WO2023116374A1 - 带状疱疹疫苗组合物 - Google Patents

带状疱疹疫苗组合物 Download PDF

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WO2023116374A1
WO2023116374A1 PCT/CN2022/135376 CN2022135376W WO2023116374A1 WO 2023116374 A1 WO2023116374 A1 WO 2023116374A1 CN 2022135376 W CN2022135376 W CN 2022135376W WO 2023116374 A1 WO2023116374 A1 WO 2023116374A1
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adjuvant
herpes zoster
varicella
vaccine
composition according
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PCT/CN2022/135376
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French (fr)
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张超
鲁阳
朱钱钧
张玲莉
杨蕴琦
郭慧丽
王紫琰
胡冬冬
钱大伟
刘海平
刁美君
沈巧英
周晨亮
刘革
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上海泽润生物科技有限公司
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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
    • A61K39/12—Viral antigens
    • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
    • A61K39/25—Varicella-zoster virus
    • 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
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • 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
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
    • C12N2710/16734—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 present invention relates to the field of vaccines.
  • the present invention relates to herpes zoster vaccine compositions.
  • VZV Varicella-zoster virus
  • human herpesvirus type 3 Varicella-zoster virus
  • Varicella-zoster virus is highly infectious and widely distributed in the global population, but so far only one virus serotype has been found, and VZV only infects humans in nature. After VZV infects humans, usually the primary infection causes chickenpox in childhood, after which the virus can remain latent in the human brain ganglia and dorsal root ganglia for a long time. VZV can reactivate and cause shingles, among other complications, when the body's immunity declines with age or otherwise becomes compromised.
  • Herpes zoster is clinically manifested as a unilateral vesicular rash. Patients may have obvious pain and discomfort. Symptoms may last for weeks or months, and even years in severe cases, resulting in decreased quality of life. Herpes zoster can also occur without a rash. Complications develop in about 25 percent of people with shingles, and this increases with age at onset.
  • PPN post-herpetic neuralgia
  • HZO herpes zoster ophthalmicus
  • herpes zoster is a disease induced by the reactivation of latent VZV, the main cause of its pathogenesis is that the latent state of the virus is broken due to the decline of host immunity or immune deficiency. Usually, the main cause of decreased immunity comes from increasing age, immune function deficiency or immunosuppression. Thus, the incidence of herpes zoster and PHN tends to increase with age, and the severity and duration of associated pain also increase with age. The annual incidence of herpes zoster in people aged 40-49 is about (2-3)/1000 people, and the annual incidence increases rapidly from the age of 50, and more than 65% of herpes zoster cases occur in people aged 50 and above among adults.
  • herpes zoster also has a high incidence and recurrence rate in organ or stem cell transplant recipients who require immunosuppressive drugs and in immunocompromised patients due to cancer chemotherapy or human immunodeficiency virus infection.
  • Herpes zoster has not yet been included in my country's national Class A and B notifiable infectious diseases category, and its epidemiological data are still insufficient. Some studies have reported that the combined annual incidence of herpes zoster among people over 50 years old in Guangdong, my country, was about 4.3/1000 between 2011 and 2013; the annual incidence of herpes zoster in Beijing increased rapidly from 40 years old during 2012 to 2013, The age of 60-69 is the main high-incidence age. The comprehensive annual incidence rate of herpes zoster in groups over 50 years old is about 4.7/1000 people, and the incidence rate of women is higher than that of men.
  • Herpes zoster and postherpetic neuralgia increase the number of doctor visits and hospitalizations, seriously affect the quality of life of patients, and bring a huge social and economic burden.
  • VZV surface glycoproteins There are 9 types of VZV surface glycoproteins identified so far, namely gB, gC, gE, gH, gI, gK, gL, gM and gN.
  • gE glycoprotein is the glycoprotein with the highest expression level of VZV, which plays a major role in the replication and assembly of the virus, and also mediates the spread of the virus between cells.
  • VZV gE which can induce the body to produce an immune response against VZV, it has become one of the main candidate antigens for VZV subunit vaccines and DNA vaccines.
  • gE protein alone cannot induce a strong cellular immune response in animal models, and an adjuvant must be used to enhance the immune response of gE.
  • an object of the present invention is to provide a herpes zoster vaccine composition that can effectively enhance antibody response and T cell immune response.
  • the present invention relates to a herpes zoster vaccine composition, said composition comprises varicella-zoster virus antigen and compound adjuvant, wherein said compound adjuvant comprises CpG oligodeoxynucleotide, QS-21 and liposome , wherein the CpG oligodeoxynucleotide is CpG7909.
  • a herpes zoster vaccine composition which induces a strong antibody response and T cell immune response in the body.
  • Fig. 1 shows the particle size detection result (Z-average: 113.5nm, PDI 0.196) of the compound adjuvant sample particle size of one embodiment of the present invention.
  • Fig. 2 shows the particle size test result (Z-average: 96.7nm, PDI 0.164) of the composite adjuvant sample of another embodiment of the present invention.
  • Figure 3 shows the non-denaturing PAGE image of gE protein in the fermentation supernatant of CHO cells ( Figure 3A) and the SDS-PAGE image of purified gE protein ( Figure 3B).
  • Figure 4 shows the antibody response induced by immunizing mice after recombinant gE protein and different compound adjuvant components were co-prepared.
  • Figure 5 shows the T cell response induced by the immunized mice after the recombinant gE protein was combined with different complex adjuvant components.
  • Figure 6 shows the antibody response induced by immunizing mice with different doses of recombinant gE protein and different components of the compound adjuvant.
  • Fig. 7 shows the T cell response induced by immunizing mice after different doses of recombinant gE protein and different components of the compound adjuvant in combination preparations.
  • Figure 8 compares the antibody responses induced by recombinant gE protein and different compound adjuvant vaccines and commercial Shingrix vaccines in mice.
  • Figure 9 compares the T cell responses induced by recombinant gE protein and different compound adjuvant vaccines and commercial Shingrix vaccines in mice.
  • Figure 10 compares the antibody responses induced by the recombinant herpes zoster vaccine of the present invention and the commercial Shingrix vaccine in cynomolgus monkeys.
  • Figure 11 compares the T cell responses induced by the recombinant herpes zoster vaccine of the present invention and the commercial Shingrix vaccine in cynomolgus monkeys.
  • the present invention provides a herpes zoster vaccine composition, said composition comprises varicella-zoster virus antigen and compound adjuvant, wherein said compound adjuvant comprises CpG oligodeoxynucleotide, QS-21 and liposome , wherein the CpG oligodeoxynucleotide is CpG7909.
  • CpG oligodeoxynucleotide is a synthetic short single-stranded DNA molecule that contains unmethylated cytosine guanine dinucleotide sequence, which can simulate bacterial DNA binding and activate Toll in mammals including humans TLR9 directly activates B cells and monocytes, indirectly activates various immune effector cells such as NK cells and T cells, enhances their function and secretion of cytokines, and induces Th1 immune responses.
