to reduced vaccine efficacy. In the vaccine delivery vehicle of the present invention, the antigen may be encapsulated inside a polymer. When administered in this form, removal of the antigen can be avoided because the antigen is protected by protonation of the polymer. Therefore, when using the vaccine delivery vehicle of the present invention, there is an advantage that the antigen delivery rate can be maximized.
본 발명에서, 용어 "면역증강제(adjuvant)"는 면역보조제, 항원보강제 또는 아쥬번트(adjuvant)로 불리는 물질을 의미하며, 보다 구체적으로 백신에 대한 면역 반응을 높여 면역 시스템에 의해 항체가 많이 생성될 수 있도록 도와주는 물질을 의미한다. 상기 면역증강제는 항원의 생물학적 또는 면역학적 반감기 증가; 항원 제공 서열로의 항원 전달 개선; 항원 제공 세포에 의한 항원 프로세싱 및 제공의 개선; 및 면역조절성 사이토카인의 생산 유도를 포함하는 하나 이상의 기작과 같은 여러 기작을 통해 작용할 수 있다.In the present invention, the term "immune adjuvant" refers to a substance called an adjuvant, adjuvant, or adjuvant. More specifically, it increases the immune response to the vaccine, causing the immune system to produce more antibodies. It means a substance that helps. The adjuvant increases the biological or immunological half-life of the antigen; improving antigen delivery to antigen presenting sequences; improving antigen processing and presentation by antigen presenting cells; and one or more mechanisms including inducing the production of immunomodulatory cytokines.
본 발명의 일 구현예에서, 상기 면역증강제는 Triacylated lipoproteins (Pam3CSK4), dsRNA Polyinosinic:polycytidylic acid (poly(I:C)), Lipopolysaccharides (LPS), Flagellin, Imidazoquinolines (R848), Diacylated lipoproteins (FSL-1), Guanosine analogs (Loxoribine), CpG-ODN(cytosine-guanosine oligonucleotide), profilin-like protein 및 이들의 조합으로 이루어진 군으로부터 선택되는 것일 수 있다. In one embodiment of the present invention, the immune enhancer is Triacylated lipoproteins (Pam3CSK4), dsRNA Polyinosinic:polycytidylic acid (poly(I:C)), Lipopolysaccharides (LPS), Flagellin, Imidazoquinolines (R848), Diacylated lipoproteins (FSL-1) ), Guanosine analogs (Loxoribine), CpG-ODN (cytosine-guanosine oligonucleotide), profilin-like protein, and combinations thereof.
본 발명의 일 구현예에서, 상기 양이온성 폴리 아미노산 고분자와 상기 음이온성 고분자의 혼합비는 질량비율 10:1 내지 1:10, 예컨대 7:1 내지 1:7, 5:1 내지 1:5, 3:1 내지 1:3, 또는 1:1 내지 1:3일 수 있으나 이에 제한되는 것은 아니다. In one embodiment of the present invention, the mixing ratio of the cationic poly amino acid polymer and the anionic polymer is a mass ratio of 10:1 to 1:10, such as 7:1 to 1:7, 5:1 to 1:5, 3. :1 to 1:3, or 1:1 to 1:3, but is not limited thereto.
본 발명의 백신 전달체는 양이온성 폴리 아미노산 고분자 및 음이온성 고분자가 혼합된 나노 복합체에 항원 및 면역증강제를 탑재하여 구성되는 것일 수 있다. 본 발명의 일 구현예에서, 상기 폴리 아미노산 고분자 및 면역증강제의 혼합비는 질량비율 1:1 내지 100:1일 수 있다. 바람직하게는 상기 혼합비는 질량비율 1:1 내지 100:1, 1:1 내지 90:1, 1:1 내지 80:1, 1:1 내지 70:1, 1:1 내지 60:1, 1:1 내지 50:1, 1:1 내지 40:1, 10:1 내지 40:1, 20:1 내지 40:1, 또는 20:1 내지 30:1일 수 있으나, 이에 제한되는 것은 아니다.. The vaccine delivery vehicle of the present invention may be constructed by loading an antigen and an adjuvant into a nanocomposite of a cationic poly amino acid polymer and an anionic polymer. In one embodiment of the present invention, the mixing ratio of the poly amino acid polymer and the immune enhancer may be 1:1 to 100:1 by mass. Preferably, the mixing ratio is 1:1 to 100:1, 1:1 to 90:1, 1:1 to 80:1, 1:1 to 70:1, 1:1 to 60:1, 1:1 by mass ratio. It may be 1 to 50:1, 1:1 to 40:1, 10:1 to 40:1, 20:1 to 40:1, or 20:1 to 30:1, but is not limited thereto.
본 발명에서, "엔도좀"은 세포내 섭취(endocytosis)를 통해서 세포질에 형성된 생체막으로 둘러싸인 소낭을 말하며, 좀 더 넓은 의미에서는 이렇게 들어온 소낭이 원형질막과 골지체 사이를 이동하며, 융합하고 분리되는 막으로 싸인 낭을 총칭한다. 상기 엔도좀은 세포 내에서 세포소기관들 사이 또는 세포내부와 세포 외부와의 물질의 이동에서 중요한 역할을 한다. In the present invention, "endosome" refers to a vesicle surrounded by a biological membrane formed in the cytoplasm through endocytosis. In a broader sense, such an entered vesicle moves between the plasma membrane and the Golgi apparatus, and is a membrane that fuses and separates. This is the general term for the encircled sac. The endosome plays an important role in the movement of substances between organelles within a cell or between the inside and outside of the cell.
사람 혈청의 pH는 7.4인 반면, 세포내 평균 pH는 약 7.1 내지 7.3의 범위로, 약간 더 낮다. 상기 엔도좀은 일반적으로 5 이상 7 이하, 또는 5.5 이상 6.5 이하의 pH를 가지므로, 본 발명의 pH 감응형 고분자는 세포 내 엔도좀 환경 (약산성 pH 조건)에서 특이적으로 이온화될 수 있다. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Since the endosome generally has a pH of 5 or more and 7 or less, or 5.5 or more and 6.5 or less, the pH-sensitive polymer of the present invention can be specifically ionized in the intracellular endosomal environment (weakly acidic pH conditions).
따라서, 본 발명의 일 구현예에서 상기 나노 복합체를 포함하는 백신 전달체는 pH 5 이상 7 이하, 바람직하게는 pH 5.5 이상 7 이하 또는 5.5이상 6.5 이하의 조건에서 이온화되는 것일 수 있다. Therefore, in one embodiment of the present invention, the vaccine carrier containing the nanocomposite may be ionized under conditions of pH 5 or more and 7 or less, preferably pH 5.5 or more and 7 or less, or 5.5 or more and 6.5 or less.
