WO2024114650A1 - 一种重组带状疱疹疫苗及其制备与应用 - Google Patents

一种重组带状疱疹疫苗及其制备与应用 Download PDF

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WO2024114650A1
WO2024114650A1 PCT/CN2023/134838 CN2023134838W WO2024114650A1 WO 2024114650 A1 WO2024114650 A1 WO 2024114650A1 CN 2023134838 W CN2023134838 W CN 2023134838W WO 2024114650 A1 WO2024114650 A1 WO 2024114650A1
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adjuvant
vaccine
protein
cpg
herpes zoster
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PCT/CN2023/134838
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English (en)
French (fr)
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王希良
程晋霞
王莉
李世崇
司炳银
王立博
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北京吉诺卫生物科技有限公司
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Priority claimed from CN202211533170.7A external-priority patent/CN116064548B/zh
Priority claimed from CN202211523443.XA external-priority patent/CN116059337B/zh
Priority claimed from CN202211535908.3A external-priority patent/CN116023510B/zh
Application filed by 北京吉诺卫生物科技有限公司 filed Critical 北京吉诺卫生物科技有限公司
Publication of WO2024114650A1 publication Critical patent/WO2024114650A1/zh

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  • the present invention claims priority to three applications in China, with application number 202211523443.X, application date December 1, 2022, and invention name “Novel adjuvant recombinant shingles vaccine and its preparation and application”; in China, with application number 202211535908.3, application date December 1, 2022, and invention name “Dual adjuvant recombinant shingles vaccine”; and in China, with application number 202211533170.7, application date December 1, 2022, and invention name “A novel CpG vaccine adjuvant and its application”.
  • the above applications are introduced into the present invention as a whole and serve as a part of the present invention.
  • the invention belongs to the field of biotechnology and relates to a vaccine preparation, in particular to a recombinant herpes zoster vaccine and the preparation and application thereof.
  • Herpes zoster is an infectious skin disease caused by the reactivation of latent varicella-zoster virus (VZV). It is common in middle-aged and elderly people and often presents as a unilateral rash along the area innervated by sensory nerves, accompanied by neuropathic pain. The most common complication is postherpetic neuralgia (PHN). With the increasing trend of aging population in my country, the incidence of herpes zoster has shown a significant upward trend. The prevention and treatment of herpes zoster has become an important public health issue. Vaccination with herpes zoster vaccine is the most effective means to prevent the disease.
  • VZV latent varicella-zoster virus
  • VZV Varicella zoster virus belongs to the genus Herpesvirus, family Herpesviridae.
  • the VZV genome has 71 genes, encoding 67 different proteins, including 6 glycoproteins (gpI ⁇ gpVI), which are now uniformly named gE, gB, gH, gI, gC and gL.
  • gE is the most abundant in the viral envelope and is the most important glycoprotein recognized by the host immune system. It is encoded by the orf68 gene and is located in the short region of the vzv genome. It contains 623 amino acids, including 544 in the hydrophilic membrane extracellular region, 17 in the transmembrane region, and 62 in the intracellular region.
  • VZV gE monoclonal antibodies can mediate antibody-dependent cellular toxicity and neutralize the infectivity of the virus.
  • the current challenge is that gE can only induce low levels of humoral immunity and cannot meet the requirements for vaccine use.
  • the herpes zoster vaccine products that have been launched globally include Zostavax (Merck), Shingrix (GSK) and NBP608 (SK Chemical, only sold in the Korean market).
  • Zostavax and SkyZoster are live attenuated vaccines that are processed to achieve structural changes, reduce toxicity but retain immunogenicity, thereby establishing an immune response;
  • Shingrix is a recombinant subunit vaccine that produces and enhances immune responses in vivo through varicella-zoster glycoprotein E antigen and AS01B adjuvant.
  • Clinical data show that Shingrix is 97% effective in people aged 50 to 69, and still as high as 91% in people over 70 years old; Zostavax is 70% effective in people aged 50 to 69, and the overall effectiveness is 51%.
  • Shingrix is more effective than Zostavax.
  • the Shingrix vaccine formula is complex, the antigen needs to be freeze-dried and separated from the adjuvant, the adjuvant ASO1B, especially the QS21 in it, cannot be chemically synthesized, the raw materials are scarce and expensive, resulting in insufficient supply.
  • CpG ODN cytosine phosphate guanidine oligodeoxynucleotide
  • CpG ODN 7909 was used as an adjuvant for the commercialized multivalent inactivated influenza vaccine (Fluarix).
  • the results of its randomized double-blind Phase I clinical trial showed that CpG ODN is a safe and reliable adjuvant that can reduce the dosage of the multivalent inactivated influenza vaccine (Fluarix).
  • CpG ODN as a new type of highly effective immune activator, has shown a good immune adjuvant effect.
  • CpG ODN has different immune stimulating effects on different species, cells and antigen components, and its immune stimulating effect is closely related to its sequence structure. Therefore, it is very necessary to make corresponding improvements and designs on CpG ODN for different application scenarios and antigen components.
  • a fusion protein comprising VZV-gE protein and the Fc fragment of human IgG4.
  • the VZV-gE is directly or indirectly linked to the Fc fragment.
  • the indirect connection comprises a linker.
  • the linker is a flexible linker.
  • the flexible linker is selected from GGGGS (SEQ ID NO.12), GGGGSGGGGS (SEQ ID NO.13), and GSGGGSGGGGSGGGGSA (SEQ ID NO.14).
  • the flexible polypeptide is GSGGGSGGGGSGGGGSA (SEQ ID NO.14).
  • the VZV-gE protein is any of the following:
  • (a2) a fusion protein obtained by connecting a tag to the N-terminus and/or C-terminus of the protein described in (a1);
  • (a3) a protein having the same function obtained by replacing and/or deleting and/or adding one or more amino acid residues of any one of (a1) to (a2);
  • the human IgG4 Fc protein is any of the following:
  • (b2) a fusion protein obtained by connecting a tag to the N-terminus and/or C-terminus of the protein described in (b1);
  • (b4) A protein having 80% or more identity with any of (b1) to (b2) and having the same function.
  • the fusion protein is any of the following:
  • (c2) a fusion protein obtained by connecting a tag to the N-terminus and/or C-terminus of the protein described in (c1);
  • (c3) a protein having the same function obtained by replacing and/or deleting and/or adding one or more amino acid residues of any of (c1) to (c2);
  • the amino acid sequence has an amino acid sequence with 80% or more identity, preferably an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence with 98% or 99% or more identity.
  • Another aspect of the present invention provides a nucleic acid encoding the above fusion protein.
  • the nucleic acid encoding the fusion protein comprises a nucleotide sequence that is 80% or more identical to the nucleotide sequence shown in SEQ ID NO.4, preferably a nucleotide sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably a nucleotide sequence that is 98% or 99% or more identical.
  • the nucleic acid encoding the fusion protein is the nucleic acid shown in SEQ ID NO.4.
  • the nucleic acid is mRNA.
  • Another aspect of the present invention provides an expression vector comprising the above nucleic acid.
  • the expression vector is selected from a prokaryotic expression vector or a eukaryotic expression vector.
  • the expression vector is a eukaryotic expression vector.
  • the eukaryotic expression vector is an adenoviral vector.
  • the eukaryotic expression vector is a vector expressed in 293 cells or CHO cells.
  • Another aspect of the present invention provides a host cell, which expresses any of the above-mentioned fusion proteins, or contains any of the above-mentioned nucleic acids and/or contains any of the above-mentioned expression vectors.
  • the host cell is a prokaryotic cell or a eukaryotic cell.
  • the prokaryotic cell is a bacterial cell.
  • the prokaryotic cell is an E. coli cell.
  • the eukaryotic cell is selected from the group consisting of a yeast cell, an insect cell, and a mammalian cell.
  • the mammalian cell is selected from CHO, HEK293, SP2/0, BHK, C127, etc.
  • the eukaryotic cell is a CHO cell.
  • Another aspect of the present invention provides use of any of the above-mentioned fusion proteins, nucleic acids, expression vectors and/or host cells in the preparation of a vaccine for treating or preventing diseases or conditions associated with varicella-zoster virus.
  • VZV fusion protein vaccine comprising any one of the above fusion proteins, nucleic acid, expression vector and/or host cell, and one or more adjuvants.
  • the adjuvant is selected from one or more of aluminum hydroxide, CpG, aluminum phosphate, saponin, such as QuilA, QS-21, GPI-0100, water-in-oil emulsion, oil-in-water emulsion, and water-in-oil-in-water emulsion.
  • the adjuvant is a dual adjuvant of aluminum hydroxide and CpG.
  • the ratio of the fusion protein, aluminum adjuvant and CpG adjuvant is 1-8:5-240:1-20.
  • the ratio of the fusion protein, aluminum adjuvant and CpG adjuvant is 1-4:20-60:1-10.
  • the CpG adjuvant is any CpG adjuvant, such as CpG1018, or the CpG adjuvant is the nucleotide shown in any sequence in SEQ ID NO:5-9, and all nucleotide residues are thiolated.
  • Another aspect of the present invention provides a herpes zoster vaccine, comprising an antigen and an adjuvant;
  • the adjuvant is an aluminum adjuvant, a CpG adjuvant or a combination of the two;
  • the antigen is from the varicella zoster virus.
  • the CpG adjuvant is a nucleotide shown in any one of SEQ ID NO:5-9, and all nucleotide residues are thiolated.
  • the vaccine is an inactivated vaccine, live attenuated vaccine, viral vector vaccine, subunit vaccine, recombinant vaccine, polysaccharide vaccine, conjugate vaccine, toxoid vaccine, or nucleic acid vaccine for herpes zoster;
  • the nucleic acid vaccine is an mRNA vaccine or a DNA vaccine.
  • the nucleic acid vaccine is an mRNA vaccine or a DNA vaccine.
  • the aluminum adjuvant is AL(OH)3 or ALPO4.
  • the adjuvant consists of an aluminum adjuvant and a CpG adjuvant
  • the aluminum adjuvant is AL(OH)3;
  • the CpG adjuvant is a nucleic acid molecule shown in any one of SEQ ID NO:5-9, and all nucleotide residues are thiolated.
  • the antigen and the adjuvant are packaged mixedly or separately.
  • the mass ratio of the antigen, the AL(OH)3(AL) and the CpG adjuvant is 2.5-30:10-200:1-10.
  • the mass ratio of the antigen, the AL(OH)3(AL) and the CpG adjuvant is 7.5:50:5.
  • the above-mentioned vaccine can be dispensed into 2 ml vials (or pre-filled glass syringes), 0.5 ml (or 1.0 ml) per vial.
  • the antigen content in every 100 ⁇ l of double adjuvant vaccine preparation is 2.5-30 ⁇ g
  • the aluminum hydroxide adjuvant is 10-200 ⁇ g
  • the CpG adjuvant is 1-10 ⁇ g, all of which are preferred.
  • the antigen content in every 100 ⁇ l of double adjuvant vaccine preparation is 5 ⁇ g
  • the aluminum hydroxide adjuvant is 50 ⁇ g
  • the CpG adjuvant is 2 ⁇ g.
  • the antigen content is 75 ⁇ g
  • the aluminum hydroxide adjuvant is 500 ⁇ g
  • the CpG adjuvant is 50 ⁇ g, which are all better.
  • the antigen content is 7.5 ⁇ g
  • the aluminum hydroxide adjuvant is 50 ⁇ g
  • the CpG adjuvant is 5 ⁇ g, which are all better.
  • the antigen is derived from any of the above-mentioned fusion proteins, nucleic acids, expression vectors and/or host cells.
  • Another aspect of the present invention provides a method for preparing the above-mentioned herpes zoster vaccine, comprising the following steps: mixing the antigen and the adjuvant to obtain the herpes zoster vaccine.
  • the mixing mentioned above is to mix the antigen and the adjuvant according to the required mass ratio.
  • Another aspect of the present invention provides the above fusion protein, and the use of the aluminum adjuvant and the CpG adjuvant in any of the following:
  • the product is a vaccine.
  • Another aspect of the present invention provides the use of any of the above fusion proteins, nucleic acids, vectors or host cells in any of the following:
  • the above-mentioned herpes zoster virus antibody has the function of neutralizing the herpes zoster virus.
  • Another aspect of the present invention provides a method for inducing an immune response against varicella-zoster virus in a subject in need thereof, the method comprising: administering any of the vaccines described above to the subject.
  • the subject is a mammal or a bird.
  • the subject is a human, bovine, canine, feline, goat, sheep, porcine, horse, turkey, duck, or chicken.
  • compositions which comprises any of the above-mentioned fusion proteins, nucleic acids, expression vectors and/or host cells, and one or more pharmaceutically acceptable carriers, diluents or excipients.
  • Another aspect of the present invention provides a method for treating or preventing a disease or condition associated with varicella-zoster virus, the method comprising administering any of the above vaccines or pharmaceutical compositions to a subject.
  • Another aspect of the present invention provides an oligonucleotide comprising the sequence:
  • X1 is G or A
  • X2 is G or A
  • X 3 is A or T
  • X 4 is G or A
  • X5 is T or C
  • X 6 is T or C
  • the aforementioned oligonucleotide comprises the nucleotide sequence TGACTGAACGTTTTAACGTCAGACTGA (SEQ ID NO:5) or any variant thereof.
  • the aforementioned oligonucleotide comprises the nucleotide sequence TGACTGAACGTTTTAGCGCTAGACTGA (SEQ ID NO:6) or any variant thereof.
  • the aforementioned oligonucleotide comprises the nucleotide sequence TGACTAGTCGTTTTAACGTCAGACTGA (SEQ ID NO:7) or any variant thereof.
  • the aforementioned oligonucleotide comprises the nucleotide sequence TGACTAGTCGTTTTAGCGCTAGACTGA (SEQ ID NO:8) or any variant thereof.
  • the aforementioned oligonucleotide comprises the nucleotide sequence TGACTGTGAACGTTCGAGATGA (SEQ ID NO:9) or any variant thereof.
  • the aforementioned oligonucleotide comprises a nucleotide sequence as shown in any one of SEQ ID NO:5-9.
  • the present invention provides a CpG adjuvant, wherein the adjuvant comprises the aforementioned oligonucleotide.
  • it comprises the aforementioned oligonucleotide as an effective ingredient.
  • the adjuvant further comprises one or more selected from mineral salt adjuvants, oil emulsion adjuvants, microorganisms and plant extracts and derivatives targeting pattern recognition receptors, particulate antigen presentation system adjuvants or cytokine adjuvants.
  • the mineral salt adjuvants are one or more of aluminum adjuvants or magnesium adjuvants.
  • the aforementioned oil emulsion adjuvant is selected from one or more of saponin adjuvants, oil-in-water and water-in-oil emulsions.
  • the aforementioned microorganisms and plant extracts and derivative adjuvants targeting pattern recognition receptors are one or more of phospholipid A adjuvants and CpG adjuvants.
  • the aforementioned microparticle antigen presentation system adjuvant is one or more of poly(lactide-glycolide) (PLG) and liposomes.
  • the aforementioned cytokine adjuvant is one or more of cytokines IL-1, IL-2, and IL-12.
  • the adjuvant comprises the aforementioned oligonucleotide and an aluminum adjuvant as active ingredients.
  • the adjuvant comprising the aforementioned oligonucleotide and the mineral salt is a composite adjuvant, wherein the mineral salt adjuvant is one or more of an aluminum adjuvant or a magnesium adjuvant.
  • the aluminum adjuvant of the present invention includes aluminum hydroxide and aluminum phosphate.
  • Another aspect of the present invention provides a vaccine comprising the aforementioned oligonucleotide or adjuvant.
  • the aforementioned vaccine further contains an antigen.
  • the aforementioned antigen is coronavirus RBD-Fc.
  • the aforementioned coronavirus is the novel coronavirus.
  • the dosage of the aforementioned adjuvant in the aforementioned vaccine is 5-3000 ⁇ g/dose.
  • the dosage of the aforementioned oligonucleotide is selected from 5 ⁇ g/dose, 10 ⁇ g/dose, 30 ⁇ g/dose or 50 ⁇ g/dose.
  • the dosage of the aforementioned oligonucleotide is 10 ⁇ g/dose.
  • the mass ratio of the aforementioned oligonucleotide to the aluminum adjuvant in each dose of vaccine is 1:8-1:100.
  • the aforementioned adjuvant comprises 10 ⁇ g/dose of the oligonucleotide and 0.5 mg/dose of the aluminum adjuvant.
  • Another aspect of the present invention provides a use of the aforementioned oligonucleotide, CpG adjuvant or composite adjuvant.
  • the present invention provides a use of the aforementioned oligonucleotide, CpG adjuvant or composite adjuvant in preparing a vaccine, or improving the immunogenicity of an antigen or a vaccine.
  • the aforementioned vaccine is a novel coronavirus vaccine.
  • the aforementioned antigen is coronavirus RBD-Fc.
  • the aforementioned coronavirus is the novel coronavirus.
  • Another aspect of the present invention provides a method for preparing the aforementioned oligonucleotide, CpG adjuvant or composite adjuvant and vaccine.
  • the oligonucleotide is a nucleotide shown in any one of SEQ ID NO:5-9, and all nucleotides of the nucleotide are thio-modified.
  • Another aspect of the present invention provides the use of the above-mentioned CpG adjuvant and aluminum adjuvant in preparing vaccines, or improving the immunogenicity of antigens or vaccines.