  • the CpG ODN used in the present invention is CpG7909, also known as CpG2006, ODN7909 or ODN2006.
  • CpG7909 has the following nucleotide sequence: TCGTCGTTTTGTCGTTTTGTCGTT (SEQ ID No.2).
  • QS-21 is an active ingredient extracted from the bark of Quillaja Saponaria, which can induce mouse CD8+ cell immune response, produce IgG1 and IgG2a antibodies, and promote T lymphocytes to secrete Th1 cytokines, interleukin 2 and ⁇ interferon.
  • QS-21 when used alone as an adjuvant, it had significant hemolytic toxicity.
  • the concentration range of CpG ODN is 0.25 ⁇ 2mg/mL composite adjuvant. In a more preferred embodiment, the concentration of CpG ODN is 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75 or 2 mg/mL complex adjuvant.
  • the concentration range of QS-21 is 25-200 ⁇ g/mL compound adjuvant. In a more preferred embodiment, the concentration of QS-21 is 25, 50, 75, 100, 125, 150, 175 or 200 ⁇ g/mL of the complex adjuvant.
  • the relative weight ratio of CpG ODN to QS-21 is 5:1 to 20:1. In a more preferred embodiment, the relative weight ratio of CpG ODN to QS-21 is 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12: 1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1 or 20:1.
  • the liposomes comprise phosphatidylcholine (PC) and cholesterol (CHOL). In a more preferred embodiment, the liposomes consist of phosphatidylcholine (PC) and cholesterol (CHOL).
  • PC Phosphatidylcholine
  • DOPC dioleoylphosphatidylcholine
  • the fatty acid of this structure is 18 carbons in length and contains 1 unsaturated double bond at the same time.
  • Cholesterol is a lipid widely present in the human body. It is an important component of cell membranes and can stabilize liposomes and reduce the permeability of lipid membranes. Cholesterol is composed of a steroidal part and a long side chain, which varies with different cholesterol derivatives. In one embodiment, cholesterol derivatives having the same steroidal moiety structure as in the following formula and a side chain length of 4-8 carbon chains are preferred. In another embodiment, cholesterol having the structure shown below is preferred.
  • the concentration range of phosphatidylcholine is 0.5-4 mg/mL compound adjuvant. In a more preferred embodiment, the concentration of phosphatidylcholine is 0.5, 1, 1.5, 2, 2.5, 3, 3.5 or 4 mg/mL complex adjuvant.
  • the concentration of cholesterol ranges from 0.125 to 1 mg/mL of the complex adjuvant. In a more preferred embodiment, the concentration of cholesterol is 0.125, 0.25, 0.375, 0.5, 0.625, 0.75, 0.875 or 1 mg/mL complex adjuvant.
  • the varicella-zoster virus antigen is the varicella-zoster virus glycoprotein gE, more preferably a fragment of the extracellular region of gE. In a most preferred embodiment, the varicella-zoster virus antigen is a fragment of the extracellular region of gE having the amino acid sequence shown in SEQ ID No.1. In one embodiment, the varicella-zoster virus antigen comprises amino acids more than 95%, more than 96%, more than 97%, more than 98% or more than 99% identical to the amino acid sequence shown in SEQ ID No.1 sequence. In one embodiment, the nucleic acid encoding the gE extracellular domain fragment having the amino acid sequence of SEQ ID No.1 has the nucleotide sequence shown in SEQ ID No.3.
  • the concentration of varicella-zoster virus antigen in the herpes zoster vaccine composition ranges from 20-400 ⁇ g/mL. In a preferred embodiment, the concentration of the varicella-zoster virus antigen in the herpes zoster vaccine composition is 50-200 ⁇ g/mL.
  • the concentration of varicella-zoster virus antigen in the herpes zoster vaccine composition is, for example, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 or 400 ⁇ g/mL.
  • the herpes zoster vaccine composition of the present invention can be obtained by dissolving varicella-zoster virus antigen in a complex adjuvant.
  • varicella-zoster virus antigen as freeze-dried powder can be dissolved in 0.5 mL of complex adjuvant liquid, mixed uniformly, and prepared as a unit dose for injection.
  • the content of varicella-zoster virus antigen per dose is 10-200 ⁇ g, more preferably 25-100 ⁇ g.
  • Example 1 Preparation of liposome complex adjuvant by ethanol injection method
  • DOPC Dioleoylphosphatidylcholine
  • CpG7909 was purchased from Guangzhou Ruibo Biotechnology Co., Ltd.
  • the product obtained in the previous step was subjected to ultrafiltration and centrifugation using an Amicon Ultra ultrafiltration tube (molecular weight cut-off 30KD), and finally the solvent was changed to buffer 2.
  • particle size of the sample was detected, as shown in Figure 1.
  • a dynamic light scattering instrument (model Zetasizer nano) produced by Malvern was used for detection. Each sample was measured in triplicate.
  • the product obtained in the previous step was subjected to ultrafiltration and centrifugation using an Amicon Ultra ultrafiltration tube (molecular weight cut-off 30KD), and finally the solvent was changed to buffer 2.
  • Embodiment 3 Expression and identification of gE protein
  • VZV Varicella-zoster virus
  • SEQ ID No. .1 the extracellular domain amino acid sequence
  • SEQ ID No. 3 the gE protein cDNA sequence
  • the constructed expression vector plasmid carrying the target gE protein gene was digested and linearized, it was electrotransfected into the eukaryotic mammalian CHO-K1 cell line. After mother clone cell line screening, monoclonal cell line screening, monoclonal cell line evaluation, and monoclonal cell line stability evaluation, a stable CHO-K1 cell line capable of stably expressing the target gE protein antigen was obtained.
  • the monoclonal cell lines are screened, they are gradually scaled up to shake flask and reactor scale for fermentation.
  • the supernatant after fermentation was collected the purified gE glycoprotein was obtained by anion chromatography, hydrophobic chromatography and ultrafiltration, and finally the obtained target protein was identified by SDS-PAGE.
  • the results showed that the target protein gE product had a high purity and a molecular weight of about 70 kDa, which was consistent with expectations ( FIG. 3B ).
  • the target protein gE is a glycoprotein, and the CHO cell expression system can retain the glycosylation modification of the protein to the greatest extent, in the PAGE gel, the band of the target protein gE presents a certain diffuse shape.
  • C57BL/6 mice were immunized with 5 ⁇ g of recombinant gE protein, combined with compound adjuvant and its components, respectively, to immunize C57BL/6 mice (provided by "Experimental Animal Management Department of Shanghai Institute of Family Planning Sciences"). All mice were immunized twice on the hind leg muscles on day 0 and day 21, and the injection volume was 0.1 mL each. On the 31st day, blood was collected, serum was separated, and antibody titer was tested. After the blood was collected, the mouse spleen was taken, and the lymphocytes were separated to detect the T cell immune response. The specific grouping information is shown in the table below.
