WO2023201881A1 - Use of lentinan in preparing sars-cov-2 respiratory mucosal vaccine - Google Patents

Use of lentinan in preparing sars-cov-2 respiratory mucosal vaccine Download PDF

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WO2023201881A1
WO2023201881A1 PCT/CN2022/101512 CN2022101512W WO2023201881A1 WO 2023201881 A1 WO2023201881 A1 WO 2023201881A1 CN 2022101512 W CN2022101512 W CN 2022101512W WO 2023201881 A1 WO2023201881 A1 WO 2023201881A1
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lentinan
hamsters
vaccine
new coronavirus
adjuvant
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赵平
郑旭
江亮亮
彭浩然
唐海琳
王文
丁翠玲
刘燕
戚中田
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中国人民解放军海军军医大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55583—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention belongs to the field of medical technology, and specifically relates to the application of lentinan in the preparation of a novel coronavirus respiratory mucosal vaccine.
  • the new coronavirus (SARS-CoV-2), hereinafter referred to as the new coronavirus, is extremely contagious and can spread rapidly among people, which can cause viral pneumonia (Coronavirus Disease 2019, COVID-19) in some infected people. . Since the outbreak was discovered in early 2020, the new coronavirus has spread rapidly around the world, and mutant strains with significant changes in transmissibility and pathogenicity have continued to emerge. The common feature of these mutant strains is that they have significant immune evasion capabilities and can break through the immune protection barriers established by the population due to previous infection with the virus or vaccination.
  • Vaccines are the most effective way for humans to prevent pathogen infection and reduce the damage caused by pathogens. Faced with the huge challenge of the new coronavirus continuing to evolve to evade population immunity, the development of new vaccines for upper respiratory tract mucosal immunization has become an important measure to prevent and control the new coronavirus epidemic. and urgent needs.
  • Current vaccines administered via intramuscular injection including inactivated vaccines, adenovirus vector vaccines, recombinant antigen subunit vaccines, and mRNA vaccines, are unable to induce respiratory mucosal immunity, thus limiting their ability to prevent viral infections.
  • the neutralizing antibodies induced by various current COVID-19 vaccines can only last for a short period of several months, and immunization with intramuscular vaccines requires a large amount of dedicated human resources to support it.
  • Vaccines administered through the respiratory mucosal route such as intranasal vaccination or nasal spray vaccination, are not only convenient to inoculate, but also can effectively induce upper respiratory mucosal immunity, which is the first barrier against the new coronavirus invading the human body, so it is a better vaccine choice.
  • the adenovirus vector vaccine can also be vaccinated through the respiratory mucosal route, the prevention and control of new coronavirus infection has to be boosted at a certain interval. This type of vaccine is not suitable for use in the same vaccination route because it induces antibodies against the coat protein of the adenovirus itself. homologous booster immunity.
  • the spike protein on the surface of the new coronavirus envelope mediates the binding of the virus to host cell membrane surface receptors and triggers the fusion of the viral envelope and the host endosomal membrane. Therefore, it is the target antigen for inducing virus neutralizing antibodies and is also the target antigen of most current new coronavirus vaccines. . mRNA vaccines, adenovirus vector vaccines and recombinant subunit vaccines based on this target antigen are all on the market and promoted globally.
  • Lentinan is an effective active ingredient extracted from high-quality Lentinus edodes fruiting bodies. It is an immunomodulator, especially effective in enhancing T cell activity. Clinical and pharmacological studies have shown that lentinan has anti-viral, anti-tumor, modulating immune function and stimulating the formation of interferon. Its active ingredient is branched ⁇ -(1-3)-D-glucan. The main chain is composed of ⁇ -(1-3)-linked glucose groups, and ⁇ -(1-) is randomly distributed along the main chain. 6) The connected glucose groups have a comb-like structure.
  • Lentinan injection and oral lentinan tablets are clinically used in many countries, including my country, for the adjuvant treatment of chronic hepatitis and tumors. They can effectively enhance patients' anti-viral and anti-tumor immune responses and are safe. Studies have also reported that lentinan can be used as a vaccine adjuvant and can effectively induce protective immune responses against Trichinella spiralis and influenza viruses when inoculated into mice via intramuscular or subcutaneous injection. References: 1.
  • Lentinan improved the efficacy of vaccine against Trichinella spiralis in an NLRP3 dependent manner.
  • the present invention aims to provide a new coronavirus recombinant subunit vaccine that uses lentinan as an adjuvant and is inoculated through the respiratory mucosa to prevent new coronavirus infection.
  • the invention provides the application of lentinan in preparing a novel coronavirus respiratory mucosal vaccine.
  • Lentinan as an effective upper respiratory tract mucosal immune adjuvant, is used as an immune adjuvant for the new coronavirus recombinant antigen or inactivated virus, and is inoculated through nasal drops or nasal spray. , used to prevent novel coronavirus infection.
  • the application provided by the present invention is characterized in that the vaccination route of the COVID-19 vaccine with lentinan as an adjuvant includes intranasal drops and nasal spray.
  • the present invention uses the new coronavirus envelope spike protein as an antigen, mixes the antigen protein solution and lentinan solution, injects intranasally into Syrian golden hamsters, then detects the antibodies in the hamster serum, and infects Syrian golden hamsters with live virus. It was detected that the intranasal vaccine can effectively induce hamsters to produce anti-new coronavirus spike protein receptor-binding functional region IgG antibodies, and effectively protect hamsters against new coronavirus infection, reduce the viral load in the upper respiratory tract of hamsters and alleviate the symptoms of hamsters. The disease occurs in mice and has application prospects.
  • Enzyme-linked immunosorbent assay was used to detect anti-RBD-specific IgG antibodies in hamster serum after the first and second immunizations, that is, on days 14 and 28 in the figure.
  • Str is the trimer of the extracellular segment of the new coronavirus envelope spike protein.
  • Ixazomib used in the embodiments of the present invention can be purchased commercially.
