KR20230095025A - Variant SARS-CoV-2 vaccine composition and use thereof - Google Patents
Variant SARS-CoV-2 vaccine composition and use thereof Download PDFInfo
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Abstract
Description
본 개시는 대한민국 보건복지부의 지원 하에서 과제번호 HV20C0132에 의해 이루어진 것으로서, 상기 과제의 연구관리 전문기관은 백신실용화 기술개발 사업단, 연구사업명은 “감염병 예방치료 기술개발”, 연구과제명은 “팬데믹에 신속하게 대응할 수 있는 새로운 mRNA 기반 백신 플랫폼 개발”, 주관기관은 아이진㈜, 연구기간은 2021.12.01-2023.06.30 이다. This disclosure was made by the project number HV20C0132 under the support of the Ministry of Health and Welfare of the Republic of Korea, and the specialized research management institution for the project is the Vaccine Commercialization Technology Development Center, the research project name is “infectious disease prevention and treatment technology development”, and the research project name is “quick response to pandemic” Development of a new mRNA-based vaccine platform that can effectively respond”, the leading institution is Igene Co., Ltd., and the research period is 2021.12.01-2023.06.30.
또한, 본 개시는 대한민국 보건복지부의 지원 하에서 과제번호 HV22C0052에 의해 이루어진 것으로서, 상기 과제의 연구관리 전문기관은 한국보건산업진흥원, 연구사업명은 “신병종 감염병대응 mRNA 백신 임상지원”, 연구과제명은 “다가 코로나19 예방 mRNA 백신 개발”, 주관기관은 아이진㈜, 연구기간은 2022.04.01-2023.03.31 이다. In addition, this disclosure was made by the project number HV22C0052 under the support of the Ministry of Health and Welfare of the Republic of Korea, and the research management specialized institution of the project is the Korea Health Industry Development Institute, the research project name is “Clinical Support for mRNA Vaccine for New Infectious Disease Response”, and the research project name is “Multivalent Development of mRNA vaccine to prevent COVID-19”, Hosted by I-Gene Co., Ltd., research period 2022.04.01-2023.03.31.
본 특허출원은 2021년 12월 20일에 대한민국 특허청에 제출된 대한민국 특허출원 제10-2021-0182914호에 대하여 우선권을 주장하며, 상기 특허출원의 개시사항은 본 명세서에 참조로서 삽입된다.This patent application claims priority to Korean Patent Application No. 10-2021-0182914 filed with the Korean Intellectual Property Office on December 20, 2021, the disclosure of which is incorporated herein by reference.
본 개시는 변이 SARS-CoV-2 백신 조성물에 관한 것으로, 더욱 자세하게는 변이 SARS-CoV-2의 S 항원을 코딩하는 mRNA를 포함하는 변이 SARS-CoV-2 백신 조성물, 이의 용도에 관한 것이다.The present disclosure relates to a mutant SARS-CoV-2 vaccine composition, and more particularly, to a mutant SARS-CoV-2 vaccine composition comprising mRNA encoding an S antigen of mutant SARS-CoV-2, and uses thereof.
코로나바이러스(coronavirus)는 RNA 바이러스의 한 종류로 유전정보가 리보핵산(RNA)으로 이뤄진 바이러스이다. 사람과 동물의 호흡기와 소화기계 감염을 유발한다. 주로, 점막전염, 비말전파 등으로 쉽게 감염되며, 사람은 일반적으로 경미한 호흡기 감염을 일으키지만 드물게 치명적인 감염을 일으키기도 한다.Coronavirus is a type of RNA virus whose genetic information is composed of ribonucleic acid (RNA). It causes respiratory and digestive tract infections in humans and animals. Mainly, it is easily infected by mucosal transmission, droplet transmission, etc., and generally causes mild respiratory infections in humans, but rarely causes fatal infections.
현재 팬데믹을 일으키고 있는 코로나바이러스감염증-19(coronavirus disease 2019, COVID-19) 원인 바이러스인 사스-코로나바이러스-2(severe acute respiratory syndrome coronavirus 2, SARS-CoV-2)는 유전적 배열(DNA sequencing)상 전도 기능(Positive sense) 단일 가닥 RNA(single-stranded RNA) 코로나바이러스이다. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19), which is currently causing the pandemic, has been genetically sequenced (DNA sequencing). ) It is a positive sense single-stranded RNA coronavirus.
SARS-CoV-2 바이러스는 표면의 스파이크 단백질(Spike protein)을 이용하여 기도의 상피세포(airway epithelial cells), 폐포 상피세포(alveolar epithelial cells), 혈관 내피 세포(vascular endothelial cells) 및 폐 내의 대식세포(macrophage) 표면에 존재하는 안지오텐신-전환 효소 2(angiotensin-converting enzyme 2, ACE2)에 결합함으로써 숙주 세포 내로 침입한다.SARS-CoV-2 virus uses the spike protein on the surface to induce airway epithelial cells, alveolar epithelial cells, vascular endothelial cells, and macrophages in the lungs. (Macrophage) Invades into the host cell by binding to angiotensin-converting enzyme 2 (ACE2) present on the surface.
SARS-CoV-2의 유전체 염기 서열 연구 결과, 스파이크 단백질 내에 사스-코로나바이러스(SARS-CoV)와 3차 구조가 상당히 유사한 수용체-결합 도메인(receptor-binding domain, RBD)이 확인되었으며, SARS-CoV-2의 RBD가 SARS-CoV의 RBD에 비해 ACE2에 대한 결합력이 높아 SARS-CoV-2의 전염성이 SARS-CoV보다 더 강한 것으로 추측되었다.As a result of the genome sequence study of SARS-CoV-2, a receptor-binding domain (RBD) with a tertiary structure very similar to that of SARS-CoV was identified in the spike protein, and SARS-CoV It was speculated that the RBD of -2 has a higher affinity for ACE2 than the RBD of SARS-CoV, so that SARS-CoV-2 is more infectious than SARS-CoV.
SARS-CoV-2의 스파이크 단백질은 S1 및 S2 두 개의 단백질로 구성되어 있다. 그 중, S1 단백질은 아미노-말단 영역(amino-terminal domain)과 RBD로 이루어져 있다. RBD가 ACE2와 결합하면 SARS-CoV-2 비리온(virion)이 엔도사이토시스(endocytosis)를 통해 세포의 엔도좀(endosome) 내로 들어가며, 그 후 융합 펩타이드(fusion peptide)가 노출되어 숙주 세포의 막으로 삽입된다. S2 단백질은 융합 펩타이드 영역(fusion peptide region, FP region)과 헵타드 반복 영역들(heptad repeat regions: HR1, HR2)로 이루어져 있다. HR1과 HR2가 서로 닿는 형태로 바이러스 막에 융합하여 SARS-CoV-2 비리온이 숙주 세포 밖으로 방출된다. S1, S2는 다른 절단 부위(cleavage site)를 가지며 각각의 프로테아제(protease)에 의해 절단되어 SARS-CoV-2의 감염이 발생하게 되므로 S1, S2 절단을 억제하거나 ACE2 또는 트랜스멤브레인 세린 프로테아제(transmembrane serine protease 2, TMPRSS2)와 바이러스 간의 결합을 방해하는 전략을 사용해 SARS-CoV-2의 치료제 및 백신을 개발 중이다.The spike protein of SARS-CoV-2 is composed of two proteins, S1 and S2. Among them, the S1 protein is composed of an amino-terminal domain and an RBD. When RBD binds to ACE2, SARS-CoV-2 virion enters the endosome of the cell through endocytosis, and then the fusion peptide is exposed and breaks down the host cell membrane. inserted into The S2 protein consists of a fusion peptide region (FP region) and heptad repeat regions (HR1, HR2). When HR1 and HR2 touch each other, they fuse to the viral membrane, releasing SARS-CoV-2 virions out of the host cell. S1 and S2 have different cleavage sites and are cleaved by each protease, resulting in SARS-CoV-2 infection. SARS-CoV-2 treatments and vaccines are being developed using a strategy to disrupt the binding between protease 2 (TMPRSS2) and the virus.
SARS-CoV-2에 감염된 사람들은 열, 기침, 호흡 곤란, 설사와 같이 경증에서 중증의 증상을 보일 수 있다. 합병증이나 병을 가진 사람들, 노인은 사망할 가능성이 크다. 특히, 심장질환 및 당뇨병 등의 기저질환 보유자가 감염에 더 취약하며, 합병증이나 장기 손상 등을 겪기 때문에 조기 발견과 치료가 매우 중요하다.People infected with SARS-CoV-2 may develop mild to severe symptoms, such as fever, cough, shortness of breath, and diarrhea. People with complications or illness, the elderly, are more likely to die. In particular, early detection and treatment are very important because those with underlying diseases such as heart disease and diabetes are more vulnerable to infections and suffer from complications or organ damage.
SARS-CoV-2와 같은 RNA 바이러스는 게놈 복제 과정에서 높은 변이 빈도로 진화하며, 공통조상에서 파생하여 유전적으로 밀접하게 관련된 바이러스 변이 그룹인 '계통'을 형성하게 된다. SARS-CoV-2도 펜데믹이 시작된 이후 지속적인 변이를 통해 계통을 형성하고 있으며, 이 중에서 변이비율과 연관된 지역범위, 돌연변이 집단이 의학적 대책에 미치는 효과, 질병의 심각성, 사람간 전파력에 미치는 잠재적 또는 알려진 영향을 고려하여, SIG 변이분류체계로 관리하고 있다.RNA viruses such as SARS-CoV-2 evolve with a high mutation frequency during genome replication, forming a 'lineage', a group of genetically closely related viral mutations derived from a common ancestor. SARS-CoV-2 has also formed a lineage through continuous mutations since the start of the pandemic, and among them, the regional range related to the mutation rate, the effect of the mutation group on medical measures, the severity of the disease, and the potential or Considering the known impact, it is managed under the SIG variant classification system.
SIG 분류체계는 모니터링 중인 변이(VBM), 관심변이(VOI), 우려변이(VOC) 및 고위험 변이(VOHC)의 4가지 클래스로 SARS-CoV-2를 정의하고 있다.The SIG taxonomy defines SARS-CoV-2 into four classes: variant under monitoring (VBM), variant of interest (VOI), variant of concern (VOC), and variant of high risk (VOHC).
이중에서 우려변이는 델타(B.1.617.2 및 AY 계통) 및 2021년 11월에 발견된 오미크론(B.1.1.529)이 해당한다. 그 외 알파(B.1.1.7 및 Q 계통), 베타(B.1.351 및 파생계통), 감마(P.1 및 파생계통), 및 엡실론(B.1.427, B1.429) 등을 포함하여 10종의 변이 바이러스가 모니터링 중인 변이로 설정되어 있다.Of these, the mutations of concern are delta (B.1.617.2 and AY lines) and omicron (B.1.1.529) discovered in November 2021. Other 10, including alpha (B.1.1.7 and Q strains), beta (B.1.351 and derivatives), gamma (P.1 and derivatives), and epsilon (B.1.427 and B1.429). Species Variation The virus is set to the variation being monitored.
변이 바이러스의 가장 큰 문제는 스파이크 단백질에 다수의 변이가 발생하기 때문에, 기존에 허가된 백신에 의한 중화 효능이 감소하는 특징이 있으며, 또한 감염력 향상에 의하여 델타의 경우는 우세종이 된 사례가 있다(Business Insider, May 2021).The biggest problem with the mutant virus is that since a number of mutations occur in the spike protein, it is characterized by a decrease in neutralization efficacy by previously licensed vaccines, and in the case of delta, there is a case of becoming a dominant species due to improved infectivity ( Business Insider, May 2021).