본 발명의 실시예에 따르면, 상기 나노 복합체를 포함하는 백신 전달체는 상기 나노 복합체를 포함하지 않는 백신 전달체에 비해 항원전달능이 50배 이상, 40배 이상, 30배 이상, 또는 20배 이상 증가될 수 있다. According to an embodiment of the present invention, the vaccine delivery vehicle containing the nanocomplex may have an antigen delivery ability of 50-fold or more, 40-fold or more, 30-fold or more, or 20-fold or more compared to the vaccine delivery vehicle that does not contain the nanocomplex. there is.
본 발명의 일 구현예에서, 상기 백신 전달체는 50nm 이상 500nm 이하의 직경을 갖는 것일 수 있다. 바람직하게는 50nm 이상 450nm 이하, 60nm 이상 400nm 이하, 70nm 이상 350nm 이하, 80nm 이상 300nm 이하, 90nm 이상 300nm 이하, 100nm 이상 250nm 이하의 직경을 갖는 것일 수 있으나, 이에 제한되는 것은 아니다. In one embodiment of the present invention, the vaccine delivery vehicle may have a diameter of 50 nm or more and 500 nm or less. Preferably, it may have a diameter of 50 nm to 450 nm, 60 nm to 400 nm, 70 nm to 350 nm, 80 nm to 300 nm, 90 nm to 300 nm, and 100 nm to 250 nm, but is not limited thereto.
본 발명은 백신 전달체 및 약학적으로 허용 가능한 담체를 포함하는 백신 조성물을 제공한다. The present invention provides a vaccine composition comprising a vaccine carrier and a pharmaceutically acceptable carrier.
상기에서 백신 전달체와 관련하여 기술한 모든 내용이 백신 조성물에 그대로 적용 또는 준용될 수 있다. All of the information described above regarding the vaccine delivery system can be directly applied or applied to the vaccine composition.
본 발명에서, 용어 "백신"은 생체에 면역을 주는 항원을 함유한 생물학적인 제제로서, 감염 및/또는 감염증의 예방을 위하여 사람이나 동물에 투여함으로써 생체에 면역이 생기게 하는 면역원 또는 항원성 물질을 말한다.In the present invention, the term "vaccine" refers to a biological agent containing an antigen that provides immunity to a living body, and is an immunogen or antigenic substance that creates immunity in a living body by administering it to a person or animal to prevent infection and/or infectious diseases. says
상기 백신은 사균백신, 약독화백신, 아단위 백신, 컨쥬게이트 백신, 재조합 백신, 단가백신, 다가백신 또는 혼합백신 형태일 수 있다.The vaccine may be in the form of a killed vaccine, attenuated vaccine, subunit vaccine, conjugate vaccine, recombinant vaccine, monovalent vaccine, multivalent vaccine, or mixed vaccine.
본 발명에서, 상기 백신은 세균 및/또는 바이러스의 감염 또는 감염 질환을 예방 또는 치료하기 위한 것일 수 있다.In the present invention, the vaccine may be for preventing or treating bacterial and/or viral infection or infectious disease.
본 발명에서, 용어 "예방"은 상기 백신 조성물의 투여로 인해 세균 및/또는 바이러스의 감염 및 상기 감염에 의한 질환 발병을 억제 또는 지연시키는 모든 행위를 의미한다.In the present invention, the term “prevention” refers to all actions that inhibit or delay bacterial and/or viral infection and the onset of disease caused by the infection by administering the vaccine composition.
본 발명에서, 용어 "치료"는 상기 백신 조성물의 투여로 인해 세균 및/또는 바이러스의 감염에 의해 이미 유발된 질환의 증세가 호전되거나 이롭게 되는 모든 행위를 의미한다.In the present invention, the term “treatment” refers to any action in which the symptoms of a disease already caused by bacterial and/or viral infection are improved or beneficial due to the administration of the vaccine composition.
"약학적으로 허용가능한 담체"란 임의의 및 모든 용매, 분산 매질, 코팅제, 항원보강제, 안정제, 희석제, 보존제, 항균제 및 항진균제, 등장성 작용제, 흡착지연제 등을 포함한다. 백신용 조성물에 포함될 수 있는 담체, 부형제, 희석제로는 락토즈, 덱스트로스, 슈크로스, 솔비톨, 만니톨, 자일리톨, 말티톨, 전분, 글리세린, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘포스페이트, 칼슘실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다.“Pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coating agents, adjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delay agents, etc. Carriers, excipients, and diluents that may be included in the vaccine composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
또한, 본 발명의 백신 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 제제화할 경우에는 보통 사용되는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제할 수 있다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 레시틴 유사 유화제에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 슈크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제할 수 있다. 또한 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용할 수 있다. 경구투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등을 사용할 수 있으며, 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비 경구투여를 위한 제제에는 멸균된 수용액, 비수용성제, 현탁제, 유제, 동결건조제제가 포함된다. 비수용성제제, 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다.In addition, the vaccine composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., and sterile injectable solutions according to conventional methods. When formulating, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain the lecithin-like emulsifier plus at least one excipient, such as starch, calcium carbonate, or sucrose. It can be prepared by mixing sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be used. may be included. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous preparations, suspensions, emulsions, and freeze-dried preparations. Non-aqueous preparations and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.
본 발명의 투여는 어떠한 적절한 방법으로 대상에게 소정의 물질을 도입하는 것을 의미하며, 투여 경로는 목적 조직에 도달할 수 있는 한 일반적인 경로를 통하여 투여될 수 있다. 예를 들어, 복강내 투여, 근육내 투여, 경구 투여, 피하 투여, 국소 투여 또는 침지 투여될 수 있으나, 이에 제한되지 않는다.Administration of the present invention means introducing a predetermined substance into a subject by any appropriate method, and the administration route may be through a general route as long as it can reach the target tissue. For example, it may be administered intraperitoneally, intramuscularly, orally, subcutaneously, topically, or by submersion, but is not limited thereto.
상기 백신 조성물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 그러나 바람직한 효과를 위해서, 본 발명의 면역증강제는 1일에 10 내지 100 mg/kg으로, 바람직하게는 50 내지 500 mg/kg으로 투여할 수 있다. 투여는 하루에 한 번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다. 상기 투여량은 개체의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출비율, 치료기간, 동시에 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다.The preferred dosage of the vaccine composition varies depending on the patient's condition and weight, degree of disease, drug type, administration route and period, but can be appropriately selected by a person skilled in the art. However, for desirable effects, the immune enhancer of the present invention can be administered at 10 to 100 mg/kg, preferably 50 to 500 mg/kg per day. Administration may be administered once a day, or may be administered several times. The above dosage does not limit the scope of the present invention in any way. The dosage depends on factors including the type and severity of the individual's disease, activity of the drug, sensitivity to the drug, administration time, administration route and excretion rate, treatment period, drugs used simultaneously, and other factors well known in the medical field. can be decided.