  • the SARS-CoV-2 virus was isolated and cultured from the nasopharyngeal swab samples of COVID-19 patients by the Biomedical Protection Teaching and Research Office of the Naval Medical University. Its gene sequence can be found in GenBank Accession No. MZ664555. The virus was cultured in Vero E6 cells. The cultured virus was infected with Vero E6 cells, and 18 hours later, immunofluorescence staining was used to detect the virus titer (focus forming units, FFU), which is the number of infectious virus particles per milliliter of virus fluid. All experimental operations involving viral infection were conducted in the P3 laboratory of the Naval Medical University.
  • FFU focus forming units
  • the vaccine antigen is the full-length extracellular segment of the new coronavirus spike envelope protein, in which the amino acid residues PRRARS of the furin site between the S1/S2 subunits are replaced by GSAS, and the leucine at position 986 and the leucine at position 987
  • the valine was mutated to proline, and the carboxyl terminus was fused to the T4 phage fibrin trimer motif.
  • the expression product forms a homotrimer (Str), is secreted and expressed in CHO cells by the Biomedical Protection Teaching and Research Office of the Newcastle Medical University.
  • Lentinan purchased from MCE (MedChemExpress) company, catalog number: HY-N6653, dissolved in DMSO.
  • Aluminum hydroxide adjuvant (Al adjuvant), purchased from InvivoGen Company, catalog number: vac-alu-250.
  • New coronavirus nucleic acid fluorescence quantitative PCR detection reagent was purchased from Shanghai Berger Biotechnology Co., Ltd.
  • RBD Receptor-binding domain
  • the negative control group was further divided into a blank control group (5 animals) and an infection control group (8 animals) during the virus challenge, with a total of 13 animals: nasal drip of PBS, nasal drip on both nostrils, 25 ⁇ l/each nostril;
  • Str+LNT intranasal drip group 8 animals: intranasal instillation into both nostrils, 25 ⁇ l/each nostril, containing 10 ⁇ g of Str protein and 200 ⁇ g of LNT;
  • Str+Al adjuvant intramuscular injection group 8 animals: intramuscular injection into both thighs, 100 ⁇ l on each side, containing 10 ⁇ g of Str protein and 80 ⁇ g of aluminum hydroxide.
  • a total of two immunizations were performed with an interval of 14 days. The second immunization was performed on the day after blood collection.
  • Antibody detection was performed according to the conventional ELISA technique.
  • the RBD protein was coated on a high-adsorption enzyme-labeled microplate, with 0.1 ⁇ g of protein per well, and placed in a refrigerator at 4°C overnight.
  • the protein solution was aspirated the next day and washed with phosphate buffer saline (PBS, pH value 7.0), wash the wells once, then block with PBS containing 3% bovine serum albumin (3% BSA-PBS) at room temperature for 2 hours, then aspirate the blocking solution, wash the wells 3 times with PBS; add two consecutive times to each well Diluted hamster serum, the diluent is 3% BSA-PBS, the volume is 100 ⁇ l/well, and placed in a refrigerator at 4°C overnight; the next day, aspirate the serum diluent and use it with PBS containing 0.05% Tween 20 (0.05% Tween 20 -PBS), wash the wells 5 times, then add 1000-fold
  • RBD is the main target of neutralizing antibodies against the new coronavirus, and the RBD antibody level in the serum represents the virus neutralizing ability.
  • the RBD IgG antibody titers in the sera of each group of hamsters are shown in Figure 1.
  • a single Str intranasal immunization that is, intranasal immunization with the trimer of the extracellular segment of the new coronavirus envelope spike protein, induced extremely low RBD IgG antibody levels, while Intranasal immunization with Str combined with lentinan can induce high levels of RBD IgG antibodies, which are similar to those induced by intramuscular immunization with Str combined with human adjuvant aluminum hydroxide.
  • each group of hamsters was challenged with the new coronavirus by intranasal instillation at a dose of 1.8*10 8 FFU.
  • 8 were used as the infected control group, and the remaining 5 were not infected with the virus and were used as the blank control group.
  • the body weight of the hamsters was measured starting from the day of virus challenge (before intranasal virus administration) and then every day thereafter.
  • 3 hamsters from each group were anesthetized with isoflurane and killed. The turbinates were cut and ground with a homogenizer.
  • Trizol LS reagent was used to extract tissue cell RNA, and reverse transcription real-time fluorescence quantification technology was used. The nucleic acid content of the nucleocapsid N gene of the new coronavirus was detected, and the relative levels of hamsters in each group were calculated. The remaining 5 hamsters in each group continued to observe changes in body weight until the 15th day.
  • the relative levels of the N gene nucleic acid of the new coronavirus in the turbinates of each group of hamsters on the 4th day after virus challenge are shown in Figure 2: If the average level of the N gene on the 4th day of the 3 hamsters in the infected control group is set to 1, then a single The relative level of the Str intranasal drip group was 0.81; the relative level of the Str + aluminum hydroxide intramuscular injection group was 0.79; the relative level of the Str + LNT intranasal injection group was 0.14. The relative level of N gene in the Str+LNT intranasal drip group was significantly lower than that in other groups (p ⁇ 0.001, p ⁇ 0.001, p ⁇ 0.01; t test).
  • the weight changes of hamsters are shown in Figure 3: the weight of hamsters in the blank control group continued to increase; the weight of hamsters in the infected control group continued to decrease after the virus challenge, with the maximum reduction reaching 14.6%, and began to rise after the 6th day; a single Str drop
  • the weight change of the nasal group was similar to that of the infected control group; the weight change of the Str+aluminum hydroxide intramuscular injection group showed a gentle increase, showing that it has a better immune protective effect; compared with the Str+aluminum hydroxide intramuscular injection group, the Str+LNT drop
  • the nose group gained greater weight, indicating better immune protection.
  • the spike protein of the new coronavirus was combined with the human adjuvant aluminum hydroxide for intramuscular immunization.