  • Antibody titer was detected by ELISA method. Specifically, the collected mouse peripheral blood serum was first stored at -20°C until use. Use coating buffer (ORIGENE, product number ZLI-9063) to dilute the gE protein stock solution and coat it on a 96-well microtiter plate overnight, then block with PBS blocking solution adding 5% skim milk powder (Deyun skim milk powder, product number 5381) 2 hours. After adding the serum to be tested and incubating at 37°C, add HRP enzyme-labeled secondary antibody (Bio-Rad, Cat. No. 170-6515) and continue to incubate for 45 minutes.
  • Use coating buffer (ORIGENE, product number ZLI-9063) to dilute the gE protein stock solution and coat it on a 96-well microtiter plate overnight, then block with PBS blocking solution adding 5% skim milk powder (Deyun skim milk powder, product number 5381) 2 hours. After adding the serum to be tested and incubating at 37°C, add
  • the prepared commercial color development solution (Sera Care, product number 5120-0038 and 5120-0049) was added to develop color at 37°C for 10-15 minutes, and finally 50 ⁇ l of 2M H 2 SO 4 stop solution was added to stop the color development.
  • Use a multifunctional microplate reader (MOLECULAR, model SpectraMax iD3) to read its OD450/620 value. The OD value of 0.105 was used as the cut-off value. When the OD value ⁇ 0.105 at a certain dilution of the serum group, it was considered positive. When the OD value was ⁇ 0.105, the previous dilution was the antibody titer of the serum.
  • the T cell immune response was detected by using the FluoroSpot method to detect the number of positive spots for cytokines IL-2, IFN- ⁇ and TNF- ⁇ produced by mouse spleen lymphocytes after gE protein stimulation.
  • a mouse spleen single cell suspension was first prepared. The mice sacrificed by neck dissection were transferred to an ultra-clean bench for aseptic dissection to remove the mouse spleen, and the spleen cell suspension was obtained by submerging and filtering with a filter. Add erythrocyte lysate (BOSTER, Cat. No. AR1118) to remove erythrocytes in the cell suspension.
  • the pre-designed group stimulation method add an appropriate amount of cell suspension, gE protein pool stimulator and anti-CD28 antibody solution to each well of the corresponding stimulation well, place in a cell culture incubator, and stimulate for about 24-36 hours at 37°C.
  • After stimulation add detection secondary antibody and tertiary antibody sequentially according to the instructions, and then add 50 ⁇ l color development solution to each well for 15 minutes at room temperature in the dark. Then discard the chromogenic solution, remove the rubber pad at the bottom of the 96-well plate, and dry the PVDF membrane at the bottom of the well in a ventilated place.
  • the spot counting data of cytokines IFN- ⁇ , TNF- ⁇ and IL-2 secreted in each well were read and analyzed with the FluoroSpot plate reader of CTL.
  • mice were immunized twice in hind leg muscles on day 0 and day 21, with an injection volume of 0.1 ml each. Blood was collected on day 31, serum was separated, and antibody titer was detected. After the blood was collected, the mouse spleen was taken, and the lymphocytes were separated to detect the T cell immune response.
  • the specific grouping information is shown in the table below.
  • Antibody titer detection adopts ELISA method, same as Example 4. T cell immune response was detected by flow cytometry and FluoroSpot method (same as Example 4).
  • Flow cytometry can detect antigen-specific CD4+ T cell immune responses that secrete specific cytokines.
  • a mouse spleen single cell suspension was first prepared. The mice sacrificed by neck dissection were transferred to an ultra-clean bench for aseptic dissection to remove the mouse spleen, and the spleen cell suspension was obtained by submerging and filtering with a filter. Add erythrocyte lysate (BOSTER, Cat. No. AR1118) to remove erythrocytes in the cell suspension. After washing with complete medium (10% FBS (Gibco, 10091-148) + RPMI 1640 medium (Gibco, 10091-148)), a small amount of suspension was taken for cell counting by a cell counter before use.
  • complete medium 10% FBS (Gibco, 10091-148) + RPMI 1640 medium (Gibco, 10091-148)
  • BD cyto-fix/cytoperm Fixation/Permeabilization kit (BD, product number 554714) to fix the cells and permeabilize the membrane for 20 minutes, then add PE-Cy7 anti-mouse IL-2 (BD, product number 560538), APC anti -Mouse IFN- ⁇ (BD, Cat. No. 554413), PE anti-mouse TNF- ⁇ (BD, Cat. No. 554419) antibodies were stained at low temperature for 30 minutes in the dark. Finally, the supernatant was centrifuged and the cells were washed with PBS. The washed cells were resuspended in PBS, and the ratio of different cytokines secreted by CD4+ T live cells was detected by FACS Canto II Flow Cytometer and data analysis was performed.
  • PE-Cy7 anti-mouse IL-2 (BD, product number 560538)
  • APC anti -Mouse IFN- ⁇ (BD, Cat. No. 554413)
  • Example 6 Comparison of immunogenicity of gE protein and different compound adjuvant vaccines and commercial Shingrix vaccines in mice
  • mice were pre-immunized with live attenuated vaccines to simulate the natural pathogenesis, so as to evaluate the efficacy of the product more reasonably . All mice were immunized with 1 dose of human varicella live attenuated vaccine by subcutaneous injection 28 days before the candidate vaccine composition, and the injection volume was 0.5 mL per mouse. On the 0th day and the 21st day, the candidate vaccine composition and the control sample were immunized twice in the hind leg muscle, and the injection volume was 0.05 mL each.
  • Antibody titer detection adopts ELISA method, same as Example 4.
  • the T cell immune response was detected using the FluoroSpot method, the same as in Example 4, but limited to the kit (Mabtech, Cat. No. FS-4142-10), only two cytokines, IL-2 and IFN- ⁇ , were detected.
  • the results are shown in Figure 8 and Figure 9.
  • the results show that under the premise of using the same recombinant gE protein, the recombinant herpes zoster vaccine prepared with the composite adjuvant of the present invention has no significant difference in the level of antibody response induced in mice relative to the vaccine prepared with the imitation AS01B composite adjuvant, but Can induce higher levels of T cell responses.
  • the recombinant herpes zoster vaccine prepared with the compound adjuvant of the present invention has no significant difference in the level of antibody response induced in mice compared with the commercial recombinant herpes zoster vaccine Shingrix, but it can also induce a higher level of T cell response , showing better vaccine immunogenicity.
  • Example 7 Comparison of the immunogenicity of the herpes zoster vaccine of the present invention and the commercial Shingrix vaccine in monkeys
  • herpes zoster vaccine composition of the present invention we compared the commercialized similar recombinant herpes zoster vaccine Shingrix (the listed recombinant herpes zoster vaccine produced by GSK Company, Chinese name: Xin'an Lishi) in monkeys. ) for a comparative immunogenicity study.