현재 허가된 백신은 SARS-CoV-2 최초 바이러스 유래 스파이크 단백질을 기반으로 제조된 백신이며, 개발사에서도 상기 효능 감소에 대한 문제를 인식하고 있기 때문에, 이의 해결을 위하여 부스터 백신 및 신규 백신 개발로 대응하고 있는 상태로, 보다 안정적으로 효과가 지속되면서 높은 면역원성을 나타내는 백신의 개발이 여전히 요구되고 있다.The currently licensed vaccine is a vaccine manufactured based on the first virus-derived spike protein of SARS-CoV-2, and since the developer is also aware of the problem of reduced efficacy, we are responding with the development of a booster vaccine and a new vaccine to solve this problem. As such, there is still a need to develop a vaccine that exhibits high immunogenicity while maintaining its effect more stably.
본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 개시가 속하는 기술 분야의 수준 및 본 개시의 내용이 보다 명확하게 설명된다.A number of papers and patent documents are referenced throughout this specification and their citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly explain the content of the present disclosure and the level of the technical field to which the present disclosure belongs.
본 발명자들은 보관 안정성이 우수하고, 체내 면역원성이 우수한 변이 SARS-CoV-2에 대한 예방 백신을 개발하고자 예의 연구 노력하였다. 그 결과, SARS-CoV-2의 스파이크 단백질에 대한 변이 항원을 코딩하는 핵산을 탑재한 mRNA 백신을 개발하고, 상기 백신이 우수한 안정성과 높은 면역원성을 나타내는 것을 규명함으로써, 본 발명을 완성하였다.The present inventors have made intensive research efforts to develop a preventive vaccine against mutant SARS-CoV-2, which has excellent storage stability and excellent immunogenicity in the body. As a result, the present invention was completed by developing an mRNA vaccine loaded with a nucleic acid encoding a mutant antigen for the spike protein of SARS-CoV-2, and confirming that the vaccine exhibits excellent stability and high immunogenicity.
따라서, 본 개시의 목적은 우수한 안정성과 높은 면역원성을 나타내는, 변이 SARS-CoV-2 백신 조성물을 제공하는 것이다.Accordingly, an object of the present disclosure is to provide a variant SARS-CoV-2 vaccine composition that exhibits excellent stability and high immunogenicity.
본 개시의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.Other objects and advantages of the present disclosure will become more apparent from the following detailed description of the invention, claims and drawings.
본 발명자들은 보관 안정성이 우수하고, 체내 면역원성이 우수한 변이 SARS-CoV-2에 대한 예방 백신을 개발하고자 예의 연구 노력하였다. 그 결과, SARS-CoV-2의 스파이크 단백질에 대한 변이 항원을 코딩하는 핵산을 탑재한 mRNA 백신을 개발하고, 상기 백신이 우수한 안정성과 높은 면역원성을 나타내는 것을 규명하였다.The present inventors have made intensive research efforts to develop a preventive vaccine against mutant SARS-CoV-2, which has excellent storage stability and excellent immunogenicity in the body. As a result, an mRNA vaccine loaded with a nucleic acid encoding a mutant antigen for the spike protein of SARS-CoV-2 was developed, and it was confirmed that the vaccine exhibits excellent stability and high immunogenicity.
본 개시는 변이 SARS-CoV-2 백신 조성물에 관한 것이다.The present disclosure relates to a variant SARS-CoV-2 vaccine composition.
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로 본 명세서에서 사용된 명명법은 본 기술분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is one well known and commonly used in the art.
이하, 본 개시를 더욱 자세히 설명하고자 한다.Hereinafter, the present disclosure will be described in more detail.
본 개시의 일 양태에 따르면, 본 개시는 변이 SARS-CoV-2(severe acute respiratory syndrome coronavirus 2 variant)의 Spike 항원을 인코딩하는 mRNA를 포함하는, 변이 SARS-CoV-2 백신 조성물을 제공한다.According to one aspect of the present disclosure, the present disclosure provides a variant SARS-CoV-2 vaccine composition comprising mRNA encoding the Spike antigen of severe acute
본 개시에서 '변이 SARS-CoV-2'는 SARS-CoV-2의 항원에서의 변이, 특히 S 항원의 수용체 결합 단백질(receptor binding domain, RBD) 등의 변이가 일어난 모든 SARS-CoV-2를 포함하는 의미로 사용된다.In the present disclosure, 'mutated SARS-CoV-2' includes all SARS-CoV-2 mutations in the antigen of SARS-CoV-2, in particular, mutations such as the receptor binding domain (RBD) of the S antigen. used in the sense of
상기 변이는 오미크론(Omicron) 변이이다.The shift is an Omicron shift.
본 개시의 일 구현예에 따르면, 본 개시의 변이 SARS-CoV-2 백신 조성물은 필요에 따라 상기 변이에 기반한 백신, 및/또는 다른 전염(감염)병을 예방하는 백신을 조합(혼합)한 다가(multivalent) 형태로 제조될 수 있다.According to one embodiment of the present disclosure, the mutant SARS-CoV-2 vaccine composition of the present disclosure is a multivalent vaccine that combines (mixes) a vaccine based on the mutation and/or a vaccine to prevent other infectious (infectious) diseases, if necessary. (multivalent) form can be prepared.
이에 따라, 본 개시의 변이 SARS-CoV-2 백신 조성물은 여러 종류의 감염인자를 동시에 방어함으로써 더욱 효과적인 면역 반응을 유도할 수 있다.Accordingly, the mutant SARS-CoV-2 vaccine composition of the present disclosure can induce a more effective immune response by simultaneously protecting various types of infectious factors.
상기 Omicron 변이(lineage B.1.1.529)는 S 항원에 A67V, △69-70, T95I, G142D/△143-145, △211/L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493K, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, 및 L981F 돌연변이/결실을 가진 변이 SARS-CoV-2이다.The Omicron mutation (lineage B.1.1.529) is A67V, Δ69-70, T95I, G142D/Δ143-145, Δ211/L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493K, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F mutations/ Mutations with deletions SARS-CoV- 2.
본 개시에서 'Spike 항원' 또는 'S 항원'은 SARS-CoV-2에 대한 항체 생성을 유발하는 스파이크 단백질(Spike protein) 및 이의 서열변경, 단편 또는 융합 등의 변형에 의한 유사 단백질(항원)을 포함하는 의미로 사용된다.In the present disclosure, 'Spike antigen' or 'S antigen' refers to a spike protein that induces antibody production against SARS-CoV-2 and similar proteins (antigens) by modifications such as sequence alteration, fragmentation, or fusion thereof. used in the sense of inclusion.
상기 변이 SARS-CoV-2의 S 항원을 코딩하는 mRNA는 Omicron-PQ SARS-CoV-2 S 단백질을 코딩하는 서열번호 1의 mRNA일 수 있다. 서열번호 1의 mRNA는 추가적으로 K986P, V987P, 682-QQAQ-685의 치환된 서열을 가지는 Omicron 변이의 S 단백질을 코딩할 수 있다.The mRNA encoding the S antigen of the mutant SARS-CoV-2 may be the mRNA of SEQ ID NO: 1 encoding the Omicron-PQ SARS-CoV-2 S protein. The mRNA of SEQ ID NO: 1 may additionally encode an Omicron mutant S protein having a substituted sequence of K986P, V987P, or 682-QQAQ-685.
상기 변이 SARS-CoV-2의 S 항원은 서열번호 2의 아미노산 서열을 포함할 수 있으나, 이에 한정되는 것은 아니다.The S antigen of the mutant SARS-CoV-2 may include the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.
상기 변이 SARS-CoV-2의 S 항원을 코딩하는 mRNA는 전형적으로 mRNA와 함께 제공되는 세포 또는 유기체에 의해 번역될 수 있는 적어도 하나의 오픈 리딩 프레임을 갖는 mRNA일 수 있다. 이 번역의 산물은 항원, 바람직하게 면역원으로 작용할 수 있는 펩타이드 또는 단백질이다. 이 산물은 또한, 둘 이상의 면역원으로 이루어진 융합 단백질, 예를 들어 동일하거나 상이한 바이러스 단백질로부터 유래된 둘 이상의 에피토프, 펩타이드 또는 단백질로 이루어지는 융합 단백질일 수 있고, 이때, 이 에피토프, 펩타이드 또는 단백질은 링커 서열에 의해 연결될 수 있다.The mRNA encoding the S antigen of the mutant SARS-CoV-2 may be an mRNA having at least one open reading frame that can be translated by a cell or organism, which is typically provided together with the mRNA. The product of this translation is an antigen, preferably a peptide or protein capable of serving as an immunogen. The product may also be a fusion protein consisting of two or more immunogens, for example a fusion protein consisting of two or more epitopes, peptides or proteins derived from the same or different viral proteins, wherein the epitopes, peptides or proteins comprise a linker sequence can be connected by
또한, 상기 변이 SARS-CoV-2의 S 항원을 코딩하는 mRNA는 인공 mRNA, 즉 자연적으로 발생하지 않는 mRNA 분자인 것으로 이해될 수 있다. 인공 mRNA 분자는 비-천연 mRNA 분자로서 이해될 수 있다. 이러한 mRNA 분자는 (자연적으로 발생하지 않는) 개별 서열 및/또는 자연적으로 발생하지 않는 다른 변경, 예컨대 뉴클레오티드의 구조적 변경으로 인해 비-천연적일 수 있다. 인공 mRNA 분자는 뉴클레오티드의 원하는 인공 서열(이종 서열)에 상응하는 유전자 조작 방법에 의해 설계 및/또는 생성될 수 있다. 즉, 상기 변이 SARS-CoV-2 바이러스 S 항원을 코딩하는 mRNA는 적어도 하나의 뉴클레오티드가 야생형 서열과 다르다. 여기에서 '야생형'은 자연적으로 발생하는 서열로서 이해될 수 있다.In addition, it can be understood that the mRNA encoding the S antigen of the mutant SARS-CoV-2 is an artificial mRNA, that is, a non-naturally occurring mRNA molecule. An artificial mRNA molecule can be understood as a non-natural mRNA molecule. Such mRNA molecules may be non-natural due to (non-naturally occurring) individual sequences and/or other non-naturally occurring alterations, such as structural alterations of nucleotides. Artificial mRNA molecules can be designed and/or produced by genetic engineering methods that correspond to a desired artificial sequence of nucleotides (heterologous sequence). That is, the mRNA encoding the mutant SARS-CoV-2 virus S antigen differs from the wild-type sequence in at least one nucleotide. A 'wild type' herein may be understood as a naturally occurring sequence.
본 개시의 구체적인 구현예에 있어서, 본 개시의 mRNA에 포함되는 우리딘(uridine)은 5-메톡시-우리딘(5-methoxy-uridine)으로 치환된 것일 수 있다.In a specific embodiment of the present disclosure, uridine included in the mRNA of the present disclosure may be substituted with 5-methoxy-uridine.
나아가, 상기 변이 SARS-CoV-2의 S 항원을 코딩하는 mRNA는 생체 내 분해(예를 들어 엑소- 또는 엔도-뉴클레아제에 의한 분해) 및/또는 생체 외 분해(예를 들어 백신 투여 전 제조 과정에 의해, 예를 들어 투여되는 백신 용액의 제조 과정에서)에 대한 내성을 증가시키는 변형을 나타낼 수 있다. RNA의 안정화는 예를 들어 5'-CAP 구조, 폴리-A-꼬리, 또는 임의의 기타 UTR 변형의 제공에 의해 달성될 수 있다. 또한, RNA의 안정화는 화학적 변형 또는 핵산의 G/C 함량의 변형에 의해 달성될 수 있다. 다양한 다른 방법이 당해 분야에 공지되어 있으며, 본 개시의 적용이 가능하다.Furthermore, the mRNA encoding the S antigen of the mutant SARS-CoV-2 is degraded in vivo (e.g., by exo- or endo-nuclease) and/or ex vivo (e.g., prepared prior to vaccine administration). process, for example in the preparation of vaccine solutions to be administered). Stabilization of the RNA can be achieved, for example, by providing a 5'-CAP structure, poly-A-tail, or any other UTR modification. Stabilization of RNA can also be achieved by chemical modification or modification of the G/C content of nucleic acids. A variety of other methods are known in the art and are applicable to the present disclosure.