  • the weight changes of the hamsters showed that this immunization method also had a significant protective effect.
  • the replication level of the new coronavirus in the turbinates of the hamsters combined with intramuscular injection of aluminum hydroxide was higher, and the weight gain was lower.
  • lentinan can be used as an effective immune adjuvant for the upper respiratory tract mucosa.
  • the new coronavirus envelope spike protein combined with lentinan intranasal inoculation can prevent new coronavirus infection.

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Abstract

The present invention relates to the technical field of pharmaceutics, and particularly, to use of lentinan in preparing an SARS-CoV-2 respiratory mucosal vaccine. The SARS-CoV-2 respiratory mucosal vaccine is a vaccine using lentinan as the only immunologic adjuvant or using a composite adjuvant comprising lentinan administered by means of nasal drop or nasal spray to prevent SARS-CoV-2 infection.

Description

香菇多糖在制备新型冠状病毒呼吸道粘膜疫苗中的应用Application of Lentinan in the Preparation of Novel Coronavirus Respiratory Mucosal Vaccine 技术领域Technical field
本发明属于医药技术领域，具体涉及香菇多糖在制备新型冠状病毒呼吸道粘膜疫苗中的应用。The invention belongs to the field of medical technology, and specifically relates to the application of lentinan in the preparation of a novel coronavirus respiratory mucosal vaccine.
背景技术Background technique
新型冠状病毒(SARS-CoV-2)，下文中简称为新冠病毒，具有极高的传染性和快速的人群间传播能力，可导致部分感染者出现病毒性肺炎(Coronavirus Disease 2019，COVID-19)。自2020年初疫情被发现以后，新冠病毒迅速在全球传播，并不断出现传播力和致病力发生明显改变的突变株。这些突变株的共同特征是具备显著的免疫逃逸能力，能突破人群因先前感染病毒或接种疫苗而建立的免疫保护屏障。尤其是2021年11月在南非首次被发现的奥密克戎突变株，具有极强的传染性、传播力和免疫逃逸能力，迅速在全球范围内取代先前流行的主要突变株-德尔塔突变株。未来，人类将长期面对新冠病毒的威胁。The new coronavirus (SARS-CoV-2), hereinafter referred to as the new coronavirus, is extremely contagious and can spread rapidly among people, which can cause viral pneumonia (Coronavirus Disease 2019, COVID-19) in some infected people. . Since the outbreak was discovered in early 2020, the new coronavirus has spread rapidly around the world, and mutant strains with significant changes in transmissibility and pathogenicity have continued to emerge. The common feature of these mutant strains is that they have significant immune evasion capabilities and can break through the immune protection barriers established by the population due to previous infection with the virus or vaccination. In particular, the Omicron mutant strain, which was first discovered in South Africa in November 2021, is extremely contagious, transmissible, and immune evasion capable, and quickly replaced the previously circulating main mutant strain - the Delta mutant strain - globally. . In the future, mankind will face the threat of the new coronavirus for a long time.
疫苗是人类防止病原体感染和减轻病原体引起病损的最有效方式，面临新冠病毒通过持续进化以逃避人群免疫的巨大挑战，开发经上呼吸道粘膜免疫接种的新型疫苗成为当前防控新冠疫情的重要举措和迫切需求。当前以肌肉注射途径接种的疫苗，包括灭活疫苗、腺病毒载体疫苗、重组抗原亚单位疫苗、mRNA疫苗，均不能诱导呼吸道粘膜免疫，因而限制了其预防病毒感染的能力。此外，当前各种新冠疫苗诱导的中和抗体均仅可维持数月的较短时间，且肌注疫苗的免疫接种需要大量专门的人力资源进行支撑。通过呼吸道粘膜途径接种，如滴鼻接种、鼻腔喷雾接种的疫苗，不仅接种方便，而且可以高效诱导上呼吸道粘膜免疫，从抵抗新冠病毒侵入人体的第一道屏障，因而是更好的疫苗选择。虽 然腺病毒载体疫苗也可通过呼吸道粘膜途径接种，但防控新冠病毒感染不得不间隔一定时间即进行加强免疫，该类型疫苗因诱导针对腺病毒本身外壳蛋白的抗体而不适合用于相同接种途径的同源加强免疫。Vaccines are the most effective way for humans to prevent pathogen infection and reduce the damage caused by pathogens. Faced with the huge challenge of the new coronavirus continuing to evolve to evade population immunity, the development of new vaccines for upper respiratory tract mucosal immunization has become an important measure to prevent and control the new coronavirus epidemic. and urgent needs. Current vaccines administered via intramuscular injection, including inactivated vaccines, adenovirus vector vaccines, recombinant antigen subunit vaccines, and mRNA vaccines, are unable to induce respiratory mucosal immunity, thus limiting their ability to prevent viral infections. In addition, the neutralizing antibodies induced by various current COVID-19 vaccines can only last for a short period of several months, and immunization with intramuscular vaccines requires a large amount of dedicated human resources to support it. Vaccines administered through the respiratory mucosal route, such as intranasal vaccination or nasal spray vaccination, are not only convenient to inoculate, but also can effectively induce upper respiratory mucosal immunity, which is the first barrier against the new coronavirus invading the human body, so it is a better vaccine choice. Although the adenovirus vector vaccine can also be vaccinated through the respiratory mucosal route, the prevention and control of new coronavirus infection has to be boosted at a certain interval. This type of vaccine is not suitable for use in the same vaccination route because it induces antibodies against the coat protein of the adenovirus itself. homologous booster immunity.
新冠病毒包膜表面的刺突蛋白介导病毒和宿主细胞膜表面受体结合以及触发病毒包膜和宿主内体膜融合，因而是诱导病毒中和抗体的靶抗原，也是当前多数新冠疫苗的靶抗原。基于该靶抗原的mRNA疫苗、腺病毒载体疫苗以及重组亚单位疫苗都有上市，并在全球推广。The spike protein on the surface of the new coronavirus envelope mediates the binding of the virus to host cell membrane surface receptors and triggers the fusion of the viral envelope and the host endosomal membrane. Therefore, it is the target antigen for inducing virus neutralizing antibodies and is also the target antigen of most current new coronavirus vaccines. . mRNA vaccines, adenovirus vector vaccines and recombinant subunit vaccines based on this target antigen are all on the market and promoted globally.