  • the vaccine is used to treat and/or prevent diseases or conditions caused by herpes zoster virus or coronavirus. More preferably, the coronavirus includes the new coronavirus.
  • Another aspect of the present invention provides a method for treating or preventing a disease or condition, the method comprising administering any of the above-mentioned vaccines or pharmaceutical compositions to a subject.
  • the disease or condition includes a disease or condition caused by herpes zoster virus or coronavirus, and more preferably, the coronavirus includes a new coronavirus.
  • the term “about” or “approximately” means within plus or minus 10% of a given value or range. Where an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.
  • fusion protein refers to a natural or synthetic molecule consisting of one or more molecules, in which two or more peptide- or protein-based (including glycoprotein) molecules with different specificities are optionally fused together through chemical or amino acid-based linker molecules.
  • the connection can be achieved by C-N fusion or N-C fusion (in 5' ⁇ 3' direction), preferably C-N fusion.
  • antibody or “immunoglobulin” has the broadest meaning, specifically including intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) composed of at least two intact antibodies, and antibody fragments, as long as they show the desired biological activity.
  • This term generally includes hybrid antibodies composed of two or more antibodies or antibody fragments with different binding specificities linked together.
  • the term "Fc region” is used herein to define the C-terminal region of an immunoglobulin heavy chain, including a native sequence Fc region and a variant Fc region.
  • the human IgG heavy chain Fc region is generally defined as extending from position Cys226, or from the amino acid residue at Pro230 to the carboxyl end of the heavy chain.
  • the C-terminal lysine (residue 447 according to the EU numbering system) in the Fc region can be removed, for example, during the production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody. Therefore, the composition of the complete antibody can include an antibody population from which all K447 residues have been removed, an antibody population from which no K447 residues have been removed, and an antibody population with a mixture of antibodies with and without the K447 residue.
  • sequence “identity” or “identity” has a meaning recognized in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using invented techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule. Although there are many methods for measuring the identity between two polynucleotides or polypeptides, the term “identity” is well known to technicians (Carrillo, H. & Lipman, D., SIAM J Applied Math 48: 1073 (1988)).
  • disease or “condition” refers to a state of life or health of a patient or individual that can be treated with the fusion proteins, pharmaceutical compositions or methods provided herein.
  • the term "vaccine” is a purified antigen vaccine or immunogenic composition, a subunit vaccine or immunogenic composition, an inactivated whole virus vaccine or immunogenic composition, or an attenuated virus vaccine or immunogenic composition.
  • the vaccine or immunogenic composition is a purified fusion protein.
  • treating refers to any indicator of successful treatment or improvement of an injury, disease, pathology, or condition, including any objective or subjective parameter, such as elimination; relief; alleviation of symptoms or making the injury, pathology, or condition more tolerable to the patient; slowing the rate of degeneration or decline; or making the end point of degeneration less declining; improving the patient's physical or mental health.
  • the treatment or improvement of symptoms can be based on objective or subjective parameters; including the results of a physical examination, neuropsychiatric examination, and/or psychiatric assessment.
  • treatment and its conjugates can include preventing an injury, pathology, condition, or disease. In an embodiment, treatment is prevention. In an embodiment, treatment does not include prevention.
  • treating also broadly encompasses any method for obtaining a beneficial or desired result (including a clinical result) in a subject's condition.
  • beneficial or desired clinical results may include, but are not limited to, alleviation or improvement of one or more symptoms or conditions, alleviation of the extent of the disease, stabilization (i.e., non-exacerbation) of the disease state, prevention of disease transmission or spread, delay or slowing of disease progression, improvement or alleviation of the disease state, reduction of disease recurrence, and alleviation (whether partial or total, and whether detectable or undetectable).
  • treatment encompasses any cure, improvement, or prevention of a disease. Treatment may prevent the occurrence of a disease; inhibit the spread of a disease; alleviate the symptoms of a disease, completely or partially remove the underlying cause of a disease, shorten the duration of a disease, or a combination of these things.
  • treatment includes preventive treatment.
  • the treatment method includes administering a therapeutically effective amount of an active agent to a subject.
  • the administration step may consist of a single administration, or may include a series of administrations.
  • the length of the treatment period depends on a variety of factors, such as the severity of the condition, the age of the patient, the concentration of the active agent, the activity of the composition used in the treatment, or a combination thereof.
  • the effective dose of the agent used for treatment or prevention may increase or decrease during a specific treatment or prevention regimen. By standard diagnostic assays known in the art, changes in dosage may occur and become apparent. In some cases, chronic administration may be required.
  • the composition is administered to a subject in an amount sufficient to treat the patient, and for a sufficient duration.
  • prevention refers to reducing the occurrence of disease symptoms in a patient. As described above, prevention can be complete (no detectable symptoms) or partial, such that fewer symptoms are observed than would occur in the absence of treatment.
  • patient or “subject in need” refers to a living organism that suffers from or is susceptible to a disease or condition that can be treated by administering a pharmaceutical composition as provided herein.
  • Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goats, sheep, cattle, deer, and other non-mammals.
  • the patient is a human.
  • co-administration refers to "co-administration" of a fusion protein or vaccine of the present invention with a known drug (or other compound, or other vaccine), so that both have a therapeutic or diagnostic effect.
  • co-administration may include administering the drug (or other compound, or other vaccine) in parallel (i.e., simultaneously), before, or sequentially relative to the administration of the fusion protein or vaccine of the present invention.
  • One of ordinary skill in the art will be able to easily determine the appropriate administration time, sequence, and dosage of a particular drug (or other compound, or other vaccine) and the combination of the present invention.
  • the term "effective amount” is an amount sufficient to achieve the stated purpose (e.g., to achieve the effect it is administered to achieve, treat a disease, reduce enzyme activity, increase enzyme activity, reduce protein function, alleviate one or more symptoms of a disease or condition).
  • An example of an “effective amount” is an amount sufficient to facilitate treatment, prevention, or reduction of one or more symptoms of a disease, which amount may also be referred to as a "therapeutically effective amount.”
  • “Reduction" of one or more symptoms means reducing the severity or frequency of one or more symptoms, or eliminating one or more symptoms.
  • the "preventive effective amount" of a drug is the amount of the drug that will have the expected preventive effect when administered to a subject, such as preventing or delaying the onset (or recurrence) of damage, disease, pathology, or condition, or reducing the possibility of the onset (or recurrence) of damage, disease, pathology, or condition, or its symptoms.
  • a complete preventive effect does not necessarily occur by administering a single dose, and may occur after only administering a series of doses. Therefore, a preventive effective amount may be administered in the form of one or more administrations.
  • a therapeutically effective amount refers to the amount of a therapeutic agent sufficient to improve a condition as described above.
  • a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%.
  • the therapeutic efficacy can also be expressed as a "fold" increase or decrease.
  • a therapeutically effective amount can have an effect of at least 1.2 times, 1.5 times, 2 times, 5 times, or more relative to a control.
  • the dosage can vary depending on the patient's needs and the fusion protein or vaccine used.
  • the dosage administered to the patient is The amount should be sufficient to produce a beneficial therapeutic response in the patient over time.
  • the size of the dosage will also be determined by the presence, nature and extent of any adverse side effects. It is within the skill of the practitioner to determine the appropriate dosage for a particular situation.
  • treatment begins with a smaller dose than the optimal dose of the fusion protein or vaccine. Thereafter, the dosage is increased in small increments until the optimal effect in these cases is reached.
  • the amount and interval of administration can be adjusted individually to provide an effective level of the administered fusion protein or vaccine for the specific clinical indication being treated. This will provide a treatment regimen commensurate with the severity of the individual disease state.
  • administer means oral administration to a subject, administration in the form of a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or implantation of a slow release device (e.g., a mini-osmotic pump).
  • Administered by any route including parenteral and transmucosal (e.g., buccal, sublingual, palate, gums, nose, vagina, rectum or transdermal).
  • Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular and intracranial administration.
  • Other modes of delivery include, but are not limited to, use of liposome formulations, intravenous infusions, transdermal patches, etc.
  • administration does not include administration of any activating agent other than the narrated activating agent.
  • the human IgG4Fc mutant is used in the vaccine of the present invention, which reduces the formation of single molecules and reduces the ADCC effect, overcomes the problem of terminal heterogeneity in the production process, and ensures that the vaccine of the present invention is safer and more stable;
  • VZV-gE and human IgG4Fc to form a dimer are beneficial to promote expression and purification, while the antigen presentation to DC cells improves the level of immune response.
  • the formation of gE dimers increases the molecular weight, which is beneficial to increase the immunogenicity of gE.
  • the present invention uses the extracellular region of VZV gE to fuse with the human IgG4Fc fragment to obtain a highly efficiently expressed secretory gE-Fc fusion protein in mammalian cells (CHO), with an expression level of 3g/L;
  • the recombinant VZV-gE-IgG4Fc fusion protein expressed by CHO cells of the present invention is a glycosylated protein, which maintains the spatial structure of the natural gE protein, has good immunogenicity, and is effective in cell-mediated immune detection of IL-2 and IFN- ⁇ ; and the immunogenicity of the fusion protein is significantly increased when used in combination with a double adjuvant;
  • the CpG adjuvant of the present invention plays an important role in cellular immunity.
  • the combination of the CpG adjuvant of the present invention and the aluminum adjuvant has a significant improvement effect in stimulating cellular immunity.
  • the vaccine of the present invention uses a conventional aluminum adjuvant and a CpG adjuvant in combination, which is cheap and easy to obtain, and overcomes the defects of the Shingrix vaccine developed by GSK, such as the complex formula, the need for lyophilization of the antigen and the need to separate it from the adjuvant, and the scarcity of adjuvant raw materials and the high price.
  • the Shingrix (GSK-shingrix) antigen developed by GSK needs to be lyophilized and separated from the adjuvant, it is more troublesome in the preparation of antigens, the preparation process and the subsequent use.
  • the antigen and adjuvant of the present invention can be directly mixed, and the pre-filled syringe can be packaged into a preparation, which is very convenient.
  • CpG-cjx1 had a similar immune stimulation effect to the positive control Dynavax's ISS1018 (CpG-1018), and showed good immunostimulatory effects on mouse spleen cells and human PBMC.
  • mice were immunized, spleen lymphocytes were isolated, and the levels of IFN- ⁇ , TNF- ⁇ , IL-2, and IL-4 expressed by CD4+/CD8+T cells were measured.
  • the results showed that the cellular immune effect of the CpG group and the group combined with aluminum adjuvant of the present invention was significantly higher than that of the single antigen group without adjuvant or the single aluminum adjuvant group. It can be seen that the CpG adjuvant screened by the present invention plays an important role in cellular immunity. At the same time, the adjuvant of the present invention combined with aluminum adjuvant has a significant improvement effect in stimulating cellular immunity.
  • the ELISA method and cytopathic inhibition method were used to measure the binding antibody and neutralizing antibody titers of the serum of Balb/c mice 14 days after immunization with the recombinant novel coronavirus fusion protein vaccine in four groups twice.
  • the results showed that the vaccine groups with CpG adjuvant were better than those without CpG adjuvant, and the neutralizing antibody titer of the double adjuvant group was at least 10 times higher than that of the other groups. It can be seen that the combination of aluminum adjuvant and CpG adjuvant has a synergistic effect in stimulating humoral immunity and cellular immunity.
  • the CpG of the present invention is used as an adjuvant in vaccines, and the effects of different dosages are not much different. In the mouse animal model, CpG above 10 ⁇ g/dose can achieve good immune effects.
  • FIG1 shows the purification results of VZV-gE-Fc.
  • Figure 2 is a comparison of the immune effects of mice immunized with recombinant VZV-gE-Fc vaccines formulated with different aluminum salt adjuvants.
  • Figure 3 is a comparison of the immune effects of different doses of aluminum hydroxide adjuvant.
  • FIG. 4 is the detection result of CpG ODN stimulating mouse spleen cells in vitro by MTT method.
  • Figure 5 shows the detection results of CpG ODN stimulating human PBMC in vitro using the MTT method.
  • FIG6 is a comparison of the immune effects of different types of CpG adjuvants used in combination with aluminum adjuvants.
  • FIG. 7 is a comparison of the immune effects of different doses of CpG adjuvant and aluminum adjuvant.
  • Figure 8 is a comparison of the immune effects of dual adjuvant vaccines with different doses of antigen.
  • Figure 9 is a comparison of the immune effects of different vaccine components (ELISA).
  • FIG. 10 is an evaluation of the ELISPOT cell immune effect after immunization with a double adjuvant vaccine, wherein FIG. 10a is the detection result of IFN- ⁇ , and FIG. 10b is the detection result of IL-2.
  • Figure 11 is a comparison of the immune effects of mice immunized with the double adjuvant VZV-gE-Fc recombinant herpes zoster vaccine (VZV-Genevax) and GlaxoSmithKline Shingrix (GSK-Shingrix), wherein Figure 11a is the binding antibody titer after the first and second immunizations determined by ELISA, and Figure 11b is the detection result of ELISPOT cell immunization IFN- ⁇ .
  • Figure 12 is a comparison of the immune effects of crab-eating macaques immunized with the double adjuvant VZV-gE-Fc recombinant shingles vaccine (VZV-Genevax) and GlaxoSmithKline Shingrix (GSK-Shingrix), wherein Figure 12a is the binding antibody titer after the first and second immunizations determined by ELISA, Figure 12b is the flow cytometry test to determine the levels of IFN- ⁇ , IL-2, TNFa (Th1) and IL-4 (Th2) cytokines expressed by CD4/CD8T cells, and Figure 12c is the detection result of ELISPOT cell immunity to IFN- ⁇ .
  • VZV-Genevax double adjuvant VZV-gE-Fc recombinant shingles vaccine
  • GSK-Shingrix GlaxoSmithKline Shingrix
  • mice in the following examples were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.
  • the recombinant herpes zoster subunit vaccine antigen target of this embodiment is VZV-gE (SEQ ID NO.1).
  • VZV-gE human IgG4Fc fragment
  • SEQ ID NO.2 a human IgG4Fc fragment
  • the VZV-gE protein and the human IgG Fc fragment are fused through a connecting peptide to obtain a VZV-gE-Fc fusion protein, the amino acid sequence of which is shown in SEQ ID NO.3.
  • Two identical VZV-gE-Fc fusion proteins are connected to form homodimers through disulfide bonds.
  • the sequence of the gene encoding the VZV-gE-Fc fusion protein with high expression biased towards CHO-K1 cells was designed, and its nucleotide sequence is shown in SEQ ID NO.4.
  • Gene fragment VZV-gE-Fc (SEQ ID NO. 4) was synthesized by GenScript Biotech Co., Ltd. and HindIII and Not I restriction sites were added at both ends.
  • the gene fragment VZV-gE-Fc and PKS001 empty vector (purchased from Zhongshan Kangsheng Biological Co., Ltd., product number A13201) were digested by HindIII and Not I.
  • the target band and the large vector fragment were recovered after digestion, and the two were connected and transformed into Escherichia coli top10 competent cells.
  • the transformed bacteria were rejuvenated with LB liquid screening medium containing ampicillin and expanded.
  • the recombinant expression vector successfully constructed was named PKS001gEFc after sequencing identification.
  • the recombinant expression vector PKS001gEFc containing the VZV-gE-Fc gene was transfected into CHO-K1 (Zhongshan Kangsheng Biological Co., Ltd., Catalog No. A14101) cells by electroporation. After transfection, the CHO-K1 cells were cultured in the culture medium CD04 (Zhongshan Kangsheng Biological Co., Ltd., Catalog No. A11004).
  • the transfected CHO-K1 cells were replaced with medium CD04 (i.e., selection medium) containing 25 ⁇ M methionine sulfoximine (MSX, Sigma-Aldrich product, catalog number: M5379), and 10,000 cells/200 ⁇ l were inoculated into 96-well cell culture plates per well, and placed in a 37°C cell culture incubator for static culture for 2-3 weeks.
  • the culture wells of monoclonal cells were observed and marked under an optical microscope. When the cells grew to more than two-thirds of the area of the culture wells, 15 cell lines with higher expression were screened by ELISA.
  • the supernatants of these 15 monoclonal cells were harvested after shake flask feeding culture and purified by Protein A affinity chromatography in one step, and then subjected to physical and chemical analysis (SEC was used to detect and analyze the content of target VZV-gE-Fc protein aggregates, dimers and small molecule fragments) based on yield and The quality of the cells was finally determined to be three cell lines, 1E11, 3B7 and 2D10, as candidate cell lines for subsequent development.
  • the 3B7 cell line was finally determined as the production cell line (using cell target gene sequencing analysis, no base insertion, deletion, or mutation was found, and DNA sequence comparison with the full-length sequence of the target gene confirmed that the DNA sequence of the target gene in the cell was completely correct).
  • the 3B7 cells were revived and cultured in a shake flask. Feed and glucose were added to the cells on days 3, 5, 7, 9, and 11. The cell culture temperature was lowered to 33°C starting on day 6. When the cell viability dropped to about 50%, cells and cell debris were removed by high-speed centrifugation at 12,000 r/min for 30 minutes, and the cell culture supernatant was collected. The culture supernatant was tested and the final product expression level reached more than 3 g/L.
  • the culture supernatant of the above 3B7 cells was filtered through a 0.45 ⁇ m filter membrane and then affinity purified using a Protein A gel chromatography column. After low pH virus inactivation, hydrophobic chromatography was used, and then gradient elution was performed using DEAE Sepharose 4 Fast Flow with different concentrations of NaCl solutions to obtain the target protein solution.
