WO2022242446A1 - 一种植物乳杆菌及在制备新冠疫苗免疫增强剂中的应用 - Google Patents

一种植物乳杆菌及在制备新冠疫苗免疫增强剂中的应用 Download PDF

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WO2022242446A1
WO2022242446A1 PCT/CN2022/089867 CN2022089867W WO2022242446A1 WO 2022242446 A1 WO2022242446 A1 WO 2022242446A1 CN 2022089867 W CN2022089867 W CN 2022089867W WO 2022242446 A1 WO2022242446 A1 WO 2022242446A1
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vaccine
lactobacillus plantarum
antibody
strain
cells
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徐建国
徐建青
任志鸿
曹康丽
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上海市公共卫生临床中心
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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
    • A61K39/12—Viral antigens
    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • A61K2039/55588—Adjuvants of undefined constitution
    • A61K2039/55594—Adjuvants of undefined constitution from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • 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
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to a preserved strain of Lactobacillus plantarum and its application, belonging to the field of microorganisms and vaccines.
  • the novel coronavirus has a strong transmission ability, a wide range of transmission, and multiple routes of infection. There have been outbreaks in 222 countries and regions around the world, with more than 110 million confirmed cases reported. Vaccines are a powerful weapon to control the spread of the new crown virus. There are more than 100 new coronavirus vaccines under development worldwide. To evaluate the immune protection effect obtained after natural infection or vaccination with the new coronavirus, neutralizing antibodies are often used as reference indicators. Some patients infected with the new coronavirus can produce neutralizing antibodies 1-4 days after the onset of the disease, and the antibody level begins to increase significantly after the 10-15 day, and the antibody level reaches a peak on the 31-40 day and then maintains a stable state or decreases slightly.
  • a randomized, double-blind, placebo-controlled human trial in Spain showed that 60 hospitalized volunteers aged 65-85 who had been vaccinated with trivalent influenza vaccine received skim milk powder containing Lactobacillus plantarum for 3 months after oral administration , can increase the level of influenza-specific IgA and IgG antibodies.
  • a randomized, double-blind, placebo-controlled human clinical trial including 50 volunteers showed that oral administration of Lactobacillus fermentum (CECT5716) can enhance the immune response against influenza vaccine, and can enhance the helper T cell type I Response and virus neutralizing antibodies enhance the body's protection against infection. Limited research suggests that some probiotic strains may have adjuvant-like functions, which can stimulate and enhance the specific antibody levels produced by certain bacterial vaccines or certain viral vaccines, and enhance immune protection.
  • the object of the present invention is to provide a kind of Lactobacillus plantarum that can up-regulate the serum neutralizing antibody titer of virus vaccines such as new crown and bacterial vaccines such as Shigella, enhance the immune effect, and prolong the protection time, so as to solve the problem of serum neutralization of current new crown virus vaccines. And the problem of the rapid decline of antibodies and the rapid decline of protection.
  • virus vaccines such as new crown and bacterial vaccines such as Shigella
  • the present invention firstly provides a strain of Lactobacillus plantarum, the preservation number of which is CGMCC NO.21720, the preservation date is January 22, 2021, and the preservation classification is named Lactobacillus Plantarum (Lactobacillus plantarum) GUANKE strain, the preservation unit is the General Microbiology Center of the China Committee for the Collection of Microbial Cultures.
  • sequence of the 16S rDNA of the bacterial strain is as SEQ ID NO.1 is shown.
  • the present invention also provides the application of the above bacterial strains in the preparation of vaccine immune enhancers.
  • the vaccines include hepatitis B vaccine, BCG, live attenuated polio vaccine, acellular diphtheria combined vaccine, measles-mumps combined vaccine, hepatitis A vaccine, meningococcal polysaccharide vaccine, Japanese encephalitis Vaccines, measles vaccine, new coronavirus vaccine, influenza vaccine, rabies vaccine, AIDS vaccine, Ebola vaccine, West Nile virus vaccine, yellow fever vaccine, rotavirus vaccine, chickenpox vaccine, typhoid Vi polysaccharide vaccine, pneumococcal vaccine , Haemophilus influenzae type B conjugate vaccine, bacillary dysentery vaccine, or HPV vaccine.
  • the vaccine is a novel coronavirus vaccine.
  • the novel coronavirus vaccine is a novel coronavirus recombinant plasmid vaccine, a novel coronavirus recombinant adenovirus vector vaccine or a novel coronavirus cell vector vaccine.
  • the present invention also provides a composition containing the above-mentioned bacterial strain, and the composition contains a pharmaceutically acceptable carrier.
  • the composition is prepared as injection, capsule, lyophilized powder, spray, suspension or tablet.
  • the composition also contains targeted drugs or thymosin.
  • the targeted drug includes targeting PD-1/PD-L1 (Programmed cell death protein), CTLA-4 (Cytotoxic-T-lymphocyte-Antigen-4), CD24, EGFR (Epidermal Growth Factor Receptor), VEGFR (Vascular Endothelial Growth Factor Receptor), HER-2 (Human Epidermal Growth Factor Receptor 2), Claudin18.2, glypican-3, FAP (Fibroblast Activation Protein), PSMA (Prostate-Specific Membrane Antigen), PSA (Prostate-Specific Antigen), CEA (Carcinoembryonic Antigen), AXL, CD20, CD19, BCMA (B-cell Maturation Antigen ), CD22, ROR1, CD24, mTOR (Mechanistic Target of Rapamycin), ALK (Anaplastic Lymphoma Kinase), c-kit or MUC1 (mucin1).
  • PD-1/PD-L1 Programmed cell death protein
  • CTLA-4 Cytotoxic
  • the invention separates and purifies probiotic plant Lactobacillus with immune enhancing effect from healthy human feces.
  • the isolated Lactobacillus plantarum is harmless to animals, and it has been confirmed by animal experiments that it has the titer of specific neutralizing antibodies in the serum of individuals vaccinated with the new coronavirus vaccine.
  • the Lactobacillus plantarum GUANKE can effectively maintain the neutralizing antibody level titer in the serum of mice after inoculation, showing excellent application prospects in the preparation of new crown vaccine immune enhancement preparations.
  • Figure 1- Figure 6 shows the application of Lactobacillus plantarum GUANKE to enhance the anti-Shigella specific antibody of Shigella vaccine immunized mice, and the mice used in the experiment were 18-22 g SPF grade Balb/c mice are divided into male and female, and the immunogen is Shigella vaccine.
  • Figure 1 ELISA method to detect the titer of anti-Shigella dysenteriae IgG binding antibody titer in the feces of Lactobacillus plantarum host 30 days after intragastric administration of Shigella vaccine; the abscissa is the time point, and the ordinate is the binding antibody The titer of *** means p ⁇ 0.001.
  • Figure 2 ELISA method to detect the titer of anti-Shigella dysenteriae IgA binding antibody titer in the feces of Lactobacillus plantarum host 30 days after intragastric administration of Shigella vaccine; the abscissa is the time point, and the ordinate is the binding antibody The titer of *** means p ⁇ 0.001.
  • Figure 3 ELISA method to detect the titer of anti-Shigella dysenteriae IgM binding antibody titer in the feces of Lactobacillus plantarum host 30 days after intragastric administration of Shigella vaccine; the abscissa is the time point, and the ordinate is the binding antibody titer.
  • Figure 4 ELISA method to detect the titer of anti-Shigella dysenteriae IgG binding antibody in the serum of Lactobacillus plantarum host 30 days after intragastric administration of Shigella vaccine; the abscissa is the time point, and the ordinate is the binding antibody ** means p ⁇ 0.01, *** means p ⁇ 0.001.
  • Figure 5 ELISA method to detect the titer of anti-Shigella dysenteriae IgA binding antibody in the serum of Lactobacillus plantarum host 30 days after intragastric administration of Shigella vaccine; the abscissa is the time point, and the ordinate is the binding antibody titer.
  • Figure 6 ELISA method to detect the titer of anti-Shigella dysenteriae IgM binding antibody in serum of Lactobacillus plantarum host 30 days after intragastric administration of Shigella vaccine; the abscissa is the time point, and the ordinate is the binding antibody ** means p ⁇ 0.01, *** means p ⁇ 0.001.