또한, 본 발명은 상기 백신 조성물을 개체에 투여하는 단계를 포함하는, 세균, 바이러스 및/또는 미생물의 감염 및 감염 질환을 예방 또는 치료하는 방법을 제공한다. 상기에서 백신 전달체 및 백신 조성물과 관련하여 기술한 모든 내용이 상기 방법에 그대로 적용 또는 준용될 수 있다.Additionally, the present invention provides a method for preventing or treating bacterial, viral and/or microbial infections and infectious diseases, comprising administering the vaccine composition to an individual. All of the information described above regarding the vaccine delivery system and vaccine composition may be directly applied or applied to the method.
본 발명에서, 용어 "개체"는 세균, 바이러스 및/또는 미생물의 감염 및 감염 질환이 발병되거나 발병할 위험이 있는 쥐, 가축, 인간 등을 포함하는 포유동물, 양식어류 등을 제한 없이 포함할 수 있다. 상기 개체는 인간을 제외하는 것일 수 있다.In the present invention, the term "individual" may include, without limitation, mammals, including mice, livestock, humans, etc., that are infected or at risk of developing infectious diseases and infections by bacteria, viruses, and/or microorganisms, and farmed fish. there is. The entity may be excluding humans.
상기 백신 조성물은 약학적으로 유효한 양으로 단일 또는 다중 투여될 수 있다. 이때, 조성물은 액제, 산제, 에어로졸, 주사제, 수액제(링겔), 캡슐제, 환제, 정제, 좌제 또는 패치의 형태로 제형화되어 투여할 수 있다.The vaccine composition can be administered single or multiple times in a pharmaceutically effective amount. At this time, the composition can be formulated and administered in the form of a solution, powder, aerosol, injection, infusion solution (injection), capsule, pill, tablet, suppository, or patch.
상기 세균, 바이러스 및/또는 미생물의 감염 및 감염 질환을 예방 또는 치료하기 위한 백신 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여도 투여될 수 있다.The route of administration of the vaccine composition for preventing or treating infection and infectious diseases by bacteria, viruses and/or microorganisms may be administered through any general route as long as it can reach the target tissue.
상기 백신 조성물은 목적하는 바에 따라 복강내 투여, 정맥내 투여, 근육내 투여, 피하 투여, 피내 투여, 경피패치투여, 경구 투여, 비내 투여, 폐내 투여, 직장내 투여 등의 경로를 통해 투여 될 수 있다. 다만, 경구 투여 시에는 제형화되지 않은 형태로도 투여할 수 있고, 위산에 의하여 상기 백신 조성물이 변성 또는 분해될 수 있기 때문에 경구용 조성물은 활성 약제를 코팅하거나 위에서의 분해로부터 보호되도록 제형화된 형태 또는 경구용 패치형태로 구강내에 투여할 수도 있다. 또한, 상기 조성물은 활성 물질이 표적세포로 이동할 수 있는 임의의 장치에 의해 투여될 수 있다.The vaccine composition can be administered through routes such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, intranasal administration, intrapulmonary administration, and intrarectal administration, depending on the purpose. there is. However, during oral administration, it can be administered in an unformulated form, and since the vaccine composition may be denatured or decomposed by stomach acid, the oral composition is formulated to coat the active agent or protect it from decomposition in the stomach. It can also be administered intraorally in the form of a form or oral patch. Additionally, the composition can be administered by any device that allows the active substance to move to target cells.
본 발명의 이점 및 특징, 그리고 그것들을 달성하는 방법은 상세하게 후술되어있는 실시예들을 참조하면 명확해질 것이다. 그러나 본 발명은 이하에서 개시되는 실시예들에 한정되는 것이 아니라 서로 다른 다양한 형태로 구현될 것이며, 단지 본 실시예들은 본 발명의 개시가 완전하도록 하고, 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 발명의 범주를 완전하게 알려주기 위해 제공되는 것이며, 본 발명은 청구항의 범주에 의해 정의될 뿐이다.The advantages and features of the present invention and methods for achieving them will become clear with reference to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below and will be implemented in various different forms. The present embodiments are merely provided to ensure that the disclosure of the present invention is complete and to provide common knowledge in the technical field to which the present invention pertains. It is provided to fully inform those who have the scope of the invention, and the present invention is only defined by the scope of the claims.
[실시예] [Example]
실시예 1: PLI의 합성 및 분석Example 1: Synthesis and analysis of PLI
1) 이미다졸 개질 양이온성 폴리 아미노산 고분자 (PLI) 합성1) Synthesis of imidazole-modified cationic polyamino acid polymer (PLI)
이미다졸 그룹이 개질된 양이온성 폴리 아미노산 고분자 (PLI) 중합을 위해, H-Lys(Z)-OH와 트리포스젠(Triphosgene)과의 반응을 통해 Lys(Z)-NCA를 합성하였다. 주어진 물질들을 50ml의 THF(tetrahydrofuran)에 녹여 혼합한 후, 3시간 동안 50℃에서 반응을 진행하였다. 반응 종결 후 용액을 헥산(hexane)에 침전시켜 감압 증류 및 건조를 통해 Lys(Z)-NCA를 분리하였다. 합성된 물질은 35ml DMF(dimethylformamide) 상에서 헥실아민(Hexylamine)과 혼합되어 40℃, 48시간 동안 중합반응을 진행하였으며, 에틸 에테르(ethyl ether)를 사용한 침전 및 감압 증류로 Poly-L-lysine(Z) (PLL(Z))를 분리하였다. 이후 HBr/acetic acid 용액을 처리하여 Cbz 그룹을 탈그룹화 하여 아민기를 노출시킨 형태인 PLL을 제조하였고, 수용액 기반에서 EDC/HOBt 법을 이용하여 PLL에 4-이미다졸아세트산(4-imidazoleacetic acid)을 결합하였다. 결합 후 혼합용액에 대한 투석(dialysis)을 진행하여 여분의 용매와 시료들을 제거하였으며, 동결 건조를 통해 최종적인 고분자 (PLI)를 수득하였다. PLI의 합성 과정을 도 1에 나타내었다.For polymerization of imidazole group-modified cationic poly amino acid polymer (PLI), Lys(Z)-NCA was synthesized through reaction of H-Lys(Z)-OH with Triphosgene. The given materials were dissolved and mixed in 50 ml of THF (tetrahydrofuran), and then the reaction was carried out at 50°C for 3 hours. After completion of the reaction, the solution was precipitated in hexane and Lys(Z)-NCA was separated through reduced pressure distillation and drying. The synthesized material was mixed with hexylamine in 35ml DMF (dimethylformamide) and polymerized at 40°C for 48 hours. Poly-L-lysine (Z) was obtained through precipitation using ethyl ether and distillation under reduced pressure. ) (PLL(Z)) was isolated. Afterwards, PLL in the form of an amine group was prepared by treating the HBr/acetic acid solution to degroup the Cbz group, and 4-imidazoleacetic acid was added to PLL using the EDC/HOBt method in an aqueous solution. combined. After combining, the mixed solution was subjected to dialysis to remove excess solvent and samples, and the final polymer (PLI) was obtained through freeze-drying. The synthesis process of PLI is shown in Figure 1.