본 개시의 일 구현예에 있어서, 상기 mRNA 는 5'UTR을 추가로 포함한다. 상기 5' UTR은 서열번호 3의 서열을 포함한다. In one embodiment of the present disclosure, the mRNA further includes a 5'UTR. The 5' UTR includes the sequence of SEQ ID NO: 3.
본 개시의 일 구현예에 있어서, 상기 mRNA는 3' UTR을 추가로 포함한다. 상기 3' UTR은 서열번호 4의 서열을 포함한다. In one embodiment of the present disclosure, the mRNA further includes a 3' UTR. The 3' UTR includes the sequence of SEQ ID NO: 4.
본 개시의 일 구현예에 있어서, 상기 mRNA는 5'Cap 구조를 포함할 수 있다. 상기 5' Cap은 m7G(5')ppp(5')(2'OMeA)pG Cap, m7G(5')ppp(5')(2'OMeA)pU Cap, m7(3'OMeG)(5')ppp(5')(2'OmeA)pG Cap, 3´-O-Me-m7G(5')ppp(5')G Cap, G(5')ppp(5')G Cap, 또는 m7G(5')ppp(5')G Cap 일 수 있으나, 이에 한정되는 것은 아니다.In one embodiment of the present disclosure, the mRNA may include a 5'Cap structure. The 5' Cap is m7G(5')ppp(5')(2'OMeA)pG Cap, m7G(5')ppp(5')(2'OMeA)pU Cap, m7(3'OMeG)(5' )ppp(5')(2'OmeA)pG Cap, 3'-O-Me-m7G(5')ppp(5')G Cap, G(5')ppp(5')G Cap, or m7G( 5')ppp(5')G Cap may be, but is not limited thereto.
본 개시의 일 구현예에 있어서, 상기 mRNA는 50 내지 150 nt의 길이를 갖는 poly A tail을 추가로 포함한다. 구체적인 구현예에 있어서, 상기 poly A tail은 50 내지 150 nt, 50 내지 120 nt, 50 내지 100 nt, 50 내지 90 nt, 50 내지 80 nt, 50 내지 70 nt, 50 내지 60 nt, 60 내지 150 nt, 60 내지 120 nt, 60 내지 100 nt, 60 내지 90 nt, 60 내지 80 nt, 60 내지 70 nt, 70 내지 150 nt, 70 내지 120 nt, 70 내지 100 nt, 70 내지 90 nt, 70 내지 80 nt, 50 nt, 60 nt, 70 nt, 80 nt, 90 nt, 또는 100 nt일 수 있으나, 이에 한정되는 것은 아니다. In one embodiment of the present disclosure, the mRNA further includes a poly A tail having a length of 50 to 150 nt. In specific embodiments, the poly A tail is 50 to 150 nt, 50 to 120 nt, 50 to 100 nt, 50 to 90 nt, 50 to 80 nt, 50 to 70 nt, 50 to 60 nt, 60 to 150 nt , 60 to 120 nt, 60 to 100 nt, 60 to 90 nt, 60 to 80 nt, 60 to 70 nt, 70 to 150 nt, 70 to 120 nt, 70 to 100 nt, 70 to 90 nt, 70 to 80 nt , 50 nt, 60 nt, 70 nt, 80 nt, 90 nt, or 100 nt, but is not limited thereto.
본 개시의 일 구현예에 있어서, 상기 mRNA 는 SARS-CoV-2의 S 항원에 융합된 신호 펩타이드(signal peptide)를 인코딩하는 ORF를 갖는 RNA를 추가적으로 포함할 수 있다. In one embodiment of the present disclosure, the mRNA may additionally include RNA having an ORF encoding a signal peptide fused to the S antigen of SARS-CoV-2.
단백질의 N-말단에 10-60개 아미노산을 포함하는 신호 펩타이드는 전형적으로 분비성 경로상의 막을 가로지르는 전위에 필요하며, 따라서 진핵생물 및 원핵생물 모두에서 대부분의 단백질의 분비성 경로로의 진입을 보편적으로 제어한다. 진핵생물에서, 초기 전구체 단백질(전-단백질)의 신호 펩타이드는 리보솜을 조면 소포체(ER) 막으로 안내하고, 처리를 위해 막을 가로지르는 성장하는 펩타이드 사슬의 수송을 개시한다. ER 처리는 성숙한 단백질을 생성하고, 상기 신호 펩타이드는 전형적으로 숙주 세포의 ER-상주 신호 펩티다아제에 의해 전구체 단백질로부터 절단되거나, 이들은 절단되지 않은 채로 남아있고 막 앵커로서 기능한다. 신호 펩타이드는 또한 단백질의 세포막으로의 표적화를 용이하게 할 수 있다.A signal peptide comprising 10-60 amino acids at the N-terminus of a protein is typically required for transmembrane translocation on the secretory pathway and thus permits entry of most proteins into the secretory pathway in both eukaryotes and prokaryotes. universally controlled. In eukaryotes, the signal peptide of the nascent precursor protein (pre-protein) guides the ribosome to the rough endoplasmic reticulum (ER) membrane and initiates transport of the growing peptide chain across the membrane for processing. ER processing results in mature proteins, and the signal peptides are typically cleaved from the precursor proteins by the host cell's ER-resident signal peptidases, or they remain uncleaved and function as membrane anchors. Signal peptides can also facilitate targeting of proteins to cell membranes.
신호 펩타이드는 10-60개의 아미노산의 길이를 가질 수 있다. 예를 들어, 신호 펩타이드는 10개, 11개, 12개, 13개, 14개, 15개, 16개, 17개, 18개, 19개, 20개, 21개, 22개, 23개, 24개, 25개, 26개, 27개, 28개, 29개, 30개, 31개, 32개, 33개, 34개, 35개, 36개, 37개, 38개, 39개, 40개, 41개, 42개, 43개, 44개, 45개, 46개, 47개, 48개, 49개, 50개, 51개, 52개, 53개, 54개, 55개, 56개, 57개, 58개, 59개, 또는 60개의 아미노산의 길이를 가질 수 있다.Signal peptides can be 10-60 amino acids in length. For example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 signal peptides. 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 , 58, 59, or 60 amino acids in length.
본 개시의 일 구현예에 있어서, 상기 신호 펩타이드는 서열번호 5의 아미노산서열을 포함한다. In one embodiment of the present disclosure, the signal peptide includes the amino acid sequence of SEQ ID NO: 5.
본 개시의 일 구현예에 있어서, 상기 서열번호 5의 아미노산 서열을 포함하는 신호 펩타이드는 서열번호 6의 뉴클레오타이드로 표시되는 mRNA에 의하여 인코딩될 수 있으나, 이에 한정되는 것은 아니다. In one embodiment of the present disclosure, the signal peptide comprising the amino acid sequence of SEQ ID NO: 5 may be encoded by mRNA represented by the nucleotide of SEQ ID NO: 6, but is not limited thereto.
본 개시의 일 구현예에 있어서, 폴리뉴클레오타이드를 암호화하는 cDNA는 시험관내 전사(IVT) 시스템을 사용하여 전사될 수 있다. RNA의 시험관내 전사는 당업계에 공지되어 있고, 국제 공개 WO 2014/152027호에 기재되어 있으며, 이는 그 전체가 본원에 참조로 포함된다.In one embodiment of the present disclosure, cDNA encoding the polynucleotide can be transcribed using an in vitro transcription (IVT) system. In vitro transcription of RNA is known in the art and is described in International Publication No. WO 2014/152027, which is incorporated herein by reference in its entirety.
일부 구현예에서, RNA 전사체는 RNA 전사체를 생성하기 위해 시험관내 전사 반응에서 비-증폭된, 선형화된 DNA 주형을 사용하여 생성된다. 일부 구현예에서, 주형 DNA는 단리된 DNA이다. 일부 구현예에서, 주형 DNA는 cDNA이다. 일부 구현예에서, cDNA는 RNA 폴리뉴클레오티드, 이에 제한되지는 않으나, 예를 들어, 코로나바이러스 mRNA의 역전사에 의해 형성된다. 일부 구현예에서, 세포, 예를 들어, 박테리아 세포, 예를 들어, E. coli, 예를 들어, DH-1 세포는 플라스미드 DNA 주형으로 형질감염된다. 일부 구현예에서, 형질감염된 세포는 플라스미드 DNA를 복제하기 위해 배양되며, 이는 이후 단리되고 정제된다. 일부 구현예에서, DNA 주형은 관심 유전자의 5'에 위치하고 작동가능하게 연결된 RNA 폴리머라제 프로모터, 예를 들어, T7 프로모터를 포함한다.In some embodiments, RNA transcripts are generated using a non-amplified, linearized DNA template in an in vitro transcription reaction to generate RNA transcripts. In some embodiments, template DNA is isolated DNA. In some embodiments, template DNA is cDNA. In some embodiments, cDNA is formed by reverse transcription of RNA polynucleotides, such as but not limited to coronavirus mRNA. In some embodiments, a cell, eg, a bacterial cell, eg, E. coli, eg, a DH-1 cell, is transfected with a plasmid DNA template. In some embodiments, transfected cells are cultured to replicate plasmid DNA, which is then isolated and purified. In some embodiments, the DNA template is located 5' to the gene of interest and includes an operably linked RNA polymerase promoter, eg, the T7 promoter.
본 개시의 변이 SARS-CoV-2 백신 조성물은 리포좀 또는 지질 나노입자(lipid nanoparticle, LNP)를 추가로 포함할 수 있다. 구체적으로, 본 개시의 백신 조성물에 포함된 변이 SARS-CoV-2의 S 항원을 코딩하는 mRNA는 리포좀 또는 지질 나노입자의 외부에 흡착(adsorption) 또는 회합되거나, 내부에 캡슐화 또는 봉입(encapsulation)된 상태일 수 있다.The variant SARS-CoV-2 vaccine composition of the present disclosure may further include liposomes or lipid nanoparticles (LNPs). Specifically, the mRNA encoding the S antigen of the mutant SARS-CoV-2 included in the vaccine composition of the present disclosure is adsorbed or associated with the outside of liposomes or lipid nanoparticles, or encapsulated or encapsulated inside. may be in a state
본 개시에서 '리포좀'은 생리활성 성분(예를 들어, mRNA)을 안정하게 전달하고, 침투 효과를 극대화하는 데에 사용되는 물질을 의미한다.In the present disclosure, 'liposome' refers to a material used to stably deliver physiologically active components (eg, mRNA) and maximize the penetration effect.
본 개시에서 '지질 나노입자'는 1종 이상의 지질을 포함하는, 대략 나노미터(예컨대, 1 내지 1,000 nm)의 적어도 하나의 규모를 갖는 입자를 지칭한다. 이러한 지질 나노입자는 양이온성 지질, 중성 지질, 하전된 지질, 스테로이드 및 폴리머 접합 지질로부터 선택된 1종 이상의 부형제를 포함할 수 있다. In the present disclosure, a 'lipid nanoparticle' refers to a particle having at least one dimension on the order of nanometers (eg, 1 to 1,000 nm), comprising one or more lipids. Such lipid nanoparticles may contain one or more excipients selected from cationic lipids, neutral lipids, charged lipids, steroids and polymer conjugated lipids.
상기 지질 나노입자는 임의의 특정 형태에 한정되지 않으며, 예컨대, 수성 환경에서 및/또는 핵산 화합물의 존재 하에서, 양이온성 지질 또는 이온성 지질, 및 선택적으로 1종 이상의 추가적인 지질이 조합될 때 생성된 임의의 형태를 포함하는 것으로 해석해야 한다. 예를 들어, 지질 복합체, 리포플렉스(lipoplex) 등은 지질 나노입자의 범위 내에 있다.The lipid nanoparticle is not limited to any particular form, and is produced when, for example, a cationic lipid or an ionic lipid, and optionally one or more additional lipids are combined in an aqueous environment and/or in the presence of a nucleic acid compound. should be construed to include any form. For example, lipid complexes, lipoplexes, etc. are within the scope of lipid nanoparticles.