香菇多糖(Lentinan，LNT)是从优质香菇子实体中提取的有效活性成分，是一种免疫调节剂，尤其是可有效增强T细胞活性。临床与药理研究表明，香菇多糖具有抗病毒、抗肿瘤、调节免疫功能和刺激干扰素形成等作用。其活性成分是具有分支的β-(1-3)-D-葡聚糖，主链由β-(1-3)-连接的葡萄糖基组成，沿主链随机分布着由β-(1-6)连接的葡萄糖基，呈梳状结构。香菇多糖注射液和口服香菇多糖片在包括我国在内的多个国家被临床用于慢性肝炎和肿瘤的辅助治疗，可有效增强患者的抗病毒和抗肿瘤免疫应答，安全性好。也有研究报道，香菇多糖可作为疫苗佐剂，通过肌肉注射、皮下注射接种小鼠，可有效诱导针对旋毛虫和流感病毒的保护性免疫应答。参考文献：1、Lentinan improved the efficacy of vaccine against Trichinella spiralis in an NLRP3 dependent manner.Jin X,Liu X,Ding J,Zhang L,Yang Y,Wang X,Yang Y,Liu M.PLoS Negl Trop Dis.2020 Sep 25；14(9):e0008632；2、Immune-adjuvant activity of lentinan-modified calcium carbonate microparticles on a H5N1 vaccine.He J,Liu Z,Jiang W,Zhu T,Wusiman A,Gu P,Liu J,Wang D.Int J Biol Macromol.2020 Nov 15；163:1384-1392.Lentinan (LNT) is an effective active ingredient extracted from high-quality Lentinus edodes fruiting bodies. It is an immunomodulator, especially effective in enhancing T cell activity. Clinical and pharmacological studies have shown that lentinan has anti-viral, anti-tumor, modulating immune function and stimulating the formation of interferon. Its active ingredient is branched β-(1-3)-D-glucan. The main chain is composed of β-(1-3)-linked glucose groups, and β-(1-) is randomly distributed along the main chain. 6) The connected glucose groups have a comb-like structure. Lentinan injection and oral lentinan tablets are clinically used in many countries, including my country, for the adjuvant treatment of chronic hepatitis and tumors. They can effectively enhance patients' anti-viral and anti-tumor immune responses and are safe. Studies have also reported that lentinan can be used as a vaccine adjuvant and can effectively induce protective immune responses against Trichinella spiralis and influenza viruses when inoculated into mice via intramuscular or subcutaneous injection. References: 1. Lentinan improved the efficacy of vaccine against Trichinella spiralis in an NLRP3 dependent manner.Jin X, Liu X, Ding J, Zhang L, Yang Y, Wang X, Yang Y, Liu M.PLoS Negl Trop Dis.2020 Sep 25;14(9):e0008632;2. Immune-adjuvant activity of lentinan-modified calcium carbonate microparticles on a H5N1 vaccine.He J, Liu Z, Jiang W, Zhu T, Wusiman A, Gu P, Liu J, Wang D.Int J Biol Macromol.2020 Nov 15;163:1384-1392.
发明内容Contents of the invention
本发明旨在提供一种以香菇多糖为佐剂，经呼吸道粘膜接种的新冠病毒重组亚单位疫苗，用于预防新冠病毒感染。The present invention aims to provide a new coronavirus recombinant subunit vaccine that uses lentinan as an adjuvant and is inoculated through the respiratory mucosa to prevent new coronavirus infection.
本发明提供了香菇多糖在制备新型冠状病毒呼吸道粘膜疫苗中的应用。The invention provides the application of lentinan in preparing a novel coronavirus respiratory mucosal vaccine.
进一步地，本发明提供的应用，其特征在于：香菇多糖作为一种有效的上呼吸道粘膜免疫佐剂，用作新型冠状病毒重组抗原或灭活病毒的免疫佐剂，通过滴鼻或鼻腔喷雾接种，用于预防新型冠状病毒感染。Further, the application provided by the present invention is characterized in that: Lentinan, as an effective upper respiratory tract mucosal immune adjuvant, is used as an immune adjuvant for the new coronavirus recombinant antigen or inactivated virus, and is inoculated through nasal drops or nasal spray. , used to prevent novel coronavirus infection.
进一步地，本发明提供的应用，其特征在于：所述以香菇多糖为佐剂的新冠疫苗，其接种途径包括滴鼻、鼻腔喷雾。Further, the application provided by the present invention is characterized in that the vaccination route of the COVID-19 vaccine with lentinan as an adjuvant includes intranasal drops and nasal spray.
本发明利用新冠病毒包膜刺突蛋白为抗原，将抗原蛋白溶液与香菇多糖溶液混匀，滴鼻接种叙利亚金黄地鼠，随后检测地鼠血清中的抗体，并用活病毒感染叙利亚金黄地鼠，检测到滴鼻疫苗可有效诱导地鼠产生抗新型冠状病毒刺突蛋白受体结合功能区IgG抗体，并有效保护地鼠抵抗新型冠状病毒感染，降低地鼠上呼吸道中的病毒载量和减轻地鼠发病，具有应用前景。The present invention uses the new coronavirus envelope spike protein as an antigen, mixes the antigen protein solution and lentinan solution, injects intranasally into Syrian golden hamsters, then detects the antibodies in the hamster serum, and infects Syrian golden hamsters with live virus. It was detected that the intranasal vaccine can effectively induce hamsters to produce anti-new coronavirus spike protein receptor-binding functional region IgG antibodies, and effectively protect hamsters against new coronavirus infection, reduce the viral load in the upper respiratory tract of hamsters and alleviate the symptoms of hamsters. The disease occurs in mice and has application prospects.