2) FT-IR 분석2) FT-IR analysis
PLI 합성 결과를 푸리에변환적외선분광기(Fourier Transform Infrared Spectroscopy, FT-IR) 분석을 통해 확인하여 도 2에 나타내었다. H-Lys(Z)-OH를 트리포스젠(Triphosgene)과 반응시켜 Lys(z)-NCA 리신 무수물을 합성하였으며, FT-IR 분석을 통해 1800nm-1에 존재하는 NCA 피크와 1200nm-1에 존재하는 Cbz 피크를 확인하여 적절하게 합성된 것을 확인하였다. 합성된 Lys(z)-NCA는 헥실아민(Hexylamine)을 이용해 중합하여 양이온성 폴리 아미노산 고분자(PLL(z), Poly-l-lysine(z))를 합성하였고, 이는 FT-IR 데이터에서 NCA 피크가 사라진 것을 통해 중합의 성공을 확인하였다. 이후 HBr/acetic acid 용액을 이용하여 아민기를 보호하고 있는 Cbz 보호그룹을 제거하고, FT-IR 데이터에서 Cbz 피크가 사라진 것을 통해 폴리 리신(Poly-l-lysine)의 합성을 확인하였다. The PLI synthesis results were confirmed through Fourier Transform Infrared Spectroscopy (FT-IR) analysis and are shown in Figure 2. Lys(z)-NCA lysine anhydride was synthesized by reacting H-Lys(Z)-OH with Triphosgene, and through FT-IR analysis, NCA peak existed at 1800nm -1 and 1200nm -1 It was confirmed that it was properly synthesized by checking the Cbz peak. The synthesized Lys(z)-NCA was polymerized using hexylamine to synthesize cationic poly amino acid polymers (PLL(z), Poly-l-lysine(z)), which showed the NCA peak in FT-IR data. The success of polymerization was confirmed by the disappearance of . Afterwards, the Cbz protecting group protecting the amine group was removed using an HBr/acetic acid solution, and the disappearance of the Cbz peak in FT-IR data confirmed the synthesis of poly-l-lysine.
3) NMR 분석3) NMR analysis
보다 정확한 확인을 위해, PLI 합성 결과를 핵자기공명(Nuclear Magnetic Resonance, NMR) 분석을 통해 확인하여 도 3에 나타내었다. H-Lys(Z)-OH를 트리포스젠(Triphosgene)과 반응시켜 Lys(z)-NCA 리신 무수물을 합성하였으며 NMR 분석을 통해 NCA와 Cbz의 피크를 확인하여 적절히 합성된 것을 확인하였다. 합성된 Lys(z)-NCA 리신 무수물은 헥실아민(Hexylamine)을 이용해 중합하여 양이온성 폴리 아미노산 고분자(PLL(z), Poly-l-lysine(z))를 합성하였고, 이는 NMR 데이터에서 NCA 피크가 사라진 것을 통해 중합의 성공을 확인하였다. 이후 HBr/acetic acid 용액을 이용하여 아민기를 보호하고 있는 Cbz 보호그룹을 제거하고, NMR 분석 데이터에서 Cbz 피크가 약해지고 사라진 것을 통해 양이온성 아미노산 고분자인 폴리 리신(Poly-l-lysine)의 합성을 확인하였다. 또한, EDC/HOBt 법을 활용하여 4-이미다졸아세트산(4-imidazoleacetic acid)을 폴리 리신에 결합시켜 폴리 리신의 아민 기가 이미다졸 기로 개질될 수 있도록 하였다. 이의 결과로 NMR 분석 데이터에서 이미다졸 기의 피크가 생긴 것을 통해 중합되었음을 확인하였다.For more accurate confirmation, the PLI synthesis results were confirmed through nuclear magnetic resonance (NMR) analysis and are shown in Figure 3. Lys(z)-NCA lysine anhydride was synthesized by reacting H-Lys(Z)-OH with Triphosgene, and the peaks of NCA and Cbz were confirmed through NMR analysis to confirm that it was properly synthesized. The synthesized Lys(z)-NCA lysine anhydride was polymerized using hexylamine to synthesize a cationic poly amino acid polymer (PLL(z), Poly-l-lysine(z)), which showed the NCA peak in NMR data. The success of polymerization was confirmed by the disappearance of . Afterwards, the Cbz protecting group protecting the amine group was removed using HBr/acetic acid solution, and the Cbz peak weakened and disappeared in the NMR analysis data, confirming the synthesis of poly-l-lysine, a cationic amino acid polymer. did. In addition, using the EDC/HOBt method, 4-imidazoleacetic acid was bound to polylysine so that the amine group of polylysine could be modified with an imidazole group. As a result, it was confirmed that polymerization occurred through the appearance of an imidazole group peak in the NMR analysis data.
실시예 2: PLI-HA-면역증강제 복합체의 제조Example 2: Preparation of PLI-HA-immune adjuvant complex
1) PLI:CpG-ODN 몰비율에 따른 나노입자 분석1) Nanoparticle analysis according to PLI:CpG-ODN molar ratio
PLI-면역증강제 복합체의 제조를 위하여, 이미다졸이 개질된 양이온성 폴리 아미노산 고분자 (PLI) 및 톨-유사 수용체 9(Toll-like receptor 9, TLR-9)에 작용하는 면역증강제인 CpG-ODN을 사용하였다. 1ml HEPES 버퍼(10mM, pH 7.4)에 PLI와 CpG-ODN의 몰 비율이 정해진 비율 (1-10)이 되도록 투입하였다. 혼합한 용액은 교반기를 사용하여 30분 간 강도 3으로 교반하였다. 입자가 제조된 후에는 ELSZ-2000(오츠카전자)을 사용하여 입도 분석 및 제타 전위 측정을 진행하여 도 4에 나타내었다. 면역증강제에 대한 고분자의 몰 비율이 2.5 이상인 경우부터 300nm 이하의 크기를 가지는 균일하고 안정적인 입자가 제조되었음을 확인하였고, 입자의 제타전위는 양이온성 고분자 비율이 상승함에 따라 증가하는 것을 확인하였다.To prepare the PLI-immune adjuvant complex, imidazole-modified cationic polyamino acid polymer (PLI) and CpG-ODN, an adjuvant that acts on Toll-like receptor 9 (TLR-9), were used. used. The molar ratio of PLI and CpG-ODN was added to 1ml HEPES buffer (10mM, pH 7.4) at a set ratio (1-10). The mixed solution was stirred at intensity 3 for 30 minutes using a stirrer. After the particles were manufactured, particle size analysis and zeta potential measurement were performed using ELSZ-2000 (Otsuka Electronics), as shown in Figure 4. It was confirmed that uniform and stable particles with a size of 300 nm or less were produced when the molar ratio of polymer to immune enhancer was 2.5 or more, and the zeta potential of the particles was confirmed to increase as the cationic polymer ratio increased.