상기 지질 나노입자는 약 30 nm 내지 약 200 nm, 40 nm 내지 약 200 nm, 약 50 nm 내지 약 200 nm, 약 60 nm 내지 약 200 nm, 약 70 nm 내지 약 200 nm, 약 80 nm 내지 약 200 nm, 약 90 nm 내지 약 200 nm, 약 100 nm 내지 약 200 nm, 약 110 nm 내지 약 200 nm, 약 120 nm 내지 약 200 nm, 약 130 nm 내지 약 200 nm, 약 140 nm 내지 약 200 nm, 약 150 nm 내지 약 200 nm, 약 30 nm 내지 약 180 nm, 약 40 nm 내지 약 180 nm, 약 50 nm 내지 약 180 nm, 약 60 nm 내지 약 180 nm, 약 70 nm 내지 약 180 nm, 약 80 nm 내지 약 180 nm, 약 90 nm 내지 약 180 nm, 약 100 nm 내지 약 180 nm, 약 110 nm 내지 약 180 nm, 약 120 nm 내지 약 180 nm, 약 130 nm 내지 약 180 nm, 약 140 nm 내지 약 180 nm, 약 30 nm 내지 약 160 nm, 약 40 nm 내지 약 160 nm, 약 50 nm 내지 약 160 nm, 약 60 nm 내지 약 160 nm, 약 70 nm 내지 약 160 nm, 약 80 nm 내지 약 160 nm, 약 90 nm 내지 약 160 nm, 약 100 nm 내지 약 160 nm, 약 110 nm 내지 약 160 nm, 약 120 nm 내지 약 160 nm, 약 130 nm 내지 약 160 nm, 약 140 nm 내지 약 160 nm, 약 30 nm 내지 약 150 nm, 약 40 nm 내지 약 150 nm, 약 50 nm 내지 약 150 nm, 약 60 nm 내지 약 150 nm, 약 70 nm 내지 약 150 nm, 약 80 nm 내지 약 150 nm, 약 90 nm 내지 약 150 nm, 약 100 nm 내지 약 150 nm, 약 110 nm 내지 약 150 nm, 약 120 nm 내지 약 150 nm, 약 130 nm 내지 약 150 nm, 약 140 nm 내지 약 150 nm, 약 30 nm 내지 약 150 nm, 약 40 nm 내지 약 150 nm, 약 50 nm 내지 약 150 nm, 약 60 nm 내지 약 150 nm, 약 70 nm 내지 약 150 nm, 약 80 nm 내지 약 150 nm, 약 90 nm 내지 약 150 nm, 약 100 nm 내지 약 150 nm, 약 110 nm 내지 약 150 nm, 약 120 nm 내지 약 150 nm, 약 130 nm 내지 약 150 nm, 약 140 nm 내지 약 150 nm, 약 60 nm 내지 약 130 nm, 약 70 nm 내지 약 110 nm, 약 70 nm 내지 약 100 nm, 약 80 nm 내지 약 100nm, 약 90 nm 내지 약 100 nm, 약 70 내지 약 90 nm, 약 80 nm 내지 약 90 nm, 약 70 nm 내지 약 80 nm, 또는 약 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 또는 150 nm의 평균 직경을 가지며, 실질적으로 무독성이다. 평균 직경은 동적 광산란에 의해 결정한 z-평균값으로 표현될 수 있다.The lipid nanoparticles are about 30 nm to about 200 nm, 40 nm to about 200 nm, about 50 nm to about 200 nm, about 60 nm to about 200 nm, about 70 nm to about 200 nm, about 80 nm to about 200 nm. nm, about 90 nm to about 200 nm, about 100 nm to about 200 nm, about 110 nm to about 200 nm, about 120 nm to about 200 nm, about 130 nm to about 200 nm, about 140 nm to about 200 nm, About 150 nm to about 200 nm, about 30 nm to about 180 nm, about 40 nm to about 180 nm, about 50 nm to about 180 nm, about 60 nm to about 180 nm, about 70 nm to about 180 nm, about 80 nm to about 180 nm, about 90 nm to about 180 nm, about 100 nm to about 180 nm, about 110 nm to about 180 nm, about 120 nm to about 180 nm, about 130 nm to about 180 nm, about 140 nm to About 180 nm, about 30 nm to about 160 nm, about 40 nm to about 160 nm, about 50 nm to about 160 nm, about 60 nm to about 160 nm, about 70 nm to about 160 nm, about 80 nm to about 160 nm nm, about 90 nm to about 160 nm, about 100 nm to about 160 nm, about 110 nm to about 160 nm, about 120 nm to about 160 nm, about 130 nm to about 160 nm, about 140 nm to about 160 nm, About 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 150 nm, about 70 nm to about 150 nm, about 80 nm to about 150 nm, about 90 nm nm to about 150 nm, about 100 nm to about 150 nm, about 110 nm to about 150 nm, about 120 nm to about 150 nm, about 130 nm to about 150 nm, about 140 nm to about 150 nm, about 30 nm to About 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 150 nm, about 70 nm to about 150 nm, about 80 nm to about 150 nm, about 90 nm to about 150 nm nm, about 100 nm to about 150 nm, about 110 nm to about 150 nm, about 120 nm to about 150 nm, about 130 nm to about 150 nm, about 140 nm to about 150 nm, about 60 nm to about 130 nm, About 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm , 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm in average diameter, and is substantially non-toxic. The average diameter can be expressed as a z-average value determined by dynamic light scattering.
본 개시의 일 구현예에 있어서, 동적 광산란에 의해 결정한 제타전위는 -50 내지 -150 mV, -50 내지 -140 mV, -50 내지 -130 mV, -50 내지 -120 mV, -50 내지 -110 mV, -50 내지 -100 mV, -50 내지 -90 mV, -50 내지 -80 mV, -60 내지 -150 mV, -60 내지 -140 mV, -60 내지 -130 mV, -60 내지 -120 mV, -60 내지 -110 mV, -60 내지 -100 mV, -60 내지 -90 mV, -60 내지 -80 mV, -70 내지 -150 mV, -70 내지 -140 mV, -70 내지 -130 mV, -70 내지 -120 mV, -70 내지 -110 mV, -70 내지 -100 mV, -70 내지 -90 mV, -70 내지 -80 mV, -75 내지 -150 mV, -75 내지 -140 mV, -75 내지 -130 mV, -75 내지 -120 mV, -75 내지 -110 mV, -75 내지 -100 mV, -75 내지 -90 mV, 또는 -75 내지 -80 mV 일 수 있으나, 이에 한정되는 것은 아니다. In one embodiment of the present disclosure, the zeta potential determined by dynamic light scattering is -50 to -150 mV, -50 to -140 mV, -50 to -130 mV, -50 to -120 mV, -50 to -110 mV, -50 to -100 mV, -50 to -90 mV, -50 to -80 mV, -60 to -150 mV, -60 to -140 mV, -60 to -130 mV, -60 to -120 mV , -60 to -110 mV, -60 to -100 mV, -60 to -90 mV, -60 to -80 mV, -70 to -150 mV, -70 to -140 mV, -70 to -130 mV, -70 to -120 mV, -70 to -110 mV, -70 to -100 mV, -70 to -90 mV, -70 to -80 mV, -75 to -150 mV, -75 to -140 mV, - 75 to -130 mV, -75 to -120 mV, -75 to -110 mV, -75 to -100 mV, -75 to -90 mV, or -75 to -80 mV, but is not limited thereto. .
본 개시에서, 변이 SARS-CoV-2의 S 항원을 코딩하는 mRNA 또는 이의 부분은 상기 리포좀 또는 지질 나노입자의 지질 부분에 흡착되어, 또는 리포좀 또는 지질 나노입자의 일부 또는 모든 지질 부분에 의해 둘러싸인 수성 공간에 캡슐화되어, 효소적 분해 또는, 숙주 유기체 또는 세포의 메커니즘, 예컨대, 부정적인 면역 반응에 의해 유도된 기타 원하지 않는 효과로부터 mRNA 또는 이의 부분이 보호될 수 있다.In the present disclosure, mRNA encoding the S antigen of mutant SARS-CoV-2 or a portion thereof is adsorbed to the lipid portion of the liposome or lipid nanoparticle, or an aqueous solution surrounded by part or all of the lipid portion of the liposome or lipid nanoparticle. Encapsulated in space, mRNA or portions thereof may be protected from enzymatic degradation or other undesirable effects induced by mechanisms of the host organism or cell, such as negative immune responses.
상기 리포좀 또는 지질 나노입자는 양이온성 지질을 포함할 수 있다.The liposome or lipid nanoparticle may contain a cationic lipid.
본 개시에서 '양이온성 지질'은 pH 변화의 영향 없이 지속적으로 양이온성을 가지는 지질 또는 pH 변화에 의하여 양이온성으로 전환되는 이온성 지질을 포함한다.In the present disclosure, 'cationic lipid' includes a lipid having cationic property continuously without the influence of pH change or an ionic lipid that is converted to cationic property by pH change.
상기 양이온성 지질은 1,2-디올레오일-3-트리메틸암모늄프로페인(DOTAP), 디메틸디옥타데실암모늄 브로마이드(DDA), 3β-[N-(N',N'-디메틸아미노에테인 카바모일 콜레스테롤(3β-[N-(N',N'-dimethylaminoethane) carbamoyl cholesterol, DC-Chol), 1,2-디올레오일옥시-3-디메틸암모늄프로페인(DODAP),1,2-디-O-옥타데세닐-3-트리에틸암모늄 프로페인(1,2-di-O-octadecenyl-3-trimethylammonium propane, DOTMA), 1,2-디미리스토레오일-sn-글리세로-3-에틸포스포콜린(1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine, 14:1 Etyle PC), 1-팔미토일-2-올레오일-sn-글리세로-3-에틸포스포콜린(1-palmitoyl-2-oleoyl-snglycero-3-ethylphosphocholine, 16:0-18:1 Ethyl PC), 1,2-디올레오일-sn-글리세로-3-에틸포스포콜린(1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, 18:1 Ethyl PC), 1,2-디스테아로일-sn-글리세로-3-에틸포스포콜린(1,2-distearoyl-sn-glycero-3-ethylphosphocholin, 18:0 Ethyl PC), 1,2-디팔미토일-sn-글리세로-3-에틸포스포콜린(1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, 16:0 Ethyl PC), 1,2-디미리스토일-sn-글리세로-3-에틸포스포콜린(1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, 14:0 Ethyl PC), 1,2-디라우로일-sn-글리세로-3-에틸포스포콜린(1,2-dilauroyl-sn-glycero-3-ethylphosphocholin, 12:0 Ethyl PC), N1-[2-((1S)-1-[(3-아미노프로필)아미노]-4-[디(3-아미노-프로필)아미노]부틸카복사미도)에틸]-3,4-디[올레일옥시]-벤자마이드(N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide, MVL5), 1,2-디미리스토일-3-디메틸암모늄-프로페인(1,2-dimyristoyl-3-dimethylammonium-propane,14:0 DAP), 1,2-디팔미토일-3-디메틸암모늄-프로페인(1,2-dipalmitoyl-3-dimethylammonium-propane, 16:0DAP), 1,2-디스테아로일-3-디메틸암모늄-프로페인(1,2-distearoyl-3-dimethylammonium-propane, 18:0 DAP), N-(4-카복시벤질)-N,N-디메틸-2,3-비스(올레오일옥시)프로판-1-아미늄(N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium, DOBAQ), 1,2-스테아로일-3-트리메틸암모늄-프로페인(1,2-stearoyl-3-trimethylammoniumpropane, 18:0 TAP), 1,2-디팔미토일-3-트리메틸암모늄-프로페인(1,2-dipalmitoyl-3-trimethylammonium-propane, 16:0 TA), 1,2-디미리스토일-3-트리메틸암모늄-프로페인(1,2-dimyristoyl-3-trimethylammonium-propane, 14:0 TAP) 및/또는 N4-콜레스테릴-스퍼민(N4-Cholesteryl-Spermine, GL67)일 수 있고, 바람직하게는 1,2-디올레오일-3-트리메틸암모늄프로페인(DOTAP) 또는 C12-200일 수 있으나, 이에 한정되는 것은 아니다.The cationic lipids are 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), dimethyldioctadecylammonium bromide (DDA), 3β-[N-(N',N'-dimethylaminoethane carbamoyl Cholesterol (3β-[N-(N',N'-dimethylaminoethane) carbamoyl cholesterol, DC-Chol), 1,2-dioleoyloxy-3-dimethylammonium propane (DODAP), 1,2-di-O -Octadecenyl-3-triethylammonium propane (1,2-di-O-octadecenyl-3-trimethylammonium propane, DOTMA), 1,2-dimyristoleoyl-sn-glycero-3-ethylphos Forcolin (1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine, 14:1 Etyle PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (1-palmitoyl- 2-oleoyl-snglycero-3-ethylphosphocholine, 16:0-18:1 Ethyl PC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (1,2-dioleoyl-sn-glycero -3-ethylphosphocholine, 18:1 Ethyl PC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (1,2-distearoyl-sn-glycero-3-ethylphosphocholine, 18:0 Ethyl PC), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, 16:0 Ethyl PC), 1,2-di Myristoyl-sn-glycero-3-ethylphosphocholine (1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, 14:0 Ethyl PC), 1,2-dilauroyl-sn-glycero -3-Ethylphosphocholine (1,2-dilauroyl-sn-glycero-3-ethylphosphocholin, 12:0 Ethyl PC), N1-[2-((1S)-1-[(3-aminopropyl)amino] -4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (N1-[2-((1S)-1-[( 3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide, MVL5), 1,2-dimyristoyl-3- Dimethylammonium-propane (1,2-dimyristoyl-3-dimethylammonium-propane, 14:0 DAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (1,2-dipalmitoyl-3-dimethylammonium- propane, 16:0 DAP), 1,2-distearoyl-3-dimethylammonium-propane, 18:0 DAP, N-(4-carboxybenzyl) -N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1- aminium, DOBAQ), 1,2-stearoyl-3-trimethylammonium-propane (1,2-stearoyl-3-trimethylammoniumpropane, 18:0 TAP), 1,2-dipalmitoyl-3-trimethylammonium- Propane (1,2-dipalmitoyl-3-trimethylammonium-propane, 16:0 TA), 1,2-dimyristoyl-3-trimethylammonium-propane, 14:0 TAP) and/or N4-Cholesteryl-Spermine (GL67), preferably 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) or C12 -200, but is not limited thereto.