  • the target protein solution was subjected to SDS-PAGE electrophoresis detection. The results are shown in FIG1 . It can be seen that a target protein of about 110 KD was obtained, and its size indicated that the VZV-gE-Fc fusion protein formed a homodimer, and the target protein was a VZV-gE-Fc fusion protein dimer.
  • the VZV-gE-Fc fusion protein dimer purified in Example 1 was used as the immunogen. Specifically, the VZV-gE-Fc fusion protein dimer purified in Example 1 was diluted with a dilution buffer solution containing 20 mM histidine hydrochloride, 140 mM arginine hydrochloride and 0.02% (volume percentage) polysorbate 80 (the solvent of the solution was water, the solutes were histidine hydrochloride, arginine hydrochloride and polysorbate 80 at corresponding concentrations, and the pH value was 6.0) to obtain 0.5 mg/ml diluted antigen.
  • a dilution buffer solution containing 20 mM histidine hydrochloride, 140 mM arginine hydrochloride and 0.02% (volume percentage) polysorbate 80 (the solvent of the solution was water, the solutes were histidine hydrochloride, arginine hydrochloride and polysorbate 80 at corresponding concentrations, and the pH
  • diluted antigen and aluminum hydroxide aluminum adjuvant suspension or aluminum phosphate adjuvant suspension are placed in a glass bottle equipped with a rotor at 150 r/min and mixed evenly to obtain different aluminum salt adjuvant vaccines, wherein the mass ratio of the antigen to the adjuvant is shown in Table 1 or Table 2.
  • the vaccine prepared above is aseptically dispensed into 2 ml vials (or pre-filled glass syringes), 0.5 ml (or 1.0 ml) per vial, and stored at 2-8°C away from light after sealing.
  • the vaccine prepared above was taken out and immunogenicity study was carried out using C57BL/6 mice (purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.) as an animal model.
  • Blood samples were collected at 3 and 5 weeks after immunization, and spleen samples were collected at 5 weeks.
  • the ELISA method was used to detect the antibody titer (i.e., total IgG) against VZV-gE-Fc protein in the serum.
  • the results showed that the vaccine combination prepared by the VZV gE-human IgG Fc protein (VZV-gE-Fc) of the present invention had very good immunogenicity and could be used as a potential candidate recombinant herpes zoster vaccine antigen.
  • the specific operation was as follows:
  • ELISA coating solution (1X) Take ELISA coating solution (10X) (purchased from Solebol, product number: C1055) and dilute it to 1X with sterile distilled water.
  • PBS preparation Take out PBS powder (purchased from Solebao, product number: P1003) and dissolve each bag with 2L of sterile distilled water.
  • PBST PBS containing 0.05% Tween-20
  • Washing solution PBST (PBS containing 0.05% Tween-20), prepared as follows: Measure 1L of filtered PBS into a blue-capped bottle, add 500 ⁇ L Tween-20, mix thoroughly, and store at 2-8°C for later use. According to actual conditions, prepare the required volume of PBST according to this method. Note: PBST should be prepared on the day of use.
  • TMB colorimetric solution 1 was purchased from Huzhou Yingchuang Biotechnology Co., Ltd., catalog number TMB-S-001.
  • Stop solution was purchased from Solebro, product number C1058.
  • VZV-gE-his antigen a protein obtained by adding 6 his to the C-terminus of VZV-gE shown in SEQ ID NO.1, prepared by Beijing Geneway Products Co., Ltd., with a concentration of 2 mg/ml
  • ELISA coating solution (1X) a concentration of 2 mg/ml
  • Blocking After taking out the coated plate from 4°C, wash the plate three times, each time with a washing volume of 300 ⁇ l/well. If there is any washing solution left in the wells after washing, pat it dry on absorbent paper; then add the pre-prepared blocking solution to the coated wells, 300 ⁇ l/well, cover with sealing film, and incubate at 37°C for 90min.
  • Serum dilution Dilute the serum to be tested to a suitable concentration using a sample diluent in a centrifuge tube to obtain a sample to be tested.
  • Color development Wash the 96-well plate three times, with a wash volume of 300 ⁇ l/well each time. If there is any wash solution left in the wells after washing, pat them dry on absorbent paper, add single-component TMB color development solution 1 (taken out from 4°C in advance and equilibrated to room temperature), 100 ⁇ l/well, and develop at 25°C in the dark for 15 min.
  • Termination Add stop solution immediately after color development to terminate the reaction, 50 ⁇ l/well, and shake gently to mix.
  • Table 1 shows the immunization scheme for mice immunized with recombinant VZV-gE-Fc vaccines prepared with different aluminum salt adjuvants
  • Group 1 in the above table was injected intramuscularly with PBS;
  • Group 2 and Group 3 in the above table were given intramuscular injections of different aluminum adjuvant vaccines.
  • mice Twenty-five C57BL/6 female mice aged 6-8 weeks were randomly divided into 5 groups. The animals were immunized according to the immunization scheme designed in Table 2. The optimal dose of aluminum adjuvant was screened by detecting the antibody titer 21 days after the first immunization (the day of the first immunization was recorded as Day 0 of the first immunization) and 14 days after the second immunization (the day of the second immunization was recorded as Day 0 of the second immunization) in groups 4, 5, 6, 7, 8, and 9.
  • Table 2 is the immunization scheme for screening the best aluminum adjuvant dosage
  • Group 4 in the above table received intramuscular injection of PBS
  • Groups 5 to 9 in the above table were injected intramuscularly with different doses of aluminum adjuvant vaccines.
  • Sequence number 1018 is a positive control, which is the ISS1018 sequence (CpG1018) of Dynavax.
  • Table 3 shows the CpG ODN sequences.
  • RPMI 1640 culture medium (Sigma product, catalog number: R8758) containing L-glutamine, 10% calf serum, penicillin, streptomycin and ⁇ -mercaptoethanol, stored at 4°C for future use
  • MTT Sigma product, catalog number: 11465007001: prepared with PBS to a liquid with a mass concentration of 5 mg/mL, filtered and sterilized, and stored at 4°C for future use
  • lymphocyte separation fluid was purchased from Dakoway Biotechnology Co., Ltd.
  • mice 4-6 week-old female Balb/c mice were purchased from Beijing Sibeifu (Beijing) Biotechnology Co., Ltd.
  • mice kill the mice by pulling their necks, disinfect their surfaces with 75% alcohol, and cut the fur on the upper and middle part of the left side of the mice with scissors under sterile conditions to expose the subcutaneous connective tissue. Cut the tissue to expose the spleen, and try to remove unnecessary components such as fat. Place the intact spleens into the wells of a six-well plate filled with 2 mL of culture medium, and mark the lid;
  • the synthetic CpG ODN sequences shown in Table 3 were diluted with deionized water and added to the spleen cells obtained in 7) as stimuli at a final concentration of 20 ⁇ g/mL. After culturing at 37°C and 5% CO2 for 72 hours, the proliferation level of mouse spleen cells induced by CpG ODN was detected by MTT method (A value of each group was detected) to screen and analyze the immunomodulatory activity of CpG ODN adjuvants. PBS was added as control.
  • Human PBMCs are rich in T and B lymphocytes, monocytes and dendritic cells, which are the main functional cells for the human body to respond to infection by foreign pathogens.
  • Normal healthy human PBMC were separated from fresh whole blood with Ficoll separation solution, suspended in 1640 medium containing 10% FBS, and inoculated in 24-well plates, 5 ⁇ 105 cells/well.
  • the four synthetic CpG ODN sequences shown in Table 3 were diluted with deionized water and added to PBMC cells at a final concentration of 20 ⁇ g/mL as stimuli, with 3 replicates for each sequence.
  • the MTT method was used to detect the level of PBMC cell proliferation induced by CpG ODN (detecting the A value of each group), and the immunomodulatory activity of the self-designed CpG ODN adjuvant was screened and analyzed.
  • PBS was added as a control.
  • C57BL/6 female mice Twenty-five 6-8 week old C57BL/6 female mice were randomly divided into 5 groups, and the C57BL/6 female mice were immunized according to the method shown in Table 4.
  • the best CpG adjuvant was screened by detecting the antibody titer of groups 10-14 at 21 days after the first immunization (the day of the first immunization was recorded as day 0 of the first immunization) and 14 days after the second immunization (the day of the second immunization was recorded as day 0 of the second immunization).
  • a and B were used to screen out the best aluminum adjuvant AL(OH)3 and its dose, and the candidate CpG adjuvant screened by C above was used as a dual adjuvant, where the CpG adjuvant was CpG-cjx1, Dynavax’s ISS1018 sequence (CpG1018) and CpG7909 (SEQ ID NO.10: 5’-TCG TCG TTT TGT CGT TTT GTC GTT-3’, Bio-Pharmaceuticals).
  • Table 4 shows the immunization scheme for screening the best CpG adjuvant
  • Groups 11 to 13 in the above table were injected intramuscularly with different double-adjuvant vaccines.
  • Table 5 shows the immunization scheme with the best CpG adjuvant dose screened
  • Group 15 in the above table received intramuscular injection of PBS
  • Groups 16 to 19 in the above table were injected intramuscularly with different double-adjuvant vaccines.
  • mice Twenty-five C57BL/6 female mice aged 6-8 weeks were randomly divided into 5 groups. The animals were immunized according to the immunization scheme designed in Table 6. The optimal antigen dose was screened by detecting the binding antibody titer 21 days after the first immunization and 14 days after the second immunization in groups 20, 21, 22, 23, 24, and 25.
  • Table 6 shows the immunization scheme for exploring the optimal antigen dose in mice
  • Group 20 in the above table was injected intramuscularly with PBS;
  • Groups 21 to 25 in the above table were injected intramuscularly with different doses of double-adjuvant vaccines.
  • mice Twenty-five C57BL/6 female mice aged 6-8 weeks were randomly divided into 5 groups. The animals were immunized according to the immunization scheme designed in Table 7. The animals were immunized twice by intramuscular injection on days 0 and 21. The spleens were collected 14 days after the second immunization and the spleen lymphocytes were isolated. The expression of IFN- ⁇ and IL-2 factors in spleen lymphocytes of groups 26, 27, 28, 29, and 30 14 days after the second immunization was detected by ELISPOT.
  • mice lymphocyte separation solution to spleen tissue, grind gently, filter through 200-mesh nylon mesh into a clean centrifuge tube, gently add 1 ml of PBS above the liquid surface, centrifuge at 800g for 30 min, with fast ascent and slow descent, and collect the middle lymphocyte layer.
  • the peptide library was purchased from Novozymes Biotechnology Co., Ltd., product name Varicella-Zoster Virus peptide pool, product number: DD9120.
  • the vaccine prescription was further screened by testing the binding antibody titers of groups 26, 27, 28, 29, and 30 after the first vaccination at 21 days and the second vaccination at 14 days.
  • Table 7 shows the design of cell-mediated immunity program
  • Groups 27 to 30 in the above table were injected intramuscularly with different types of vaccines.
  • the binding antibody titer results are shown in Figure 9.
  • the binding antibody titer of the double adjuvant vaccine (Group 30) is superior to that of the single adjuvant vaccine group (Groups 28 and 29) and the single antigen vaccine group (Group 27).
  • the detection results of IFN- ⁇ and IL-2 are shown in Figures 10a and 10b.
  • the stimulation of the double adjuvant vaccine group is significantly better than that of the single adjuvant group and the antigen group without adjuvant.
  • the double adjuvant vaccine group has the best immunization effect
  • the antigen content is 2.5-30 ⁇ g
  • the aluminum hydroxide adjuvant is 10-200 ⁇ g
  • the CpG adjuvant is 1-10 ⁇ g, which are all better.
  • the antigen content is 5 ⁇ g
  • the aluminum hydroxide adjuvant is 50 ⁇ g
  • the CpG adjuvant is 2 ⁇ g.
  • VZV-Genevax VZV-Genevax
  • Shingrix VZV-GSK
  • herpes zoster there is no herpes zoster vaccine on the market in China.
  • Shingrix developed by GSK is the best vaccine among the herpes zoster vaccines currently approved for use.
  • the US CDC recommends Shingrix to replace Zostavax, the herpes zoster vaccine of Merck.
  • the adjuvant used in GSK's VZV vaccine Shingrix is AS01B, in which the limited supply of QS21 ingredients causes the price of Shingrix to reach 150-200 US dollars per dose.
  • the antigen recombinant gE antigen (CHO cell expression) needs to be freeze-dried and packaged separately, and then AS01B is packaged separately, and the adjuvant needs to be mixed with the antigen before injection into the human body.
  • the vaccine of the present invention takes effect faster after one immunization, and the serum antibody titer of Shingrix developed by GSK is also improved after two immunization tests.
  • the results of Elispot test of IFN-r factor showed that the cellular immunity of the vaccine of the present invention was superior to Shingrix developed by GSK.
  • VZV-Genevax The human double-adjuvant VZV-gE-Fc recombinant shingles vaccine (VZV-Genevax) was prepared as follows per 0.5 ml: 1.5 ml 0.5 mg/ml diluted antigen VZV-gE-Fc, 0.5 ml aluminum adjuvant (aluminum hydroxide suspension (with an aluminum content of 10 mg/ml, purchased from Changchun Institute of Biological Products), and 50 ⁇ l 10 mg/ml CpG-cjx1 solution were mixed evenly at room temperature to obtain a double-adjuvant vaccine, in which the content of antigen VZV-gE-Fc was 75 ⁇ g/500 ⁇ l, the content of AL(OH)3 was 500 ⁇ g/500 ⁇ l, and the content of CpG-cjx1 was 50 ⁇ g/500 ⁇ l.
  • the immunization dose of VZV-Genevax and Shingrix developed by GSK in mice is 1/10 of the human dose, that is, 50 ⁇ l per mouse.
  • VZV-Genevax The 50 ⁇ l VZV-gE-Fc recombinant herpes zoster vaccine (VZV-Genevax) used to immunize mice contained 7.5 ⁇ g VZV-gE-Fc, 50 ⁇ g AL(OH)3 and 5 ⁇ g CpG-cjx1, and the preparation method was the same as the above-mentioned human double adjuvant VZV-gE-Fc.
  • the mass ratio of antigen to aluminum adjuvant and CpG-cjx1 adjuvant is 7.5:50:5.
  • mice Eighteen 6-8 week old C57BL/6 female mice were randomly divided into three groups, with 6 mice in each group.
  • the animals were immunized according to the immunization scheme designed in Table 8, and the VZV specific IgG binding antibody titer was detected by ELISA method 21 days after the first immunization and 14 days after the second immunization; the expression of IFN- ⁇ factor in spleen lymphocytes of groups 31, 32, and 33 at 14 days after the second immunization was detected by ELISPOT (the method is the same as above).
  • Table 8 shows the dual adjuvant VZV-gE-Fc recombinant zoster vaccine and the shingrix mouse immunization scheme developed by GSK
  • the ELISA titer results are shown in Figure 11a, and the ELISPOT results are shown in Figure 11b.
  • the results show that the immune effect of VZV-Genevax of the present invention is better than that of GSK-Shingrix.
  • Table 9 shows the dual adjuvant VZV-gE-Fc recombinant herpes zoster vaccine and the shingrix cynomolgus monkey immunization scheme developed by GSK
  • Vaccine test group (VZV-Genevax): human double adjuvant VZV-gE-Fc recombinant herpes zoster vaccine (75 ⁇ g VZV-gE-Fc+0.5mgAL(OH)3+0.05mgCpG-cjx1/0.5mL), 0.5mL/dose/animal, immunized 5 animals;
  • Control vaccine test group 50 ⁇ g VZV-gE-Fc+ASO1B/0.5 mL, 0.5 mL/dose/animal, immunized 5 animals;
  • PBS control group 0.5mgAL(OH)3+0.05mgCpG-cjx1/0.5mL (compared with the human double-adjuvant VZV-gE-Fc recombinant herpes zoster vaccine, only without adding antigen), 0.5mL/dose/animal, 5 animals were immunized.
  • Immunization was performed twice, on day 0 (the day of the first immunization was recorded as day 0) and day 28 (the day of the first immunization was recorded as day 0), and the VZV-specific IgG binding antibody titer was detected by ELISA method 21 days after the first immunization and 14 days after the second immunization; 5 mL of hind limb venous blood was collected 14 days after the last immunization and sent to The cells were collected from Nanning Weierkai Biotechnology Co., Ltd., lymphocytes were isolated, and the expression of IFN- ⁇ , IL-2, TNFa (Th1), and IL-4 (Th2) cytokines by CD4/CD8 T cells was determined by flow cytometry.
  • the expression of IFN- ⁇ factor in peripheral blood lymphocytes of groups 34, 35, and 36 was detected by ELISPOT at 14 days after the second immunization.
  • the ELISPOT detection kit catalog number: MBF3421M-4AST-2, brand: Mabtech
  • MBF3421M-4AST-2 catalog: Mabtech
  • Table 10 is the experimental reagents
  • the ascending speed is 2
  • the descending speed is 2
  • the centrifugal force is 1000g and centrifuged for 30 minutes.
  • the cells were collected and centrifuged to discard the supernatant.
  • the cells were resuspended in 100 ⁇ l of PBS containing 1% FBS, and CD3, CD4, and CD8 antibodies were added, mixed, and incubated at 4°C for 20 min.