  • Antibody titer detection adopts the ELISA method, the same as in Example 4, but the detection secondary antibody is replaced by the corresponding monkey secondary antibody (Bethyl Laboratories, Cat. No. A140-102P).
  • the T cell immune response was detected using the FluoroSpot method, the same as in Example 4, but the detection kit was replaced with a corresponding monkey detection kit (Mabtech, catalog number FSP-212822-10). PBMC were separated by Ficoll density gradient centrifugation.

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Abstract

提供了一种带状疱疹疫苗组合物，所述组合物包含水痘-带状疱疹病毒抗原和复合佐剂，其中所述复合佐剂包含CpG寡脱氧核苷酸、QS-21和脂质体，其中CpG寡脱氧核苷酸是CpG7909。所述组合物可诱导强的抗体反应和T细胞免疫反应，用于预防水痘-带状疱疹病毒感染。

Description

带状疱疹疫苗组合物 技术领域
本发明涉及疫苗领域。具体而言，本发明涉及带状疱疹疫苗组合物。
背景技术
水痘-带状疱疹病毒(Varicella-zoster virus，VZV)是八种人类疱疹病毒之一，也叫做3型人疱疹病毒。水痘-带状疱疹病毒具有很强的传染性，在全球人群中广泛分布，但迄今只发现一种病毒血清型，且在自然界中VZV仅感染人类。VZV感染人体后，通常原发性感染在儿童期导致水痘，之后病毒可长期潜伏于人体脑神经节和背根神经节中。当机体随着年龄的增长或其他原因导致免疫力下降或免疫功能受损时，VZV可以重新激活并引发带状疱疹及其他并发症。
带状疱疹临床表现为单侧性水泡样皮疹，患者可有明显疼痛和不适，症状可持续数周或数月，在重症患者中甚至可持续数年，导致生活质量下降，极少数情况下带状疱疹也可无皮疹出现。约25％患带状疱疹的人会发生并发症，并且随着发病年龄的增长而增加。最常见的严重并发症是带状疱疹后神经痛(post-herpetic neuralgia，PHN)，即疱疹急性期后仍持续存在的疼痛，在带状疱疹患者中发生率为10％-30％，疼痛可持续数月甚至数年，严重影响患者生活质量；其次是眼部带状疱疹(herpes zoster ophthalmicus，HZO)，在带状疱疹患者中有10％-20％的发生率；其他并发症包括运动神经损伤和其他神经系统并发症如脑膜炎等。
由于带状疱疹是由潜伏的VZV再次激活所诱发的疾病，其发病的主要原因是由于宿主免疫力下降或免疫缺陷导致病毒的潜伏状态被打破。而通常情况下，免疫力下降的主要原因来自于年龄的增加、免 疫功能的缺陷或免疫抑制。因此，年龄越大，带状疱疹和PHN的发病率往往越高，且相关疼痛的严重程度和持续时间也随着年龄的增加而增加。在40-49岁人群中带状疱疹的年发病率约为(2-3)/1000人，从50岁开始年发病率快速增加，超过65％的带状疱疹病例发生在50岁及以上的成年人中。人在一生中发生带状疱疹的风险约为25％-30％，但在80岁以上的人群中这一风险增加到50％。临床上带状疱疹在需要服用免疫抑制药物的器官或干细胞移植者以及由于肿瘤化疗或人类免疫缺陷病毒感染导致免疫功能受损的患者中也具有较高的发病率和复发率。
带状疱疹目前尚未被我国纳入国家甲乙类法定报告传染病范畴，其流行病学数据尚不充分。部分研究报道了我国广东地区2011-2013年间50岁以上群体中带状疱疹综合年发病率约为4.3/1000人；北京地区2012-2013年间带状疱疹年发病率从40岁开始快速升高，60-69岁为主要的高发年龄，50岁以上群体中带状疱疹综合年发病率约为4.7/1000人，且女性发病率高于男性。从以上研究数据可以看出，我国的带状疱疹发病情况不容乐观，尤其随着中国人口的老龄化将更加加剧带状疱疹的疾病负担。在全世界其他地区，带状疱疹的发病情况和中国略有差别，但同样不容乐观。在亚太地区，韩国、台湾、日本等地区发病率高峰在70岁左右，而澳大利亚、新西兰等地区发病率高峰在80岁左右。并且韩国是整个亚太地区发病率相对较高的国家，其带状疱疹最高年发病率超过了20/1000人。在欧洲和美国，其带状疱疹在老年人中最高年发病率主要在10/1000人左右。
带状疱疹及带状疱疹后神经痛增加了患者就诊次数和住院次数，严重影响了患者的生活质量，带来了极大的社会经济负担。已有研究估算2010-2012年间国内带状疱疹患者人均医疗花费达840元，估计在2010-2012年间，国内每年带状疱疹患者医疗总费用达到13.14亿元。因此，面对如此严重的疾病负担，急需更多更好的疾病治疗和预防药物，尤其是预防性疫苗，以降低带状疱疹发病率。
目前确定的VZV病毒表面糖蛋白有9种，分别是gB、gC、gE、gH、gI、gK、gL、gM和gN。其中gE糖蛋白是VZV表达量最高的糖蛋白，在病毒的复制和组装过程中起到主要作用，也介导病毒在细胞间的传播。鉴于VZV gE具有很强的免疫原性，能诱导机体产生针对VZV的免疫反应，它已成为VZV亚单位疫苗和DNA疫苗的主要候选抗原之一。
目前已知的是，单独的gE蛋白在动物模型中并不能够诱导强烈的细胞免疫反应，必须通过佐剂才能增强gE的免疫反应。
然而，目前常用的铝佐剂的免疫增强效果较弱，且主要产生体液免疫，无法满足带状疱疹疫苗的开发需求。
发明内容
鉴于以上情况，本发明的一个目的在于，提供一种可有效增强抗体反应和T细胞免疫反应的带状疱疹疫苗组合物。
本发明涉及一种带状疱疹疫苗组合物，所述组合物包含水痘-带状疱疹病毒抗原和复合佐剂，其中所述复合佐剂包含CpG寡脱氧核苷酸、QS-21和脂质体，其中CpG寡脱氧核苷酸是CpG7909。
根据本发明，可提供在机体内诱导强的抗体反应和T细胞免疫反应的带状疱疹疫苗组合物。