2) Gel retardation assay2) Gel retardation assay
제조한 PLI/CpG-ODN의 몰 비율에 따른 DNA 탈출능을 평가하기 위해 gel retardation assay를 진행하였다. 1% Agarose gel에 각기 다른 몰 비율의 입자를 처리하고 100V 하에서 20분 간 전기 영동을 진행하였다. 이후 CYBR Gold 용액을 15분간 처리하여 DNA를 staining하고 Typhoon™ laser-scanner platform을 통해 분석하고 도 5에 나타내었다. 면역증강제에 대한 고분자의 몰 비율이 0.5 이상인 경우부터 면역증강제의 방출이 관찰되지 않았으며, 이를 통해 고분자와 면역증강제의 정전기적 인력에 의하여 안정적으로 입자가 형성된다는 것을 확인하였다.A gel retardation assay was performed to evaluate the DNA escape ability according to the molar ratio of the prepared PLI/CpG-ODN. Particles of different molar ratios were treated on 1% agarose gel and electrophoresis was performed at 100V for 20 minutes. Afterwards, DNA was stained by treating it with CYBR Gold solution for 15 minutes and analyzed using Typhoon™ laser-scanner platform, as shown in Figure 5. When the molar ratio of the polymer to the adjuvant was 0.5 or more, release of the adjuvant was not observed, confirming that particles were formed stably due to the electrostatic attraction between the polymer and the adjuvant.
3) HA/PLI/CpG-ODN 입자 합성3) HA/PLI/CpG-ODN particle synthesis
1ml HEPES 버퍼(10mM, pH 7.4)에 PLI와 CpG-ODN의 몰 비율이 10이 되도록 투입하였다. 이와 동시에, 히알루론산의 질량이 PLI의 3배가 되도록 투입하고 교반기를 사용하여 30분 간 강도 3으로 교반하였다. 입자가 제조된 후에는 ELSZ-2000(오츠카전자)를 사용하여 입도 분석하여 도 6에 나타내었다. 히알루론산이 포함된 나노구조체 또한 균일하고 안정한 상태에 있음을 확인하였다.1ml HEPES buffer (10mM, pH 7.4) was added so that the molar ratio of PLI and CpG-ODN was 10. At the same time, the mass of hyaluronic acid was added so that it was three times the PLI and stirred at intensity 3 for 30 minutes using a stirrer. After the particles were manufactured, the particle size was analyzed using ELSZ-2000 (Otsuka Electronics) and is shown in Figure 6. It was confirmed that the nanostructure containing hyaluronic acid was also in a uniform and stable state.
4) HA/PLI/CpG-ODN/OVA (PLI-HA-면역증강제-항원 복합체)의 제조 및 입도분석4) Preparation and particle size analysis of HA/PLI/CpG-ODN/OVA (PLI-HA-immune enhancer-antigen complex)
기존 나노복합체에 표준항원 모델인 오발부민(Ovalbumin, OVA)을 함께 포함하는 입자를 제조하였다. PLI와 CpG-ODN의 몰 비율을 1:10, PLI와 HA의 질량 비율을 1:3으로 하여 투입하였고, OVA의 질량 (5-20ug) 및 투입 시기를 조절하여 입자를 제조하였다. 항원의 질량 및 투입 시기는 도 7에 상세히 나타내었다. 제조된 입자는 ELSZ-2000(오츠카전자)을 사용하여 입도 분석하여 도 8에 나타내었다. 다양한 항원 투입량에 대해 입자의 안정성 및 균일성이 유지되었으며, 항원 투입 시점과 상관없이 균일한 입자 제조가 가능함을 확인하였다.Particles containing ovalbumin (OVA), a standard antigen model, were manufactured in an existing nanocomposite. PLI and CpG-ODN were added at a molar ratio of 1:10 and PLI and HA at a mass ratio of 1:3, and particles were prepared by adjusting the mass of OVA (5-20ug) and the time of addition. The mass of the antigen and the timing of injection are shown in detail in Figure 7. The manufactured particles were analyzed for particle size using ELSZ-2000 (Otsuka Electronics) and are shown in Figure 8. It was confirmed that the stability and uniformity of the particles were maintained for various antigen dosages, and that uniform particle production was possible regardless of the time of antigen injection.
실시예 3: PLI의 proton sponge capacity 확인Example 3: Confirmation of proton sponge capacity of PLI
PLI의 proton sponge capacity의 확인을 위해, 150mM 염화나트륨(NaCl) 및 PLL, PLI, 4-이미다졸아세트산(4-imidazoleacetic acid)에 대한 적정 실험을 진행 및 분석하여 도 9에 나타내었다. 3ml의 150mM NaCl 용액 또는 이 용액을 용매로 한 PLL, PLI, 4-이미다졸아세트산 혼합 용액에 1N 수산화나트륨(NaOH)을 적당량 더하여 최초 pH를 12.00으로 설정하였다. 이후, 0.1N 염산(HCl)을 50ul씩 순차적으로 투입하고 염산 투입량에 따른 pH 변화를 측정하였다. 염화나트륨과 PLL에 비해, 이미다졸 그룹이 개질된 PLI에서 더 강한 proton sponge capacity를 가지는 것을 확인하였다.To confirm the proton sponge capacity of PLI, a titration experiment was performed and analyzed with 150mM sodium chloride (NaCl), PLL, PLI, and 4-imidazoleacetic acid, and is shown in Figure 9. An appropriate amount of 1N sodium hydroxide (NaOH) was added to 3 ml of 150mM NaCl solution or a mixed solution of PLL, PLI, and 4-imidazole acetic acid using this solution as a solvent, and the initial pH was set to 12.00. Afterwards, 50ul of 0.1N hydrochloric acid (HCl) was sequentially added, and the pH change according to the amount of hydrochloric acid added was measured. Compared to sodium chloride and PLL, it was confirmed that PLI modified with the imidazole group had a stronger proton sponge capacity.