구체적으로, 상기 'DOTAP(Dioleoyl-3-trimethylammonium propane)'는 하기 화학식 1의 구조를 가지는 양이온성 유화제로, 섬유 유연제로 사용되고 있으며, 최근에는 리포좀을 형성하는 핵산 운반체로 사용되고 있다.Specifically, the 'DOTAP (Dioleoyl-3-trimethylammonium propane)' is a cationic emulsifier having a structure of Chemical Formula 1, used as a fabric softener, and recently used as a nucleic acid carrier for forming liposomes.
[화학식 1][Formula 1]
상기 리포좀 또는 지질 나노입자는 중성 지질을 추가로 포함할 수 있다.The liposome or lipid nanoparticle may further include a neutral lipid.
본 개시에서 '중성 지질'은 pH 변화의 영향 없이 지속적으로 중성을 가지는 지질 또는 pH 변화에 의하여 중성으로 전환되는 이온성 지질을 포함한다.In the present disclosure, 'neutral lipids' include lipids that have neutrality continuously without the influence of pH change or ionic lipids that are converted to neutrality by pH change.
상기 중성 지질은 1,2-디올레오일-sn-글리세로-3-포스포에탄올아민(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE), 1,2-디미리스토일-sn-글리세로-3-포스파티딜콜린(1,2-Dimyristoyl-sn-glycero-3-phosphorylcholine, DMPC), 1,2-디올레오일-sn-글리세로-3-포스포콜린(1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC), 1,2-디팔미토일-sn-글리세로-3-포스포콜린(1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC), 1,2-디스테아로일-sn-글리세로-3-포스포콜린(1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC), 1,2-디리노레오일-sn-글리세로-3-포스포콜린(1,2-dilinoleoyl-sn-glycero-3-phosphocholine, DLPC), 포스파티딜세린(PS), 포스포에탄올라민(PE), 포스파티딜글리세롤(PG), 포스포릭액시드(PA) 및/또는 포스파티딜콜린(PC)일 수 있고, 바람직하게는 1,2-디올레오일-sn-글리세로-3-포스포에탄올아민(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE)일 수 있으나, 이에 한정되는 것은 아니다.The neutral lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE), 1,2-dimyristoyl- sn-glycero-3-phosphatidylcholine (1,2-Dimyristoyl-sn-glycero-3-phosphorylcholine, DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (1,2-dioleoyl -sn-glycero-3-phosphocholine, DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC), 1, 2-distearoyl-sn-glycero-3-phosphocholine (1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC), 1,2-dilinoleoyl-sn-glycero-3 -Phosphocholine (1,2-dilinoleoyl-sn-glycero-3-phosphocholine, DLPC), phosphatidylserine (PS), phosphoethanolamine (PE), phosphatidylglycerol (PG), phosphoric acid (PA) and / or may be phosphatidylcholine (PC), preferably 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE) It may, but is not limited thereto.
구체적으로, 상기 'DOPE(1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine)'는 하기 화학식 2의 구조를 가지며, 양이온성 리포좀용 보조 지질로 사용된다.Specifically, 'DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine)' has a structure represented by
[화학식 2][Formula 2]
본 개시에서, 상기 양이온성 지질과 중성 지질의 중량비는 1:9 내지 9.5:0.5, 바람직하게는 2:8 내지 9:1, 더욱 바람직하게는 3:7 내지 8:2, 가장 바람직하게는 4:6 내지 7:3일 수 있으나, 이에 한정되는 것은 아니다. In the present disclosure, the weight ratio of the cationic lipid to the neutral lipid is 1:9 to 9.5:0.5, preferably 2:8 to 9:1, more preferably 3:7 to 8:2, most preferably 4 :6 to 7:3, but is not limited thereto.
상기 중량 비율을 벗어나는 경우, mRNA 전달 효율이 현저히 떨어질 수 있다.If the weight ratio is out of the range, mRNA delivery efficiency may be significantly reduced.
또한, 리포좀 또는 지질 나노입자는 콜레스테롤을 추가적으로 포함할 수 있다.In addition, liposomes or lipid nanoparticles may additionally contain cholesterol.
본 개시에서, 상기 양이온성 지질과 콜레스테롤의 중량비는 6:1 내지 1:3, 바람직하게는 4:1 내지 1:2.5, 더욱 바람직하게는 3:1 내지 1:2, 가장 바람직하게는 2:1 내지 1:1.5일 수 있으나, 이에 한정되는 것은 아니다.In the present disclosure, the weight ratio of the cationic lipid to cholesterol is 6:1 to 1:3, preferably 4:1 to 1:2.5, more preferably 3:1 to 1:2, most preferably 2: It may be 1 to 1:1.5, but is not limited thereto.
본 개시의 일 구현예에 있어서, 리포좀 또는 지질 나노입자는 PEG 변형된 지질을 추가적으로 포함할 수 있다. 상기 PEG 변형된 지질은 PEG-변형된 포스파티딜에탄올아민, PEG-변형된 포스파티드산, PEG-변형된 세라마이드, PEG-변형된 디알킬아민, PEG-변형된 디아실글리세롤, PEG-변형된 디알킬글리세롤, 또는 이들의 조합을 포함한다. 일 구현예에서, PEG-변형된 지질은 DMG-PEG, PEG-c-DOMG(PEG-DOMG로도 지칭됨), PEG-DSG, PEG-DPG, 또는 이들의 조합이나, 이에 한정되는 것은 아니다.In one embodiment of the present disclosure, the liposome or lipid nanoparticle may additionally include a PEG-modified lipid. The PEG modified lipid is PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkyl glycerol, or combinations thereof. In one embodiment, the PEG-modified lipid is, but is not limited to, DMG-PEG, PEG-c-DOMG (also referred to as PEG-DOMG), PEG-DSG, PEG-DPG, or combinations thereof.
또한, 본 개시에 따른 리포좀 또는 지질 나노입자에 양이온성 지질, 중성 지질 및 콜레스테롤이 모두 포함되는 경우, 상기 양이온성 지질, 중성 지질 및 콜레스테롤의 중량비는 1 내지 9.5:9 내지 0.5:0.05 내지 3, 바람직하게는 3 내지 8:7 내지 1:0.45 내지 7.0, 더욱 바람직하게는 1 내지 3.5:1 내지 3.5:0.5 내지 3일 수 있으나, 이에 한정되는 것은 아니다.In addition, when the liposome or lipid nanoparticle according to the present disclosure contains both cationic lipid, neutral lipid and cholesterol, the weight ratio of the cationic lipid, neutral lipid and cholesterol is 1 to 9.5:9 to 0.5:0.05 to 3, Preferably 3 to 8:7 to 1:0.45 to 7.0, more preferably 1 to 3.5:1 to 3.5:0.5 to 3, but is not limited thereto.
상기 중량 비율을 벗어나는 경우, mRNA 전달 효율이 현저히 떨어질 수 있다.If the weight ratio is out of the range, mRNA delivery efficiency may be significantly reduced.
예시적으로, 콜레스테롤이 추가로 포함될 경우, DOTAP:DOPE=1:1에 대해 콜레스테롤을 0.2 내지 0.85, 바람직하게는 0.5 내지 0.85의 중량비의 비율로 혼합하여 리포좀을 제조할 수 있다.Illustratively, when cholesterol is further included, liposomes may be prepared by mixing cholesterol at a weight ratio of 0.2 to 0.85, preferably 0.5 to 0.85, with respect to DOTAP:DOPE=1:1.
나아가, 상기 리포좀 또는 지질 나노입자는 protamine, albumin, transferrin, PTD(protein transduction domains), CPP(cell penetrating peptide) 및 Macrophage targeting moiety 등의 하나 이상의 전달용 인자를 추가적으로 포함할 수 있다.Furthermore, the liposome or lipid nanoparticle may additionally contain one or more delivery factors such as protamine, albumin, transferrin, protein transduction domains (PTD), cell penetrating peptide (CPP), and macrophage targeting moiety.
본 개시의 변이 SARS-CoV-2 백신 조성물에 있어서, 리포좀 또는 지질 나노입자와 mRNA의 혼합비율은 N:P ratio로 표시할 수 있으며, N:P ratio에 따라 발현 및 전달체의 안정성에 영향을 미친다.In the mutant SARS-CoV-2 vaccine composition of the present disclosure, the mixing ratio of liposomes or lipid nanoparticles and mRNA can be expressed as N:P ratio, and the N:P ratio affects the expression and stability of the delivery system .
본 개시의 변이 SARS-CoV-2 백신 조성물은 추가적으로 면역증강제가 포함될 수 있다.The mutant SARS-CoV-2 vaccine composition of the present disclosure may additionally include an immune enhancer.
상기 면역증강제는 비독성 리포올리고사카라이드(detoxied lipooligosaccharide, dLOS), 병원체연관 분자유형(Pathogen-associated molecular pattern, PAMP)에 해당하여 패턴인식수용체(Pathogen recognition receptor, PRR)에 반응하는 물질군, CpG DNA, lipoprotein, flagella, poly I:C, 사포닌, 스쿠알렌(Squalene), 트리카프린(tricaprin) 및/또는 3D-MPL일 수 있으나, 이에 한정되는 것은 아니다.The immune enhancer corresponds to non-toxic lipooligosaccharide (dLOS), Pathogen-associated molecular pattern (PAMP) and responds to the pattern recognition receptor (PRR), CpG. It may be DNA, lipoprotein, flagella, poly I:C, saponin, squalene, tricaprin and/or 3D-MPL, but is not limited thereto.