附图说明
图1显示本发明的一个实施方案的复合佐剂样品粒径检测结果(Z-average：113.5nm，PDI 0.196)。
图2显示本发明的另一个实施方案的复合佐剂样品粒径检测结果(Z-average：96.7nm，PDI 0.164)。
图3显示CHO细胞发酵上清液中的gE蛋白非变性PAGE胶图像(图3A)和纯化的gE蛋白的SDS-PAGE图像(图3B)。
图4显示重组gE蛋白与不同的复合佐剂成分联合制剂后免疫小鼠诱导的抗体反应。
图5显示重组gE蛋白与不同的复合佐剂成分联合制剂后免疫小 鼠诱导的T细胞反应。
图6显示不同剂量重组gE蛋白与复合佐剂不同成分剂量配比联合制剂后免疫小鼠诱导的抗体反应。
图7显示不同剂量重组gE蛋白与复合佐剂不同成分剂量配比联合制剂后免疫小鼠诱导的T细胞反应。A流式细胞术检测CD4+T细胞反应结果；B FluoroSpot方法检测T细胞反应结果。
图8比较重组gE蛋白与不同复合佐剂联合制剂后疫苗以及商品化Shingrix疫苗在小鼠体内诱导的抗体反应。
图9比较重组gE蛋白与不同复合佐剂联合制剂后疫苗以及商品化Shingrix疫苗在小鼠体内诱导的T细胞反应。
图10比较本发明重组带状疱疹疫苗和商品化Shingrix疫苗在食蟹猴体内诱导的抗体反应。
图11比较本发明重组带状疱疹疫苗和商品化Shingrix疫苗在食蟹猴体内诱导的T细胞反应。
具体实施方式
除非另外定义，本文所用的所有技术和科学术语具有本领域技术人员通常理解的相同含义。在冲突的情况下，以包括定义在内的本文件为准。下面描述优选的方法和材料，但是与本文所述那些类似或等同的方法和材料可用于实施或测试本发明。本文公开的材料、方法和实例仅是说明性的，而非旨在限制。
对于本公开内容涉及的所有数值范围，应理解为公开了该范围内所有具体数值，以及该范围内任意两个数值限定的子范围。例如，对于1％-10％，应理解为公开了1％、2％、3％、3.5％、4.5％、10％等具体数值，以及1％-5％，2％-6％，3.5％-7.5％等子范围。
本发明提供一种带状疱疹疫苗组合物，所述组合物包含水痘-带状疱疹病毒抗原和复合佐剂，其中所述复合佐剂包含CpG寡脱氧核苷酸、QS-21和脂质体，其中CpG寡脱氧核苷酸是CpG7909。
CpG寡脱氧核苷酸(CpG ODN)是人工合成的短单链DNA分子， 包含了未甲基化的胞嘧啶鸟嘌呤二核苷酸序列，可以模拟细菌DNA结合并激活哺乳动物包括人的Toll样受体9(TLR9)，直接激活B细胞和单核细胞，间接激活NK细胞和T细胞等多种免疫效应细胞，增强其功能和细胞因子的分泌，诱导Th1型免疫应答。用于本发明中的CpG ODN是CpG7909，也称为CpG2006、ODN7909或ODN2006。CpG7909具有以下核苷酸序列：TCGTCGTTTTGTCGTTTTGTCGTT(SEQ ID No.2)。
QS-21是从南美皂树(Quillaja Saponaria)树皮中提取的一种活性成分，能够引起小鼠CD8+细胞免疫应答，产生IgG1和IgG2a抗体，促进T淋巴细胞分泌Th1细胞因子，白介素2和γ干扰素。但是，在将QS-21单独用作佐剂时，具有显著的溶血毒性。
在一个优选的实施方案中，CpG ODN的浓度范围是0.25～2mg/mL复合佐剂。在一个更优选的实施方案中，CpG ODN的浓度是0.25、0.5、0.75、1、1.25、1.5、1.75或2mg/mL复合佐剂。
在一个优选的实施方案中，QS-21的浓度范围是25～200μg/mL复合佐剂。在一个更优选的实施方案中，QS-21的浓度是25、50、75、100、125、150、175或200μg/mL复合佐剂。
在一个优选的实施方案中，CpG ODN与QS-21的相对重量比为5∶1至20∶1。在一个更优选的实施方案中，CpG ODN与QS-21的相对重量比为5∶1、6∶1、7∶1、8∶1、9∶1、10∶1、11∶1、12∶1、13∶1、14∶1、15∶1、16∶1、17∶1、18∶1、19∶1或20∶1。
本发明人发现，将QS-21和脂质体载体联合配制，使QS-21吸附于脂质体载体上，可显著降低其潜在的机体内溶血毒性副作用。
在一个优选的实施方案中，脂质体包含磷脂酰胆碱(PC)和胆固醇(CHOL)。在一个更优选的实施方案中，脂质体由磷脂酰胆碱(PC)和胆固醇(CHOL)组成。
磷脂酰胆碱(PC)是一种由胆碱经磷酸根和两条脂肪酸结合形成酯键的结构。胆碱上的结构上季胺和羟基直接可以相隔多个碳原子。 两条脂肪酸的长度为12-18个碳原子，同时可能存在一个或多个不饱和双键。其中优选的是具有如下所示结构的二油酰基磷脂酰胆碱(DOPC)，该结构脂肪酸为18个碳的长度同时含有1个不饱和双键。
Figure PCTCN2022135376-appb-000001
胆固醇是一种广泛存在于人体内的类脂，是构成细胞膜的重要组成部分，可以起到稳定脂质体，降低脂质膜的通透性等功能。胆固醇是由甾体部分和一条长的侧链组成，不同的胆固醇衍生物各有不同。在一个实施方案中，优选甾体部分结构和下式中相同，侧链长度为4-8个碳链的胆固醇衍生物。在另一个实施方案中，优选具有如下所示结构的胆固醇。
Figure PCTCN2022135376-appb-000002
在一个优选的实施方案中，磷脂酰胆碱的浓度范围是0.5～4mg/mL复合佐剂。在一个更优选的实施方案中，磷脂酰胆碱的浓度是0.5、1、1.5、2、2.5、3、3.5或4mg/mL复合佐剂。
在一个优选的实施方案中，胆固醇的浓度范围是0.125～1mg/mL复合佐剂。在一个更优选的实施方案中，胆固醇的浓度是0.125、0.25、0.375、0.5、0.625、0.75、0.875或1mg/mL复合佐剂。
在一个优选的实施方案中，水痘-带状疱疹病毒抗原是水痘-带状 疱疹病毒糖蛋白gE，更优选gE的胞外区片段。在一个最优选的实施方案中，水痘-带状疱疹病毒抗原是具有SEQ ID No.1所示的氨基酸序列的gE胞外区片段。在一个实施方案中，水痘-带状疱疹病毒抗原包含与SEQ ID No.1所示的氨基酸序列具有95％以上、96％以上、97％以上、98％以上或99％以上的同一性的氨基酸序列。在一个实施方案中，编码具有SEQ ID No.1的氨基酸序列的gE胞外区片段的核酸具有SEQ ID No.3所示的核苷酸序列。