  • Figure 12b shows that the expression of IFN- ⁇ , IL-2, and TNFa in peripheral blood lymphocyte CD4/CD8T cells of the vaccine of the present invention (VZV-Genevax) is significantly higher than that of Shingrix (GSK-shingrix) developed by GSK, indicating that the vaccine of the present invention (VZV-Genevax) triggers a strong CD4+ and CD8+T cell response, induces a Th1 immune response, and has an outstanding advantage in activating cellular immune responses by vaccines;
  • the detection of IFN- ⁇ in Figure 12c shows that the vaccine of the present invention (VZV-Genevax) stimulates cells to secrete more IFN- ⁇ than Shingrix (GSK-shingrix) developed by GSK, indicating that the vaccine of the present invention (VZV-Genevax) has a higher cellular immunity level and can effectively play an antiviral and immune regulatory role.
  • Example 4 Verification of the in vivo effect of CpG ODN as an adjuvant for COVID-19 vaccine in mice
  • mice healthy Balb/c mice, 6-8 weeks old, 18-20 g, female, SPF grade, purchased from Spef (Beijing) Biotechnology Co., Ltd.; animal certificate number: NO.110324210104634878, receipt date: 2021.9.5, kept in the animal laboratory of Beijing Medilibon Biotechnology Co., Ltd.
  • Vero-E6 cell line SARS-CoV-2 (hCoV-19/China/CAS-B001/2020, GISAID No.EPI_ISL_514256-7), prepared and provided by the Institute of Microbiology, Chinese Academy of Sciences.
  • DMEM Gibco, batch number: 8120287
  • FBS Gibco
  • penicillin-streptomycin New Saimei Biotechnology Co., Ltd., batch number 2009021031
  • 96-well cell culture plates were provided by the Institute of Microbiology, Chinese Academy of Sciences
  • RBD-FC stock solution Beijing Zhaoyin Derivative Technology Co., Ltd., batch number BDS201302
  • aluminum hydroxide adjuvant (Changchun Institute of Biological Products Co., Ltd., batch number 2P18-003-202005); CpG-cjx1 adjuvant designed by the present invention
  • diluent Beijing Zhaoyin Derivative Technology Co., Ltd., batch number 2P18-003-202005
  • recombinant SARS-CoV-2 RBD-his protein Beijing Medilibon Biotechnology Co., Ltd.
  • mice were immunized twice by intraperitoneal injection at 0d and 21d. Blood and spleen were collected at 28d, spleen lymphocytes were isolated, and the expression levels of IFN- ⁇ , TNF- ⁇ , IL-2, and IL-4 by CD4 + /CD8 + T cells were determined.
  • Immunization was performed twice by intraperitoneal injection at 0d and 21d, and blood was collected from the tail vein on 35d and serum was separated. Flow cytometry was used to determine the cellular immune effect of mice immunized with recombinant new coronavirus fusion protein vaccine (CHO cells) or recombinant new coronavirus fusion protein vaccine preparations.
  • the specific scheme is shown in Table 11.
  • the ELISA method and cytopathic inhibition method were used to detect the binding antibody titer and neutralizing antibody titer produced by immunized mice, respectively.
  • the sample numbers are shown in Table 12. Each sample serum was frozen at about 0.2mL. Specifically, 25 serum samples of Balb/c mice were collected, including 20 serum samples from the vaccine test group and 5 serum samples from the PBS control group.
  • CBS carbonate buffer
  • FCS 10% bovine serum
  • Vero-E6 cells were inoculated into a 96-well cell plate, with 2 ⁇ 10 5 to 4 ⁇ 10 5 cells per well.
  • the culture medium was DMEM containing 10% FBS, and the culture conditions were 37°C, 5% CO 2 , and saturated humidity.
  • the mouse serum was diluted with DMEM containing 2% FBS by the double dilution method, and the original strain of the new coronavirus was added to each serum dilution liquid at 100CCID 50 /200 ⁇ L, and placed at 37°C for 1.0h.
  • the culture medium was discarded and washed 3 times with PBS; finally, the mixed serum-virus solution was added, and each dilution was repeated for 4 wells, with 200 ⁇ L added to each well.
  • the cells continued to be cultured at 37°C, 5% CO 2 , and saturated humidity, and the cell lesions were observed and recorded every 24h, and observed continuously for 72h.
  • the neutralizing titer of serum is calculated by Reed-Muench method (the highest dilution of serum that protects 50% of cells from lesions is the NT50 of the serum); the calculation is performed by Reed-Muench method, and the results are expressed as mean plus minus standard deviation.
  • ELISA and cytopathic inhibition assays were used to determine the binding antibody and neutralizing antibody titers of the serum of 4 groups of recombinant novel coronavirus fusion protein vaccines 14 days after immunization of Balb/c mice twice, 1) 25 ⁇ g RBD-Fc; 2) 25 ⁇ g RBD-Fc+0.5mgAL(OH) 3 ; 3) 25 ⁇ g RBD-Fc+10 ⁇ g CpG-cjx1; 4) 25 ⁇ g RBD-Fc+0.5mgAL(OH) 3 +10 ⁇ g CpG-cjx1.
  • Table 14 The results are shown in Table 14.
  • CpG-cjx1 of the present invention has little difference in effect between different dosages when used as an adjuvant in vaccines.
  • CpG-cjx1 above 10 ⁇ g/0.5 mL/dose can achieve good immune effects.

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Abstract

一种带状疱疹疫苗，包括抗原和佐剂；所述抗原为含有VZV-gE蛋白和人IgG4 Fc的融合蛋白；所述佐剂为铝佐剂、CpG佐剂或二者的结合。疫苗使用常规的铝佐剂和CpG佐剂或者新型CpG佐剂联合，价格低廉容易获得；抗原和佐剂可以直接混合，预灌充注射器分装制剂。

Description

一种重组带状疱疹疫苗及其制备与应用
交叉引用
本发明要求在中国的、申请号为202211523443.X、申请日为2022年12月01日、发明名称为“新型佐剂重组带状疱疹疫苗及其制备与应用”；在中国的、申请号为202211535908.3、申请日为2022年12月01日、发明名称为“双佐剂重组带状疱疹疫苗”以及在中国的、申请号为202211533170.7、申请日为2022年12月01日、发明名称为“一种新型CpG疫苗佐剂及其应用”的三个申请为优先权，上述申请整体引入到本发明中，并作为本发明的一部分。
技术领域
本发明属于生物技术领域，涉及一种疫苗制剂，特别涉及一种重组带状疱疹疫苗及其制备与应用。
背景技术
带状疱疹是由潜伏的水痘-带状疱疹病毒(VZV)重新激活引起的感染性皮肤病，好发于中老年人，常表现为沿感觉神经支配的区域形成单侧皮疹，并伴有神经病理性疼痛，最普遍的并发症是带状疱疹后遗神经痛(PHN)。随着我国人口老龄化趋势日益加重，带状疱疹的发病率呈显著上升趋势，带状疱疹的防治已经成为重要的公共卫生问题，接种带状疱疹疫苗来防止该病发生是最为行之有效的手段。
水痘带状疱疹病毒(VZV)属于疱疹病毒属A疱疹病毒科，VZV基因组有71个基因，编码67个不同蛋白，包括6种糖蛋白(gpⅠ～gpⅥ)，现统一分别命名为gE、gB、gH、gI、gC和gL。其中gE在病毒包膜含量最丰富，是宿主免疫系统识别的最主要糖蛋白，由orf68基因编码，位于vzv基因组的短片区，含623个氨基酸，其中亲水膜外区544个，跨膜区17个，胞内区62个。已有研究表明以gE为抗原诱导的免疫反应，能保护动物免受病毒攻击。VZV gE单克隆抗体可介导抗体依赖性的细胞毒性，能中和病毒的感染性。目前的挑战是gE仅可以诱导低水平的体液免疫，无法满足疫苗使用的要求。
全球已上市的带疱疫苗产品有Zostavax(默沙东)、Shingrix(GSK)以及NBP608(SK化学，仅韩国市场销售)。Zostavax与SkyZoster是减毒活疫苗，通过处理实现结构改变、毒性减弱但保留免疫原性，从而建立免疫反应；Shingrix为重组亚单位疫苗，通过水痘-带状疱疹糖蛋白E抗原和AS01B佐剂在体内产生和增强免疫反应。临床数据表明，Shingrix在50至69岁有效率为97％，70岁以上有效率仍然高达为91％；Zostavax在50至69岁有效率为70％，整体有效率为51％，Shingrix的有效性要优于Zostavax。但Shingrix疫苗配方复杂，抗原需要冻干并且需要和佐剂分开，佐剂ASO1B，特别是其中的QS21不能化学合成，原材料稀缺，价格贵，导致供应不足。
因此有必要开发安全、有效，且配方简单、成本低的VZV疫苗。
近些年，在疫苗的研制过程中人们逐渐认识到，佐剂的使用对疫苗的成功制备至关重要。因此，疫苗佐剂的研究一直是疫苗研究过程中的重要环节，研究佐剂与疫苗成份的配伍，使二者形成稳定、安全、具有免疫原性的疫苗复合物，使疫苗佐剂作为非特异性免疫增强剂对疫苗接种后诱导有效的免疫应答发挥作用。目前有许多物质被尝试作为疫苗佐剂，但只有铝盐佐剂，MA59(水包油型乳剂)，MPL(糖脂)，病毒样颗粒(viral like particle,VLP)，免疫增强的再造流感病毒小体(immunopotentiating reconsitituted influenza virosome,IRIV)和霍乱肠毒素(cholera toxin,CT)被批准应用于人类疫苗中。但在实际应用中，上述佐剂常出现注射部位肿胀、出现肉芽肿、发烧、疼痛、发生过敏等副反应。此外，有些佐剂成本高昂。因此，研制广谱、安全、高效，同时便于生产和使用的疫苗佐剂迫在眉睫。
胞嘧啶－鸟嘌呤寡脱氧核苷酸(cytosine phosphate guanidine oligodeoxynucleotide，CpG ODN)作为一种新型、安全、高效的免疫佐剂，展现出了良好的应用前景。其可以诱发机体产生多种免疫学效应，提高系统免疫和黏膜免疫水平，具有安全性高、耐受性强等特点。2017年，Dynavax Technologies公司旗下的HEPLISAV-B药物作为世界首个含CpG佐剂获批的疫苗，其包含CpG ODN 1018与市售乙肝疫苗的联用(http://investors.dynavax.com/events-presentations)。此外，Coley公司将CpG ODN 7909作为商品化多价流感灭活疫苗(Fluarix)的佐剂，其随机双盲Ⅰ期临床试验结果表明，CpG ODN是一种安全、可靠的佐剂，可降低多价流感灭活疫苗(Fluarix)的用量。国内外大量研究都证实了CpG ODN作为一种新型高效的免疫激活剂，呈现出了良好的免疫佐剂效应。而CpG ODN对不同的种属、细胞和抗原成分体现出不同的免疫刺激效果，其免疫刺激效果也与其序列结构紧密相关。因此，十分有必要针对不同的应用场景和抗原成分对CpG ODN做出相应的改进和设计。
发明内容
本发明的一方面，提供了一种融合蛋白，所述融合蛋白包括VZV-gE蛋白和人IgG4的Fc片段。
在一些实施方案中，所述VZV-gE与Fc片段直接或者间接连接。
所述间接连接包含接头。