  • Figure 7- Figure 14 shows the effect of intragastric administration of Lactobacillus plantarum GUANKE on the antibody titer of mice five months after immunization with the new crown vaccine.
  • the mice used in the experiment were 6-8 week old female ICR, and the immunogen was the cell carrier vaccine K562- S.
  • Figure 7 ELISA method to detect IgG-binding antibody titers in mouse serum at a series of time points after immunization and before gavage; the abscissa is the time point, and the ordinate is the titer of the binding antibody.
  • Figure 8 ELISA method to detect the titer of IgG-binding antibody in mouse serum at a series of time points after gavage; the abscissa is the time point, and the ordinate is the titer of the binding antibody, * indicates p ⁇ 0.05, ** indicates p ⁇ 0.01.
  • Figure 9 The titer of IgG-binding antibody in mouse serum at a series of time points after gavage was detected by ELISA method; Ratios, ** indicates p ⁇ 0.01.
  • Figure 10 ELISA method to detect the titer of IgG-binding antibody in mouse serum at a series of time points after gavage; the abscissa is the time point, and the ordinate is the average value of the binding antibody of the Lactobacillus plantarum group and the PBS group at this time point ratio.
  • FIG. 11 293T-ACE2 cells detect the titer of neutralizing antibody in mouse serum at a series of time points before gavage after immunization; the abscissa is the time point, and the ordinate is the titer of neutralizing antibody (ID50).
  • FIG. 12 293T-ACE2 cells detect the titer of neutralizing antibody in mouse serum at a series of time points after immunization; the abscissa is the time point, and the ordinate is the titer of neutralizing antibody (ID50).
  • FIG. 13 293T-ACE2 cells detect the titer of neutralizing antibody in mouse serum at a series of time points after immunization; the abscissa is the time point, and the ordinate is the titer of neutralizing antibody at this time point and the neutralizing antibody before gavage
  • FIG. 14 293T-ACE2 cells detect the titer of neutralizing antibody in mouse serum at a series of time points after immunization; the abscissa is the time point, and the ordinate is the average value of neutralizing antibody in the Lactobacillus plantarum group and the PBS group at this time point Ratio to the antibody mean.
  • Figure 15- Figure 20 shows the effect of intragastric administration of Lactobacillus plantarum GUANKE on the antibody titer and T cell response of mice immediately after immunization with the new crown vaccine.
  • the mice used in the experiment were 6-8-week-old female ICR, and the immunogen was DNA-S and AdC68-RHAF.
  • Figure 15 ELISA method to detect the titer of IgG-binding antibody in mouse serum at a series of time points after gavage; the abscissa is the time point, and the ordinate is the titer of the binding antibody, * indicates p ⁇ 0.05, ** indicates p ⁇ 0.01.
  • Figure 16 ELISA method to detect IgG-binding antibody titers in mouse serum at a series of time points after gavage; the abscissa is the time point, and the ordinate is the ratio of the binding antibody titer at this time point to the binding antibody titer before gavage Ratio, * indicates p ⁇ 0.05.
  • Figure 17 ELISA method to detect the titer of IgG-binding antibody in mouse serum at a series of time points after gavage; the abscissa is the time point, and the ordinate is the average value of the binding antibody of the Lactobacillus plantarum group and the PBS group at this time point ratio.
  • FIG. 18 293T-ACE2 cells detect the titer of neutralizing antibody in mouse serum at a series of time points after immunization; the abscissa is the time point, and the ordinate is the titer of neutralizing antibody (ID50), ** means p ⁇ 0.01.
  • Figure 19 ELISPOT method to detect the T cell response of mouse splenocytes at various time points after immunization; the abscissa is the time point, and the ordinate is the number of cells secreting IFN- ⁇ per million splenocytes; * means p ⁇ 0.05 .
  • Figure 20 ELISPOT method to detect T cell responses in mouse lung lavage fluid at various time points after immunization.
  • the abscissa is the time point, and the ordinate is the number of IFN- ⁇ -secreting cells per million splenocytes; * indicates p ⁇ 0.05.
  • the culture medium containing 25% glycerol is used as the bacteria-preserving solution to carry out the cryopreservation of the strains, and the method is as follows:
  • Lactobacillus is a facultative anaerobic bacterium, which grows well under anaerobic conditions, and the colonies are milky white with a smooth surface; under aerobic conditions, Lactobacillus can also grow, most of the colony surface is rough, and the colony color is mostly milky white .
  • the morphology of Lactobacillus cells observed under the microscope is pleomorphic, mostly in the shape of slender rods, thicker rods, clubs, etc., arranged in fences, chains, etc.
  • a single colony was inoculated on BHI medium, cultured anaerobically overnight at 37°C, and DNA was extracted according to the instructions of the Bacterial Genomic DNA Extraction Kit (TIANGEN).
  • Bacterial 16S rRNA identification Bacterial genomic DNA was extracted, the 16S rDNA PCR product of Lactobacillus universal primer was amplified and sequenced, and the sequences were compared by BLAST on NCBI for preliminary identification.
  • the bacterial 16S rDNA PCR amplification primers used in this experiment and the conditions of the PCR reaction are as follows.
  • the preservation information of the strain is: the preservation number of the strain is CGMCC NO.21720, the preservation date is January 22, 2021, the preservation classification is named Lactobacillus Plantarum GUANKE, and the preservation unit is China Microbial Cultures Preservation Committee General Microbiology Center, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, postal code: 100101.
  • Lactobacillus plantarum GUANKE enhances the application of Shigella vaccine-immunized mice with anti-Shigella specific antibodies
  • the antibody detection method is as follows.
  • Serum IgM was significantly higher than that of the PBS treatment group after three days, one week and two weeks of probiotic treatment, reached a peak after one week of treatment, gradually decreased, and decreased to no significant difference with the untreated group after four weeks (Figure 6). Serum IgA did not change significantly during bacterin immunization and probiotic immune modulation (Fig. 5).
  • Example 3 The effect of gavage of Lactobacillus plantarum GUANKE on the antibody titer of mice five months after immunization with the new crown vaccine
  • the experimental animals, immunization methods, immunogens, pseudoviruses and detection methods involved in the experiment are as follows.
  • mice 6-8 weeks old female ICR mice, purchased from Beijing Weishang Lituo Technology Co., Ltd.
  • K562-S as a bone marrow erythroid cell line, does not express transplant rejection antigens (MHC class I and class II), nor does it express blood group antigens, so the human body has great potential for such cell products well tolerated.
  • K562 cells were used as vectors to express and display abundant novel coronavirus spike protein (Spike, S) immunogen on its surface (Genebank: NC_045512.2), thus constructing K562-S new crown vaccine.
  • Immunization dose Refer to Example 1 for the preparation of the immunogen, and the immunogen immunization dose used in the example is as follows.
  • Cell-based vaccine K562-S (dissolved in sterile PBS) and aluminum adjuvant (Aluminum, InvivoGen, Cat. No. 5200) were mixed at a volume ratio of 1:1 for immunization, 1E6 cells/mouse, 100 ⁇ L.
  • Immunization interval See the description below for specific immunization intervals.
  • mice Peripheral whole blood of mice was collected by the method of orbital vein blood collection, collected at 1.5 In mL EP tubes, let stand at room temperature to allow the blood to coagulate naturally, and centrifuge the coagulated mouse serum at 7000 g for 15 min. Transfer the mouse serum to a new 1.5 mL EP tube. The samples need to be inactivated at 56°C for 30 min before the experiment to destroy the complement activity in the serum. Centrifuge briefly before inactivation to avoid residual sample on the tube wall and bottle cap. The liquid level of the water bath should not exceed the liquid level of the sample, but not exceed the cap of the bottle.
  • ELISA sample diluent (0.5% skimmed milk powder, dissolved in PBST), starting from 1:100, perform 2-fold dilution. Sera from unimmunized mice were used as negative controls. Set up a blank well, add only the sample diluent, and make 2 duplicate wells for each sample, with a final volume of 100 ⁇ L in each well, and incubate at room temperature for 3 h.