附图说明Description of the drawings
图1.疫苗免疫叙利亚金黄地鼠诱导的血清抗新冠病毒刺突蛋白中的受体结合功能区(RBD)IgG抗体Figure 1. Serum anti-receptor binding domain (RBD) IgG antibodies in the spike protein of the new coronavirus induced by vaccine immunization of Syrian golden hamsters
用酶联免疫吸附试验检测第1次和第2次免疫以后，即图中第14天和28天时地鼠血清中抗RBD特异性IgG抗体。Str为新冠病毒包膜刺突蛋白胞外段三聚体。Enzyme-linked immunosorbent assay was used to detect anti-RBD-specific IgG antibodies in hamster serum after the first and second immunizations, that is, on days 14 and 28 in the figure. Str is the trimer of the extracellular segment of the new coronavirus envelope spike protein.
图2.新冠病毒攻击后第4天地鼠鼻甲中新冠病毒N基因核酸的相对含量Figure 2. Relative content of SARS-CoV-2 N gene nucleic acid in turbinates of hamsters on day 4 after COVID-19 challenge
在病毒攻击后第4天，分别处死各组3只地鼠，取鼻甲，研磨，抽提组织细胞RNA，用逆转录-定量PCR检测新冠病毒N基因相对表达水平。On the 4th day after the virus challenge, 3 hamsters in each group were killed, and the turbinates were removed, ground, and tissue cell RNA was extracted. Reverse transcription-quantitative PCR was used to detect the relative expression level of the new coronavirus N gene.
图3.新冠病毒感染后地鼠体重变化Figure 3. Changes in body weight of hamsters after COVID-19 infection
每组各5只地鼠在病毒攻击以后每天的体重变化，其中空白对照组未进行病毒攻击。Daily weight changes of 5 hamsters in each group after virus challenge. The blank control group was not challenged with the virus.
具体实施方式Detailed ways
为了更清楚地说明本发明，下面结合优选实施例对本发明做进一步的说明。本领域技术人员应当理解，下面所具体描述的内容是说明性的而非限制性的，不应以此限制本发明的保护范围。In order to illustrate the present invention more clearly, the present invention will be further described below with reference to preferred embodiments. Those skilled in the art should understand that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of the present invention.
本发明实施例所用的伊沙佐米可以通过市售方式购买获得。Ixazomib used in the embodiments of the present invention can be purchased commercially.
一、病毒、药物、试剂及其他材料1. Viruses, drugs, reagents and other materials
1.病毒：SARS-CoV-2病毒由海军军医大学生物医学防护教研室从COVID-19患者鼻咽拭子样本中分离培养出，其基因序列参见GenBank Accession No.MZ664555，用Vero E6细胞培养病毒。培养的病毒感染Vero E6细胞，18小时以后用免疫荧光染色检测病毒的滴度(focus forming units,FFU)，即每毫升病毒液中感染性病毒颗粒的数量。涉及病毒感染的实验操作均在海军军医大学P3实验室中进行。1. Virus: The SARS-CoV-2 virus was isolated and cultured from the nasopharyngeal swab samples of COVID-19 patients by the Biomedical Protection Teaching and Research Office of the Naval Medical University. Its gene sequence can be found in GenBank Accession No. MZ664555. The virus was cultured in Vero E6 cells. The cultured virus was infected with Vero E6 cells, and 18 hours later, immunofluorescence staining was used to detect the virus titer (focus forming units, FFU), which is the number of infectious virus particles per milliliter of virus fluid. All experimental operations involving viral infection were conducted in the P3 laboratory of the Naval Medical University.
2.疫苗抗原新冠病毒刺突包膜蛋白全长胞外段，其中S1/S2亚基之间的弗林蛋白酶位点氨基酸残基PRRARS替换为GSAS，第986位的亮氨酸和第987位的缬氨酸均突变为脯氨酸，羧基末端融合T4噬菌体纤维蛋白三聚体基序。表达产物形成同源三聚体(Str)，分泌表达，由海军军医大学生物医学防护教研室用CHO细胞表达。2. The vaccine antigen is the full-length extracellular segment of the new coronavirus spike envelope protein, in which the amino acid residues PRRARS of the furin site between the S1/S2 subunits are replaced by GSAS, and the leucine at position 986 and the leucine at position 987 The valine was mutated to proline, and the carboxyl terminus was fused to the T4 phage fibrin trimer motif. The expression product forms a homotrimer (Str), is secreted and expressed in CHO cells by the Biomedical Protection Teaching and Research Office of the Naval Medical University.
3.香菇多糖(LNT)，购自MCE(MedChemExpress)公司，目录号：HY-N6653，用DMSO溶解。3. Lentinan (LNT), purchased from MCE (MedChemExpress) company, catalog number: HY-N6653, dissolved in DMSO.
4.氢氧化铝佐剂(Al佐剂)，购自InvivoGen公司，目录号：vac-alu-250。4. Aluminum hydroxide adjuvant (Al adjuvant), purchased from InvivoGen Company, catalog number: vac-alu-250.
5.新冠病毒核酸荧光定量PCR检测试剂，购自上海伯杰生物科技有限公司。5. New coronavirus nucleic acid fluorescence quantitative PCR detection reagent was purchased from Shanghai Berger Biotechnology Co., Ltd.
6.叙利亚金黄地鼠，雄性、8周龄，北京维通利华实验动物技术有限公司。6. Syrian golden hamster, male, 8 weeks old, Beijing Vitong Lever Experimental Animal Technology Co., Ltd.