本领域技术人员理解，对于水痘-带状疱疹病毒抗原在带状疱疹疫苗组合物中的用量没有特别限制，只要是能够作为带状疱疹疫苗组合物起作用即可。在一个实施方案中，水痘-带状疱疹病毒抗原在带状疱疹疫苗组合物中的浓度范围为20-400μg/mL。在一个优选的实施方案中，水痘-带状疱疹病毒抗原在带状疱疹疫苗组合物中的浓度为50-200μg/mL。在另一个实施方案中，水痘-带状疱疹病毒抗原在带状疱疹疫苗组合物中的浓度例如为20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、280、290、300、310、320、330、340、350、360、370、380、390或400μg/mL。
作为一个非限制性实例，本发明的带状疱疹疫苗组合物可通过将水痘-带状疱疹病毒抗原溶解于复合佐剂中得到。例如，可在0.5mL的复合佐剂液体中溶解作为冻干粉末的水痘-带状疱疹病毒抗原，混合均匀，制成单位剂量后用于注射。在一个实施方案中，水痘-带状疱疹病毒抗原的每剂含量是10-200μg，更优选25-100μg。
本领域技术人员会理解，虽然上文对于本发明的带状疱疹疫苗组合物中的各组分的含量等进行了详细说明，但是它们可由本领域技术人员适当地确定，而不限于以上所列举的具体实例。
实施例
以下提供具体的实施例以进一步说明本发明。
实施例1：采用乙醇注入法制备脂质体复合佐剂
二油酰基磷脂酰胆碱(DOPC)和胆固醇(CHOL)购自艾伟拓(上海)医药科技有限公司，QS-21购自Desert King International(美国)，CpG7909购买自广州锐博生物有限公司。
缓冲液1配制：
分别称量Na 2HPO 4 12H 2O 0.6446g、KH 2PO 4 1.1207g、NaCl 1.1702g于容器中，加入注射用水200ml，搅拌溶解，即得9mM Na 2HPO4、41mM KH 2PO 4、100mM NaCl、pH6.1的缓冲液。标记为缓冲液1。除菌过滤备用。
缓冲液2配制：
分别称量Na 2HPO 4 12H 2O 0.1432g、KH 2PO 4 0.2177g、NaCl 1.755g于容器中，加入注射用水200ml，搅拌溶解，即得2mM Na 2HPO 4、8mM KH 2PO 4、150mM NaCl、pH6.1的缓冲液。标记为缓冲液2。除菌过滤备用。
有机相溶液配制：
精密称量DOPC 0.400g、CHOL 0.100g于EP管中，移液器吸取20ml无水乙醇加入其中，超声溶解，即得20mg/ml DOPC、5mg/ml CHOL的混合溶液。
QS-21溶液配制：
精密称取适量QS-21后加入缓冲液1，超声溶解，配制QS-21浓度为2mg/ml。
CpG7909溶液配制：
精密称量CpG7909冻干粉0.402g，加入50ml PBS溶解后，用超微量分光光度计检测其浓度为5.242mg/ml。
精密量取10ml浓度为5.242mg/ml的CpG7909溶液、5ml浓度为2mg/ml的QS-21溶液和75ml缓冲液1于玻璃小瓶中，搅拌混合均匀，作为水相。
精密量取10ml浓度为20mg/ml DOPC和5mg/ml CHOL的混合溶液，逐滴加入至水相中，边滴加边搅拌，搅拌转速600rpm，温度为室 温。待全部滴加完成后，继续搅拌30分钟。即为脂质体复合佐剂中间品。
取脂质体复合佐剂中间品100ml使用高压均质机在600巴的压力下均质处理10min后收集产物。
将上一步所得产物使用Amicon Ultra超滤管(截留分子量30KD)进行超滤离心，最终将溶剂换液至缓冲液2中。
最后检测样品的粒径大小，如图1所示。使用了马尔文公司所生产的动态光散射仪器(型号Zetasizer nano)进行检测。每个样品重复测量三次。
实施例2：采用薄膜水化法制备脂质体复合佐剂
有机相溶液配制：
精密称量DOPC 0.400g、CHOL 0.100g于EP管中，移液器吸取20ml无水乙醇加入其中，超声溶解，即得20mg/ml DOPC、5mg/ml CHOL的混合溶液。
QS-21溶液配制：
精密称取适量QS-21加入缓冲液1，超声溶解，配制QS-21浓度为2mg/ml。
CpG7909溶液配制：
精密称量CpG7909冻干粉0.402g，加入50ml PBS溶解后，用超微量分光光度计检测其浓度为5.242mg/ml。
精密量取10ml浓度为5.242mg/ml的CpG7909溶液、5ml浓度为2mg/ml的QS-21溶液和75ml缓冲液1于玻璃小瓶中，搅拌混合均匀，作为水相。
精密量取10ml浓度为20mg/ml DOPC和5mg/ml CHOL的混合溶液，加入圆底烧瓶后，使用旋转蒸发仪除去溶剂后再抽真空1小时除去残留有机溶剂，形成脂质薄膜。
在室温下将水相缓慢加入烧瓶中进行水化，继续搅拌30分钟后超声5min，即为脂质体复合佐剂中间品。
取脂质体复合佐剂中间品100ml使用高压均质机在600巴的压力下均质处理10min后收集产物。
将上一步所得产物使用Amicon Ultra超滤管(截留分子量30KD)进行超滤离心，最终将溶剂换液至缓冲液2中。
最后检测样品的粒径大小，如图2所示。
实施例3：gE蛋白的表达和鉴定
水痘-带状疱疹病毒(VZV)表面糖蛋白gE抗原是来自水痘-带状疱疹病毒Ellen株(GenBakn序列号：JQ972913.1)表面糖蛋白E截短后的胞外区氨基酸序列(SEQ ID No.1)。依据氨基酸序列，针对哺乳动物CHO细胞密码子偏好进行密码子优化后，委托苏州金唯智生物科技有限公司进行gE蛋白cDNA序列(SEQ ID No.3)合成。合成后的cDNA序列分别插入到真核表达载体中，并进行测序鉴定。
将构建完成的、携带目的gE蛋白基因的表达载体质粒经酶切线性化后，电转染至真核哺乳动物CHO-K1细胞株中。分别经过母克隆细胞株筛选、单克隆细胞株筛选、单克隆细胞株评估和单克隆细胞株稳定性评估后，获得能稳定表达目的gE蛋白抗原的稳定CHO-K1细胞株。
在细胞株构建过程中，为了验证目的gE蛋白的表达，取少量的发酵上清液进行gE蛋白表达检测。按照试剂盒(上海生工生物工程，货号C601104)说明，采用Precast-Glgel 4-15％非变性电泳Hepes预制胶对发酵上清液进行跑胶检测。结果显示，目的蛋白gE为二聚体结构，分子量大小约为理论大小的二倍(图3A)。