실시예 4: 복합체의 항원전달능 확인Example 4: Confirmation of antigen delivery ability of complex
1) OVA-FITC의 세포내 전달능 확인 (도 10)1) Confirmation of intracellular delivery ability of OVA-FITC (Figure 10)
3가지 조건 i) 면역항원 모델 OVA-FITC와 면역증강제 CpG-ODN 혼합투여 (OVA-FITC/CpG-ODN) ii) 면역항원 모델 OVA-FITC와 면역증강제 CpG-ODN, 양이온성 아미노산 고분자 PLI 혼합투여 (OVA-FITC/PLI/CpG-ODN), iii) 면역항원 모델 OVA-FITC, 양이온성 아미노산 고분자 PLI, 음이온성 고분자 HA, 면역증강제 CpG-ODN 혼합투여 (HA/ OVA-FITC /PLI/CpG-ODN)로 실험을 진행하였으며, 면역항원 모델 OVA-FITC는 26 μg 과 CpG-ODN은 500 pmol 으로 백신제제를 제조하여 confocal dish 에서 실험을 수행하였다. FITC 형광 라벨링이 되어있는 면역항원 모델 OVA-FITC(초록)를 사용하였다. 형광 이미징을 위해 핵(파랑)과 lysosome(빨강)을 hoechst33342, lysotracker red-99 dnd를 사용하여 염색하였다. 형광 이미지 분석 결과 iii) 면역항원 모델 OVA-FITC, 양이온성 아미노산 고분자 PLI, 음이온성 고분자 HA, 면역증강제 CpG-ODN 혼합투여 (HA/ OVA-FITC /PLI/CpG-ODN) 에서 OVA-FITC가 세포내 세포질에 많이 분포하는 것을 강한 초록형광으로 확인 할 수 있었다. 즉, 항원의 세포내 전달능이 향상된 것을 확인할 수 있었다 (도 10).Three conditions i) Mixed administration of immune antigen model OVA-FITC and immune enhancer CpG-ODN (OVA-FITC/CpG-ODN) ii) Mixed administration of immune antigen model OVA-FITC, immune enhancer CpG-ODN, and cationic amino acid polymer PLI (OVA-FITC/PLI/CpG-ODN), iii) Immune antigen model OVA-FITC, cationic amino acid polymer PLI, anionic polymer HA, immune enhancer CpG-ODN mixed administration (HA/ OVA-FITC /PLI/CpG- ODN), the vaccine preparation was prepared with 26 μg of the immune antigen model OVA-FITC and 500 pmol of CpG-ODN, and the experiment was performed in a confocal dish. The immunoantigen model OVA-FITC (green) with FITC fluorescence labeling was used. For fluorescence imaging, nuclei (blue) and lysosomes (red) were stained using hoechst33342 and lysotracker red-99 dnd. Fluorescent image analysis results iii) In the mixed administration of immune antigen model OVA-FITC, cationic amino acid polymer PLI, anionic polymer HA, and immune enhancer CpG-ODN (HA/ OVA-FITC /PLI/CpG-ODN), OVA-FITC was observed in cells. It was confirmed by strong green fluorescence that it was widely distributed in my cytoplasm. In other words, it was confirmed that the intracellular delivery ability of the antigen was improved (FIG. 10).
2) CpG-ODN-FITC의 세포내 전달능 확인 (도 11)2) Confirmation of intracellular delivery ability of CpG-ODN-FITC (Figure 11)
3가지 조건 i) 면역항원 모델 OVA 와 면역증강제 CpG-ODN-FITC 혼합투여 (OVA/CpG-ODN-FITC), ii) 면역항원 모델 OVA 와 면역증강제 CpG-ODN-FITC, 양이온성 아미노산 고분자 PLI 혼합투여 (OVA/PLI/CpG-ODN-FITC), iii) 면역항원 모델 OVA, 양이온성 아미노산 고분자 PLI, 음이온성 고분자 HA, 면역증강제 CpG-ODN-FITC 혼합투여 (HA/OVA/PLI/CpG-ODN-FITC)로 실험을 진행하였으며, 면역항원 모델 OVA 는 26 μg 과 CpG-ODN-FITC은 500 pmol 으로 백신제제를 제조하여 confocal dish에서 실험을 수행하였다. FITC 형광 라벨링이 되어있는 면역증강제 CpG-ODN-FITC(초록)를 사용하였다. 형광 이미징을 위해 핵(파랑)과 lysosome(빨강)을 hoechst33342, lysotracker red-99 dnd를 사용하여 염색하였다. 형광 이미지 분석 결과 iii) 면역항원 모델 OVA, 양이온성 아미노산 고분자 PLI, 음이온성 고분자 HA, 면역증강제 CpG-ODN-FITC 혼합투여 (HA/OVA/PLI/CpG-ODN-FITC) 에서 CpG-ODN-FITC가 세포내 세포질내 lysosome에 많이 분포하는 것을 강한 초록 형광으로 확인 할 수 있었다. 즉, 면역증강제의 세포내 전달능이 향상된 것을 확인할 수 있었다 (도 11).Three conditions i) mixed administration of immune antigen model OVA and immune enhancer CpG-ODN-FITC (OVA/CpG-ODN-FITC), ii) mixture of immune antigen model OVA, immune enhancer CpG-ODN-FITC, and cationic amino acid polymer PLI Administration (OVA/PLI/CpG-ODN-FITC), iii) Immune antigen model OVA, cationic amino acid polymer PLI, anionic polymer HA, immune enhancer CpG-ODN-FITC mixed administration (HA/OVA/PLI/CpG-ODN -FITC), the vaccine preparation was prepared with 26 μg of immune antigen model OVA and 500 pmol of CpG-ODN-FITC, and the experiment was performed in a confocal dish. The adjuvant CpG-ODN-FITC (green), which is FITC fluorescently labeled, was used. For fluorescence imaging, nuclei (blue) and lysosomes (red) were stained using hoechst33342 and lysotracker red-99 dnd. Fluorescent image analysis results iii) CpG-ODN-FITC in immune antigen model OVA, cationic amino acid polymer PLI, anionic polymer HA, and immune enhancer CpG-ODN-FITC mixed administration (HA/OVA/PLI/CpG-ODN-FITC) It was confirmed by strong green fluorescence that it was widely distributed in lysosomes within the cytoplasm of cells. In other words, it was confirmed that the intracellular delivery ability of the adjuvant was improved (FIG. 11).