  • luciferase substrate purchased from Promega, Cat#E1501
  • GloMax® 96 microplate luminescence detector to detect luciferase activity.
  • Example 2 we found that for bacterial vaccines, administration of Lactobacillus plantarum GUANKE can increase the binding antibody titers in mouse feces and serum. So we wanted to see if the same effect would work in viral vaccines.
  • Orbital venous blood was collected before and 1, 2, 3, and 6 weeks after gavage of Lactobacillus plantarum GUANKE. Thirty weeks after immunization, ampicillin pretreatment and immunomodulation of Lactobacillus plantarum GUANKE were repeated, and orbital venous blood was collected 1, 2, 3, and 4 weeks after immunomodulation to detect antibody responses. The results showed that the binding antibody and neutralizing antibody in the sera of the immunized mice reached the peak at the second week, and then declined slowly (Figure 7, 11). After the two interventions, the titers of serum binding antibodies and neutralizing antibodies in each group of mice are shown in Figures 8 and 12.
  • the experimental animals, immunization methods, immunogens, pseudoviruses and detection methods involved in the experiment are as follows.
  • Experimental animals 6-8 week-old female ICR mice, purchased from Beijing Weishang Lituo Technology Co., Ltd.
  • Immunization method intramuscular injection into the left and right hind limbs of mice; or intranasal drip. Specific dosage see embodiment.
  • DNA vaccines will produce reverse mutations, and are safer than inactivated vaccines; they are flexible in use and can act on muscle (skeletal muscle), subcutaneous or mucosal tissues; stable at room temperature, can maintain activity for a long time, and are convenient for storage and transportation . Therefore, we used the traditional expression vector pcDNA3.1 to insert the new crown spike protein (Spike, S) immunogen (Genebank: NC_045512.2), thus constructing the pcDNA3.1-S new crown vaccine.
  • Spike, S new crown spike protein
  • AdC68-RHAF Chimpanzee adenovirus vector vaccine AdC68-RHAF Chimpanzee adenovirus vector
  • AdC68 is a linear DNA virus with a genome size of 26-45 kb.
  • Ad5 and Ad26 adenoviruses
  • its pre-existing immunity in the human population is low , will avoid the weakening of the vaccine effect brought by the carrier itself, and at the same time, the combination of Hexon of chimpanzee adenovirus and coagulation factor X is unstable and does not accumulate in the liver, which is safer.
  • the doses of immunogens used in the examples are as follows.
  • Recombinant plasmid vaccine 100 ⁇ g/mouse, 100 ⁇ L, dissolved in sterile saline.
  • Recombinant adenovirus vector vaccine 5E10 vp/mouse, 100 ⁇ L (intramuscular injection); 5E10 vp/mouse, 30 ⁇ L (nasal drop).
  • Immunization interval See below for specific immunization intervals.
  • the Millipore plate provided by the purified IFN- ⁇ antibody coating kit (purchased from BD, Cat. No. 551083) was coated at a ratio of 1:250 and coated overnight at 4°C.
  • a stimulating peptide library (Suzhou Qiangyao Biotechnology Co., Ltd. synthesized single peptide, each single peptide has 15 amino acids, covering RBD sequence, a total of 65 Single peptide, 5 single peptides for each peptide library, a total of 13 peptide libraries, 50 ⁇ L/well, the concentration of each peptide is 5 ⁇ g/mL.
  • RPMI 1640 complete medium to the negative control well; positive control well Add 50 ⁇ L of phorbol ester polyclonal stimulator (PMA, purchased from Sigma, product number FXP012) (final concentration 100ng/mL) and RPMI 1640 complete medium with ionomycin (final concentration 2 ⁇ g/mL) .
  • PMA phorbol ester polyclonal stimulator