구체적으로, 상기 비독성 리포올리고사카라이드(dLOS)는 대한민국 등록특허 제1509456호 또는 대한민국 등록특허 제2042993호에 개시된 물질일 수 있지만, 이에 한정되는 것은 아니다.Specifically, the non-toxic lipooligosaccharide (dLOS) may be a substance disclosed in Korean Patent Registration No. 1509456 or Korean Patent Registration No. 2042993, but is not limited thereto.
본 개시의 다른 일 양태에 따르면, 본 개시는 변이 SARS-CoV-2의 S 항원을 코딩하는 mRNA; 및 리포좀 또는 지질 나노입자를 혼합하는 단계를 포함하는, 변이 SARS-CoV-2(severe acute respiratory syndrome coronavirus 2 variant) 백신 조성물의 제조방법을 제공한다.According to another aspect of the present disclosure, the present disclosure provides mRNA encoding the S antigen of the mutant SARS-CoV-2; And it provides a method for preparing a severe acute
상기 변이 SARS-CoV-2의 S 항원을 코딩하는 mRNA, 및/또는 리포좀 또는 지질 나노입자는 동결건조된 상태의 분말 형태로 제공될 수도 있고, 적절한 용액이나 버퍼에 용해된 상태로 제공될 수 있다. 변이 SARS-CoV-2의 S 항원을 코딩하는 mRNA, 및/또는 리포좀 또는 지질 나노입자가 동결건조된 상태로 제공될 경우, 적절한 용액이나 버퍼에 용해시켜 사용될 수 있다.The mRNA encoding the S antigen of the mutant SARS-CoV-2, and/or liposomes or lipid nanoparticles may be provided in the form of a lyophilized powder or dissolved in an appropriate solution or buffer. . When mRNA encoding the S antigen of mutant SARS-CoV-2 and/or liposome or lipid nanoparticles are provided in a lyophilized state, they can be used after being dissolved in an appropriate solution or buffer.
본 개시의 변이 SARS-CoV-2 백신 조성물의 제조방법은 면역증강제를 혼합하는 단계를 추가로 포함할 수 있다.The method for preparing the mutant SARS-CoV-2 vaccine composition of the present disclosure may further include mixing an immune enhancer.
상기 면역증강제는 동결건조된 상태의 분말 형태로 제공될 수도 있고, 적절한 용액이나 버퍼에 용해된 상태로 제공될 수 있다. 면역증강제가 동결건조된 상태로 제공될 경우, 적절한 용액이나 버퍼에 용해시켜 사용될 수 있다.The adjuvant may be provided in the form of a lyophilized powder or dissolved in an appropriate solution or buffer. When the immunostimulant is provided in a lyophilized state, it may be used after being dissolved in an appropriate solution or buffer.
본 개시의 변이 SARS-CoV-2의 S 항원을 코딩하는 mRNA, 및 리포좀 또는 지질 나노입자는 상술한 변이 SARS-CoV-2 백신 조성물의 유효 성분이므로, 중복되는 내용에 대해서는 본 명세서의 과도한 복잡성을 피하기 위하여 그 기재를 생략한다.Since the mRNA encoding the S antigen of the mutant SARS-CoV-2 of the present disclosure and the liposome or lipid nanoparticle are active ingredients of the above-described mutant SARS-CoV-2 vaccine composition, the excessive complexity of the present specification is avoided for redundant information. To avoid that, the description is omitted.
본 개시의 또 다른 일 양태에 따르면, 본 개시는 상술한 변이 SARS-CoV-2 백신을 대상체에 투여하는 단계를 포함하는 변이 SARS-CoV-2 감염증의 예방방법을 제공한다. According to another aspect of the present disclosure, the present disclosure provides a method for preventing a mutant SARS-CoV-2 infection comprising administering the above-described mutant SARS-CoV-2 vaccine to a subject.
상기 본 개시의 일 양태에 따른 변이 SARS-CoV-2 감염증의 예방방법은 상술한 본 발명의 일 양태에 따른 변이 SARS-CoV-2 백신 조성물을 구성요소로 포함하므로, 양 발명 간에 중첩되는 사항은 동일하게 적용된다. Since the method for preventing mutant SARS-CoV-2 infection according to one aspect of the present disclosure includes the mutant SARS-CoV-2 vaccine composition according to one aspect of the present invention as a component, overlapping matters between the two inventions The same applies.
상기 변이 SARS-CoV-2 백신 조성물의 투여 경로는 제한되지 않으나, 진피내, 근육내, 피하투여, 또는 비강내 투여를 포함한다. 상기 백신 조성물의 투여로 인한 변이 SARS-CoV-2 바이러스로부터의 예방 효과는 투여 후 적어도 7일, 8일, 9일, 10일, 11일, 12일, 13일, 또는 14일 후에 획득될 수 있다. The route of administration of the mutant SARS-CoV-2 vaccine composition includes, but is not limited to, intradermal, intramuscular, subcutaneous, or intranasal administration. The preventive effect from the mutant SARS-CoV-2 virus due to administration of the vaccine composition may be obtained after at least 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after administration. there is.
상기 투여 횟수는 1회, 2회, 3회, 4회 또는 그 이상 투여될 수 있으나, 1회의 투여로 충분한 예방 효과를 획득할 가능성이 있다. The number of administrations may be once, twice, three times, four times or more, but there is a possibility that a sufficient preventive effect can be obtained with one administration.
본 개시의 일 구현예에 있어서, 상기 백신 조성물은 대상체에서 항원 특이적 면역 반응(예를 들어, 코로나바이러스 항원에 특이적인 항체의 생산)을 생성하기 위한 유효량으로 제제화된다. "유효량"은 항원-특이적 면역 반응을 생성하는데 효과적인 RNA의 용량이다. In one embodiment of the present disclosure, the vaccine composition is formulated in an effective amount to generate an antigen-specific immune response (eg, production of antibodies specific to coronavirus antigens) in a subject. An "effective amount" is a dose of RNA effective to generate an antigen-specific immune response.
상기 면역 반응은 체액성 면역반응 및 세포성 면역반응을 포함한다.The immune response includes a humoral immune response and a cellular immune response.
"체액성" 면역 반응은 예를 들어, 분비(IgA) 또는 IgG 분자를 포함하는, 항체 분자에 의해 매개되는 면역반응을 지칭하는 반면, "세포성" 면역 반응은 T-림프구(예를 들어, CD4+ 헬퍼 및/또는 CD8+ T 세포(예를 들어, CTL)) 및/또는 기타 백혈구에 의해 매개되는 면역 반응이다.A "humoral" immune response refers to an immune response mediated by antibody molecules, including, for example, secretory (IgA) or IgG molecules, whereas a "cellular" immune response refers to a T-lymphocyte (eg, It is an immune response mediated by CD4+ helper and/or CD8+ T cells (eg, CTL)) and/or other leukocytes.
본 개시에 따른 변이 SARS-CoV-2 백신은 우수한 안정성과 in vivo에서 높은 면역원성을 나타내어, 백신의 보관 및 사용이 간편하고, 기존 백신으로는 감염 방어효과가 낮아지는 SARS-CoV-2의 변이 바이러스에 대한 뛰어난 예방 효과를 기대할 수 있다.The mutant SARS-CoV-2 vaccine according to the present disclosure exhibits excellent stability and high immunogenicity in vivo , making it easy to store and use the vaccine, and a SARS-CoV-2 mutant that has a low protective effect against infection with existing vaccines. Excellent preventive effect against viruses can be expected.
도 1은 본 개시의 mRNA인 EG-COVARo mRNA와 야생형인 Omicron WT mRNA의 2차 구조를 나타낸 도이다.
도 2는 본 개시의 EG-COVARo mRNA를 용량별로 투여한 경우, IgG 항체가를 측정한 결과를 나타낸 도이다.
도 3은 본 개시의 EG-COVARo mRNA를 용량별로 투여한 경우, 생성되는 중화항체 형성능을 나타낸 도이다.
도 4는 본 개시의 EG-COVARo mRNA를 용량별로 투여한 경우, 생성된 항체의 중화능을 확인하기 위하여 FRNT 결과를 나타낸 도이다. 1 is a diagram showing the secondary structures of EG-COVARo mRNA, which is an mRNA of the present disclosure, and Omicron WT mRNA, which is a wild type.
Figure 2 is a diagram showing the results of measuring the IgG antibody titer when the EG-COVARo mRNA of the present disclosure was administered by dose.
3 is a diagram showing the ability to form neutralizing antibodies produced when EG-COVARo mRNA of the present disclosure is administered by dose.
4 is a diagram showing FRNT results to confirm the neutralizing ability of the antibody produced when the EG-COVARo mRNA of the present disclosure was administered by dose.
실시예Example
이하, 실시예를 통하여 본 개시를 더욱 상세히 설명하고자 한다. 이들 실시 예는 오로지 본 개시를 보다 구체적으로 설명하기 위한 것으로, 본 개시의 범위가 이들 실시 예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present disclosure will be described in more detail through examples. These examples are only for explaining the present disclosure in more detail, and it will be apparent to those skilled in the art that the scope of the present disclosure is not to be construed as being limited by these examples.
준비예. mRNA 서열 선정ready yes. mRNA sequence selection
mRNA 내 구아닌(Guanine) 및 시토신(Cytosine)의 함량을 증가시켜 mRNA를 안정화시키고 인간(human)에서의 번역 효율을 증가시키기 위한 목적으로 다음의 방법을 통해 서열 최적화를 진행하였다.Sequence optimization was performed through the following method for the purpose of stabilizing mRNA by increasing the content of guanine and cytosine in mRNA and increasing translation efficiency in humans.
한편, SARS-CoV-2의 스파이크 단백질은 단백질 구조가 불안정하기 때문에 986(K) 및 987(V)의 아미노산을 프롤린 치환(proline substitution)(2P)하여 misfolding 또는 triggering을 방지함으로써 prefusion stabilized viral glycoprotein이 더 우수한 면역원으로 작용한다는 것이 선행연구를 통해 확인되었다.Meanwhile, since the protein structure of the spike protein of SARS-CoV-2 is unstable, misfolding or triggering is prevented by proline substitution (2P) of the amino acids 986(K) and 987(V), resulting in prefusion stabilized viral glycoprotein. It was confirmed through previous studies that it acts as a better immunogen.
이에, 스파이크 단백질의 단백질 구조를 안정화시키기 위해 986(K) 및 987(V)의 아미노산을 프롤린(Proline)으로, 더하여 682 내지 685의 아미노산 서열의 일부를 추가 변이시켜, GenArt codon optimization program으로 도출된 서열로 mRNA의 서열을 선택하였다. 수득한 mRNA는 "EG-COVARo mRNA"라고 명명하였다.Accordingly, in order to stabilize the protein structure of the spike protein, amino acids 986 (K) and 987 (V) were further mutated to proline, and a part of the amino acid sequence of 682 to 685 was additionally mutated, resulting in GenArt codon optimization program. The sequence of the mRNA was selected as the sequence. The obtained mRNA was named "EG-COVARo mRNA".