在一些实施方案中，所述接头为柔性接头。
在一些实施方案中，所述柔性接头选自GGGGS(SEQ ID NO.12)、GGGGSGGGGS(SEQ ID NO.13)、GSGGGSGGGGSGGGGSA(SEQ ID NO.14)。
在一些实施方案中，所述柔性多肽为GSGGGSGGGGSGGGGSA(SEQ ID NO.14)。
在一些实施方案中，所述VZV-gE蛋白为如下任一种：
(a1)SEQ ID NO.1所示的蛋白质；
(a2)在(a1)所述蛋白质的N端或/和C端连接标签得到的融合蛋白；
(a3)将(a1)-(a2)任一种经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质；
(a4)与(a1)-(a2)任一种具有80％以上同一性且具有相同功能的蛋白质。
在一些实施方案中，所述人IgG4Fc蛋白为如下任一种：
(b1)SEQ ID NO.2所示的蛋白质；
(b2)在(b1)所述蛋白质的N端或/和C端连接标签得到的融合蛋白；
(b3)将(b1)-(b2)任一种经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质；
(b4)与(b1)-(b2)任一种具有80％以上同一性且具有相同功能的蛋白质。
在具体的实施方案中，所述融合蛋白为如下任一种：
(c1)SEQ ID NO.3所示的蛋白质；
(c2)在(c1)所述蛋白质的N端或/和C端连接标签得到的融合蛋白；
(c3)将(c1)-(c2)任一种经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质；
(c4)与(c1)-(c2)任一种具有80％以上同一性且具有相同功能的蛋白质。
所述氨基酸序列具有80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列。
本发明的另一方面，提供一种编码上述融合蛋白的核酸。
在一些实施方案中，编码融合蛋白的核酸包含与SEQ ID NO.4所示核苷酸序列具有80％或以上同一性的核苷酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的核苷酸列，更优选具有98％或99％以上同一性的核苷酸序列。
在一些实施方案中，编码所述融合蛋白的核酸为SEQ ID NO.4所示的核酸。
在一些实施方案中，所述核酸是mRNA。
本发明的另一方面，提供一种包含上述核酸的表达载体。
在一些实施方案中，所述表达载体选自原核表达载体或真核表达载体。
在一些实施方案中，所述表达载体为真核表达载体。
在一些实施方案中，所述真核表达载体是腺病毒载体。
在一些实施方案中，所述真核表达载体为在293细胞或CHO细胞中表达的载体。
本发明的另一方面，提供一种宿主细胞，其表达上述任一融合蛋白，或包含上述任一核酸和/或包含上述任一表达载体。
在一些实施方案中，所述宿主细胞是原核细胞或真核细胞。
在一些实施方案中，所述原核细胞是细菌细胞。
在一些实施方案中，所述原核细胞是大肠杆菌细胞。
在一些实施方案中，所述真核细胞选自酵母细胞、昆虫细胞和哺乳动物细胞。
在一些实施方案中，所述哺乳动物细胞选自CHO、HEK293、SP2/0、BHK、C127等。
在一些实施方案中，所述真核细胞为CHO细胞。
本发明的另一方面，提供上述任一的融合蛋白、核酸、表达载体和/或宿主细胞在制备治疗或预防与水痘带状疱疹病毒相关的疾病或病状的疫苗中的用途。
本发明的另一方面，提供一种VZV融合蛋白疫苗，其包含上述任一的融合蛋白、核酸、表达载体和/或宿主细胞，以及一种或多种佐剂。
在一些实施方案中，所述佐剂选自氢氧化铝、CpG、磷酸铝、皂苷，例如QuilA、QS-21、GPI-0100、油包水型乳状液、水包油型乳状液、水包油包水型乳状液中的一种或多种。
在一些实施方案中，所述佐剂为氢氧化铝和CpG双佐剂。
在一些实施方案中，所述融合蛋白、铝佐剂和CpG佐剂的比例为1-8:5-240：1-20。
在一些实施方案中，所述融合蛋白、铝佐剂和CpG佐剂的比例为1-4:20-60:1-10。
在一些实施方案中，所述CpG佐剂为任一的CpG佐剂，例如CpG1018，或，所述CpG佐剂为SEQ ID NO:5-9中任一条序列所示的核苷酸，且所有核苷酸残基均硫代修饰。
本发明的另一方面，提供一种带状疱疹疫苗，其包括抗原和佐剂；
所述佐剂为铝佐剂、CpG佐剂或二者的结合；
所述抗原来自水痘带状疱疹病毒。
在一些实施方案中，所述CpG佐剂为SEQ ID NO:5-9中任一条序列所示的核苷酸，且所有核苷酸残基均硫代修饰。
在一些实施方案中，所述疫苗为带状疱疹的灭活疫苗、减毒活疫苗、病毒载体疫苗、亚单位疫苗、重组疫苗、多糖疫苗、结合疫苗、类毒素疫苗、核酸疫苗；所述核酸疫苗为mRNA疫苗、DNA疫苗。
上述中，所述核酸疫苗为mRNA疫苗或DNA疫苗。
上述带状疱疹疫苗中，所述铝佐剂为AL(OH)3或ALPO4。
上述带状疱疹疫苗中，所述佐剂由铝佐剂和CpG佐剂组成；
所述铝佐剂为AL(OH)3；
所述CpG佐剂为SEQ ID NO:5-9任一所示的核酸分子，且所有核苷酸残基均硫代修饰。
上述带状疱疹疫苗，所述抗原和所述佐剂混合包装或单独包装。
上述带状疱疹疫苗中，所述抗原、所述AL(OH)3(AL)和所述CpG佐剂质量比为2.5-30：10-200：1-10。
上述带状疱疹疫苗中，所述带状疱疹疫苗中，所述抗原、所述AL(OH)3(AL)和所述CpG佐剂质量比为7.5：50：5。
上述的疫苗可以分装入2ml西林瓶内(或预填充玻璃注射器内)，每瓶0.5ml(或1.0ml)。
在本发明的实施例中，每100μl双佐剂疫苗制剂中，抗原含量2.5-30μg，氢氧化铝佐剂10-200μg，CpG佐剂1-10μg均较佳，具体举例每100μl双佐剂疫苗制剂中，抗原含量5μg，氢氧化铝佐剂50μg，CpG佐剂2μg。
每500μl双佐剂疫苗制剂中，抗原含量75μg，氢氧化铝佐剂500μg，CpG佐剂50μg均较佳。
每50μl双佐剂疫苗制剂中，抗原含量7.5μg，氢氧化铝佐剂50μg，CpG佐剂5μg均较佳。
在一些实施方案中，所述抗原来源上述任一的融合蛋白、核酸、表达载体和/或宿主细胞。
本发明的另一方面，提供一种制备上述带状疱疹疫苗的方法，包括如下步骤：将所述抗原和所述佐剂混匀，得到带状疱疹疫苗。
上述所述混匀为将所述抗原和所述佐剂按照上述所需质量比混匀。
本发明的另一方面，提供上述融合蛋白，和，所述铝佐剂和所述CpG佐剂在如下任一中的应用：
1)制备预防或治疗带状疱疹的产品；
2)制备中和带状疱疹病毒的产品；
3)制备诱导机体对带状疱疹病毒产生体液免疫和/或细胞免疫的产品；
4)制备带状疱疹病毒抗体。
上述的应用中，所述产品为疫苗。
本发明的另一方面，提供上述任一的融合蛋白、核酸、载体或宿主细胞在如下任一中的应用：
1)作为带状疱疹疫苗抗原；
2)制备预防或治疗带状疱疹的产品；
3)制备中和带状疱疹病毒的产品；
4)制备诱导机体对带状疱疹病毒产生体液免疫和/或细胞免疫的产品；
5)制备带状疱疹病毒抗体。
上述带状疱疹病毒抗体具有中和带状疱疹病毒的功能。
本发明的另一方面，提供一种在有相应需要的受试者中诱导针对水痘带状疱疹病毒的免疫应答的方法，该方法包括：向受试者施用所述任一的疫苗。
在一些实施方案中，所述受试者是哺乳动物或鸟类。
在一些实施方案中，所述受试者是人类、牛、犬、猫、山羊、绵羊、猪、马、火鸡、鸭或鸡。
本发明的另一方面，提供一种药物组合物，所述药物组合物包括上述任一的融合蛋白、核酸、表达载体和/或宿主细胞，以及一种或多种药学上可接受载体、稀释剂或赋形剂。
本发明的另一方面，提供一种治疗或预防与水痘带状疱疹病毒相关的疾病或病状的方法，所述方法包括向受试者施用上述任一的疫苗或药物组合物。
本发明的另一方面，提供一种寡核苷酸，所述寡核苷酸包含序列：
TGACTX1X2X3CGTTTTAX4CGX5X6AGACTGA(SEQ ID NO:11)；
其中：
X1为G或A；
X2为G或A；
X3为A或T；
X4为G或A；
X5为T或C；并且
X6为T或C，
所述序列的全部核苷酸均为硫代修饰。
在一些实施方案中，前述寡核苷酸包含核苷酸序列TGACTGAACGTTTTAACGTCAGACTGA(SEQ ID NO:5)或其任何变体。
在一些实施方案中，前述寡核苷酸包含核苷酸序列TGACTGAACGTTTTAGCGCTAGACTGA(SEQ ID NO:6)或其任何变体。
在一些实施方案中，前述寡核苷酸包含核苷酸序列TGACTAGTCGTTTTAACGTCAGACTGA(SEQ ID NO:7)或其任何变体。
在一些实施方案中，前述寡核苷酸包含核苷酸序列TGACTAGTCGTTTTAGCGCTAGACTGA(SEQ ID NO:8)或其任何变体。
在一些实施方案中，前述寡核苷酸包含核苷酸序列TGACTGTGAACGTTCGAGATGA(SEQ ID NO:9)或其任何变体。
在一些实施方案中，前述寡核苷酸包含如SEQ ID NO:5-9任一所示的核苷酸序列。
本发明的另一方面，本发明提供了一种CpG佐剂，所述佐剂包含前述寡核苷酸。
在一些实施方案中，其包含前述寡核苷酸作为有效成分。
在一些实施方案中，所述佐剂，还包含选自矿物盐类佐剂、油乳剂佐剂、靶向于模式识别受体的微生物和植物提取物以及衍生物类佐剂、微粒抗原递呈系统佐剂或细胞因子类佐剂中的一种或多种。其中，前述矿物盐类佐剂是铝佐剂或镁佐剂中的一种或多种。
前述油乳剂佐剂选自皂角苷类佐剂、水包油类和油包水类乳剂中的一种或多种。前述靶向于模式识别受体的微生物和植物提取物以及衍生物类佐剂是磷酰脂A佐剂、CpG佐剂中的一种或多种。前述微粒抗原递呈系统佐剂是聚乙丙交酯(PLG)、脂质体中的一种或多种。前述细胞因子类佐剂是细胞因子IL-1、IL-2、IL-12中的一种或多种。
在一些实施方案中，该佐剂包含前述寡核苷酸和铝佐剂作为有效成分。
包含前述寡核苷酸和所述矿物盐类的佐剂是复合佐剂。其中，所述矿物盐类佐剂是铝佐剂或镁佐剂中的一种或多种。
本发明的铝佐剂包括氢氧化铝、磷酸铝。
本发明的另一方面，提供一种疫苗，其包含前述寡核苷酸或佐剂。
在一些实施方案中，前述疫苗中还含有抗原。
在一些优选的实施方案中，其中前述抗原为冠状病毒RBD-Fc。
在一些优选的实施方案中，前述冠状病毒为新型冠状肺炎病毒。
在一些实施方案中，前述疫苗中前述佐剂的剂量为5-3000μg/剂。
在一些优选的实施方案中，前述寡核苷酸的剂量选自5μg/剂、10μg/剂、30μg/剂或50μg/剂。
在一些优选的实施方案中，前述寡核苷酸的剂量为10μg/剂。
在一些实施方案中，每剂疫苗中前述寡核苷酸和铝佐剂的质量比为1:8-1:100。
在一些优选的实施方案中，前述佐剂包含所述寡核苷酸10μg/剂，以及所述铝佐剂0.5mg/剂。
本发明的另一方面，提供一种前述寡核苷酸、CpG佐剂或复合佐剂的应用。
在一些实施方案中，本发明提供了一种前述寡核苷酸、CpG佐剂或复合佐剂在制备疫苗，或者提高抗原或疫苗的免疫原性中的用途。
在一些实施方案中，前述疫苗为新型冠状肺炎病毒疫苗。
在一些实施方案中，前述抗原为冠状病毒RBD-Fc。
在一些实施方案中，前述冠状病毒为新型冠状肺炎病毒。
本发明的另一方面，提供一种前述寡核苷酸、CpG佐剂或复合佐剂以及疫苗的制备方法。
在一些实施方案中，所述寡核苷酸为SEQ ID NO:5-9中任一条序列所示的核苷酸，所述核苷酸的全部核苷酸均为硫代修饰。
本发明的另一方面，提供上述CpG佐剂和铝佐剂在制备疫苗，或者提高抗原或疫苗的免疫原性中的用途，优选的，所述疫苗用于治疗和/或预防带状疱疹病毒或冠状病毒引起的疾病或病症，更优选的，所述冠状病毒包括新型冠状病毒。
本发明的另一方面，提供一种治疗或预防疾病或病状的方法，所述方法包括向受试者施用上述任一所述的疫苗或上述的药物组合物，优选的，所述疾病或病状包括带状疱疹病毒或冠状病毒引起的疾病或病症，更优选的，所述冠状病毒包括新型冠状病毒。
如本文使用的和除非另作说明，术语“约”或“大约”是指在给定值或范围的加或减10％之内。在需要整数的情况下，该术语是指在给定值或范围的加或减10％之内、向上或向下舍入到最接近的整数。
如本文使用的和除非另作说明，术语“包含”，“包括”，“具有”，“含有”，包括其语法上的等同形式，通常应当理解为开放式且非限制性的，例如，不排除其他未列举的要素或步骤。
如本文所使用的，术语“融合蛋白”是指由一种或多种分子组成的天然或合成分子，其中具有不同特异性的两种或多种基于肽或蛋白质(包括糖蛋白)的分子任选的通过化学的或基于氨基酸的接头分子融合在一起。该连接可通过C-N融合或N-C融合(以5′→3′方向)，优选C-N融合而实现。
如本文所使用的，术语“抗体”或“免疫球蛋白”有最广义的含义，特别包括完整的单克隆抗体、多克隆抗体、由至少2个完整抗体构成的多特异性抗体(例如双特异性抗体)以及抗体片段，只要其显示出具有所需的生物学活性即可。此术语一般包括由2个或多个具有不同结合特异性的抗体或抗体片段连接在一起构成的杂合抗体。
如本文所使用的，术语“Fc区”在本文中用于定义免疫球蛋白重链的C端区，包括天然序列Fc区和变体Fc区。尽管免疫球蛋白重链的Fc区的边界可以变化，但人IgG重链Fc区通常定义为自位置Cys226，或自Pro230处的氨基酸残基延伸至重链的羧基端。可以除去Fc区的C端赖氨酸(依照EU编号系统的残基447)，例如在抗体的产生或纯化期间，或通过重组工程化改造编码抗体重链的核酸。因此，完整抗体的组合物可以包含已除去所有K447残基的抗体群体，未除去任何K447残基的抗体群体，和具有有和无K447残基的抗体混合物的抗体群体。
如本文所使用的，序列“相同性”或“同一性”具有本领域公认的含义，并且可以利用发明的技术计算两个核酸或多肽分子或区域之间序列相同性的百分比。可以沿着多核苷酸或多肽的全长或者沿着该分子的区域测量序列相同性。虽然存在许多测量两个多核苷酸或多肽之间的相同性的方法，但是术语“相同性”是技术人员公知的(Carrillo,H.&Lipman,D.,SIAM J Applied Math 48:1073(1988))。
如本文所使用的，术语“疾病”或“病状”是指能够用本文提供的融合蛋白、药物组合物或方法治疗的患者或个体的生存状态或健康状态。
术语“疫苗”是纯化的抗原疫苗或免疫原性组合物，亚基疫苗或免疫原性组合物，灭活的整体病毒疫苗或免疫原性组合物，或减毒病毒疫苗或免疫原性组合物。在一些实施方案中，疫苗或免疫原性组合物是纯化的融合蛋白。
如本文所使用的，术语“治疗(treating或treatment)”是指成功治疗或改善损伤、疾病、病理或病状(condition)的任何指标，包含任何客观或主观参数，如，消除；缓解；减轻症状或使得损伤、病理或病状对患者而言更易忍受；减缓退化或衰退的速度；或使退化的最终点较少衰退；改善患者的身体或精神健康。症状的治疗或改善可以基于客观或主观参数；包含身体检查、神经精神病学检查和/或精神病学评估的结果。术语“治疗”及其缀合可以包含预防损伤、病理、病状或疾病。在实施例中，治疗是预防。在实施例中，治疗不包含预防。
如本文所使用的(并且在本领域中被充分理解的)，“治疗(treating或treatment)”还广泛地包含用于在受试者的病状中获得有益的或期望的结果(包含临床结果)的任何方法。有益的或期望的临床结果可以包含但不限于：减轻或改善一种或多种症状或病状、减轻疾病程度、稳定(即，不恶化)疾病状态、预防疾病传播或扩散、延迟或减缓疾病进展、改善或缓解疾病状态、减少疾病复发以及缓解(无论是部分的还是全部的，以及无论是可检测的还是不可检测的)。换句话说，如本文所使用的，“治疗”包含对疾病的任何治愈、改善或预防。治疗可以预防疾病发生；抑制疾病扩散；缓解疾病的症状、完全或部分去除疾病的根本原因、缩短疾病的持续时间或这些事物的组合。
如本文所使用的，“治疗(Treating和treatment)”包含预防性治疗。治疗方法包含向受试者施用治疗有效量的活性剂。施用步骤可以由单次施用组成，或者可以包含一系列施用。治疗期的长度取决于多种因素，如病状的严重程度、患者的年龄、活性剂的浓度、在治疗中所使用的组合物的活性或其组合。还应当理解，用于治疗或预防的药剂的有效剂量可以在特定治疗或预防方案的过程中增加或减少。通过本领域中已知的标准诊断测定，剂量的变化可以产生并且变得显而易见。在一些情况下，可能需要慢性施用。例如，以足以治疗患者的量向受试者施用组合物，且持续足够的持续时间。
如本文所使用的，术语“预防”是指减少患者的疾病症状的发生。如上所述，预防可以是完全的(没有可检测的症状)或部分的，使得观察到比不存在治疗时可能发生的症状更少的症状。
如本文所使用的，“患者”或“有需要的受试者”是指遭受或易于遭受可以通过施用如本文所提供的药物组合物进行治疗的疾病或病状的活生物体。非限制性实例包含人、其它哺乳动物、牛科动物、大鼠、小鼠、狗、猴、山羊、绵羊、牛、鹿和其它非哺乳动物。在一些实施例中，患者是人。
术语“联合给药”是指将本发明的融合蛋白或疫苗与已知药物(或其它化合物，或其它疫苗)进行“联合用药”，从而二者都具有治疗或诊断效果。这种联合用药可以包括相对于施用本发明的融合蛋白或疫苗而言对该药物(或其它化合物，或其它疫苗)进行并行(即同时)、在前或相继给药。本领域一般技术人员将能够很容易判断特定药物(或其它化合物，或其它疫苗)以及本发明的联合物的合适的给药时间、顺序和剂量。