  • Example 3 we found that the administration of Lactobacillus plantarum GUANKE to mice half a year after the end of immunization can significantly reduce the decline of binding antibodies and neutralizing antibodies, and even increase the antibody level again. Therefore, we want to observe whether the administration of Lactobacillus plantarum GUANKE to mice by gavage immediately after the immunization will improve the T cell response and antibody level induced by the vaccine.
  • mice were sacrificed on the 3rd, 7th, and 14th days after the probiotics were instilled, and the blood, lung lavage fluid cells, and splenocytes were collected, and the sera on the 7th and 14th days were tested for binding antibodies and neutralizing antibodies (the detection method was the same as in Example 3 ), 3, 7, 14 days of lavage fluid cells and splenocytes to detect T cell response.
  • the experimental data show that oral administration of Lactobacillus plantarum GUANKE can increase the anti-new coronavirus binding antibody and neutralizing antibody titer, as well as the specific T cell response against new coronavirus, prolong the protection time of antibodies and T cells, and prevent new coronavirus infection prevention is of great significance.
  • the strain of Lactobacillus plantarum disclosed by the invention is easy to be industrially cultivated and prepared as a pharmaceutical preparation, and has industrial applicability.

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Abstract

本发明公开了一种植物乳杆菌冠克株，所述菌株的保藏号为CGMCC NO.21720，保藏日期为2021年 01月22日，保藏分类命名为植物乳杆菌冠克株（Lactobacillus plantarum guanke），保藏单位为中国微生物菌种保藏管理委员会普通微生物中心。本发明提供的菌株发可显著提高新冠病毒血清中和抗体滴度，延长保护时间。本发明还提供了所述菌株在制备疫苗免疫增强剂中的应用。

Description

一种植物乳杆菌及在制备新冠疫苗免疫增强剂中的应用 技术领域
本发明涉及一种植物乳杆菌保藏菌株及应用，属于微生物和疫苗领域。
 背景技术
新冠病毒传播力强、传播范围广、传染途径多，全球已经有222个国家、地区发生疫情，报告确诊病例数超过1.1亿，疫苗是控制新冠病毒传播的有力武器。全球在研新冠病毒疫苗超百种。评价新冠病毒自然感染或疫苗接种后获得的免疫保护效力，多用中和抗体作参考指标。部分新冠病毒感染者发病后第1-4天可产生中和抗体，第10-15天后抗体水平开始显著升高，第31-40天抗体水平达到峰值后维持稳定状态或略微下降。60%左右的感染者（包括确诊病例和无症状感染者）约发病2个月左右，中和抗体水平开始下降。新冠疫苗免疫后刺激产生的血清中和抗体能够在高位持续多久，公开发表的数据很少，尚无明确结论，但是，应该和新冠病毒感染者产生的保护力相似。普遍认为，需要增加免疫次数，才可延长免疫保护时间。
在西班牙开展的一项随机、双盲、安慰剂对照的人体试验表明，接种了三价流感疫苗的60名65-85岁的住院志愿者，在口服含有植物乳杆菌的脱脂奶粉3个月后，可使流感特异性IgA和IgG抗体水平升高。一项随机、双盲、安慰剂对照的包括50名志愿者的人体临床试验表明，口服发酵乳杆菌（ Lactobacillus fermentum CECT5716）可增强抗流感疫苗的免疫应答，并可通过增强辅助性T细胞I型应答和病毒中和抗体，增强机体对感染的保护作用。有限的研究提示，某些益生菌菌株可能具有佐剂样功能，能够刺激增强某些细菌性疫苗，或者某些病毒疫苗，产生的特异性抗体水平，增强免疫保护作用。
技术问题
本发明目的就是提供一种能够上调新冠等病毒疫苗及志贺菌等细菌疫苗接种者血清中和抗体滴度、增强免疫效果、延长保护时间的植物乳杆菌，解决目前新冠病毒疫苗接种者血清中和抗体迅速下降、保护力迅速下降的问题。
 技术解决方案
基于上述发明目的，本发明首先提供了一种植物乳杆菌菌株，所述菌株的保藏号为CGMCC NO.21720，保藏日期为2021年01月22日，保藏分类命名为 Lactobacillus Plantarum（植物乳杆菌）GUANKE株，保藏单位为中国微生物菌种保藏管理委员会普通微生物中心。
在一个优选的实施方案中，所述菌株的16S rDNA的序列如SEQ ID NO.1所示。
其次，本发明还提供了上述菌株在制备疫苗免疫增强剂中的应用。
在一个优选的实施方案中，所述疫苗包括乙肝疫苗、卡介苗、脊髓灰质炎减毒活疫苗、无细胞百白破联合疫苗、麻腮风联合疫苗、甲肝疫苗、脑膜炎球菌多糖疫苗、乙脑疫苗、麻疹疫苗、新型冠状病毒疫苗、流感疫苗、狂犬疫苗、艾滋疫苗、埃博拉疫苗、西尼罗河病毒疫苗、黄热病疫苗、轮状病毒疫苗、水痘疫苗、伤寒Vi多糖疫苗、肺炎球菌疫苗、B型流感嗜血杆菌结合疫苗、细菌性痢疾疫苗或者HPV疫苗。
在一个更为优选的实施方案中，所述疫苗为新型冠状病毒疫苗。
更为优选地，所述新型冠状病毒疫苗为新型冠状病毒重组质粒疫苗、新型冠状病毒重组腺病毒载体疫苗或者新型冠状病毒细胞载体疫苗。
最后，本发明还提供了含有上述的菌株组合物，所述组合物含有制药学上可接受的载体。
在一个优选的实施方案中，所述组合物被制备为注射剂、胶囊、冻干粉、喷雾剂、悬液或者片剂。
在一个更为优选的实施方案中，所述组合物还含有靶向药物或者胸腺肽。
更为优选地，所述靶向药物包括靶向PD-1/PD-L1（Programmed cell death protein）、CTLA-4（Cytotoxic-T-lymphocyte-Antigen-4）、 CD24、EGFR（Epidermal Growth Factor Receptor ）、VEGFR （Vascular Endothelial Growth Factor  Receptor）、HER-2 (Human Epidermal Growth Factor Receptor 2)、Claudin18.2、glypican-3、FAP （Fibroblast Activation Protein）、PSMA（Prostate-Specific Membrane Antigen）、 PSA （Prostate-Specific Antigen）、CEA （Carcinoembryonic Antigen）、AXL、CD20、CD19、BCMA （B-cell Maturation Antigen ）、CD22、ROR1、CD24、mTOR （Mechanistic Target of Rapamycin）、ALK （Anaplastic Lymphoma Kinase）、c-kit或者MUC1（mucin1）。
 有益效果
本发明是从健康人粪便中分离纯化得到具有免疫增强作用的益生菌植物乳酸杆菌。实验证明分离得到的植物乳酸杆菌对动物无害，且经动物实验确证具有增强接种新冠病毒疫苗个体血清特异性中和抗体的滴度。所述植物乳酸杆菌GUANKE能够有效维持接种后小鼠的血清中中和抗体水平滴度，显示出在制备新冠疫苗免疫增强制剂中的优异应用前景。
 附图说明
图1-图6为 植物乳杆菌GUANKE增强志贺菌疫苗免疫小鼠抗志贺菌特异性抗体的应用，实验所用小鼠为18-22 g SPF级Balb/c小鼠雌雄各半，免疫原为志贺菌疫苗。
图1：ELISA方法检测灌胃给予志贺菌疫苗后30天灌胃给予植物乳杆菌宿主粪便中抗I型痢疾志贺菌IgG结合抗体的滴度；横坐标为时间点，纵坐标为结合抗体的滴度，***表示p<0.001。