单克隆细胞株经过筛选后，逐步放大培养至摇瓶和反应器规模进行发酵。收集发酵后上清，通过阴离子层析、疏水层析和超滤换液获得纯化的gE糖蛋白，最后对获得的目的蛋白进行SDS-PAGE跑胶鉴定。结果显示，目的蛋白gE产物纯度较高，分子量大小约70kDa，和预期一致(图3B)。此外，由于目的蛋白gE是糖蛋白，且CHO细胞表达系统可以最大限度保留蛋白的糖基化修饰，因此在PAGE胶图中， 目的蛋白gE的条带呈现一定的弥散状。
实施例4：gE蛋白的免疫原性和佐剂必要性研究
我们对gE目的抗原蛋白进行了免疫原性研究，并通过联合不同的佐剂成分，评估复合佐剂成分的必要性以及每个成分对疫苗免疫原性的影响。
我们选择拟用人用剂量的1/10在C57BL/6小鼠体内进行疫苗免疫原性研究。用5μg重组gE蛋白分别与复合佐剂及其各组分分别联合制剂成疫苗，以免疫C57BL/6小鼠(由“上海市计划生育科学研究所实验动物经营部”提供)。所有小鼠于第0天、21天后腿肌肉免疫两次，注射体积为每只0.1mL。第31天采血，分离血清，进行抗体滴度检测。取血完毕后取小鼠脾脏，分离淋巴细胞，进行T细胞免疫反应检测。具体分组信息如下表所示。
表1.小鼠体内免疫原性实验动物分组
Figure PCTCN2022135376-appb-000003
抗体滴度检测采用ELISA方法。具体而言，先将采集的小鼠外周血分离血清保存至-20℃待用。使用包被缓冲液(ORIGENE，货号ZLI-9063)将gE蛋白原液稀释后包被于96孔酶标板过夜，再用加入5％脱脂奶粉(德运脱脂奶粉，货号5381)的PBS封闭液封闭2小时。加入待测血清37℃孵育后，加入HRP酶标二抗(Bio-Rad，货号170-6515)继续孵育45分钟。洗板后加入配制好的商品化显色液(Sera Care，货号5120-0038和5120-0049)37℃显色10-15分钟，最后加入50μl 2M H 2SO 4终止液终止显色。使用多功能酶标仪(MOLECULAR，型号SpectraMax iD3)读取其OD450/620值。以OD值0.105作为截止(cut-off) 值，当血清组某稀释度下，OD值≥0.105时则为阳性，当OD值＜0.105时则上一稀释度为该血清的抗体滴度。
T细胞免疫反应检测是采用FluoroSpot方法，检测小鼠脾脏淋巴细胞在gE蛋白刺激后产生的细胞因子IL-2、IFN-γ和TNF-α阳性斑点数。具体而言，先制备小鼠脾脏单细胞悬液。将断颈处死的小鼠转移至超净台中无菌解剖取出小鼠脾脏，通过淹没和滤网过滤获得脾脏细胞悬液。加入红细胞裂解液(BOSTER，货号AR1118)去除细胞悬液中的红细胞。用完全培养基(10％FBS(Gibco，10091-148)+RPMI 1640培养基(Gibco，10091-148))清洗后，取少量悬液通过细胞计数仪(Life Technologies，型号Countess II)进行细胞计数后备用。取一块FluoroSpot板，每孔用35％的乙醇溶液润湿底层膜后，按试剂盒(Mabtech，货号FSX-41A-1-10+FSX-42B-1-10+FSX-45W-1-10+3654-4-1-10)说明书加入包被一抗溶液包被过夜，再用含血清的完全培养基室温封闭至少30分钟。按预先设计的分组刺激方式，在对应的刺激孔中每孔加入适量的细胞悬液和gE蛋白池刺激物和抗CD28抗体溶液，放置于细胞培养箱中，37℃刺激约24-36小时。刺激结束后，按说明书依次加入检测二抗和三抗，之后每孔加入50μl显色液室温避光显色15分钟。然后弃除显色液，去掉96孔板底部胶垫，通风处晾干孔底PVDF膜。用CTL的FluoroSpot读板仪读取各孔中分泌细胞因子IFN-γ、TNF-α和IL-2的斑点计数数据并进行分析。
结果如图4和图5所示。复合佐剂对照组的抗体反应和T细胞反应均与PBS阴性对照组无显著差异，说明复合佐剂本身无抗体和细胞因子刺激活性。重组gE蛋白与单独脂质体联合制剂后免疫小鼠只能诱导极低水平的抗体和T细胞免疫反应，说明脂质体本身无明显的佐剂刺激活性。重组gE蛋白与脂质体联合单独的CpG7909或QS-21制剂后疫苗免疫小鼠诱导的抗体和T细胞反应均显著低于重组gE蛋白联合复合佐剂制备的疫苗。这说明，复合佐剂对于重组带状疱疹疫苗较高的免疫原性而言具有显著的优越性和必要性。
实施例5：重组带状疱疹疫苗免疫剂量探索
我们对疫苗的抗原gE蛋白和复合佐剂成分CpG7909和QS-21的剂量配比进行了摸索，通过在小鼠体内的免疫原性研究探索最佳的疫苗剂量配比。
我们选择拟用人用剂量的1/10在C57BL/6小鼠体内进行疫苗免疫原性研究，分别对gE蛋白、CpG7909和QS-21进行了多个剂量配比分组。所有小鼠于第0天、21天后腿肌肉免疫两次，注射体积为每只0.1ml，第31天采血，分离血清，进行抗体滴度检测。取血完毕后取小鼠脾脏，分离淋巴细胞，进行T细胞免疫反应检测。具体分组信息如下表所示。
表2.小鼠体内免疫原性实验动物分组
Figure PCTCN2022135376-appb-000004
抗体滴度检测采用ELISA方法，同实施例4。T细胞免疫反应检测采用流式细胞术和FluoroSpot方法(同实施例4)。
流式细胞术可检测分泌特定细胞因子的抗原特异性CD4+T细胞免疫反应。具体而言，先制备小鼠脾脏单细胞悬液。将断颈处死的小鼠转移至超净台中无菌解剖取出小鼠脾脏，通过淹没和滤网过滤获得脾脏细胞悬液。加入红细胞裂解液(BOSTER，货号AR1118)去除细胞悬液中的红细胞。用完全培养基(10％FBS(Gibco，10091-148)+RPMI 1640培养基(Gibco，10091-148))清洗后取少量悬液通过细胞计数仪进行细胞计数后备用。取一块96孔细胞培养板，每孔铺1～3百万细胞/ 孔，同时每孔加入gE蛋白池刺激物、抗CD28抗体溶液(Mabtech，货号FS-4142-10)和高尔基抑制剂溶液(BD，货号554714)，放置于细胞培养箱中，37℃刺激约6小时。刺激结束后，按抗体说明书浓度要求分别加入LIVE/DEAD Aqua Dye(Invitrogen，货号L34966)、FITC anti-mouse CD4(BD，货号553047)、PB anti-mouse CD3(Biolegend，货号100214)抗体避光低温染色30分钟。然后用BD cyto-fix/cytoperm Fixation/Permeabilization试剂盒(BD，货号554714)对细胞进行固定和破膜20分钟，之后分别加入PE-Cy7 anti-mouse IL-2(BD，货号560538)、APC anti-mouse IFN-γ(BD，货号554413)、PE anti-mouse TNF-α(BD，货号554419)抗体避光低温染色30分钟。最后离心弃上清，用PBS清洗细胞。将清洗过的细胞重悬于PBS中，通过FACS Canto II Flow Cytometer流式细胞检测器检测CD4+T活细胞分泌不同细胞因子的比例并进行数据分析。