如本文所使用的，术语“有效量”是足以实现所陈述目的的量(例如实现它被施用来达成的作用，治疗疾病，降低酶活性，增加酶活性，降低蛋白质功能，减轻疾病或病状的一种或多种症状)。“有效量”的实例是足以促成治疗、预防或减少疾病的一种或多种症状的量，所述量也可以被称为“治疗有效量”。一种或多种症状的“减少”意指降低一种或多种症状的严重程度或频率，或消除一种或多种症状。药物的“预防有效量”是当施用于受试者时，将具有预期的预防效果的药物的量，例如预防或延迟损伤、疾病、病理或病状的发作(或复发)或降低损伤、疾病、病理或病状或其症状发作(或复发)的可能性。完全预防效果不一定通过施用一次剂量发生，并且可以在仅施用一系列剂量之后发生。因此，预防有效量可以以一次或多次施用的形式施用。
如本文所使用的，术语“治疗有效量”是指如上述所描述的足以改善病症的治疗剂的量。例如，对于给定参数，治疗有效量将显示增加或降低至少5％、10％、15％、20％、25％、40％、50％、60％、75％、80％、90％或至少100％。治疗功效也可以表示为“倍数”增加或减少。例如，治疗有效量可以相对于对照具有至少1.2倍、1.5倍、2倍、5倍或更多的效果。
剂量可以根据患者的需要和所采用的融合蛋白或疫苗而变化。在本发明的上下文中，向患者施用的剂 量应足以随时间推移而在患者体内产生有益治疗反应。剂量的大小也将通过任何不良副作用的存在、性质和程度确定。针对特定情况来确定适当的剂量是在执业者的技能之内的。通常，治疗开始于比融合蛋白或疫苗的最优剂量小的较小剂量。其后，剂量以小增量增加直到达到在这些情况下的最优效果。可以单独调整给药的量和间隔，以提供所施用融合蛋白或疫苗的对所治疗的特定临床适应症有效的水平。这将提供与个体疾病状态的严重程度相称的治疗方案。
如本文所使用的，术语“施用”意指向受试者口服施用、以栓剂形式施用、局部接触、静脉内、肠胃外、腹膜内、肌肉内、病灶内、鞘内、鼻内或皮下施用，或植入缓慢释放装置(例如，微型渗透泵)。通过任何途径进行施用，包含肠胃外和经粘膜(例如，颊、舌下、腭、牙龈、鼻、阴道、直肠或经皮)。肠胃外施用包含例如静脉内、肌肉内、动脉内、皮内、皮下、腹膜内、心室内和颅内施用。其它递送模式包含但不限于使用脂质体调配物、静脉内输注、经皮贴剂等。在实施例中，施用不包含施用除了所叙述的活性剂之外的任何活性剂。
本发明具有如下的有益效果：
1、本发明的疫苗中使用人IgG4Fc突变体，减少单分子形成的同时减少了ADCC效应、克服生产过程中出现末端不均一的问题，保证了本发明疫苗更安全和稳定；
2、VZV-gE与人IgG4Fc连接形成二聚体有利于促进表达和纯化，同时抗原提呈给DC细胞提高免疫反应水平，此外gE形成二聚体增加了分子量有利于增加gE的免疫原性；
3、本发明使用VZV gE的膜外区与人IgG4Fc片段融合，在哺乳类动物细胞(CHO)获得了高效表达的分泌型的gE-Fc融合蛋白，表达量达到3g/L；
4、本发明的CHO细胞表达的重组VZV-gE-IgG4Fc融合蛋白质为糖基化蛋白，保持了天然gE蛋白质的空间结构，免疫原性好，细胞免疫检测IL-2和IFN-γ均有作用；且该融合蛋白与双佐剂组合使用免疫原性显著增加；
5、本发明CpG佐剂在细胞免疫方面发挥了重要作用，同时，本发明CpG佐剂与铝佐剂联用在激发细胞免疫方面具有显著提高作用。
6、本发明的疫苗使用常规的铝佐剂和CpG佐剂联合，价格低廉容易获得，克服了GSK研制的Shingrix疫苗配方复杂，抗原需要冻干并且需要和佐剂分开，以及佐剂原材料稀缺，价格昂贵等缺陷。同时，由于GSK研制的Shingrix(GSK-shingrix)抗原需要冻干并且需要和佐剂分开，导致在制备抗原、制剂工艺以及后续的使用上均较为麻烦。本发明的抗原和佐剂可以直接混合，预灌充注射器分装制剂，非常便利。
7.利用设计出的4条不同CpG ODN序列对小鼠脾细胞和人PBMC进行体外刺激，结果表明4条CpG-ODN序列对小鼠脾细胞和人PBMC体外刺激A值均高于阴性对照组，其中CpG-cjx1与阳性对照Dynavax公司的ISS1018(CpG-1018)刺激免疫效果相当，对小鼠脾细胞和人PBMC均显示出较好的免疫刺激作用。
8.在进一步的方案中通过免疫Balb/c小鼠，分离脾淋巴细胞，测定CD4+/CD8+T细胞表达IFN-γ、TNF-α、IL-2、IL-4水平，结果表明本发明的CpG组和与铝佐剂联用组的细胞免疫效果明显高于比不加佐剂单抗原组或者单一铝佐剂组。可见，本发明所筛选的CpG佐剂在细胞免疫方面发挥了重要作用。同时，本发明的佐剂与铝佐剂联用在激发细胞免疫方面具有显著提高作用。
9.采用ELISA方法和细胞病变抑制法测定4个组的重组新型冠状病毒融合蛋白疫苗分别免疫Balb/c小鼠2次后14d血清结合抗体和中和抗体效价结果表明加入CpG佐剂的疫苗组均优于不加CpG佐剂的疫苗组，并且双佐剂组比其他组中和抗体效价高至少10倍。可见，铝佐剂和CpG佐剂联用在激发机体体液免疫和细胞免疫方面具有协同作用。
10.本发明的CpG作为佐剂在疫苗的使用中，其不同使用剂量的效果差异不大。其中，在小鼠动物模型中，10μg/剂以上的CpG均能取得较好的免疫效果。
附图说明
图1为VZV-gE-Fc纯化结果。
图2为不同铝盐佐剂配制的重组VZV-gE-Fc疫苗免疫小鼠的免疫效果对比。
图3为不同剂量氢氧化铝佐剂的免疫效果对比。
图4为CpG ODN体外刺激小鼠脾细胞通过MTT法的检测结果。
图5为CpG ODN体外刺激人PBMC通过MTT法的检测结果。
图6为不同种类CpG佐剂与铝佐剂配合使用的免疫效果对比。
图7为不同剂量CpG佐剂与铝佐剂配合使用的免疫效果对比。
图8为不同剂量抗原的双佐剂疫苗的免疫效果对比。
图9为不同疫苗组分免疫效果对比(ELISA)。
图10为双佐剂疫苗免疫后ELISPOT细胞免疫效果评价，其中，图10a为IFN-γ的检测结果，图10b为IL-2的检测结果。
图11为双佐剂VZV-gE-Fc重组带状疱疹疫苗(VZV-Genevax)和葛兰素史克欣安立适Shingrix(GSK-Shingrix)免疫小鼠免疫效果对比，其中，图11a为ELISA测定的一免和二免后的结合抗体效价，图11b为ELISPOT细胞免疫IFN-γ的检测结果。
图12为双佐剂VZV-gE-Fc重组带状疱疹疫苗(VZV-Genevax)和葛兰素史克欣安立适Shingrix(GSK-Shingrix)免疫食蟹猴免疫效果对比，其中，图12a为ELISA测定的一免和二免后的结合抗体效价，图12b为流式细胞术试验测定CD4/CD8T细胞表达IFN-γ、IL-2、TNFa(Th1)和IL-4(Th2)细胞因子水平，图12c为ELISPOT细胞免疫IFN-γ的检测结果。
具体实施方式
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。
下述实施例中的C57BL/6雌性小鼠均购自斯贝福(北京)生物技术有限公司。
实施例1、融合蛋白VZV-gE-Fc的制备
本实施例的重组带状疱疹亚单位疫苗抗原靶点选择VZV-gE(SEQ ID NO.1)。在抗原结构设计时，加入了人IgG4Fc片段(SEQ ID NO.2)，将VZV-gE蛋白和人IgG Fc片段通过连接肽融合，得到VZV-gE-Fc融合蛋白，该融合蛋白的氨基酸序列如SEQ ID NO.3所示。两条相同的VZV-gE-Fc融合蛋白通过二硫键连接成同源二聚体。
设计偏向CHO-K1细胞的高表达的VZV-gE-Fc融合蛋白编码基因的序列，其核苷酸序列如SEQ ID NO.4所示。
1、重组载体的制备
通过金斯瑞生物技术有限公司合成基因片段VZV-gE-Fc(SEQ ID NO.4)并两端添加HindIII、Not I酶切位点，HindIII、Not I酶切合成基因片段VZV-gE-Fc和PKS001空载体(购自中山康晟生物有限公司，货号A13201)，酶切后回收目的条带和载体大片段，将两者连接，转化至大肠杆菌top10感受态细胞，转化菌用含有氨苄的LB液体筛选培养基复壮后扩大培养，37℃振荡培养过夜后用无内毒素质粒大提试剂盒(增强型)(天根生化科技(北京)有限公司，货号DP120-01)提取质粒，经测序鉴定无误，成功构建的重组表达载体命名为PKS001gEFc。
2、融合蛋白VZV-gE-Fc的表达
将含有VZV-gE-Fc基因的重组表达载体PKS001gEFc，采用电转方法转染CHO-K1(中山康晟生物有限公司，货号A14101)细胞，转染后CHO-K1细胞在培养基CD04(中山康晟生物有限公司,货号A11004)中培养。转染48小时后，用含有25μM蛋氨酸亚砜亚胺(Methionine Sulphoximine，简称：MSX sigma-Aldrich公司产品，货号：M5379)的培养基CD04(即选择培养基)置换转染后的CHO-K1细胞，按照每孔10000个细胞/200μl接种至96孔细胞培养板中，放置于37℃细胞培养箱中静置培养2-3周，在光学显微镜下查看并标记单克隆细胞的培养孔，当细胞长满至超过培养孔面积的三分之二时，通ELISA筛选出15株表达较高的细胞株，这15株单克隆细胞通过摇瓶补料培养后收获的上清经Protein A亲和层析一步纯化后，进行理化分析(采用SEC检测分析目的VZV-gE-Fc蛋白聚集体、二聚体及小分子碎片的含量)基于产量和 质量最终确定三株细胞1E11、3B7和2D10作为后续开发的候选细胞株。
根据细胞的连续传代稳定性研究、生长状态、倍增时间及表达量，最终确定将3B7细胞株作为生产用细胞株(采用细胞目的基因测序分析，未见碱基插入、缺失、突变，与目的基因全长序列进行DNA序列比对，确认在细胞中目的基因DNA序列完全正确)。
复苏3B7细胞至摇瓶中培养，在第3、5、7、9、11天，给细胞添加补料和葡萄糖，第6天开始细胞培养温度降至33℃，当细胞活率降至50％左右，12000r/min高速离心30min时间去除细胞及细胞碎片，收集细胞培养物上清液。培养上清液经过检测最终的产物表达量达到3g/L以上。
将上述3B7细胞的培养物上清液经0.45μm滤膜过滤后采用Protein A凝胶色谱柱亲和纯化，低pH病毒灭活后采用疏水层析、然后再用DEAE Sepharose 4Fast Flow用不同浓度的NaCl溶液进行梯度洗脱，得到目标蛋白溶液。
目标蛋白溶液经过SDS-PAGE电泳检测，结果如图1所示，可以看出，得到大小为110KD左右的目标蛋白，其大小表明VZV-gE-Fc融合蛋白形成了同源二聚体，目标蛋白为VZV-gE-Fc融合蛋白二聚体。
进一步WB检测和质谱测序确定得到了目标蛋白VZV-gE-Fc，经SEC检测分析目标蛋白的纯度达到97％以上。
实施例2、疫苗组的筛选及免疫原性研究
一、疫苗的制备
以实施例1纯化得到的VZV-gE-Fc融合蛋白二聚体为免疫原，具体为将实施例1纯化得到的VZV-gE-Fc融合蛋白二聚体用含有20mM组氨酸盐酸、140mM精氨酸盐酸盐和0.02％(体积百分含量)聚山梨酯80的稀释缓冲溶液(该溶液的溶剂为水，溶质为对应浓度的组氨酸盐酸、精氨酸盐酸盐和聚山梨酯80，pH值为6.0)稀释，得到0.5mg/ml稀释抗原。
1、铝佐剂疫苗制备
室温条件下将0.5mg/ml的稀释抗原与氢氧化铝铝佐剂混悬液或磷酸铝佐剂混悬液(其中铝含量10mg/ml，溶质为氢氧化铝或磷酸铝，溶剂为PBS；购自长春生物制品研究所)按照不同体积置于加有转子的玻璃瓶中150r/min混合均匀，得到不同铝盐佐剂疫苗，其中抗原与佐剂的质量比如表1或表2所示。
2、CpG佐剂疫苗制备
室温条件下将0.5mg/ml的稀释抗原和10mg/ml表3所示各个CpG的溶液(溶剂为生理盐水，溶质为CpG)按照不同体积置于加有转子的玻璃瓶中低速混合均匀，得到不同CpG佐剂疫苗，其中抗原与佐剂的质量比如表7所示。
3、双佐剂疫苗制备
室温条件下将0.5mg/ml的稀释抗原、铝佐剂(氢氧化铝混悬液(其中铝含量10mg/ml)、10mg/ml浓度CpG的溶液按照不同体积置于加有转子的玻璃瓶中低速混合均匀，得到不同双佐剂疫苗，其中抗原与佐剂的质量比如表4或表5或表6或表7所示。
将上述制备的疫苗，无菌条件下分装入2ml西林瓶内(或预填充玻璃注射器内)，每瓶0.5ml(或1.0ml)，密封后放置于2～8℃避光保存。
二、免疫
取出上述一制备的疫苗，以C57BL/6小鼠(购自斯贝福(北京)生物技术有限公司)为动物模型开展了免疫原性研究。
选择6-8周龄C57BL/6小鼠随机分组，每组5只小鼠，肌肉注射上述一制备的疫苗，并设置疫苗组、蛋白组和佐剂组(具体按照下述四中的各组设置)，在0、3周(以第一次免疫当天记作第0天，免疫当天起的一周，记作免疫的第1周)进行二次免疫(免疫剂量和方式如下述四中所示)，二次免疫间隔21天。
在免疫第3、5周采血，第5周取脾脏。
三、ELISA法检测血清中抗VZV-gE-Fc蛋白的抗体滴度(即总IgG)
采用ELISA法检测血清中的抗VZV-gE-Fc蛋白的抗体滴度(即总IgG)，结果显示本发明的VZV gE-人IgG Fc蛋白(VZV-gE-Fc)所制备得到的疫苗组合免疫原性非常好，可作为潜在的重组带状疱疹候选疫苗抗原，具体操作如下：
I、ELISA试剂配制
ELISA包被液(1X)配制：取ELISA包被液(10X)(购自索莱宝，货号：C1055)用无菌蒸馏水稀释至1X。
PBS配制：取出PBS powder(购自索莱宝，货号：P1003)，每袋用2L无菌蒸馏水溶解。
洗液：为PBST(含0.05％Tween-20的PBS)，按照如下制备：量取1L过滤后的PBS到蓝盖瓶中，加入500μLTween-20，充分混匀，存于2-8℃备用。根据实际情况，按此方法配制所需体积的PBST。注：PBST现用现配，当天使用。
封闭液或样品稀释液(含5％脱脂奶粉的PBS)：称取PBS体积的5％的脱脂奶粉(奶粉质量/g＝PBS体积/mLx5％)，将从2-8℃取出的PBS平衡至室温，量取所需体积的PBS溶液至已加入奶粉的离心管中，然后充分溶解，备用。注：封闭液和样品稀释液现用现配，当天使用。
稀释羊抗鼠二抗：将规格为1mL的二抗(购自CST，货号7076s)平衡至室温，然后进行分装，放置在-20℃±5℃保存。每次使用之前取出，按1:4000进行稀释，此处稀释液为PBS。注：稀释后的二抗，当天使用。
TMB显色液1购自湖州英创生物科技有限公司，货号TMB-S-001。
终止液，购自索莱宝，货号C1058。
II、ELISA检测血清结合抗体滴度：
1)包被：取VZV-gE-his抗原(SEQ ID NO.1所示的VZV-gE的C末端添加6个his得到的蛋白，北京吉诺卫制品有限公司制备，浓度为2mg/ml)用ELISA包被液(1X)稀释到1000ng/mL，包被酶标版，100μl/孔，4℃放置过夜。
2)封闭：将包被板从4℃取出后，洗板3次，每次洗液体积为300μl/孔，洗完后若孔内残留洗液，则要在吸水纸上拍干；然后在已包被孔中加入预先配制的封闭液，300μl/孔，盖封板膜，37℃，90min。
3)血清稀释：在离心管内用样品稀释液将待检血清稀释至合适浓度，得到待检样品。
4)加样：将封闭后的包被板洗板3次，每次洗液体积为300μl/孔，洗完后若孔内残留洗液，则要在吸水纸上拍干。将已稀释好的各个浓度的待检样品依次加入至样品孔中，100μl/孔；加入100μl样品稀释液作为空白对照(Blk)，设置5个复孔，盖上封板膜，37℃孵育60min。
5)加二抗：弃去样品，洗板3次，每次洗液体积为300μl/孔，洗完后若孔内残留洗液，则要在吸水纸上拍干；加入稀释羊抗鼠二抗，100μl/孔，盖封板膜，37℃孵育60min。
6)显色：将96孔板洗板3次，每次洗液体积为300μl/孔，洗完后若孔内残留洗液，则要在吸水纸上拍干，加单组分TMB显色液1(提前从4℃取出，平衡至室温)，100μl/孔，25℃避光显色，15min。
7)终止：显色后立即加入终止液终止反应，50μl/孔，轻晃混匀。
8)检测：将酶标板放入酶标仪，在450nm波长下测吸光度值。
9)判定：大于阴性小鼠OD值的2.1倍判为阳性。
注：以下检测结合抗体效价的ELISA方法都同上。
四、ELISA检测结果
A：筛选最佳的铝佐剂
取6-8周龄C57BL/6雌性小鼠15只，随机分为3组，按表1设计的免疫方案免疫动物，通过检测1、2、3组二免14d(以第二次免疫当天记作二免第0天)ELISA效价，筛选到最佳铝佐剂。
表1为不同铝盐佐剂配制的重组VZV-gE-Fc疫苗免疫小鼠的免疫方案
上表中的组1为肌肉注射PBS；
上表中的组2和组3分别为肌肉注射不同铝佐剂疫苗。
结果见图2，可以看出，不同铝佐剂疫苗中的两种常用的铝佐剂使用无显著性差异，采用AL(OH)3的铝佐剂疫苗在二免时IgG滴度略高。PBS免疫效果过低不在图中显示。
B：筛选最佳的铝佐剂剂量
取6-8周龄C57BL/6雌性小鼠25只，随机分为5组，按表2设计的免疫方案免疫动物，通过检测4、5、6、7、8、9组一免21d(以第一次免疫当天记作一免第0天)和二免14d(以第二次免疫当天记作二免第0天)结合抗体效价，筛选到铝佐剂最佳剂量。
表2为筛选最佳的铝佐剂剂量的免疫方案
上表中的组4为肌肉注射PBS；
上表中的组5-组9分别为肌肉注射不同剂量的铝佐剂疫苗。
结果如图3所示，含有10μg-200μg铝的铝佐剂疫苗的免疫效果没有无显著性差异。PBS免疫效果过低不在图中显示。
C：筛选最佳的CpG佐剂
1、CpG ODN序列的设计与合成