图2：ELISA方法检测灌胃给予志贺菌疫苗后30天灌胃给予植物乳杆菌宿主粪便中抗I型痢疾志贺菌IgA结合抗体的滴度；横坐标为时间点，纵坐标为结合抗体的滴度，***表示p<0.001。
图3：ELISA方法检测灌胃给予志贺菌疫苗后30天灌胃给予植物乳杆菌宿主粪便中抗I型痢疾志贺菌IgM结合抗体的滴度；横坐标为时间点，纵坐标为结合抗体的滴度。
图4：ELISA方法检测灌胃给予志贺菌疫苗后30天灌胃给予植物乳杆菌宿主血清中抗I型痢疾志贺菌IgG结合抗体的滴度；横坐标为时间点，纵坐标为结合抗体的滴度，**表示p<0.01，***表示p<0.001。
图5：ELISA方法检测灌胃给予志贺菌疫苗后30天灌胃给予植物乳杆菌宿主血清中抗I型痢疾志贺菌IgA结合抗体的滴度；横坐标为时间点，纵坐标为结合抗体的滴度。
图6：ELISA方法检测灌胃给予志贺菌疫苗后30天灌胃给予植物乳杆菌宿主血清中抗I型痢疾志贺菌IgM结合抗体的滴度；横坐标为时间点，纵坐标为结合抗体的滴度，**表示p<0.01，***表示p<0.001。
图7-图14为新冠疫苗免疫五个月后植物乳杆菌GUANKE灌胃对小鼠的抗体滴度的影响，实验所用小鼠为6-8周龄雌性ICR，免疫原为细胞载体疫苗K562-S。
图7：ELISA方法检测免疫结束后，灌胃之前一系列时间点小鼠血清中IgG结合抗体的滴度；横坐标为时间点，纵坐标为结合抗体的滴度。
图8：ELISA方法检测灌胃结束后一系列时间点小鼠血清中IgG结合抗体的滴度；横坐标为时间点，纵坐标为结合抗体的滴度，*表示p<0.05，**表示p<0.01。
图9：ELISA方法检测灌胃结束后一系列时间点小鼠血清中IgG结合抗体的滴度；横坐标为时间点，纵坐标为该时间点结合抗体滴度与灌胃前结合抗体滴度的比值，**表示p<0.01。
图10：ELISA方法检测灌胃结束后一系列时间点小鼠血清中IgG结合抗体的滴度；横坐标为时间点，纵坐标为该时间点植物乳杆菌组结合抗体均值与PBS组结合抗体均值的比值。
图11：293T-ACE2细胞检测免疫结束后，灌胃之前一系列时间点小鼠血清中中和抗体的滴度；横坐标为时间点，纵坐标为中和抗体的滴度（ID50）。
图12：293T-ACE2细胞检测免疫结束后一系列时间点小鼠血清中中和抗体的滴度；横坐标为时间点，纵坐标为中和抗体的滴度（ID50）。
图13：293T-ACE2细胞检测免疫结束后一系列时间点小鼠血清中中和抗体的滴度；横坐标为时间点，纵坐标为该时间点中和抗体滴度与灌胃前中和抗体滴度的比值，**表示p<0.01。
图14：293T-ACE2细胞检测免疫结束后一系列时间点小鼠血清中中和抗体的滴度；横坐标为时间点，纵坐标为该时间点植物乳杆菌组中和抗体均值与PBS组中和抗体均值的比值。
图15-图20为新冠疫苗免疫后立刻植物乳杆菌GUANKE灌胃对小鼠的抗体滴度及T细胞应答的影响，实验所用小鼠为6-8周龄雌性ICR，免疫原为DNA-S及AdC68-RHAF。
图15：ELISA方法检测灌胃结束后一系列时间点小鼠血清中IgG结合抗体的滴度；横坐标为时间点，纵坐标为结合抗体的滴度，*表示p<0.05，**表示p<0.01。
图16：ELISA方法检测灌胃结束后一系列时间点小鼠血清中IgG结合抗体的滴度；横坐标为时间点，纵坐标为该时间点结合抗体滴度与灌胃前结合抗体滴度的比值，*表示p<0.05。
图17：ELISA方法检测灌胃结束后一系列时间点小鼠血清中IgG结合抗体的滴度；横坐标为时间点，纵坐标为该时间点植物乳杆菌组结合抗体均值与PBS组结合抗体均值的比值。
图18：293T-ACE2细胞检测免疫结束后一系列时间点小鼠血清中中和抗体的滴度；横坐标为时间点，纵坐标为中和抗体的滴度（ID50），**表示p<0.01。
图19：ELISPOT方法检测免疫结束后各个时间点小鼠脾细胞的T细胞应答；横坐标为时间点，纵坐标为每百万个脾细胞中分泌IFN-γ的细胞数量；*表示p<0.05。
图20：ELISPOT方法检测免疫结束后各个时间点小鼠肺灌洗液中的T细胞应答。横坐标为时间点，纵坐标为每百万个脾细胞中分泌IFN-γ的细胞数量；*表示p<0.05。
 本发明的实施方式
下面结合具体实施案例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的保护范围构成任何限制。
实施例1 植物乳杆菌GUANKE的分离、保藏和鉴定
1. 植物乳杆菌GUANKE的分离
1）从保菌管中取出人粪便来源的粪便标本100 μL，加入到预装900 μL无菌PBS的EP管中，依次对样本进行梯度稀释，粪便标本浓度稀释至10 -6倍。
2）取不同稀释度的样品100 μL涂布于MRS培养基上，放入培养箱中。
3）在37℃，0.5% CO2环境中培养48 h。
4）取出培养皿，用无菌接种环挑取不同形态特征的菌落，转接至新的MRS固体培养基中进行纯化，37℃厌氧培养48 h，连续转接3次，将纯化菌株在pH=3.5的液体MRS中培养，筛选耐酸生长优良的菌株可用于实验或冷冻保藏。
2. 菌种保藏
本实验室用含25%甘油的培养基作为保菌液进行菌种的冷冻保藏，方法如下：
1）将容量为2 mL的保菌管经121℃，15 min高压灭菌处理后以备使用。
2）细菌在固体培养基上连续转接3次后，向培养皿上加入1.5 mL的无菌保菌液。
3）用L棒对培养皿进行刮涂，使菌落充分融入保菌液中。
4）将菌液转移到保菌管中，混匀后于-80℃保藏。
3. 菌落外观和菌体形态观察
乳杆菌属兼性厌氧菌，在厌氧条件下生长良好，菌落呈乳白色、表面光滑；在有氧条件下，乳杆菌亦可生长，大部分菌种菌落表面较粗糙，菌落颜色多为乳白色。镜下观察乳杆菌细胞形态为多形性，多呈细长杆状、较粗杆状、球杆状等，排列呈栅栏状、链状等。
4. 细菌总DNA的提取
将单个菌落接种于BHI培养基上，37℃厌氧过夜培养，按照细菌基因组DNA提取试剂盒（TIANGEN）说明书操作，提取DNA。
5. 细菌通用引物16S rDNA PCR扩增
细菌16S rRNA 鉴定：提取细菌基因组DNA，扩增乳杆菌通用引物16S rDNA PCR产物并测序，序列在NCBI上进行BLAST比对，进行初步鉴定。
本实验所用的细菌16S rDNA PCR扩增的引物以及PCR反应的条件如下。
通用引物16S rDNA PCR 扩增条件：
95℃ 5 min; 95℃ 45 s, 54℃ 45 s 72℃ 1.5 min (32个循环)；72℃ 10 min。
引物序列：
27F ：       5'-AGAGTTTGATCMTGGCTCAG-3'。
1492R：    5'-TACGGYTACCTTGTTACGACTT-3。
实验全部用50 μL的PCR体系。通过对待测乳杆菌通用引物16S rDNA PCR产物测序结果在NCBI上进行BLAST比对，鉴定。挑选具有代表性的样本PCR产物测序。经测序，获得一株16S rDNA如SEQ ID NO.1的菌株，基因组大小为3.3M，编码基因2000个。选择NCBI-16S ribosome RNA sequences (Bacteria and Archaea)数据库，利用BLASTn在线比对，完成对植物乳杆菌的初步鉴定。比对结果显示，该菌株的16S rDNA序列与植物乳杆菌4333的序列一致性分别为100%。该菌株的保藏信息为：菌株保藏号为CGMCC NO.21720，保藏日期为2021年01月22日，保藏分类命名为植物乳杆菌（ Lactobacillus Plantarum GUANKE），保藏单位为中国微生物菌种保藏管理委员会普通微生物中心，地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，邮政编码：100101。
实施例2 植物乳杆菌GUANKE增强志贺菌疫苗免疫小鼠抗志贺菌特异性抗体的应用
体重18-22 g SPF级Balb/c小鼠雌雄各半，每只小鼠灌胃5×10 8CFU表达I型痢疾志贺菌脂多糖抗原的志贺菌苗DOM3（林继胜，志贺氏菌基因工程-微生态疫苗初探。中国预防医学科学院 硕士研究生论文，1998年）进行免疫，30天后灌胃给与NaHCO3溶液和5×10 9 CFU植物乳酸杆菌GUANKE进行免疫调节，对照组给与0.4 mL 0.25 mol/L NaHCO 3溶液。每只小鼠尾静脉取血50 mL稀释20倍2,000 rpm离心10 min收获血清。粪便10倍稀释后匀浆液12,000 rpm离心15 min用于多种志贺菌特异性抗体检测。抗体检测方法如下。                           
（1）用每孔100 mL 浓度为1×10 8 CFU/mL的 S.dysenteriae I  112菌液包被96孔板4℃过夜。
（2）用PBST洗三遍后，每孔加200 mL封闭液（PBST中含3%脱脂奶）37℃ 封闭1 h后洗三遍。
（3）每孔加入100 mL稀释的血清或粪便匀浆液，37℃孵育1 h。
（4）洗三遍后，结合辣根过氧化物酶的羊-抗小鼠IgA, IgG, IgM (Sigma chemical co., St.Louis, Mo)稀释1:16,000倍，每孔加100 mL，37℃孵育1 h。