실시예 5: in vitro 상의 사이토카인 발현 확인 (도 12)Example 5: Confirmation of cytokine expression in vitro (FIG. 12)
마우스 대식세포주 RAW264.7 세포에서 개발된 백신 나노복합체를 6시간 이상 처리하여 발현된 사이토카인을 ELISA를 통해 확인하여 나타내었다. 7가지 조건 i)무처리군(N.C.: PBS 50㎕ 투여), ii) COVID19 spike protein의 receptor binding domain 재조합단백질 (RBD) 단독, iii) 면역증강제 CpG-ODN 단독, iv) 양이온성 아미노산 고분자 PLI, 음이온성 고분자 HA 혼합 (HA/PLI) v) RBD와 면역증강제 CpG-ODN 혼합투여 (RBD + CpG-ODN) vi) RBD와 면역증강제 CpG-ODN, 양이온성 아미노산 고분자 PLI 혼합투여 (RBD+PLI/CpG-ODN), vii) RBD, 양이온성 아미노산 고분자 PLI, 음이온성 고분자 HA, 면역증강제 CpG-ODN 혼합투여 (HA/RBD/PLI/CpG-ODN)로 실험을 진행하였으며, 항원 RBD는 0.87 μg 과 CpG-ODN은 16.67 pmol 으로 백신제제를 제조하여 96well plate에서 well당 처리하여 실험을 수행하였다.Cytokines expressed by treating the vaccine nanocomplex developed in mouse macrophage cell line RAW264.7 cells for more than 6 hours were confirmed through ELISA. 7 conditions i) untreated group (N.C.: administered 50㎕ of PBS), ii) COVID19 spike protein receptor binding domain recombinant protein (RBD) alone, iii) immune enhancer CpG-ODN alone, iv) cationic amino acid polymer PLI, Mixed administration of anionic polymer HA (HA/PLI) v) Mixed administration of RBD and immune enhancer CpG-ODN (RBD + CpG-ODN) vi) Mixed administration of RBD and immune enhancer CpG-ODN and cationic amino acid polymer PLI (RBD+PLI/ CpG-ODN), vii) RBD, cationic amino acid polymer PLI, anionic polymer HA, and immune enhancer CpG-ODN mixed administration (HA/RBD/PLI/CpG-ODN). The antigen RBD was administered at 0.87 μg and CpG-ODN was tested by preparing a vaccine preparation of 16.67 pmol and treating it per well in a 96-well plate.
6시간 처리 이후 세포를 제외한 상층액을 ELISA 실험을 진행하여 사이토카인 IL-6, TNF-alpha 양을 비교한 결과, vii) RBD, 양이온성 아미노산 고분자 PLI, 음이온성 고분자 HA, 면역증강제 CpG-ODN 혼합투여 (HA/RBD/PLI/CpG-ODN)에서 가장 많은 양의 사이토카인이 발현된 것으로 확인할 수 있었다.After 6 hours of treatment, the supernatant excluding cells was subjected to an ELISA experiment to compare the amounts of cytokines IL-6 and TNF-alpha. vii) RBD, cationic amino acid polymer PLI, anionic polymer HA, and immune enhancer CpG-ODN. It was confirmed that the highest amount of cytokines was expressed in mixed administration (HA/RBD/PLI/CpG-ODN).
실시예 6: 동물 모델에서 항체 생성능 확인 (도 13)Example 6: Confirmation of antibody production ability in animal model (FIG. 13)
근육 주사 접종 대상으로 마우스(BALB/c)를 2주 간격으로 2번 주사 접종을 실시하였고, 2주차 4주차에 채혈하여 항원 특이적 항체기반 면역반응(humoral immunity)을 확인하기 위해 ELISA를 통해 면역 활성 측정 실험을 진행하였다. 마우스의 면역 활성 측정은 채혈한 혈청을 1:64으로 희석하여 IgG와 그 isotype(IgG1, IgG2a)의 양을 ELISA(Enzyme-linked immunosorbent assay)를 통해 확인하여 나타내었다. 5가지 조건 i) 무처리군(N.C.: PBS 50㎕ 투여), ii) COVID19 spike protein의 receptor binding domain 재조합단백질 (RBD) 단독, iii) RBD와 면역증강제 CpG-ODN 혼합투여 iv) RBD와 면역증강제 CpG-ODN, 양이온성 아미노산 고분자 PLI 혼합투여, v) RBD, 양이온성 아미노산 고분자 PLI, 음이온성 고분자 HA, 면역증강제 CpG-ODN 혼합투여로 실험을 진행하였으며, 항원 RBD는 10 μg 과 CpG-ODN은 1 nmol 으로 백신제제를 제조하여 실험을 수행하였다. 이때, 각 조건 당 8마리의 마우스를 대상으로 실험하였고, 면역 백신 항원은 COVID19 spike protein의 receptor binding domain 재조합단백질 (RBD)를 사용하였다.For intramuscular injection, mice (BALB/c) were inoculated twice at 2-week intervals, and blood was collected at the 2nd and 4th weeks and immunized through ELISA to confirm antigen-specific antibody-based immune response (human immunity). An activity measurement experiment was conducted. To measure the immune activity of mice, collected serum was diluted 1:64 and the amount of IgG and its isotypes (IgG1, IgG2a) was confirmed through ELISA (Enzyme-linked immunosorbent assay). Five conditions i) Untreated group (N.C.: administered 50㎕ of PBS), ii) COVID19 spike protein receptor binding domain recombinant protein (RBD) alone, iii) RBD and adjuvant CpG-ODN mixed administration iv) RBD and adjuvant An experiment was conducted with mixed administration of CpG-ODN, cationic amino acid polymer PLI, v) RBD, cationic amino acid polymer PLI, anionic polymer HA, and immune enhancer CpG-ODN. 10 μg of antigen RBD and 10 μg of CpG-ODN were administered. A vaccine preparation was prepared at 1 nmol and the experiment was performed. At this time, the experiment was conducted on 8 mice per condition, and the receptor binding domain recombinant protein (RBD) of COVID19 spike protein was used as the immunization vaccine antigen.
ELISA 실험을 진행하여 흡광도(450nm)에서 IgG, IgG1, IgG2a의 양을 확인한 결과, 실험군(HA/RBD/PLI/CpG-ODN)에서 다른 대조군에 비해 가장 많이 가장 생성된 것을 확인할 수 있었다. 또한, 2주차, 4주차 혈청에서 IgG 양 비교시 4주차에 더 많은 항체가 생성되어 부스팅 효과를 확인할 수 있었다.As a result of conducting an ELISA experiment to check the amount of IgG, IgG1, and IgG2a at absorbance (450 nm), it was confirmed that the experimental group (HA/RBD/PLI/CpG-ODN) produced the most compared to the other control groups. Additionally, when comparing the amount of IgG in the serum at the 2nd and 4th weeks, more antibodies were produced at the 4th week, confirming the boosting effect.