제조예. mRNA-리포좀 복합체(mRNA-liposome) 제조manufacturing example. Preparation of mRNA-liposome complex (mRNA-liposome)
1. mRNA 합성1. mRNA synthesis
Genscript사(미국)에 의뢰하여 선정된 mRNA 서열에 해당하는 DNA 서열을 포함하는 플라스미드를 제조하였다. 상기 플라스미드는 pmRNA_T7_13_Kan_80A (2925 bp)로, 유전자 삽입부에 T7, 5'-UTR, MCS, 3'-UTR, 및 poiy A tail(80)으로 이루어진 것으로, 상기 MCS 부위에 변이 SARS-CoV-2 항원을 인코딩하는 염기서열을 가진 뉴클레오타이드, 예컨대, 서열번호 1의 염기서열로 표시되는 mRNA 서열이 삽입되어 사용된다. 상기 플라스미드를 이용하여 in vitro transcription(IVT) 방법을 통해 mRNA를 수득하였다. SARS-CoV-2 omicron mRNA는 mRNA 자체의 항원성을 감소시키기 위하여 모든 우리딘을 5-메톡시-우리딘 (5-methoxyuridine, 5moU, TriLink BioTechnologies)로 치환하여 합성하였다. 1 M Tris-HCl (pH 8), 1 M Dithiothreitol (pH 8), 200 mM Spermidine, 10% Triton X-100, 1 M Magnesium acetate, 주사용수를 혼합하여 10X transcription buffer를 제조하였다.A plasmid containing a DNA sequence corresponding to the selected mRNA sequence was prepared by requesting Genscript (USA). The plasmid is pmRNA_T7_13_Kan_80A (2925 bp), consisting of T7, 5'-UTR, MCS, 3'-UTR, and poiy A tail (80) at the gene insertion part, and the SARS-CoV-2 antigen mutated at the MCS site A nucleotide having a nucleotide sequence encoding , for example, an mRNA sequence represented by the nucleotide sequence of SEQ ID NO: 1 is inserted and used. Using the plasmid, mRNA was obtained through an in vitro transcription (IVT) method. SARS-CoV-2 omicron mRNA was synthesized by replacing all uridine with 5-methoxyuridine (5moU, TriLink BioTechnologies) to reduce the antigenicity of the mRNA itself. A 10X transcription buffer was prepared by mixing 1 M Tris-HCl (pH 8), 1 M Dithiothreitol (pH 8), 200 mM Spermidine, 10% Triton X-100, 1 M Magnesium acetate, and water for injection.
상기 10X transcription buffer와 5moU, ATP, CTP, GTP, CleanCap AG, 플라스미드, RNase inhibitor, Pyrophosphate, T7 Polymerase를 혼합한 후 37℃ 항온수조에서 반응시켰다.After mixing the 10X transcription buffer with 5moU, ATP, CTP, GTP, CleanCap AG, plasmid, RNase inhibitor, Pyrophosphate, and T7 Polymerase, they were reacted in a constant temperature water bath at 37°C.
상기 혼합물에 DNA 분해효소 I (DNase I)과 DNase I reaction buffer를 첨가하고 37℃ 항온수조에서 반응시켜 DNA를 제거하였다.DNase I (DNase I) and DNase I reaction buffer were added to the mixture and reacted in a constant temperature water bath at 37° C. to remove DNA.
상기 혼합물에 주사용수, 10% SDS solution, 1 M Dithiothreitol (pH 8), Proteinase K를 첨가하고 37℃ 항온수조에서 반응시켜 단백질을 제거하였다.Water for injection, 10% SDS solution, 1 M Dithiothreitol (pH 8), and Proteinase K were added to the mixture and reacted in a constant temperature water bath at 37° C. to remove proteins.
Cation-exchange-HPLC 및 Diafiltration system을 통해 mRNA를 정제하고 완충액을 치환한 후 제균여과하여 mRNA를 수득하였다.The mRNA was purified through Cation-exchange-HPLC and a diafiltration system, the buffer was replaced, and the mRNA was obtained by sterilization filtration.
2. 리포좀 제조(Film method)2. Liposome preparation (Film method)
DOTAP(Merck & Cie/CH2900014), DOPE(Avanti Polar Lipid) 및/또는 콜레스테롤(Avanti Polar Lipid)에 클로로포름을 각각 혼합하여 3 mg/mL이 되도록 하고, 37 ℃에서 10분간 완전히 용해시켜 액상 용액으로 제조하였다.DOTAP (Merck & Cie/CH2900014), DOPE (Avanti Polar Lipid), and/or cholesterol (Avanti Polar Lipid) are mixed with chloroform, respectively, to a concentration of 3 mg/mL, and completely dissolved at 37 °C for 10 minutes to prepare a liquid solution. did
둥근바닥 플라스크에 상기 액상 용액을 일정 중량비로 혼합하여 지질 혼합물(lipid mixture)을 만들고, Rotary evaporator(Buchi/B491_R200)에서 60℃, 30분간 휘발시켜 클로로포름을 날려 플라스크 벽면에 지질막 필름을 제작하였다.A lipid mixture was prepared by mixing the liquid solution at a certain weight ratio in a round bottom flask, and volatilized at 60 ° C. for 30 minutes in a rotary evaporator (Buchi / B491_R200) to blow chloroform to form a lipid film on the flask wall.
상기 지질막 필름이 제작된 플라스크에 4% (w/v) 슈크로오스를 함유하는 20 mM HEPES 버퍼(pH 7.4)를 넣고 60℃에서 지질막을 녹여, 리포좀의 농도가 7.5 mg/mL이 되도록 리포좀을 형성하였다. 형성된 리포좀은 동적광산란 분석장비로 입자의 사이즈, 제타포텐셜, 분산도를 측정하였다. 제조된 나머지 리포좀은 시험 전까지 4℃에서 보관하였다.Add 20 mM HEPES buffer (pH 7.4) containing 4% (w/v) sucrose to the flask in which the lipid film film was prepared, melt the lipid film at 60 ° C, and prepare liposomes to a liposome concentration of 7.5 mg / mL. formed. The formed liposomes were measured for particle size, zeta potential, and dispersion using dynamic light scattering analysis equipment. The remaining prepared liposomes were stored at 4°C until testing.
3. mRNA-리포좀 복합체 제조3. Preparation of mRNA-liposome complexes
상온에서 4% 슈크로오스를 함유한 20 mM HEPES 버퍼(pH 7.4)에 상기에서 제조한 리포좀(LP-DOTAP/DOPE/Chol(40:40:20, w/w/w)) 및 mRNA를 혼합하여 mRNA-리포좀 복합체(100 ㎕)를 제조하였다. 리포좀과 mRNA의 혼합비율인 N/P 비율(N/P ratio)은 다음의 계산식으로 계산하였다.Mix the prepared liposome (LP-DOTAP/DOPE/Chol (40:40:20, w/w/w)) and mRNA in 20 mM HEPES buffer (pH 7.4) containing 4% sucrose at room temperature. Thus, an mRNA-liposome complex (100 μl) was prepared. The mixing ratio of liposome and mRNA, N/P ratio, was calculated by the following formula.
[식 1][Equation 1]
제조된 복합체는 하기와 같다:The composites prepared are as follows:
- Omicron-PQ SARS-CoV-2 S 단백질 mRNA(서열번호 1)-리포좀 복합체- Omicron-PQ SARS-CoV-2 S protein mRNA (SEQ ID NO: 1)-liposome complex
실시예 1. mRNA 2차 구조 분석Example 1. mRNA secondary structure analysis
Zuker 등이 도입한 루프 기반 에너지 모델과 동적 프로그래밍 알고리즘을 사용하여 EG-COVARo mRNA의 2차 구조를 분석하였다.The secondary structure of EG-COVARo mRNA was analyzed using the loop-based energy model and dynamic programming algorithm introduced by Zuker et al.
결과는 도 1에 나타내었다.The results are shown in Figure 1.
도 1에 나타낸 바와 같이, Omicron WT mRNA와 EG-COVARo mRNA (codon optimized) minimum free energy(MFE) 비교시 본 개시의 EG-COVARo가 -1344.50 kcal/mol으로 -1067.60kcal/mol의 WT보다 훨씬 열역학적으로 안정하였다. 또한, 구조 이미지 분석결과에서도 본 개시의 EG-COVARo에서는 빨강, 노랑, 초록색의 분포가 더 높아, 파랑 및 초록의 분포가 더 높은 WT보다 base pair probabilities가 높았다. 따라서, 본 개시의 EG-COVARo가 WT보다 안정적인 2차 구조를 더 잘 유지하는 것으로 나타났다.As shown in FIG. 1, when comparing Omicron WT mRNA and EG-COVARo mRNA (codon optimized) minimum free energy (MFE), EG-COVARo of the present disclosure is -1344.50 kcal/mol, which is much more thermodynamic than WT of -1067.60 kcal/mol. was stable. In addition, in the structural image analysis results, EG-COVARo of the present disclosure had higher distributions of red, yellow, and green, and thus had higher base pair probabilities than WT, which had higher distributions of blue and green. Thus, it was shown that EG-COVARo of the present disclosure retains a stable secondary structure better than WT.
실시예 2. mRNA-리포좀 복합체의 DLS (Dynamic Light Scattering) 분석Example 2. DLS (Dynamic Light Scattering) analysis of mRNA-liposome complexes
상기 제조예에서 제조된 mRNA-리포좀 복합체를 4% 슈크로오스를 함유하는 20 mM HEPES 버퍼(pH 7.4)로 1/10 희석하였다. Zetasizer Nano ZSP(Malvern Pnanlytical)로 DLS 분석을 진행하여, 복합체의 크기(size), 분산도(polydispersity index, PDI), 제타전위(Zeta potential)를 측정하였다.The mRNA-liposome complex prepared in Preparation Example was diluted 1/10 with 20 mM HEPES buffer (pH 7.4) containing 4% sucrose. DLS analysis was performed with a Zetasizer Nano ZSP (Malvern Pnanlytical) to measure the size, polydispersity index (PDI), and zeta potential of the complex.
결과는 하기 표 1에 나타내었다.The results are shown in Table 1 below.
표 1에 나타낸 바와 같이, 본 개시의 mRNA-리포좀 복합체는 크기 약 149 nm의 입자들이 상대적으로 고르게 분포하고 있으며, 분산도가 0.1에 가깝고 제타전위가 낮아 응집되지 않고 분산되어 구조적 안정도가 높은 것으로 나타났다.As shown in Table 1, the mRNA-liposome complex of the present disclosure showed that particles of about 149 nm in size were relatively evenly distributed, and the degree of dispersion was close to 0.1 and the zeta potential was low, so it was dispersed without aggregation and had high structural stability. .
실시예 3. mRNA-리포좀 복합체의 면역원성(immunogenicity) 확인 (IgG 항체 역가의 확인)Example 3. Confirmation of immunogenicity of mRNA-liposome complex (confirmation of IgG antibody titer)
상기 제조예에서 제조된 리포좀-mRNA 복합체에 대하여, 다음의 실험을 진행하였다. 각 실험에서, 6주령 암컷 마우스(B6C3F1/slc, 중앙실험동물)에 서로 다른 용량의 mRNA를 포함하도록 제조된 리포좀-mRNA 복합체를 마우스 왼쪽 뒷다리 대퇴부에 근육 투여 경로로 투여하였다. 구체적인 그룹별 투여 물질, 투여 용법, 및 투여 용량은 표 2에 나타내었다.With respect to the liposome-mRNA complex prepared in the above Preparation Example, the following experiment was conducted. In each experiment, liposome-mRNA complexes prepared to contain different doses of mRNA were administered to 6-week-old female mice (B6C3F1/slc, central experimental animal) by intramuscular administration to the left hind thigh of the mouse. Table 2 shows the substance to be administered, the administration method, and the administration dose for each specific group.