（5）洗三次后，每孔加入含0.02% H 2O 2 的邻苯二胺（ o-phenylenediamine)溶液 (0.4mg/ml 柠檬酸盐缓冲液pH 5.0) 100 mL，37℃ 孵育20 min。
（6）每孔加用50 mL的2 mol/L H 2SO 4终止反应，BioTekthe酶标仪读取492nm的吸光度值表示不同样本之间抗体水平的相对高低。
结果显示：小鼠免疫I型痢疾志贺菌疫苗3天后在粪便中可检测出抗I型痢疾志贺菌的特异性IgG、IgA、IgM并持续增高至14天达到高峰，所有免疫小鼠的特异性抗体水平均显著高于未免疫空白组(p<0.01)。之后三种抗体水平逐渐降低，30天接近免疫前水平。免疫后30天，分别经口给与植物乳杆菌GUANKE和PBS，一周后益生菌组小鼠粪便中抗I型痢疾志贺菌的IgG（图1）和IgA（图2）达到高峰，IgG两周后、IgA四周后降低至乳杆菌处理前的抗体水平。IgM水平无显著变化（图3）。
小鼠免疫I型痢疾志贺菌疫苗后3天至30天血清中仅可检测出很低水平的抗志贺菌的特异性IgG、IgA、IgM，所有免疫小鼠血清中的三种特异性抗体水平与未免疫空白组均无显著差异。免疫后30天，分别经口给与植物乳杆菌GUANKE和PBS，益生菌组小鼠血清中抗I型痢疾志贺菌的IgG在益生菌处理一周和两周显著高于PBS处理组，两周达到高峰，四周后降低至与未处理组无显著差异（图4）。血清IgM在益生菌处理三天、一周和两周均显著高于PBS处理组，处理一周达到高峰后逐渐降低，四周后降低至与未处理组无显著差异（图6）。血清IgA在菌苗免疫和益生菌免疫调节过程中无显著变化（图5）。
实施例3 新冠疫苗免疫五个月后植物乳杆菌GUANKE灌胃对小鼠的抗体滴度的影响
实验中涉及到的实验动物、免疫方式、免疫原、假病毒及检测方法如下。
1. 实验动物：6-8周龄雌性ICR小鼠，购自北京唯尚立拓科技有限公司。
2. 免疫方式：对小鼠左、右后肢分别进行肌肉注射，具体剂量见实施例。
3.免疫原的选择：细胞载体疫苗：K562-S作为骨髓红细胞系细胞株，不表达移植排斥抗原（MHC I类与II类），也不表达血型抗原，因此人体对于这样的细胞产品具有很好的耐受性。以K562细胞为载体，在其表面表达并展示丰富的新冠棘突蛋白（Spike, S）免疫原（Genebank: NC_045512.2），从而构建成为K562-S新冠疫苗。
4.免疫剂量：免疫原的制备参见实施例1，在实施例中所采用的免疫原免疫剂量如下。
细胞载体疫苗：K562-S(溶于无菌PBS中)与铝佐剂(Aluminium，InvivoGen，货号5200)按照体积比1:1混合后进行免疫，1E6 cells /只小鼠，100 μL。
5. 免疫间隔：具体免疫间隔见下文描述。
6. SARS-CoV2包膜假病毒(Pseudovirus)包装
（1）转染前一天准备293T细胞，用于包装质粒的转染与表达。用DMEM完全培养基将细胞稀释至5×10 6个/mL细胞，取1 mL稀释好的细胞，铺在10 cm的皿中，37℃，5% CO 2，培养过夜。
（2）吸取SARS-CoV2膜蛋白质粒pcDNA3.1-S 4 μg和pNL4-3Δenv骨架质粒8 μg (NIH AIDS Reagent Program, 3418)加入500 μL双无(无血清、无双抗，双抗为青链霉素混合液)的DMEM中，室温孵育5 min。
（3）用双无DMEM将24 μL TurboFect (Thermo Fisher Scientific)稀释，终体积为500 μL /样品，室温孵育5 min。
（4）将（2）与（3）两者混匀，1000 μL/样品终体积，室温孵育20 min，孵育结束后加到10 cm培养皿的293T细胞中。6 h后更换新鲜的15 mL完全培养基，继续在细胞培养箱中培养48 h。
（5）培养结束后，收集10 cm皿的细胞培养上清，于15 mL离心管里，然后4000 g，4℃，离心10 min，用0.45 μm的滤器过滤到新的15 mL离心管中，冻存于-80℃保存，滴定后备用。
7. 构建稳定表达hACE2受体的293T细胞
（1）人工合成人源ACE2 (hACE2)序列(Genebank#NCBI_NP_001358344.1)，序列5’端带有Age1酶切位点，3’端带有Xba1酶切位点，合成片段与载体质粒pHAGE-MCS-puro使用Age1酶切(Thermo Scientific公司，货号FD1464)与Xba1酶切(Thermo Scientific公司，FD0685)，并通过凝胶电泳后切胶回收，采用Sanprep 柱式DNA胶回收试剂盒(Promega公司，货号A9282)回收酶切片段。
（2）基因回收产物与酶切线性化载体用T4 DNA连接酶的方法连接(Thermo Scientific公司，货号2011A)：将连接产物转化至大肠杆菌E. coli Stable，在含氨苄霉素的培养板上过夜生长。第2天，随机挑取单菌落进行测序，突变位点校正，验证全部序列正确后，成功克隆出hACE2基因的慢病毒表达质粒(pHAGE-hACE2-puro)。
（3）取10 cm皿，在每个皿中接种约5×10 6个293T细胞，保证第二天转染时使细胞密度达90%为宜；将pHAGE-hACE2-puro、慢病毒包装质粒psPAX以及VSVG三种质粒，按照质量比1：2：1的比例转染293T细胞。
（4）37℃，5%的孵箱培养48 h左右，具体时间根据细胞情况而定，收集细胞上清。将收集的细胞上清用0.45 μm的滤器进行过滤，再用PEG 8000进行浓缩，即可得到较为纯化的hACE2慢病毒。
（5）提前一天铺5×10 5个的293T细胞于12孔板的一个孔内，次日向铺好的细胞中加入步骤2中浓缩的病毒500 μL，1000 g，离心2 h。
（6）离心感染结束后，继续在37℃，5%的孵箱培养12 h左右，将培养基换成添加1 μg/mL嘌呤霉素(puro)的细胞培养基培养，最后能够存活的细胞便是整合有hACE2基因的293T细胞，并通过流式分选筛选出稳定表达hACE2的293T细胞(能与S蛋白结合)。
8. 检测方法：
（1）采血：通过眼眶静脉采血的方法采集小鼠外周全血，收集于1.5 mL EP管中，室温静置使其自然凝血，凝固后的小鼠血清于7000 g，离心15 min。将小鼠血清转移至新的1.5 mL EP管中。实验前需要将样品在56℃灭活30 min，来破坏血清内的补体活性。灭活前短暂离心，避免管壁和瓶盖上的样品残存。水浴液面要没过样品液面，但不能超过瓶盖。
（2）ELSIA检测结合抗体
1）用4℃预冷的ELISA包被液稀释检测的抗原蛋白(S1，购自北京义翘神州科技有限公司；RBD，购自上海近岸生物科技有限公司)，至终浓度为1 μg/mL。在ELISA板的每孔加入100 μL包被抗原溶液，4℃过夜。
2）第二天，取出ELISA板，弃掉包被液，用0.05%的PBST缓冲液洗板3次，每次220 μL。
3）洗涤完毕后，在吸水纸上拍干，每孔用200 μL ELISA封闭液(0.5%脱脂奶粉，PBST溶解)进行封闭，室温封闭2 h。
4）封闭结束后，用0.05%的PBST洗板3次，每次220 μL。
5）对于血清或者血浆，用ELISA样品稀释液(0.5%脱脂奶粉，PBST溶解)稀释，从1:100起始，进行2倍比稀释。用未免疫的小鼠血清设置为阴性对照。设置空白孔，只加样品稀释液，每个样品需做2个复孔，每孔终体积为100 μL，室温孵育3 h。
6）样品孵育结束后，继续用PBST洗板5次，每次220 μL。
7）用ELISA封闭液(0.5%脱脂奶粉，PBST溶解)稀释相对应比例的二抗(山羊抗鼠，购自北京中杉金桥生物技术有限公司，货号ZB-2305)，每孔加入100 μL，室温孵育1-1.5 h。
8）二抗孵育结束后，用0.05%的PBST洗板5次，每次220 μL。
9） 取一对金银片OPD底物，溶解于20 mL去离子水中，随后每孔加入100 μL，避光反应5 min。
10）显色结束后，用50 μL 2 nM H 2SO 4进行终止，在酶标仪上读取OD 492-OD 630值。
11）以最后一个稀释度OD 492大于2倍的(negative mean + SD)值对应的血清稀释比的倒数作为抗体滴度。
9.  293T-ACE2细胞检测中和抗体：
（1）取96孔透明底黑板进行中和实验，第一列设置细胞对照(CC)(150 μL)，第二列设置病毒对照(VC)(100 μL)，其他均为样品孔，对血清样品进行倍比稀释，最终孔中体积为100 μL。
（2）除细胞对照组外，每孔加50 μL SARS-CoV-2假病毒稀释液，使每孔最终含假病毒为200 TCID 50。
（3）轻轻震荡混匀，将上述96孔底黑板置于细胞培养箱中，37℃，5% CO 2孵育1 h。
（4）当孵育时间至20 min时，开始准备293T-hACE2靶细胞，并用完全培养基将细胞稀释至10 5个细胞/mL。
（5）当孵育时间至1 h，向96孔透明底黑板中每孔加100 μL靶细胞，使每孔细胞为10 4个。
（6）前后左右轻轻晃动96孔透明底黑板，使孔中的细胞均匀分散，再将板子放入细胞培养箱中，37℃，5% CO 2培养48 h。