根据CpG ODN序列设计原则，共设计合成4条候选CpG ODN序列(如表3所示)。将4条候选CpG ODN序列合成后进行全链硫代修饰(用*表示)，合成后利用常规μlTRA-PAGE方法进行纯化，溶于生理盐水，-20℃冰箱中保存备用。其中序列编号1018为阳性对照，为Dynavax公司ISS1018序列(CpG1018)。
表3为CpG ODN序列
2、不同CpG ODN序列对小鼠脾细胞免疫刺激活性比较
主要实验试剂：RPMI 1640培养液(Sigma产品，货号：R8758)含L－谷氨酰胺、10％小牛血清、青霉素、链霉素和β－巯基乙醇，4℃储存备用；MTT(Sigma产品，货号：11465007001)：用PBS配成质量浓度为5mg/mL的液体，过滤除菌，4℃储存备用；淋巴细胞分离液购自达科为生物技术有限公司。
实验动物：4～6周龄雌性Balb/c小鼠，购自北京斯贝福(北京)生物技术有限公司。
实验方法：
(1)拉颈处死小鼠，使用75％酒精对其表面进行消毒，无菌条件下用剪刀剪开小鼠左侧中上部皮毛，裸露出皮下缔结组织，剪开组织使脾脏暴露，尽量去除脂肪等不必要成分，取得的完好的脾依次放入装有2mL培养基的六孔板孔内，盖子上做好标记；
(2)5mL注射器手柄在纱网包裹的单只脾脏上研磨，无肉眼可见的组织残留时，将悬液转移到15mL离心管中，并用2mL培养基冲洗六孔板底，一并转移到离心管中，对每个脾都进行如上操作；
(3)在35mm培养皿中放入4mL小鼠淋巴分离液(取用前恢复至室温并摇匀)，研磨；
(4)把悬有脾脏细胞的分离液立即转移到15mL离心管中，覆盖500μL-1000μL的RPMI 1640培养基(保持液面分界明显)；
(5)室温，水平转子800g离心力离心30min，离心结束后细胞分层；
(6)洗出淋巴细胞层，再加入10mL RPMI 1640培养基，颠倒洗涤。室温，250g离心10min收集细 胞；
(7)倾倒上清液，用培养液重悬细胞，计数。接种于24孔板，5×105个脾细胞/孔。
将表3所示的合成的CpG ODN序列用去离子水稀释后以20μg/mL的终浓度加入上述7)获得的脾细胞中作为刺激物，于37℃、5％CO2培养72h后以MTT法检测CpG ODN诱导小鼠脾细胞增殖水平(检测各组A值)，对CpG ODN佐剂的免疫调节活性进行筛选分析。以加入PBS作为对照(control)。
结果如图4所示，显示4条硫代序列中，各CpG ODN的A值均高于免疫PBS的脾细胞阴性对照组(control)，其中CpG-cjx1的A值达到0.511，与阳性对照ISS1018序列(图中记作1018s)A值(0.521)接近，显示出较好的针对小鼠脾细胞的免疫刺激作用。
3、不同CpG ODN序列对人PBMC免疫刺激活性比较
人PBMC中含有丰富的T、B淋巴细胞、单核细胞和树突状细胞，这些免疫细胞是人体应对外来病原体感染的主要功能细胞。
以Ficoll分离液将正常健康人PBMC从新鲜全血中分离，悬于含10％FBS的1640培养基中，接种于24孔板，5×105个细胞/孔。将表3所示的合成的4条CpG ODN序列用去离子水稀释后分别以20μg/mL的终浓度加入PBMC细胞作为刺激物，每个序列设3个复孔。于37℃、5％CO2培养72h后以MTT法检测CpG ODN诱导PBMC细胞增殖水平(检测各组A值)，对自行设计的CpG ODN佐剂的免疫调节活性进行筛选分析。以加入PBS作为对照(control)。
结果如图5所示，4条硫代序列中，各CpG ODN的A值均高于空白PBMC细胞阴性对照组(control)，其中CpG-cjx1的A值接近0.401，与阳性对照ISS1018序列(图中记作1018s)A值(0.393)接近，显示出较好的针对人PBMC的免疫刺激作用。
上述结果表明，利用设计出的4条不同CpG ODN序列对小鼠脾细胞和人PBMC进行体外刺激，结果表明4条CpG ODN序列对小鼠脾细胞和人PBMC体外刺激A值均高于阴性对照组，其中CpG-cjx1与阳性对照Dynavax公司的ISS1018刺激免疫效果相当，对小鼠脾细胞和人PBMC均显示出较好的免疫刺激作用，CpG-cjx1作为候选佐剂。
D：筛选最佳的双佐剂
取6-8周龄C57BL/6雌性小鼠25只，随机分为5组，按照如下表4的方式免疫C57BL/6雌性小鼠。通过检测10-14组一免21d(以第一次免疫当天记作一免0天)和二免14d(以第二次免疫当天记作二免0天)结合抗体效价，筛选到最佳的CpG佐剂。
采用A和B筛选出最佳铝佐剂AL(OH)3及其剂量与上述C筛选的候选CpG佐剂作为双佐剂，其中，CpG佐剂为CpG-cjx1，Dynavax公司ISS1018序列(CpG1018)和CpG7909(SEQ ID NO.10:5’-TCG TCG TTT TGT CGT TTT GTC GTT-3’，生工合成)。
表4为筛选最佳的CpG佐剂的免疫方案
上表中的组10和组14均为肌肉注射PBS；
上表中的组11-组13分别为肌肉注射不同双佐剂疫苗。
结果如图6所示，双佐剂均有良好的免疫效果，使用AL(OH)3配合CpG-cjx1免疫效果最佳。
D：筛选双佐剂中最佳的CpG佐剂剂量
取6-8周龄C57BL/6雌性小鼠25只，随机分为5组，按表5设计的免疫方案免疫动物，通过检测15、16、17、18、19组一免21d(以第一次免疫当天记作一免0天)和二免14d(以第二次免疫当天记作二免 0天)结合抗体效价，筛选到CpG佐剂最佳剂量。
表5为筛选到最佳的CpG佐剂剂量的免疫方案
上表中的组15为肌肉注射PBS；
上表中的组16-组19分别为肌肉注射不同双佐剂疫苗。
结果如图7所示，双佐剂中CpG-cjx1佐剂的剂量在1μg-10μg范围内无显著性差异。
E：探索最佳的抗原剂量
取6-8周龄C57BL/6雌性小鼠25只，随机分为5组，按表6设计的免疫方案免疫动物，通过检测20、21、22、23、23、24、25组一免21d和二免14d结合抗体效价，筛选到最佳抗原剂量。
表6为小鼠上探索最佳的抗原剂量的免疫方案
上表中的组20为肌肉注射PBS；
上表中的组21-组25分别为肌肉注射不同剂量的双佐剂疫苗。
结果如图8所示，双佐剂中2.5-30μg的抗原剂量免疫效果均无显著性差异。
F：体液免疫和细胞免疫测定进一步筛选疫苗处方
取6-8周龄C57BL/6雌性小鼠25只，随机分为5组，按表7设计的免疫方案免疫动物，0,21d肌肉注射免疫两次，二免14d取脾，分离脾淋巴细胞，通过ELISPOT检测26、27、28、29、30组二免14d脾淋巴细胞IFN-γ、IL-2因子表达。
小鼠脾脏ELISPOT检测方法：
根据Murine IFN-γSingle-Color Enzymatic ELISPOTAssay(CTL货号mIFNgp-2M/2)试剂盒说明书对二免14d小鼠的脾淋巴细胞检测。具体步骤如下：
1、脾组织加入5ml小鼠淋巴细胞分离液，轻轻研磨，200目尼龙网过滤至洁净离心管中，在液面以上轻轻加入1mlPBS，以800g，30min，升速快降速慢离心，收集中间淋巴细胞层。
2、加入5ml PBS洗涤细胞，500g，离心5min，弃上清。
3、CTL-Test Medium(补充L-谷氨酰胺终浓度3mM)培养基重悬细胞，细胞计数(流式细胞仪计数)后调整细胞浓度为5×106/ml。
4、准备2倍终浓度的CTL-Test Medium：在26组、27组、28组、29组、30组、的样本管加入VZV-gE-his抗原(2倍终浓度10μg/ml)、肽库(2倍终浓度10μg/ml)。铺到对应的ELISPOT板中，另外预留阴性对照孔 (不加任何刺激物)和阳性对照孔(加入PMA和Ionomycin)。37℃，5％CO2孵育20min。
其中肽库购自诺唯赞生物科技有限公司，产品名称Varicella-Zoster Virus peptide pool，货号：DD9120。
5、加入细胞100μl/孔，相当于5×105/孔，37℃，5％CO2培养48h。
6、用200μl/孔PBS清洗板2次，再用200μl/孔0.05％Tween-PBS清洗板3次。
7、加入anti-murine IFN-γ检测液80μl/孔，室温孵育2h。
8、用200μl/孔0.05％Tween-PBS清洗板3次。
9、加入Tertiary solution 80μl/孔，室温孵育30min。
10、用200μl/孔0.05％Tween-PBS清洗板2次，再用200μl/孔纯水清洗板2次。
11、加入Blue Developer Solution 80μl/孔，室温孵育15min。
12、倒掉液体，用纯水冲洗孔板3次终止反应。
13、ELISPOT板读值。
同时通过检测26、27、28、29、30组一免21d和二免14d结合抗体效价以进一步筛选疫苗处方。
表7为细胞免疫方案设计
上表中的组26为肌肉注射PBS；
上表中的组27-组30分别为肌肉注射不同种类的疫苗。
结合抗体效价结果由图9所示，双佐剂疫苗(组30)结合抗体效价优于单佐剂疫苗组(组28和29)和单抗原疫苗组(组27)。
IFN-γ和IL-2的检测结果由图10a、图10b所示，双佐剂疫苗组刺激极显著优于单佐剂组和不加佐剂的抗原组。
上述结果表明，采用双佐剂疫苗组免疫效果最好，且每100μl双佐剂疫苗制剂中，抗原含量2.5-30μg，氢氧化铝佐剂10-200μg，CpG佐剂1-10μg均较佳，具体举例可以抗原含量5μg，氢氧化铝佐剂50μg，CpG佐剂2μg。
实施例3、双佐剂VZV-gE-Fc重组带状疱疹疫苗(VZV-Genevax)和GSK研制的Shingrix(VZV-GSK)免疫效果对比
将本发明的双佐剂VZV-gE-Fc重组带状疱疹疫苗(命名为VZV-Genevax)和葛兰素史克欣安立适，即GSK研制的Shingrix(VZV-GSK)免疫效果对比。
针对带状疱疹，国内尚无带状疱疹疫苗上市，国外默克公司的ZOSTAVAX和GSK公司的Shingrix两种带状疱疹疫苗获准上市使用。其中，GSK研制的Shingrix是目前带状疱疹疫苗获准上市使用的疫苗中效果最好的，鉴于Shingrix优秀的预防效果，美国CDC推荐Shingrix取代默沙东带状疱疹疫苗Zostavax。然而，GSK的VZV疫苗Shingrix使用的佐剂是AS01B，其中QS21成分限量供应导致Shingrix每剂价格达到150-200美元。这个疫苗使用的时候需将抗原重组gE抗原(CHO细胞表达)单独冻干包装，然后将AS01B单独包装，注射人体前需要将佐剂与抗原混合。通过对比实验，发现本发明的疫苗一免后起效更快，二免检测比GSK研制的Shingrix血清抗体效价也有所提高。同过Elispot试验IFN-r因子的结果显示,本发明的疫苗细胞免疫优于GSK研制的Shingrix。
具体实验和结果如下：
一、双佐剂VZV-gE-Fc重组带状疱疹疫苗的制备
人用双佐剂VZV-gE-Fc重组带状疱疹疫苗(VZV-Genevax)每0.5ml按照如下方法制备：室温条件下将1.5ml 0.5mg/ml浓度稀释抗原VZV-gE-Fc、0.5ml铝佐剂(氢氧化铝混悬液(其中铝含量10mg/ml，购自长春生物制品研究所)、50μl 10mg/ml浓度CpG-cjx1溶液混合均匀，得到双佐剂疫苗，其中抗原VZV-gE-Fc的含量为75μg/500μl、AL(OH)3的含量为500μg/500μl和CpG-cjx1的含量为50μg/500μl。
VZV-Genevax和GSK研制的Shingrix在小鼠上的免疫剂量均为人用剂量1/10，即免疫50μl/只。
免疫小鼠的50μl VZV-gE-Fc重组带状疱疹疫苗(VZV-Genevax)疫苗里包含7.5μgVZV-gE-Fc，50μgAL(OH)3和5μgCpG-cjx1，制备方法与上述人用双佐剂VZV-gE-Fc相同。
上述双佐剂VZV-gE-Fc疫苗中，抗原和铝佐剂和CpG-cjx1佐剂质量比为7.5:50:5。
二、小鼠免疫
取6-8周龄C57BL/6雌性小鼠18只，随机分为3组，每组6只。按表8设计的免疫方案免疫动物，用ELISA方法和检测一免21d和二免14d的VZV特异性IgG结合抗体效价；通过ELISPOT检测31、32、33组二免14d脾淋巴细胞IFN-γ因子表达(方法同前)。
表8为双佐剂VZV-gE-Fc重组带状疱疹疫苗和GSK研制的shingrix小鼠免疫方案
ELISA效价结果见图11a所示，ELISPOT结果见图11b所示。结果表明，本发明的VZV-Genevax免疫效果优于GSK-Shingrix。
三、免疫食蟹猴
表9为双佐剂VZV-gE-Fc重组带状疱疹疫苗和GSK研制的shingrix食蟹猴免疫方案
取15只健康食蟹猴，雌雄随机，2-7岁，购买并试验与广西南宁灵康赛诺科生物科技有限公司。动物合格证编号：N0.45002000000032.随机分为3组，每组5只。试验分组见表9：
疫苗试验组(VZV-Genevax)：人用双佐剂VZV-gE-Fc重组带状疱疹疫苗(75μg VZV-gE-Fc+0.5mgAL(OH)3+0.05mgCpG-cjx1/0.5mL)，0.5mL/剂/只，免疫5只；
对照疫苗试验组(GSK-shingrix)：50μgVZV-gE-Fc+ASO1B/0.5mL，0.5mL/剂/只，免疫5只；
PBS对照组:0.5mgAL(OH)3+0.05mgCpG-cjx1/0.5mL(与人用双佐剂VZV-gE-Fc重组带状疱疹疫苗相比，仅不添加抗原),0.5mL/剂/只，免疫5只。
在第0天(第一次免疫当天记作第0天)和第28天(第一次免疫当天记作第0天)免疫两次，用ELISA方法检测一免21d和二免14d的VZV特异性IgG结合抗体效价；末次免疫后14d取5mL后肢静脉血，送 至南宁维尔凯生物科技有限公司，分离淋巴细胞，通过流式细胞术试验测定CD4/CD8T细胞表达IFN-γ、IL-2、TNFa(Th1)和IL-4(Th2)细胞因子水平。通过ELISPOT检测34、35、36组二免14d外周血淋巴细胞IFN-γ因子表达。ELISPOT检测试剂盒(货号：MBF3421M-4AST-2，品牌：Mabtech)，购自北京元唐盛兴科技有限公司。
上述流式细胞检测食蟹猴Th细胞实验方法如下：
1、实验试剂
表10为实验试剂
2、实验步骤
5ml血液样本和等体积生理盐水混合，倾斜45°用滴管缓缓将稀释后的样本加入到等体积淋巴细胞分离液(根据样本种属来选择合适试剂)的15ml离心管中，要求两层分明。
上升速度2，下降速度2，离心力为1000g离心30min。
用滴管将PBMC层吸至另一15ml离心管中，加入11mL 1640培养基混匀，1200rpm离心5min，弃上清。
加入5mL 1640培养基清洗一次，1200rpm离心5min，弃上清。
用无血清培养基重悬细胞，计数，将细胞密度调整为1*10e7个/mL。
设阴性孔、实验孔、阳性孔，均加入100μl细胞和FBA(阻断剂)，分别加入50μl无血清培养基、含Vzv-his(终浓度5μg/ml)和肽库(终浓度5μg/ml)的无血清培养基、含PHA的无血清培养基。37℃,5％CO2孵育过夜。
孵育结束后，收集细胞，离心弃上清。
细胞重悬于100μl含1‰Zombie Red的PBS，4℃孵育20min。
加入含1％FBS的1ml PBS，1200rpm离心5min，弃上清。
细胞重悬于100μl含1％FBS的PBS，加入CD3、CD4、CD8抗体，混匀，4℃孵育20min。
加入1ml PBS清洗一次，1200rpm离心5min，弃上清。
300μl PBS重悬，加入450ml Fix/Perm Buffer，混匀，室温避光孵育20min。
加入1ml Perm/Wash Buffer，350g离心5min，弃上清。
重复清洗一遍。
用100μl Perm/Wash Buffer重悬细胞，加入IFNg、IL-2、IL4抗体，混匀，4℃避光孵育45min。
每管加入1ml Perm/Wash Buffer，混匀，1200rpm离心5min，弃上清。
200μl PBS重悬，上机。
按表9设计的免疫方案免疫动物，用ELISA方法检测一免21d和二免14d的VZV特异性IgG结合抗体效价，结果见图12a；通过流式细胞术试验测定外周血淋巴细胞CD4/CD8T细胞表达IFN-γ、IL-2、TNFa(Th1)和IL-4(Th2)细胞因子水平，结果见图12b。通过ELISPOT检测二免14d外周血淋巴细胞IFN-γ因子表达，结果见图12c。
从上述图12a可以看出，上述食蟹猴免疫疫苗后的结果证明，本发明的疫苗(VZV-Genevax)一免后起效更快，二免检测比GSK研制的Shingrix(GSK-shingrix)血清抗体效价也有所提高。图12b显示本发明的疫苗(VZV-Genevax)比GSK研制的Shingrix(GSK-shingrix)外周血淋巴CD4/CD8T细胞表达IFN-γ、IL-2、TNFa明显升高，说明本发明的疫苗(VZV-Genevax)引发强烈的CD4+和CD8+T细胞反应，诱导Th1免疫应答，具有突出的疫苗激活细胞免疫反应优势；图12c中IFN-γ的检测显示，本发明的疫苗 (VZV-Genevax)比GSK研制的Shingrix(GSK-shingrix)刺激细胞分泌IFN-γ多，说明本发明的疫苗(VZV-Genevax)具有较高的细胞免疫水平，可有效发挥抗病毒和免疫调控的作用。