7.其他试剂：293细胞表达的原型株新冠病毒受体结合功能域(RBD)蛋白，购自上海近岸生物科技有限公司；高吸附酶标微孔板，购自Nunc公司；辣根过氧化物酶(HRP)标记的抗小鼠IgG购自Thermo Fisher，目录号：31430；酶联免疫吸附检测(ELISA)用TMB显色液购自Thermo Fisher，目录号：34024；RNA抽提试剂Trizol LS购自Thermo Fisher，目录号：10296010。7. Other reagents: Receptor-binding domain (RBD) protein of the prototype strain of the new coronavirus expressed in 293 cells, purchased from Shanghai Nearshore Biotechnology Co., Ltd.; high-adsorption enzyme-labeled microplate, purchased from Nunc Company; horseradish peroxidation HRP-labeled anti-mouse IgG was purchased from Thermo Fisher, catalog number: 31430; TMB chromogenic solution for enzyme-linked immunosorbent assay (ELISA) was purchased from Thermo Fisher, catalog number: 34024; RNA extraction reagent Trizol LS Purchased from Thermo Fisher, catalog number: 10296010.
二、实验方法：2. Experimental methods:
(一)叙利亚金黄地鼠免疫(1) Immunization of Syrian golden hamster
叙利亚金黄地鼠分为四组，先用异氟烷吸入麻醉动物，随后进行疫苗接种。Syrian golden hamsters were divided into four groups, and the animals were first anesthetized with isoflurane inhalation and subsequently vaccinated.
阴性对照组，病毒攻击时再分为空白对照组(5只)和感染对照组(8只)，共13只：滴鼻PBS，双侧鼻孔滴鼻，25微升/每侧鼻孔；The negative control group was further divided into a blank control group (5 animals) and an infection control group (8 animals) during the virus challenge, with a total of 13 animals: nasal drip of PBS, nasal drip on both nostrils, 25 μl/each nostril;
单一抗原(Str)滴鼻组，8只：双侧鼻孔滴鼻，25微升/每侧鼻孔，含Str蛋白10微克；Single antigen (Str) intranasal drip group, 8 animals: intranasal drip into both nostrils, 25 μl/each nostril, containing 10 μg of Str protein;
Str+LNT滴鼻组，8只：双侧鼻孔滴鼻，25微升/每侧鼻孔，含Str蛋白10微克以及LNT 200微克；Str+LNT intranasal drip group, 8 animals: intranasal instillation into both nostrils, 25 μl/each nostril, containing 10 μg of Str protein and 200 μg of LNT;
Str+Al佐剂肌注组，8只：双侧大腿肌内注射，每侧100微升，含Str蛋白10微克以及氢氧化铝80微克。Str+Al adjuvant intramuscular injection group, 8 animals: intramuscular injection into both thighs, 100 μl on each side, containing 10 μg of Str protein and 80 μg of aluminum hydroxide.
共免疫两次，间隔14天，第二次免疫在采血后当天进行。A total of two immunizations were performed with an interval of 14 days. The second immunization was performed on the day after blood collection.
(二)地鼠血清抗体检测(2) Hamster serum antibody detection
在第一次免疫后第14、28，用毛细管从地鼠眼眶采血，高速离心分离出血 清，冻存于-80℃冰箱。On the 14th and 28th day after the first immunization, blood was collected from the hamster orbit using a capillary tube, the serum was separated by high-speed centrifugation, and frozen in a -80°C refrigerator.
抗体检测按ELISA常规技术进行，将RBD蛋白包被于高吸附酶标微孔板，每孔蛋白0.1微克，置于4℃冰箱过夜；次日吸除蛋白溶液，用磷酸盐缓冲液(PBS，pH值7.0)洗孔一次，随后用含3％牛血清白蛋白的PBS(3％BSA-PBS)于室温封闭2小时，随后吸除封闭液，PBS洗孔3次；每孔加入连续二倍稀释的地鼠血清，稀释液为3％BSA-PBS，体积100微升/孔，置于4℃冰箱过夜；次日吸除血清稀释液，用含0.05％Tween 20的PBS(0.05％Tween 20-PBS)洗孔5次，随后加入1000倍稀释的HRP标记抗小鼠IgG，稀释液为3％BSA-PBS，体积100微升/孔，室温40分钟；吸除HRP抗体稀释液，用0.05％Tween 20-PBS洗孔5次；加TMB显色液，每孔100微升，显色10分钟，再加终止液，用酶标仪测450nm和630nm光吸收值；根据光吸收值，用Graphpad prism 5软件计算每份血清样本的抗体滴度。Antibody detection was performed according to the conventional ELISA technique. The RBD protein was coated on a high-adsorption enzyme-labeled microplate, with 0.1 μg of protein per well, and placed in a refrigerator at 4°C overnight. The protein solution was aspirated the next day and washed with phosphate buffer saline (PBS, pH value 7.0), wash the wells once, then block with PBS containing 3% bovine serum albumin (3% BSA-PBS) at room temperature for 2 hours, then aspirate the blocking solution, wash the wells 3 times with PBS; add two consecutive times to each well Diluted hamster serum, the diluent is 3% BSA-PBS, the volume is 100 μl/well, and placed in a refrigerator at 4°C overnight; the next day, aspirate the serum diluent and use it with PBS containing 0.05% Tween 20 (0.05% Tween 20 -PBS), wash the wells 5 times, then add 1000-fold diluted HRP-labeled anti-mouse IgG, the diluent is 3% BSA-PBS, the volume is 100 μl/well, room temperature for 40 minutes; aspirate the HRP antibody diluent, and use 0.05 Wash the wells 5 times with % Tween 20-PBS; add 100 microliters of TMB chromogenic solution to each well, develop the color for 10 minutes, add the stop solution, and use a microplate reader to measure the 450nm and 630nm light absorption values; according to the light absorption value, use Graphpad prism 5 software calculates the antibody titer for each serum sample.