（7）培养48 h后，从细胞培养箱中取出96孔透明底黑板，吸掉孔中上清，每孔加入100 μL PBS清洗一遍，吸去PBS，每孔加入50 μL 1×的裂解缓冲液(购自Promega公司 Cat#E153A)，室温在水平摇床上孵育30 min使细胞充分裂解。
（8）加30 μL荧光素酶的底物(购自Promega公司，Cat#E1501)于96孔黑板中，用仪器GloMax® 96微孔板发光­检测仪检测荧光素酶活性。
（9）导出荧光素读值，计算中和抑制率，结合中和抑制率结果，利用Graphpad Prism 5.0软件计算ID 50。
Figure 937812dest_path_image001
在实施例2中，我们发现针对细菌疫苗，给予植物乳杆菌GUANKE能够提高小鼠粪便及血清中的结合抗体滴度。因此我们想要验证在病毒疫苗中是否有同样的效果。我们采用6-8周雌性ICR小鼠用新冠疫苗K562-S疫苗肌肉注射免疫，4周后加强免疫一次。免疫23周后，即五个月后，饮水中给与1g/L的氨苄预处理小鼠的肠道菌群定植抗性，5天后换正常饮用水，每日经口灌胃给与5×10 9 CFU植物乳杆菌GUANKE，连续灌胃三天进行免疫调节。植物乳杆菌GUANKE灌胃前及灌胃后1、2、3、6周采集眼眶静脉血。免疫30周后，重复氨苄预处理和植物乳杆菌GUANKE的免疫调节，免疫调节后1、2、3、4周采集眼眶静脉血检测抗体应答。结果显示：免疫后小鼠的血清中的结合抗体和中和抗体在第2周达到峰值，随后缓慢下降（图7、11）。两次干预后各组小鼠血清结合抗体及中和抗体的滴度如图8、12，植物乳杆菌二次灌胃后结合抗体有明显提升，中和抗体也有上升趋势，而PBS组则缓慢下降中。同时比较灌胃后各个时间点的抗体滴度与灌胃前本底低毒的比值我们发现，植物乳杆菌灌胃后，结合抗体及中和抗体的提升相比于PBS有显著性（图9、13）。同时，我们通过比较每个点植物酸杆菌灌胃组抗体滴度与PBS灌胃组抗体滴度的比值，我们发现，和灌胃前比较，结合抗体及中和抗体的比值都在上升，提示植物乳杆菌冠克株摄入能有效阻止新冠病毒抗体下降，甚至能够提高抗体水平，对新冠感染的防治有重要意义（图10、14）。
实施例4 新冠疫苗免疫后立即植物乳杆菌GUANKE灌胃对小鼠的抗体滴度及T细胞应答的影响
实验中涉及到的实验动物、免疫方式、免疫原、假病毒及检测方法如下。
1.实验动物 ：6-8周龄雌性ICR小鼠，购自北京唯尚立拓科技有限公司。
2. 免疫方式：对小鼠左、右后肢分别进行肌肉注射；或进行滴鼻。具体剂量见实施例。
3. 免疫原的选择：重组质粒疫苗(DNA)：pcDNA3.1-S DNA疫苗不像减毒活疫苗会产生回复突变，也比灭活疫苗安全；使用灵活，可作用于肌肉（骨骼肌）、皮下或粘膜组织；常温下稳定、可以长期保持活性，便于储存、运输。因此我们采用传统的表达载体pcDNA3.1,在其多克隆位点插入新冠棘突蛋白（Spike, S）免疫原（Genebank: NC_045512.2），从而构建成为pcDNA3.1-S新冠疫苗。
重组腺病毒载体疫苗：AdC68-RHAF黑猩猩腺病毒载体AdC68是一种线性DNA病毒，基因组大小26-45 kb，相比于其他腺病毒（如Ad5和Ad26）而言，其在人群中预存免疫低，会避免载体自身带来的疫苗效果削弱，同时黑猩猩腺病毒的Hexon与凝血因子X结合后不稳定，不在肝脏富集，更为安全。我们以黑猩猩腺病毒AdC68为载体，携带含有新冠病毒受体结合域（RBD）的免疫原RHAF（Genebank: NC_045512.2，Genebank: AGI60292.1，Genebank: M97164.1），构建了AdC68-RHAF新冠疫苗。
4.免疫剂量：免疫原的制备参见实施例1。
在实施例中所采用的免疫原免疫剂量如下。
重组质粒疫苗(DNA)：100 μg /只小鼠，100 μL，溶于无菌生理盐水中。
重组腺病毒载体疫苗：5E10 vp /只小鼠，100 μL(肌肉注射)；5E10 vp /只小鼠，30 μL(滴鼻)。
5. 免疫间隔：具体免疫间隔见下文。
ELISPOT检测T细胞应答：
1. 小鼠脾脏单细胞分离：
1)    将小鼠仰卧，剖开右侧腹部皮肤，打开腹膜，取下小鼠脾脏，放入加有5 mL完全RPIM1640培养基的小平皿中。
2)    用无菌镊子将脾脏用无菌纱布包裹起来，用小弯镊夹起纱布，轻轻磨碎脾脏，可使脾细胞全部释放到培养基中。
3)    随后用5 mL移液器将脾细胞悬液经纱布吸到无菌的15 mL离心管中，800 g，离心5 min。
4)    弃掉离心后的上清，轻敲15 mL离心管重悬细胞沉淀，每个离心管中加入3 mL红细胞裂解液裂解红细胞，颠倒混匀后室温静置5 min，使红细胞充分裂解又不会损伤脾细胞。
5)    裂红结束后，用5 mLRPIM1640培养基终止裂红。800 g，离心5 min。
6)    弃掉离心后的上清，用5 mL RPIM1640培养基洗1次，800 g离心5 min。
7)    弃离心后上清，脾细胞放于冻存液(90% FBS和10%DMSO)中进行冻存备用。
2. ELISpot实验操作按照Mouse IFN-γ/Monkey IFN-γ说明书进行。
1)    用纯化IFN-γ抗体包被试剂盒(购自BD，货号551083)提供的Millipore板，比例1：250，4℃过夜包被。
2)    甩掉板中的包被抗体溶液，用200 μL RPMI 1640完全培养基洗板一遍，随后用200 μL RPMI 1640完全培养基封闭液封闭Millipore板，室温孵育2 h。
3)    弃掉孔板中的封闭液，根据不同的实验设计，在Millipore板中加入刺激肽库(苏州强耀生物科技有限公司合成单肽，每条单肽15个氨基酸，覆盖RBD序列，共65条单肽，每个肽库5条单肽，共13个肽库，50 μL/孔，每条肽的浓度为5 μg/mL。在阴性对照孔加入50 μL RPMI 1640完全培养基；阳性对照孔中加入50 μL佛波醇酯类多克隆刺激剂(PMA，购自Sigma，货号FXP012)(终浓度100ng/mL)和离子霉素(Ionomycin，终浓度2 μg/mL)的RPMI 1640完全培养基。
4)    对小鼠脾细胞进行计数，将细胞调整为4×10 6个细胞/mL，每孔加入50 μL细胞，最终使每孔的细胞数为2×10 5个细胞。将Millipore板放入湿盒，在37℃ 5% CO 2培养箱中孵育20-22 h，期间切勿摇动板子，以免造成细胞的偏移。
5)    培养孵育结束后，从培养箱中取出Millipore板，将板中的液体弃掉，用预冷的去离子水洗两遍，每次220 μL，每次清洗孵育3 min。
6)    用0.05%的PBST (PBS+0.05% Tween-20)洗板3次，每次200 μL。
7)    用10%FBS的PBS抗体稀释液稀释生物素化检测抗体(Biotinylated Detection antibody，比例1：200)，每孔加入100 μL，室温孵育2 h。
8)    孵育结束后，再用0.05%的PBST洗板3次，每次220 μL。
9)    将链霉亲和素-HRP偶联抗体用抗体稀释液进行稀释(比例1：100)，每孔加入100 μL，室温孵育1 h。
10)  孵育结束后，用0.05%的PBST洗板4次，每次220 μL。
11)  再用PBS清洗板子2次，每次220 μL。
12)  准备底物溶液(1 mL的底物缓冲液加1滴底物溶液)，每孔加入100 μL底物溶液。反应5-60 min，孵育时间根据斑点形成的情况而定。
13)  用去离子水冲洗终止反应，室温晾干后进行计数。
14)  利用ChampSpot III型酶联斑点图像分析仪进行斑点形成细胞SFC(spot forming cell，SFC)的计数以及QC。
    在实施例3中，我们发现在免疫结束后半年，给予小鼠植物乳杆菌GUANKE，能够明显减弱结合抗体及中和抗体的下降，甚至能够再次提升抗体水平。因此，我们想要观察免疫结束后立即给予小鼠植物乳杆菌GUANKE灌胃，对于疫苗引起的T细胞应答和抗体水平是否有提升。我们采用6-8周龄雌性ICR小鼠，新冠疫苗DNA-S疫苗肌肉注射进行初次免疫，12周后采用新冠疫苗AdC68-RHAF肌肉注射加滴鼻加强免疫一次。同实施例3给与氨苄预处理小鼠和植物乳杆菌GUANKE进行免疫调节。益生菌灌胃后第3、7、14天处死小鼠，采集血、肺灌洗液细胞及脾细胞，其中7、14天血清进行结合抗体及中和抗体的检测（检测方法同实施例3），3、7、14天灌洗液细胞及脾细胞进行T细胞应答的检测。结果显示：植物乳杆菌灌胃的小鼠在7天时结合抗体水平明显高于PBS灌胃组，中和抗体趋势一致（图15-18），这说明植物乳杆菌能够对抗体有一定水平的提高；同时我们检测T细胞应答发现，植物乳杆菌灌胃组脾细胞中的T细胞应答维持到14天都几乎不下降，而PBS组则在7天就有明显下降，植物乳杆菌灌胃组脾细胞T细胞应答在第7天（均值+SD vs 均值+SD，p<0.05 ）和第14 天（均值+SD，p<0.05）均显著高于PBS组（图19）；同时肺灌洗液细胞的T细胞应答，植物乳杆菌灌胃组也能够维持7天不下降，而PBS组则在14天几乎降为0，植物乳杆菌灌胃组14天的数值与PBS7天的数值接近（图20）,植物乳杆菌灌胃组肺灌洗液细胞T细胞应答在第7天（均值+SD vs 均值+SD，p<0.05 ）和第14 天（均值+SD，p<0.05）均显著高于PBS组。该实验数据显示，经口给与植物乳杆菌GUANKE，可提高抗新冠病毒的结合抗体与中和抗体滴度、以及抗新冠的特异性T细胞应答，延长抗体与T细胞保护时间，对新冠感染的防治有重要意义。