如图6和图7所示，结果表明，不同剂量的抗原组合不同剂量的复合佐剂均能在小鼠体内诱导较高的抗原特异性抗体和T细胞免疫反应。其中高低剂量的QS-21组分无显著的剂量依赖效应，但高剂量的抗原和较高水平的免疫反应有一定的相关性。
实施例6：gE蛋白与不同复合佐剂联合制剂后疫苗以及商品化Shingrix疫苗在小鼠体内免疫原性比较
我们首先按照公开的资料仿制了另一种复合佐剂AS01B，并通过联合相同的重组gE蛋白，比较用本发明复合佐剂与该仿制AS01B佐剂制备的疫苗在小鼠体内的免疫反应水平。也同时比较了购买的商品化已上市同类重组带状疱疹疫苗Shingrix。
我们选择拟用人用剂量的1/10在C57BL/6小鼠体内进行疫苗免疫原性研究。由于VZV疾病的发病机制依赖于VZV的前期潜伏感染后再激发，因此在本实验中设计对小鼠进行预先免疫减毒活疫苗，以模拟自然的发病机制，从而更合理地评估产品的药效。所有小鼠于免疫候选疫苗组合物前28天，通过皮下注射免疫1剂人用水痘减毒活 疫苗，注射体积为每只0.5mL。第0天、21天后腿肌肉免疫两次候选疫苗组合物和对照样品，注射体积为每只0.05mL，第31天采血，分离血清，进行抗体滴度检测。取血完毕后取小鼠脾脏，分离淋巴细胞，进行T细胞免疫反应检测。具体分组信息如下表所示：
表3.小鼠体内免疫原性实验动物分组
Figure PCTCN2022135376-appb-000005
抗体滴度检测采用ELISA方法，同实施例4。T细胞免疫反应检测采用FluoroSpot方法，同实施例4，但受限于试剂盒(Mabtech，货号FS-4142-10)，只检测了IL-2和IFN-γ两个细胞因子。
结果如图8和图9所示。结果表明，使用相同重组gE蛋白的前提下，用本发明复合佐剂制备的重组带状疱疹疫苗相对于用仿制AS01B复合佐剂制备的疫苗在小鼠体内诱导抗体反应水平无显著性差异，但能诱导更高水平的T细胞反应。此外，用本发明复合佐剂制备的重组带状疱疹疫苗相对于商品化重组带状疱疹疫苗Shingrix在小鼠体内诱导抗体反应水平也无显著性差异，但同样能诱导更高水平的T细胞反应，显示出更好的疫苗免疫原性。
实施例7：本发明带状疱疹疫苗与商品化Shingrix疫苗在猴子体内免疫原性比较
为了评价本发明带状疱疹疫苗组合物的免疫反应水平，我们在猴子体内与商品化已上市同类重组带状疱疹疫苗Shingrix(GSK公司生产的已上市重组带状疱疹疫苗，中文名：欣安立适)进行了免疫原性比较研究。
我们选择拟用人用剂量在食蟹猴(来源于军科正源生物医药科技有限公司)体内进行疫苗免疫原性研究，同时为了更好地模拟自然的发病机制，对每组4只食蟹猴(雌2雄2)均提前4周进行了5倍人用剂量 的水痘减毒活疫苗皮下注射预免疫，注射体积为每只2.5mL。预免疫结束后4周和12周，分两次经肌肉免疫分别注射1剂人用剂量的本发明带状疱疹疫苗(50μg gE/脂质体/500μg CpG7909/50μg QS-21)和商品化Shingrix疫苗。末次免疫结束后2周采血，分离血清和外周血单核细胞(PBMC)，分别进行gE抗原特异性抗体检测和T细胞反应检测。
抗体滴度检测采用ELISA方法，同实施例4，但检测二抗替换为对应猴二抗(Bethyl Laboratories，货号A140-102P)。T细胞免疫反应检测采用FluoroSpot方法，同实施例4，但检测试剂盒更换为对应猴检测试剂盒(Mabtech，货号FSP-212822-10)。PBMC采用Ficoll密度梯度离心分离。
结果如图10和图11所示。结果表明，本发明带状疱疹疫苗与商品化重组带状疱疹疫苗Shingrix相比，在食蟹猴体内诱导抗体反应水平和T细胞反应水平虽略低于商品化Shingrix疫苗，但无显著性差异，显示出较好的疫苗免疫原性。
虽然本发明某些特征已经在本文中阐释和描述，但本领域技术人员将想到许多修改、替代、变更和等同。因此，应理解的是，所附权利要求书意在涵盖落入本发明真实精神范围之内的所有此类修改和变更。

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  1. 一种带状疱疹疫苗组合物，所述组合物包含水痘-带状疱疹病毒抗原和复合佐剂，其中所述复合佐剂包含CpG寡脱氧核苷酸、QS-21和脂质体，其中CpG寡脱氧核苷酸是CpG7909。
  2. 根据权利要求1所述的组合物，其中，CpG寡脱氧核苷酸的浓度范围为0.25～2mg/mL复合佐剂。
  3. 根据权利要求1或2所述的组合物，其中，QS-21的浓度范围是25～200μg/mL复合佐剂。
  4. 根据权利要求1至3中任一项所述的组合物，其中，所述脂质体包含磷脂酰胆碱和胆固醇。
  5. 根据权利要求4所述的组合物，其中，所述磷脂酰胆碱的浓度范围是0.5～4mg/mL复合佐剂。
  6. 根据权利要求4或5所述的组合物，其中，所述胆固醇的浓度范围是0.125～1mg/mL复合佐剂。
  7. 根据权利要求4至6中任一项所述的组合物，其中，所述磷脂酰胆碱为二油酰基磷脂酰胆碱。
  8. 根据权利要求1至7中任一项所述的组合物，其中，所述水痘-带状疱疹病毒抗原是水痘-带状疱疹病毒糖蛋白gE。
  9. 根据权利要求8所述的组合物，其中，所述水痘-带状疱疹病毒抗原是水痘-带状疱疹病毒糖蛋白gE的胞外区片段。
  10. 根据权利要求9所述的组合物，其中，所述胞外区片段的氨基酸序列如SEQ ID No.1所示。
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