RBD是新冠病毒中和抗体的主要靶点，血清中的RBD抗体水平代表病毒中和能力。各组地鼠血清的RBD IgG抗体滴度如图1所示，单一Str滴鼻免疫，即新冠病毒包膜刺突蛋白胞外段三聚体滴鼻免疫，诱导RBD IgG抗体水平极低，而Str联合香菇多糖滴鼻免疫能诱导高水平RBD IgG抗体，与Str联合人用佐剂氢氧化铝肌注免疫诱导的抗体水平相似。加强免疫以后，两个佐剂组抗体水平均有适度提高。由于没有市售抗地鼠IgA试剂，而用抗小鼠IgA检测地鼠IgA的敏感性极低，因此未检测地鼠上呼吸道粘膜RBD IgA抗体。RBD is the main target of neutralizing antibodies against the new coronavirus, and the RBD antibody level in the serum represents the virus neutralizing ability. The RBD IgG antibody titers in the sera of each group of hamsters are shown in Figure 1. A single Str intranasal immunization, that is, intranasal immunization with the trimer of the extracellular segment of the new coronavirus envelope spike protein, induced extremely low RBD IgG antibody levels, while Intranasal immunization with Str combined with lentinan can induce high levels of RBD IgG antibodies, which are similar to those induced by intramuscular immunization with Str combined with human adjuvant aluminum hydroxide. After boosting immunization, the antibody levels in both adjuvant groups increased moderately. Since there is no commercially available anti-hamster IgA reagent and the sensitivity of using anti-mouse IgA to detect hamster IgA is extremely low, RBD IgA antibodies in the hamster upper respiratory tract mucosa were not detected.
(三)病毒攻击保护实验(3) Virus attack protection experiment
第一次免疫8周以后，以滴鼻方式对各组地鼠进行新冠病毒攻击，剂量1.8*10 8FFU。阴性对照组的13只地鼠中，取8只作为感染对照组，其余5只不感染病毒，作为空白对照组。从病毒攻击当天(滴鼻病毒前)开始称量地鼠体重，此后每天称量。在病毒攻击后第4天每组取3只地鼠以异氟烷麻醉后处死， 剪切取鼻甲，用匀浆器研磨，用Trizol LS试剂抽提组织细胞RNA，用逆转录实时荧光定量技术检测新冠病毒核衣壳N基因核酸含量，计算各组地鼠相对水平。每组其余5只地鼠继续观察体重变化，至第15天。 Eight weeks after the first immunization, each group of hamsters was challenged with the new coronavirus by intranasal instillation at a dose of 1.8*10 8 FFU. Among the 13 hamsters in the negative control group, 8 were used as the infected control group, and the remaining 5 were not infected with the virus and were used as the blank control group. The body weight of the hamsters was measured starting from the day of virus challenge (before intranasal virus administration) and then every day thereafter. On the 4th day after the virus challenge, 3 hamsters from each group were anesthetized with isoflurane and killed. The turbinates were cut and ground with a homogenizer. Trizol LS reagent was used to extract tissue cell RNA, and reverse transcription real-time fluorescence quantification technology was used. The nucleic acid content of the nucleocapsid N gene of the new coronavirus was detected, and the relative levels of hamsters in each group were calculated. The remaining 5 hamsters in each group continued to observe changes in body weight until the 15th day.
各组地鼠在病毒攻击后第4天的鼻甲中新冠病毒N基因核酸的相对水平量如图2所示：将感染对照组3只地鼠第4天N基因平均水平设置为1，则单一Str滴鼻组相对水平为0.81；Str+氢氧化铝肌注组相对水平为0.79；Str+LNT滴鼻组相对水平为0.14。Str+LNT滴鼻组的N基因相对水平显著低于其他各组(p＜0.001，p＜0.001，p＜0.01；t检验)。虽然未检测地鼠上呼吸道粘膜RBD IgA抗体，各组仓鼠鼻甲中新冠病毒N基因核酸水平的对比显示Str+LNT滴鼻免疫可有效诱导上呼吸道粘膜局部的抗新冠病毒免疫应答。The relative levels of the N gene nucleic acid of the new coronavirus in the turbinates of each group of hamsters on the 4th day after virus challenge are shown in Figure 2: If the average level of the N gene on the 4th day of the 3 hamsters in the infected control group is set to 1, then a single The relative level of the Str intranasal drip group was 0.81; the relative level of the Str + aluminum hydroxide intramuscular injection group was 0.79; the relative level of the Str + LNT intranasal injection group was 0.14. The relative level of N gene in the Str+LNT intranasal drip group was significantly lower than that in other groups (p<0.001, p<0.001, p<0.01; t test). Although RBD IgA antibodies in the upper respiratory tract mucosa of hamsters were not detected, comparison of the nucleic acid levels of the SARS-CoV-2 N gene in the turbinates of each group of hamsters showed that intranasal immunization with Str+LNT can effectively induce a local anti-COVID-19 immune response in the upper respiratory tract mucosa.
地鼠体重变化如图3所示：空白对照组地鼠体重持续增加；感染对照组地鼠在病毒攻击以后体重持续下降，最大降低幅度达14.6％，至第6天以后开始回升；单一Str滴鼻组体重与感染对照组的体重变化相似；Str+氢氧化铝肌注组体重变化表现为平缓增加，显示其具有较好的免疫保护效果；相比Str+氢氧化铝肌注组，Str+LNT滴鼻组体重增加幅度较大，显示具有更好的免疫保护效果。The weight changes of hamsters are shown in Figure 3: the weight of hamsters in the blank control group continued to increase; the weight of hamsters in the infected control group continued to decrease after the virus challenge, with the maximum reduction reaching 14.6%, and began to rise after the 6th day; a single Str drop The weight change of the nasal group was similar to that of the infected control group; the weight change of the Str+aluminum hydroxide intramuscular injection group showed a gentle increase, showing that it has a better immune protective effect; compared with the Str+aluminum hydroxide intramuscular injection group, the Str+LNT drop The nose group gained greater weight, indicating better immune protection.