 工业实用性
本发明所公开的植物乳杆菌菌株易于工业化培养并被制备为药物制剂，具有工业实用性。
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<110>  上海市公共卫生临床中心
<120>  一种植物乳杆菌及在制备新冠疫苗免疫增强剂中的应用
<160>  1    
<170>  PatentIn version 3.3
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<212>  DNA
<213>  Lactobacillus Plantarum
<400>  1
ctatacatgc agtcgaacga actctggtat tgattggtgc ttgcatcatg atttacattt     60
gagtgagtgg cgaactggtg agtaacacgt gggaaacctg cccagaagcg ggggataaca    120
cctggaaaca gatgctaata ccgcataaca acttggaccg catggtccga gtttgaaaga    180
tggcttcggc tatcactttt ggatggtccc gcggcgtatt agctagatgg tggggtaacg    240
gctcaccatg gcaatgatac gtagccgacc tgagagggta atcggccaca ttgggactga    300
gacacggccc aaactcctac gggaggcagc agtagggaat cttccacaat ggacgaaagt    360
ctgatggagc aacgccgcgt gagtgaagaa gggtttcggc tcgtaaaact ctgttgttaa    420
agaagaacat atctgagagt aactgttcag gtattgacgg tatttaacca gaaagccacg    480
gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc cggatttatt    540
gggcgtaaag cgagcgcagg cggtttttta agtctgatgt gaaagccttc ggctcaaccg    600
aagaagtgca tcggaaactg ggaaacttga gtgcagaaga ggacagtgga actccatgtg    660
tagcggtgaa atgcgtagat atatggaaga acaccagtgg cgaaggcggc tgtctggtct    720
gtaactgacg ctgaggctcg aaagtatggg tagcaaacag gattagatac cctggtagtc    780
cataccgtaa acgatgaatg ctaagtgttg gagggtttcc gcccttcagt gctgcagcta    840
acgcattaag cattccgcct ggggagtacg gccgcaaggc tgaaactcaa aggaattgac    900
gggggcccgc acaagcggtg gagcatgtgg tttaattcga agctacgcga agaaccttac    960
caggtcttga catactatgc aaatctaaga gattagacgt tcccttcggg gacatggata   1020
caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg   1080
agcgcaaccc ttattatcag ttgccagcat taagttgggc actctggtga gactgccggt   1140
gacaaaccgg aggaaggtgg ggatgacgtc aaatcatcat gccccttatg acctgggcta   1200
cacacgtgct acaatggatg gtacaacgag ttgcgaactc gcgagagtaa gctaatctct   1260
taaagccatt ctcagttcgg attgtaggct gcaactcgcc tacatgaagt cggaatcgct   1320
agtaatcgcg gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg   1380
tcacaccatg agagtttgta acacccaaag tcggtggggt aacctttagg aaccagccgc   1440
taaa                                                                                   1444

Claims (1)

  1. 一种植物乳杆菌菌株，所述菌株的保藏号为CGMCC NO.21720，保藏日期为2021年01月22日，保藏分类命名为 Lactobacillus Plantarum，保藏单位为中国微生物菌种保藏管理委员会普通微生物中心。
    2. 根据权利要求1所述的植物乳杆菌菌株，其特征在于，所述菌株的16S rDNA的序列如SEQ ID NO.1所示。
    3. 根据权利要求1或2所述的菌株在制备疫苗免疫增强剂中的应用。
    4. 根据权利要求3所述的应用，其特征在于，所述疫苗包括乙肝疫苗、卡介苗、脊髓灰质炎减毒活疫苗、无细胞百白破联合疫苗、麻腮风联合疫苗、甲肝疫苗、脑膜炎球菌多糖疫苗、乙脑疫苗、麻疹疫苗、新型冠状病毒疫苗、流感疫苗、狂犬疫苗、艾滋疫苗、埃博拉疫苗、西尼罗河病毒疫苗、黄热病疫苗、轮状病毒疫苗、水痘疫苗、伤寒Vi多糖疫苗、肺炎球菌疫苗、B型流感嗜血杆菌结合疫苗、细菌性痢疾疫苗或者HPV疫苗。
    5. 根据权利要求4所述的应用，其特征在于，所述疫苗为新型冠状病毒疫苗。
    6. 根据权利要求5所述的应用，其特征在于，所述新型冠状病毒疫苗为新型冠状病毒重组质粒疫苗、新型冠状病毒重组腺病毒载体疫苗或者新型冠状病毒细胞载体疫苗。
    7. 一种含有权利要求1或2所述的菌株的组合物，所述组合物含有制药学上可接受的载体。
    8. 根据权利要求7所述的组合物，其特征在于，所述组合物被制备为注射剂、胶囊、冻干粉、喷雾剂、悬液或者片剂。
    9. 根据权利要求8所述的组合物，其特征在于，所述组合物还含有靶向药物或者胸腺肽。
    10. 根据权利要求9所述的组合物，其特征在于，所述靶向药物包括靶向PD-1/PD-L1、CTLA-4、CD24、EGFR、VEGFR、HER-2、Claudin18.2、GPC3、FAP、PSMA、PSA、CEA、AXL、CD20、CD19、BCMA、CD22、ROR1、CD24、mTOR、ALK、c-kit或者MUC1的药物。
PCT/CN2022/089867 2021-05-18 2022-04-28 一种植物乳杆菌及在制备新冠疫苗免疫增强剂中的应用 WO2022242446A1 (zh)

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CN113308396B (zh) * 2021-05-18 2023-03-21 上海市公共卫生临床中心 一种植物乳杆菌及在制备新冠疫苗免疫增强剂中的应用
CN114028539B (zh) * 2021-09-13 2024-04-26 北京大学 粘蛋白1在抑制冠状病毒中的应用
WO2023080070A1 (ja) * 2021-11-02 2023-05-11 株式会社ヤクルト本社 Ｔｃｒクローン数の増強剤
CN115851491A (zh) * 2022-08-31 2023-03-28 西北农林科技大学 植物乳植杆菌及其应用
CN115261290B (zh) * 2022-09-26 2023-05-09 中国疾病预防控制中心传染病预防控制所 一种具有免疫调节功能的戊糖片球菌菌株及其应用
CN115595284B (zh) * 2022-10-27 2024-02-23 江苏农林职业技术学院 一株植物乳杆菌y1和巴氏毕赤酵母y2及其益生菌制剂和应用

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