근육 내 투여2 times every 3 weeks
intramuscular administration
마지막 투여 2주 후에 Avertin working solution을 250 mg/kg으로 복강에 투여하여 마취시키고, 심장 채혈을 통해 전혈을 채취하였다. 채취된 전혈을 마이크로튜브로 옮겨 상온에 3시간 동안 정치한 후, 4℃, 15,000 rpm 조건으로 10 분 동안 원심분리하고, 상층액을 새로운 마이크로튜브로 옮겨 혈청을 확보해 분석 전까지 -20℃에 보관하였다.다음으로, 1X PBS를 이용하여 SARS-CoV-2 Omicron RBD (SARS-CoV-2 receptor binding domain) 항원(Mybiosource, USA)을 1 ㎍/mL로 희석한 후 이뮤노플레이트(immunoplate)에 100 ㎕/웰씩 분주하고 실링 필름을 덮어 4℃에 하룻밤 정치하였다. ELISA washer(Tecan/Hydroflexelisa)로 각 웰의 용액을 제거하고 세척 버퍼(20X PBS를 정제수로 희석한 1 L의 1X PBS에 500 ㎕의 tween 20을 투입)를 사용하여 3회 세척하였다. 이뮤노플레이트에 시약 희석제(reagent diluent, 1% BSA, 1 g의 BSA를 100 mL의 PBS에 녹여 제조)를 200 ㎕/웰씩 분주하고 실링 필름을 덮어 37℃ 반응기에서 1시간 동안 정치하였다. ELISA washer로 각 웰의 용액을 제거하고 세척 버퍼를 사용하여 3회 세척하였다. 시약 희석제를 이뮤노플레이트에 100 ㎕/웰씩 분주하였다.Two weeks after the last administration, Avertin working solution was administered intraperitoneally at 250 mg/kg to anesthetize, and whole blood was collected through cardiac blood sampling. The collected whole blood was transferred to a microtube, allowed to stand at room temperature for 3 hours, centrifuged at 4°C and 15,000 rpm for 10 minutes, and the supernatant was transferred to a new microtube to obtain serum and stored at -20°C until analysis. Next, the SARS-CoV-2 Omicron RBD (SARS-CoV-2 receptor binding domain) antigen (Mybiosource, USA) was diluted to 1 μg/mL using 1X PBS, and then plated on an immunoplate at 100 μg/mL. It was dispensed by μl/well, covered with a sealing film, and allowed to stand overnight at 4°C. The solution in each well was removed with an ELISA washer (Tecan/Hydroflexelisa) and washed three times using a washing buffer (500 μl of
시약 희석제(1% BSA)를 이용하여 상기에서 확보한 혈청을 1:50으로 희석한 후, 이뮤노플레이트의 B ~ F의 1열에 100 ㎕로 분주하고, 웰 내에서 수회 파이펫팅하여 시료를 혼합한 후, 1열에서 100 ㎕를 취해 2번 열에 넣는 방법으로 ELISA 플레이트 상에서 시료를 12열까지 1/2 순차 희석(serial dilution)하였다. 이때, 플레이트 간 실험 조건의 동등성 확인을 위해, 시약 희석제를 이용하여 과혈청(hyper serum)을 1:100으로 희석한 뒤 각 이뮤노플레이트 H의 1열에 100 ㎕로 분주하고 상기와 같은 방법으로 1/2 순차 희석하였다.After diluting the serum obtained above 1:50 using a reagent diluent (1% BSA), dispense 100 μl in rows B to F of the immunoplate, and mix the samples by pipetting several times in the well After that, the sample was serially diluted 1/2 up to column 12 on the ELISA plate by taking 100 μl from column 1 and putting it in
이뮤노플레이트를 실링 필름으로 덮어 37℃ 반응기에서 2시간 동안 반응시켰다. ELISA washer로 각 웰의 용액을 제거하고 세척 버퍼를 사용하여 5회 세척하였다. 시약 희석제를 이용하여 goat anti-mouse IgG 항체(Jackson Laboratory)를 1:5,000으로 희석한 후, 이뮤노플레이트에 100 ㎕씩 분주하고 실링 필름을 덮어 37 ℃ 반응기에서 1시간 동안 반응시켰다. ELISA washer로 각 웰의 용액을 제거하고 세척 버퍼를 사용하여 3회 세척하였다.The immunoplate was covered with a sealing film and reacted for 2 hours in a 37° C. reactor. The solution in each well was removed with an ELISA washer and washed 5 times using wash buffer. After diluting goat anti-mouse IgG antibody (Jackson Laboratory) 1:5,000 using a reagent diluent, 100 μl of each was dispensed into an immunoplate, covered with a sealing film, and allowed to react for 1 hour in a 37 °C reactor. The solution in each well was removed with an ELISA washer and washed three times using a washing buffer.
상온과 평형화시킨 TMB 기질용액을 이뮤노플레이트에 100 ㎕/웰씩 분주하고 상온의 암소에서 3분 30초 동안 반응시켰다. 1N H2SO4 용액을 이뮤노플레이트에 100 ㎕씩 분주하여 반응을 정지하고 ELISA reader(TECAN/SPARK)를 사용하여 450 nm에서 흡광도를 측정하였다.100 μl/well of the TMB substrate solution equilibrated with room temperature was dispensed into the immunoplate and reacted for 3 minutes and 30 seconds in the dark at room temperature. The reaction was stopped by dispensing 100 μl of the 1N H 2 SO 4 solution into the immunoplate, and the absorbance was measured at 450 nm using an ELISA reader (TECAN/SPARK).
결과는 도 2에 나타내었다.The results are shown in Figure 2.
도 2에 나타낸 바와 같이, 분석 결과, 모든 용량의 EG-COVARo 투여군에서 음성대조군 대비 항체가가 유의미하게 증가하였다 (p<0.01). 특히 5 ㎍/head, 10 ㎍/head 투여군에서 용량 증가에 따라 항체가가 증가하는 경향이 관찰되었다.As shown in FIG. 2, as a result of the analysis, the antibody titer was significantly increased compared to the negative control group in the EG-COVARo administration group at all doses (p<0.01). In particular, in the 5 μg/head and 10 μg/head administration groups, an increase in antibody titer was observed with increasing dose.
실시예 4. 중화항체형성능(Neutralization)(%)Example 4. Neutralization (%)
본 개시의 EG-COVARo 백신을 투여하여 수득한 혈청 샘플에 대해 SARS-CoV-2 surrogate virus neutralization test (sVNT) kit (Genscript)를 이용하여 상기 백신에 의하여 바이러스 감염이 효과적으로 억제되는지를 확인하고자 하였다.SARS-CoV-2 surrogate virus neutralization test (sVNT) kit (Genscript) was used for serum samples obtained by administering the EG-COVARo vaccine of the present disclosure to confirm whether the vaccine effectively inhibits viral infection.
8 스트립 튜브의 1번부터 5번까지 튜브에 63㎕씩 sample dilution buffer를 분주한 후, 1번 튜브에 혈청 시료 7㎕를 담고 튜브 내에서 수회 파이펫팅하여 7㎕를 취해 다음 튜브에 넣는 방법으로 시료를 5번째 튜브까지 연속으로 희석하여 희석된 혈청 시료를 수득하였다. After dispensing 63 μl of sample dilution buffer to tubes 1 to 5 of the 8-strip tube, put 7 μl of serum sample in tube 1, pipette several times in the tube, take 7 μl, and put it into the next tube. Samples were serially diluted to the fifth tube to obtain diluted serum samples.
1.5mL 마이크로튜브에 음성 대조군, 양성 대조군 6㎕를 sample dilution buffer 54㎕와 혼합하여 1:10으로 희석하였고, 이뮤노플레이트에 희석된 혈청 샘플을 60㎕/웰씩 분주한 뒤 1:1000 diluted-HPR conjugated Omicron RBD 60㎕를 첨가한 후 37℃에서 30분 동안 반응시켰다. Microtiter test strip plate에 상기 혼합액 100 ㎕를 분주한 다음 실링 필름을 덮어 37℃에서 15분 동안 반응시켰다. In a 1.5mL microtube, 6μl of the negative control and positive control were mixed with 54μl of sample dilution buffer and diluted 1:10, and the diluted serum sample was dispensed to the immunoplate at 60μl/well, followed by 1:1000 diluted-HPR. After adding 60 μl of conjugated Omicron RBD, the mixture was reacted at 37° C. for 30 minutes. 100 μl of the mixture was dispensed on a microtiter test strip plate, covered with a sealing film, and reacted at 37° C. for 15 minutes.
각 웰의 용액을 제거하고 1X 세척 용액으로 4회 세척하였다. TMB solution를 100 ㎕/웰씩 분주하고 실링 필름을 덮어 상온의 암소에서 15분 동안 반응시킨 후, 정지 용액을 50 ㎕/웰씩 분주하여 반응을 정지시킨 다음, ELISA reader (TECAN/SPARK)를 사용하여 450nm에서 광학 밀도 (optical density)를 측정한 후, Omicron RBD 단백질 중화능 (sVNT, %)을 다음의 계산식으로 계산하였다.The solution in each well was removed and washed 4 times with 1X washing solution. After dispensing 100 μl/well of the TMB solution and reacting for 15 minutes in the dark at room temperature by covering the sealing film, stop the reaction by dispensing the stop solution by 50 μl/well, and then using an ELISA reader (TECAN/SPARK) at 450 nm After measuring the optical density, Omicron RBD protein neutralizing ability (sVNT, %) was calculated by the following formula.
[식 2][Equation 2]
Omicron RBD 단백질 중화능 (%)이 50%로 감소되는 수치 (Inhibitory Concentration 50 titer, IC50 titer)는 GraphPad Prism software의 Nonlinear regression → dose-response (Inhibition) → log (inhibitor) vs. normalized response를 통해 도출하였다.The value (Inhibitory Concentration 50 titer, IC 50 titer) at which Omicron RBD protein neutralizing ability (%) is reduced by 50% is Nonlinear regression of GraphPad Prism software → dose-response (Inhibition) → log (inhibitor) vs. It was derived through the normalized response.
결과는 도 3에 나타내었다.The results are shown in Figure 3.
도 3에 나타낸 바와 같이, 5 ㎍/head, 10 ㎍/head 투여군에서 용량 증가에 따라 Omicron RBD 단백질에 대한 중화능이 증가하는 경향이 관찰되었다.As shown in FIG. 3, in the 5 μg/head and 10 μg/head administration groups, a tendency for the neutralizing ability to Omicron RBD protein to increase with increasing dose was observed.
실시예 5. Focus Reduction Neutralization test (FRNT) 분석Example 5. Focus Reduction Neutralization test (FRNT) analysis
본 개시의 SARS-CoV-2 Omicron variant virus의 중화능을 확인하기 위해 상기 실시예 3에서 채취한 혈청을 이용하여 Focus Reduction Neutralization test (FRNT)를 수행하였다.In order to confirm the neutralizing ability of the SARS-CoV-2 Omicron variant virus of the present disclosure, a Focus Reduction Neutralization test (FRNT) was performed using the serum collected in Example 3.
Vero cell을 96 웰 플레이트에 seeding하였고, 다음날 혈청을 1:20으로 1차 희석한 후 4-fold로 연속 희석하였다. 상기 희석된 혈청과 Omicron virus (NCCP43408)를 1:1 (v/v)로 혼합하여 30분간 배양시킨 후, Vero cell을 wash한 후 혈청-Omicron virus 혼합액으로 Vero cell을 감염시켰다.Vero cells were seeded in a 96-well plate, and the next day, serum was first diluted 1:20 and serially diluted 4-fold. After mixing the diluted serum and Omicron virus (NCCP43408) at a ratio of 1:1 (v/v) and incubating for 30 minutes, the Vero cells were washed and infected with the serum-Omicron virus mixture.
감염 8시간 후 10% Formalin으로 고정하여 anti-SARS-CoV-2 NP antibody로 감염된 Vero cell을 표지한 후, TrueBlue로 발색하고 ELISPOT reader를 이용하여 foci 개수를 측정하였다.After 8 hours of infection, the infected Vero cells were fixed with 10% formalin, labeled with anti-SARS-CoV-2 NP antibody, and then colored with TrueBlue, and the number of foci was measured using an ELISPOT reader.
결과는 도 4에 나타내었다.The results are shown in FIG. 4 .
도 4에 나타낸 바와 같이, 모든 용량의 EG-COVARo 투여군에서 음성대조군 대비 중화항체역가가 유의미하게 증가하였다. (p<0.01) 특히, 투여 용량에 의존적으로 중화항체 역가가 증가하는 것이 확인되었다.As shown in FIG. 4, the neutralizing antibody titer significantly increased compared to the negative control group in the EG-COVARo administered group at all doses. (p<0.01) In particular, it was confirmed that the neutralizing antibody titer increased depending on the administered dose.
이상으로 본 개시의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 개시의 범위가 제한되는 것이 아닌 점은 명백하다.Since certain parts of the present disclosure have been described in detail above, it is clear that these specific descriptions are only preferred implementation examples for those skilled in the art, and the scope of the present disclosure is not limited thereto.
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