以上实验结果表明，将新冠病毒刺突蛋白作为抗原，联合香菇多糖佐剂，滴鼻免疫叙利亚金黄地鼠，可诱导高水平保护性抗体(RBD抗体是保护性抗体)，并能有效保护地鼠抵抗新冠病毒攻击(病毒攻击以后地鼠鼻甲中的病毒复制被显著抑制，地鼠体重未见减轻，且快速开始提升)。The above experimental results show that using the spike protein of the new coronavirus as an antigen, combined with lentinan adjuvant, to intranasally immunize Syrian golden hamsters can induce high-level protective antibodies (RBD antibodies are protective antibodies) and can effectively protect the hamsters. Resistance to the new coronavirus attack (after the virus attack, the virus replication in the turbinates of the hamsters was significantly inhibited, the hamsters did not lose weight, and quickly began to increase).
新冠病毒刺突蛋白联合人用佐剂氢氧化铝肌肉注射免疫，地鼠体重变化显示该免疫方法也有明显的保护效果。但相比联合香菇多糖佐剂滴鼻免疫组，联合氢氧化铝肌注组地鼠鼻甲中新冠病毒的复制水平较高，且体重增加幅度较低。The spike protein of the new coronavirus was combined with the human adjuvant aluminum hydroxide for intramuscular immunization. The weight changes of the hamsters showed that this immunization method also had a significant protective effect. However, compared with the nasal immunization group combined with lentinan adjuvant, the replication level of the new coronavirus in the turbinates of the hamsters combined with intramuscular injection of aluminum hydroxide was higher, and the weight gain was lower.
结果显示：香菇多糖可作为上呼吸道粘膜的有效免疫佐剂，新冠病毒包膜刺突蛋白联合香菇多糖滴鼻接种，可预防新冠病毒感染。The results show that lentinan can be used as an effective immune adjuvant for the upper respiratory tract mucosa. The new coronavirus envelope spike protein combined with lentinan intranasal inoculation can prevent new coronavirus infection.
以上所述仅是本发明的较佳实施例而已，并非对本发明作任何形式上的限制，虽然本发明已以较佳实施例揭露如上，然而并非用以限定本发明，任何熟悉本专利的技术人员在不脱离本发明技术方案范围内，当可利用上述提示的技术内容作出些许更动或修饰为等同变化的等效实施例，但凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰，均仍属于本发明方案的范围内。本发明要求保护范围由所附的权利要求书及其等同物界定。The above are only preferred embodiments of the present invention, and do not limit the present invention in any form. Although the present invention has been disclosed above in preferred embodiments, it is not intended to limit the present invention. Anyone familiar with the technology of this patent Without departing from the scope of the technical solution of the present invention, personnel can make some changes or modify the above-mentioned technical contents into equivalent embodiments with equivalent changes. In essence, any simple modifications, equivalent changes and modifications made to the above embodiments still fall within the scope of the present invention. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims (3)

  1. 香菇多糖在制备新型冠状病毒呼吸道粘膜疫苗中的应用。Application of lentinan in the preparation of novel coronavirus respiratory mucosal vaccine.
  2. 根据权利要求1所述的应用，其特征在于：香菇多糖作为一种有效的上呼吸道粘膜免疫佐剂，用作新型冠状病毒重组抗原或灭活病毒的免疫佐剂，通过滴鼻或鼻腔喷雾接种，用于预防新型冠状病毒感染。The application according to claim 1, characterized in that: Lentinan, as an effective upper respiratory tract mucosal immune adjuvant, is used as an immune adjuvant for the new coronavirus recombinant antigen or inactivated virus, and is inoculated through nasal drops or nasal spray. , used to prevent novel coronavirus infection.
  3. 根据权利要求1-2任一所述的应用，其特征在于：所述以香菇多糖为佐剂的新冠疫苗，其接种途径包括滴鼻、鼻腔喷雾。The application according to any one of claims 1-2, characterized in that: the inoculation route of the COVID-19 vaccine with lentinan as an adjuvant includes intranasal drops and nasal spray.
PCT/CN2022/101512 2022-04-20 2022-06-27 Use of lentinan in preparing sars-cov-2 respiratory mucosal vaccine WO2023201881A1 (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021236980A1 (en) * 2020-05-20 2021-11-25 Flagship Pioneering Innovations Vi, Llc Coronavirus antigen compositions and their uses
WO2021256470A1 (en) * 2020-06-16 2021-12-23 Sophy Inc. Beta-glucan for immuno-enhancement and/or immuno-balancing, and for adjuvant use
CN114344457A (en) * 2021-11-13 2022-04-15 暨南大学 Novel coronavirus protein antigen nano vaccine and preparation method and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021236980A1 (en) * 2020-05-20 2021-11-25 Flagship Pioneering Innovations Vi, Llc Coronavirus antigen compositions and their uses
WO2021256470A1 (en) * 2020-06-16 2021-12-23 Sophy Inc. Beta-glucan for immuno-enhancement and/or immuno-balancing, and for adjuvant use
CN114344457A (en) * 2021-11-13 2022-04-15 暨南大学 Novel coronavirus protein antigen nano vaccine and preparation method and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEN XIANGYAN; HAN WENWEI; WANG GUIXIANG; ZHAO XIA: "Application prospect of polysaccharides in the development of anti-novel coronavirus drugs and vaccines", INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, ELSEVIER BV, NL, vol. 164, 14 July 2020 (2020-07-14), NL , pages 331 - 343, XP086335562, ISSN: 0141-8130, DOI: 10.1016/j.ijbiomac.2020.07.106 *
HE JIN; LIU ZHENGUANG; JIANG WENMING; ZHU TIANYU; WUSIMAN ADELIJIANG; GU PENGFEI; LIU JIAGUO; WANG DEYUN: "Immune-adjuvant activity of lentinan-modified calcium carbonate microparticles on a H5N1 vaccine", INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, ELSEVIER BV, NL, vol. 163, 3 August 2020 (2020-08-03), NL , pages 1384 - 1392, XP086300294, ISSN: 0141-8130, DOI: 10.1016/j.ijbiomac.2020.08.005 *

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