본 발명은 상술한 실시예 및 실험예를 참고로 설명되었으나 이는 예시적인 것에 불과하며, 당해 기술분야에서 통상의 지식을 가진 자라면 이로부터 다양한 변형 및 균등한 다른 실시예 및 실험예가 가능하다는 점을 이해할 것이다. 따라서 본 발명의 진정한 기술적 보호 범위는 첨부된 특허청구범위의 기술적 사상에 의하여 정해져야 할 것이다.The present invention has been described with reference to the above-described examples and experimental examples, but these are merely illustrative, and those skilled in the art will understand that various modifications and equivalent other examples and experimental examples are possible therefrom. You will understand. Therefore, the true scope of technical protection of the present invention should be determined by the technical spirit of the attached patent claims.
Claims (13)
(b) 항원; 및
(c) 면역증강제를 포함하는 백신 전달체로써,
상기 양이온성 폴리 아미노산 고분자는 하기 일반식 1로 표현되는 PLI이고,
상기 음이온성 고분자는 카르복시메틸셀룰로오스, 덱스트란, 잔탄검, 알긴산, 카라기난, 젤란검, 옥시키틴 및 옥시풀루란, 콘드로이틴, 히알루론산, 카보머, 폴리갈락투론산, 폴리L-글루탐산, 폴리아스파르트산, 및 폴리아크릴산으로 이루어진 군으로부터 선택된 어느 하나이며,
상기 면역증강제는 Triacylated lipoproteins (Pam3CSK4), dsRNA Polyinosinic:polycytidylic acid (poly(I:C)), Lipopolysaccharides (LPS), Flagellin, Imidazoquinolines (R848), Diacylated lipoproteins (FSL-1), Guanosine analogs (Loxoribine), CpG-ODN(cytosine-guanosine oligonucleotide), profilin-like protein) 및 이들의 조합으로 이루어진 군으로부터 선택되고,
상기 항원은 오발부민(OVA) 또는 COVID-19 RBD이며,
상기 양이온성 폴리 아미노산 고분자와 면역 증강제의 혼합비는 몰비율 2.5:1 내지 20:1 인
백신 전달체:
[일반식 1]
XmYn
상기 일반식 1에서, X는 리신이고, Y는 이미다졸기가 개질된 리신이며, m, n은 각각 1 이상 10 이하의 정수이다.(a) a nanocomposite containing a cationic poly amino acid polymer and a pharmaceutically acceptable anionic polymer;
(b) antigen; and
(c) As a vaccine carrier containing an adjuvant,
The cationic poly amino acid polymer is PLI represented by the following general formula 1,
The anionic polymers include carboxymethylcellulose, dextran, xanthan gum, alginic acid, carrageenan, gellan gum, oxykitin and oxypullulan, chondroitin, hyaluronic acid, carbomer, polygalacturonic acid, poly L-glutamic acid, and polyaspartic acid. , and polyacrylic acid,
The immune enhancers include Triacylated lipoproteins (Pam3CSK4), dsRNA Polyinosinic:polycytidylic acid (poly(I:C)), Lipopolysaccharides (LPS), Flagellin, Imidazoquinolines (R848), Diacylated lipoproteins (FSL-1), Guanosine analogs (Loxoribine), Selected from the group consisting of CpG-ODN (cytosine-guanosine oligonucleotide), profilin-like protein) and combinations thereof,
The antigen is ovalbumin (OVA) or COVID-19 RBD,
The mixing ratio of the cationic poly amino acid polymer and the immune enhancer is a molar ratio of 2.5:1 to 20:1.
Vaccine delivery vehicle:
[General Formula 1]
XmYn
In the general formula 1,
상기 일반식 1의 m, n에 대하여, 상기 m과 n은 10:1 내지 1:10의 비율로 포함되는 것인, 백신 전달체. According to paragraph 1,
With respect to m and n of General Formula 1, m and n are contained in a ratio of 10:1 to 1:10.
상기 양이온성 폴리 아미노산 고분자는 상기 음이온성 고분자와 혼합되어 나노 복합체를 형성하는 것인, 백신 전달체.According to paragraph 1,
A vaccine delivery system in which the cationic polyamino acid polymer is mixed with the anionic polymer to form a nanocomposite.
상기 음이온성 고분자는 카르복시메틸셀룰로오스, 덱스트란, 잔탄검, 알긴산, 카라기난, 젤란검, 옥시키틴 및 옥시풀루란, 콘드로이틴, 히알루론산, 카보머, 폴리갈락투론산, 폴리L-글루탐산, 폴리아스파르트산, 및 폴리아크릴산으로 이루어진 군으로부터 선택된 어느 하나인, 백신 전달체. According to paragraph 1,
The anionic polymers include carboxymethylcellulose, dextran, xanthan gum, alginic acid, carrageenan, gellan gum, oxykitin and oxypullulan, chondroitin, hyaluronic acid, carbomer, polygalacturonic acid, poly L-glutamic acid, and polyaspartic acid. , and any one selected from the group consisting of polyacrylic acid.
상기 면역증강제는 Triacylated lipoproteins (Pam3CSK4), dsRNA Polyinosinic:polycytidylic acid (poly(I:C)), Lipopolysaccharides (LPS), Flagellin, Imidazoquinolines (R848), Diacylated lipoproteins (FSL-1), Guanosine analogs (Loxoribine), CpG-ODN(cytosine-guanosine oligonucleotide), profilin-like protein 및 이들의 조합으로 이루어진 군으로부터 선택되는 것인, 백신 전달체.According to paragraph 1,
The immune enhancers include Triacylated lipoproteins (Pam3CSK4), dsRNA Polyinosinic:polycytidylic acid (poly(I:C)), Lipopolysaccharides (LPS), Flagellin, Imidazoquinolines (R848), Diacylated lipoproteins (FSL-1), Guanosine analogs (Loxoribine), A vaccine delivery vehicle selected from the group consisting of CpG-ODN (cytosine-guanosine oligonucleotide), profilin-like protein, and combinations thereof.
상기 양이온성 폴리 아미노산 고분자와 상기 음이온성 고분자의 혼합비는 질량비율 10:1 내지 질량비율 1:10인, 백신 전달체.According to paragraph 1,
The mixing ratio of the cationic poly amino acid polymer and the anionic polymer is a mass ratio of 10:1 to 1:10.
상기 양이온성 폴리 아미노산 고분자와 면역 증강제의 혼합비는 질량비율 1:1 내지 100:1 인, 백신 전달체.According to paragraph 1,
A vaccine delivery system, wherein the mixing ratio of the cationic poly amino acid polymer and the immune enhancer is 1:1 to 100:1 by mass.
상기 백신 전달체는 pH 5 이상 7 이하의 조건에서 이온화되는 것인, 백신 전달체.According to paragraph 1,
The vaccine delivery vehicle is ionized under conditions of pH 5 or more and 7 or less.
A vaccine composition comprising the vaccine carrier of claim 1 and a pharmaceutically acceptable carrier.
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