上述表明，本发明制备的双佐剂VZV-gE-Fc重组带状疱疹疫苗可以诱发机体的体液免疫和细胞免疫。
实施例4：CpG ODN作为新冠疫苗佐剂免疫小鼠体内效果验证
1.实验材料：
(1)试验动物：健康Balb/c小鼠，6～8周龄，18-20g，雌性，SPF级，购于斯贝福(北京)生物技术有限公司；动物合格证编号：NO.110324210104634878，接收日期：2021.9.5，饲养于北京麦迪理邦生物科技有限公司动物实验室。
(2)细胞和毒株：Vero-E6细胞系，SARS-CoV-2(hCoV-19/China/CAS-B001/2020,GISAID No.EPI_ISL_514256-7)，由中国科学院微生物研究所制备与提供。
(3)试剂：DMEM(Gibco，批号：8120287)，FBS(Gibco)，青链霉素(新赛美生物科技有限公司，批号2009021031)；96孔细胞培养板(Corning)由中国科学院微生物研究所提供；RBD-FC原液(北京昭衍生物技术有限公司，批号BDS201302)；氢氧化铝佐剂(长春生物制品研究所责任有限公司，批号2P18-003-202005)；本发明所设计的CpG-cjx1佐剂；稀释液(北京昭衍生物技术有限公司，批号DM210403)；重组SARS-CoV-2RBD-his蛋白(北京麦迪理邦生物科技有限公司研发中心，批号20200501)，规格2mg/1.0mL；Anti-Mouse,IgG,HRP-LinkedAntbody鼠二抗(cst公司，货号7076s)，规格1mL；PBS注射液(北京中生奥邦生物科技有限公司，批号F210908B)，规格500mL/瓶；小鼠淋巴细胞分离液(北京达科为生物技术有限公司)；FITC anti-mouse CD4、PerCP/Cyanine5.5anti-mouse CD8a、PE anti-mouse IFN-γ、rilliantViolet421TManti-mouse IL-2、APC anti-mouse TNF-a、PE/Cyanine7anti-mouse IL-4(biolegend公司)；固定液、破膜液(biolegend公司)；重组SARS-CoV-2RBD-his蛋白(原始株)(北京麦迪理邦生物科技有限公司，批号：20200501)，规格2mg/mL。
(4)仪器：生物安全柜(AIRTECH BHC-1604IIA/B3S)；CO2细胞培养箱(Thermo Fisher 370Series)；显微镜(OLYMPUS CKX31SF)，由中国科学院微生物研究所提供；微孔板检测仪(伯腾BioTek)；低速水平离心机，安徽中科中佳提供；流式细胞仪CytoFlex S，美国贝克曼公司。
2.实验方法：
(1)免疫4组6～8周、雌性Balb/c小鼠，方案见表11，共免疫4组(0.5mL/剂/只)：
1)25μg RBD-Fc；
2)25μg RBD-Fc+0.5mgAL(OH)3；
3)25μg RBD-Fc+10μg CpG-cjx1；
4)25μg RBD-Fc+0.5mgAL(OH)3+10μg CpG-cjx1。
于0d和21d腹腔注射免疫两次，28d取血，取脾，分离脾淋巴细胞，测定CD4+/CD8+T细胞表达IFN-γ、TNF-α、IL-2、IL-4水平。
(2)免疫4组6～8周、雌性Balb/c小鼠，方案见表12，共免疫5组(0.5mL/剂/只)：
1)25μg RBD-Fc；
2)25μg RBD-Fc+0.5mgAL(OH)3；
3)25μg RBD-Fc+10μg CpG-cjx1；
4)25μg RBD-Fc+0.5mgAL(OH)3+10μg CpG-cjx1；
5)PBS对照组。
于0d和21d腹腔注射免疫两次，35d尾静脉采血并分离血清。采用流式细胞术测定重组新型冠状病毒融合蛋白疫苗(CHO细胞)或重组新型冠状病毒融合蛋白疫苗制剂免疫小鼠的细胞免疫效果，具体方案见表11。采用ELISA方法和细胞病变抑制法分别检测免疫小鼠产生的结合抗体效价和中和抗体效价，其样本编号见表12，每份样本血清0.2mL左右冻存。具体采集Balb/c小鼠血清样本25份，其中，疫苗试验组血清样本20份，PBS对照组血清样本5份。
表11：测细胞免疫实验分组方案
表12：测体液免疫实验分组方案
(3)疫苗配制：
取RBD-Fc抗原、Al(OH)3、CpG-cjx1用稀释液稀释，按照表11和表12疫苗组分配置。配置好的疫苗分别在磁力搅拌器上搅拌1.5h，4℃环境下放置1～1.5h。
(4)流式细胞术细胞免疫测定方法：
a.脾组织加入5mL小鼠淋巴细胞分离液，轻轻研磨，200目尼龙网过滤至洁净离心管中，在液面以上轻轻加入1mLPBS，以800g、30min，升速快降速慢离心，收集中间淋巴细胞层；
b.加入5mLPBS洗涤细胞，500g，离心5min，弃上清；
c.RPMI 1640完全培养基重悬细胞，细胞计数后取2×106细胞至流式管中；
d.每管加入100nM RBD-his蛋白，PMA(50ng/mL)、Ionomycin(1μg/mL)、Monensin(2μM)，37℃，5％CO2孵育5h；
e.孵育结束后，收集细胞，离心弃上清，加入抗体CD4、CD8各2μL，室温避光孵育15min；
f.加入0.5mL细胞固定液，室温避光固定40min，加入2mL 1×细胞破膜液，离心弃上清；
g.再次加入2mL 1×细胞破膜液，离心弃上清；
h.加入IFN-γ、IL-2、IL-4、TNF-α抗体各2μL，室温避光孵育30min；
i.加入2mL细胞破膜液，500g，离心5min，弃上清；
j.用0.5mL cell stainingbuffer重悬细胞，上机检测。
(5)ELISA检测血清结合抗体滴度：
取RBD-his蛋白用碳酸盐缓冲液(CBS，pH 9.6)稀释到1000ng/mL，包被酶标版，100μL/孔，4℃放置12～18h，用含10％牛血清(FCS)的PBS封闭1～2h，处理好的酶标版即可用于检测。将小鼠血清用含10％FCS的PBS进行对倍稀释；37℃温箱中孵育45min；在脱色摇床上以200rmp速度用PBST进行3×3min洗涤；加入100μL/孔HRP标记的鼠二抗工作液，37℃温箱中继续孵育45min；再次洗涤(方法同上)；加入100μL/孔TMB单组分显色液，37℃温箱中孵育15min；最后加入终止液(2M H2SO4)50μL/孔；在酶标仪上读取OD值。测量波长450nm，参比波长620nm。
(6)细胞病变抑制法检测血清中和抗体滴度：
检测步骤如下：取Vero-E6细胞接种96孔细胞板，每孔2×105～4×105个细胞，培养基为含10％FBS的DMEM液体，培养条件为37℃、5％CO2、饱和湿度。采用对倍稀释方法用含2％FBS的DMEM稀释小鼠血清，各血清稀释度液体按100CCID50/200μL加入新冠病毒原始毒株，37℃放置1.0h。待细胞铺满80％左右时，弃培养基，用PBS洗涤3次；最后加入混合处理好的血清-病毒液，每个稀释度重复4孔，每孔加液200μL。细胞继续放37℃、5％CO2、饱和湿度下培养，每24h观察记录细胞病变，连续观察72h。
(7)结果判定和统计学：
观察并记录细胞病变情况，出现细胞病变的判定为没有保护，利用Reed-Muench氏法计算血清的中和效价(保护50％细胞不产生病变的血清的最高稀释度即为该血清的NT50)；采用Reed-Muench方法进行计算，结果采用均值加减标准差表示。
3.试验结果
(1)流式细胞术测定细胞免疫效果：
采用流式细胞术测定重组新型冠状病毒融合蛋白疫苗(CHO细胞)或重组新型冠状病毒融合蛋白疫苗制剂免疫小鼠的细胞免疫效果(结果见表13)。结果表明：第4组双佐剂疫苗组CD4IFN-γ、CD4TNF-α、CD8IFN-γ、CD8TNF-α明显高于单抗原组和单铝佐剂组，偏向Th1型细胞免疫。由此表明CpG-cjx1佐剂在细胞免疫方面发挥了重要作用，铝佐剂和CpG-cjx1佐剂联用在激发机体细胞免疫方面具有协同作用。
表13：重组新型冠状病毒融合蛋白疫苗(CHO细胞)免疫小鼠细胞因子检测实验结果

(2)ELISA方法和细胞病变抑制法检测血清结合抗体滴度结果：
采用ELISA方法和细胞病变抑制法测定1)25μg RBD-Fc；2)25μg RBD-Fc+0.5mgAL(OH)3；3)25μg RBD-Fc+10μg CpG-cjx1；4)25μg RBD-Fc+0.5mgAL(OH)3+10μg CpG-cjx1，4个组的重组新型冠状病毒融合蛋白疫苗分别免疫Balb/c小鼠2次后14d血清结合抗体和中和抗体效价结果见表14。由结果可见，第4组，双佐剂的重组新型冠状病毒融合蛋白疫苗免疫小鼠2次后14d血清均产生了较高的结合抗体和中和抗体效价，第4组(双佐剂组)比不加CpG的单铝佐剂组中和抗体效价高10倍左右。
表14：ELISA方法和细胞病变抑制法检测血清结合抗体滴度结果

4.结论
(1)通过免疫Balb/c小鼠，分离脾淋巴细胞，测定CD4+/CD8+T细胞表达IFN-γ、TNF-α、IL-2、IL-4水平。结果表明本发明的CpG-cjx1组(RBD-FC+CpG-cjx1)和CpG cjx1与铝佐剂联用组(RBD-FC+AL(OH)3+CpG-cjx1)的细胞免疫效果明显高于比不加佐剂单抗原组或者单一铝佐剂组，可见，本发明所筛选的CpG-cjx1佐剂在细胞免疫方面发挥了重要作用。同时，本发明的佐剂与铝佐剂联用在激发细胞免疫方面具有显著提高作用。
(2)采用ELISA方法和细胞病变抑制法测定4个组的重组新型冠状病毒融合蛋白疫苗分别免疫Balb/c小鼠2次后14d血清结合抗体和中和抗体效价，结果表明加入CpG-cjx1佐剂的疫苗组均优于不加CpG-cjx1佐剂的疫苗组，并且第4组(双佐剂组)比其他组中和抗体效价至少高10倍。可见，矿物盐佐剂和CpG-cjx1佐剂联用在激发机体体液免疫和细胞免疫方面具有协同作用。
实施例5：CpG ODN佐剂最佳剂量的确定实验
为了进一步确定该CpG佐剂的最佳剂量，对不同剂量的CpG-cjx1佐剂(5、10、30、50μg/0.5mL/剂)配置疫苗免疫小鼠进行最佳剂量的筛选，动物免疫方案见表15。
表15：在小鼠上确定CpG佐剂剂量的动物免疫方案
对各组小鼠免疫后的结果如下表16所示：
表16：不同剂量的CpG-cjx1佐剂配制重组RBD-Fc蛋白疫苗免疫小鼠的免疫效果

由上表的结论可知，本发明的CpG-cjx1作为佐剂在疫苗的使用中，其不同使用剂量之间效果差异不大。其中，在小鼠动物模型中，10μg/0.5mL/剂以上的CpG-cjx1均能取得较好的免疫效果。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (31)

  1. 一种融合蛋白，其特征在于，所述融合蛋白包括VZV-gE蛋白和人IgG4的Fc片段。
  2. 根据权利要求1所述的融合蛋白，其特征在于，所述VZV-gE与Fc片段直接或者间接连接，优选的，所述间接连接包含接头，更优选的，所述接头为柔性接头，进一步优选的，所述柔性接头选自SEQ ID NO.12-14任一序列所示。
  3. 根据权利要求1-2任一所述的融合蛋白，其特征在于，所述VZV-gE蛋白为如下任一种：
    (a1)SEQ ID NO.1所示的蛋白质；
    (a2)在(a1)所述蛋白质的N端或/和C端连接标签得到的融合蛋白；
    (a3)将(a1)-(a2)任一种经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质；
    (a4)与(a1)-(a2)任一种具有80％以上同一性且具有相同功能的蛋白质，或者，所述人IgG4 Fc蛋白为如下任一种：
    (b1)SEQ ID NO.2所示的蛋白质；
    (b2)在(b1)所述蛋白质的N端或/和C端连接标签得到的融合蛋白；
    (b3)将(b1)-(b2)任一种经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质；
    (b4)与(b1)-(b2)任一种具有80％以上同一性且具有相同功能的蛋白质；
    优选的，所述融合蛋白为如下任一种：
    (c1)SEQ ID NO.3所示的蛋白质；
    (c2)在(c1)所述蛋白质的N端或/和C端连接标签得到的融合蛋白；
    (c3)将(c1)-(c2)任一种经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质；
    (c4)与(c1)-(c2)任一种具有80％以上同一性且具有相同功能的蛋白质。
  4. 一种编码权利要求1-3所述任一融合蛋白的核酸，优选的，所述核酸是mRNA，更优选的，所述核酸包含与SEQ ID NO.4所示核苷酸序列具有80％或以上同一性的核苷酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的核苷酸列，更优选具有98％或99％以上同一性的核苷酸序列。
  5. 一种包含权利要求4所述的核酸的表达载体；
    优选的，所述表达载体选自原核表达载体或真核表达载体；
    更优选的，所述表达载体为真核表达载体；
    更优选的，所述真核表达载体为在293细胞或CHO细胞中表达的载体；
    更优选的，所述真核表达载体是腺病毒载体。
  6. 一种宿主细胞，其表达权利要求1-3任一项所述的融合蛋白，或包含权利要求4所述的核酸和/或包含权利要求5所述的表达载体；
    优选的，所述宿主细胞是原核细胞或真核细胞；
    优选的，所述原核细胞是细菌细胞；优选的，所述原核细胞是大肠杆菌细胞；
    优选的，所述真核细胞选自酵母细胞、昆虫细胞和哺乳动物细胞；优选的，所述哺乳动物细胞选自CHO、HEK293、SP2/0、BHK、C127等；更优选的，所述真核细胞为CHO细胞。
  7. 一种药物组合物，其包含权利要求1-3任一项所述的融合蛋白、权利要求4所述核酸、权利要求5所述表达载体和/或权利要求6所述宿主细胞，以及一种或多种药学上可接受载体、稀释剂或赋形剂。
  8. 一种疫苗，其包含权利要求1-3任一项所述的融合蛋白、权利要求4所述核酸、权利要求5所述表达载体和/或权利要求6所述宿主细胞，以及一种或多种佐剂；
    优选的，所述佐剂选自氢氧化铝、CpG、磷酸铝、皂苷例如Quil A、QS-21、GPI-0100、油包水型乳状液、水包油型乳状液、水包油包水型乳状液中的一种或多种；
    优选的，所述佐剂为氢氧化铝和CpG双佐剂；
    优选的，所述融合蛋白、铝佐剂和CpG佐剂的比例为1-8:5-240:1-20；更优选的，所述融合蛋白、铝佐剂和CpG佐剂的比例为1-4:20-60:1-10，优选1:40:2。
    进一步优选的，所述CpG佐剂为SEQ ID NO:5-9中任一条序列所示的核苷酸。
  9. 权利要求1-3任一项所述的融合蛋白、权利要求4所述核酸、权利要求5所述表达载体和/或权利要求6所述宿主细胞和/或权利要求7所述的药物组合物在制备治疗或预防与水痘带状疱疹病毒相关的疾病或病状的疫苗中的用途。
  10. 一种在有相应需要的受试者中诱导针对水痘带状疱疹病毒的免疫应答的方法，所述方法包括：向受试者施用权利要求18所述的疫苗；
    优选的，所述受试者是哺乳动物或鸟类；
    优选的，所述受试者是人类、牛、犬、猫、山羊、绵羊、猪、马、火鸡、鸭或鸡。
  11. 一种带状疱疹疫苗，其包括抗原和佐剂；
    所述佐剂为铝佐剂、CpG佐剂或二者的结合；
    所述CpG佐剂为SEQ ID NO:5-9中任一条序列所示的核苷酸；
    所述带状疱疹疫苗为带状疱疹的灭活疫苗、减毒活疫苗、病毒载体疫苗、亚单位疫苗、重组疫苗、多糖疫苗、结合疫苗、类毒素疫苗或核酸疫苗。
  12. 根据权利要求11所述的带状疱疹疫苗，其特征在于：
    所述带状疱疹疫苗为亚单位疫苗，其抗原为融合蛋白；
    所述融合蛋白包括VZV-gE蛋白和人IgG4 Fc；
    所述VZV-gE蛋白为如下任一种：
    (a1)SEQ ID NO.1所示的蛋白质；
    (a2)在(a1)所述蛋白质的N端或/和C端连接标签得到的融合蛋白；
    (a3)将(a1)-(a2)任一种经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质；
    (a4)与(a1)-(a2)任一种具有98％以上同一性且具有相同功能的蛋白质；
    所述人IgG4Fc蛋白为如下任一种：
    (b1)SEQ ID NO.2所示的蛋白质；
    (b2)在(b1)所述蛋白质的N端或/和C端连接标签得到的融合蛋白；
    (b3)将(b1)-(b2)任一种经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质；
    (b4)与(b1)-(b2)任一种具有98％以上同一性且具有相同功能的蛋白质。
  13. 根据权利要求11或12所述的带状疱疹疫苗，其特征在于：
    所述铝佐剂为AL(OH)3或ALPO4。
  14. 根据权利要求11-13中任一所述的带状疱疹疫苗，其特征在于：
    所述佐剂由铝佐剂和CpG佐剂组成；
    所述铝佐剂为AL(OH)3；
    优选的，所述CpG佐剂为SEQ ID NO:5-9任一所示的核酸分子，且所有核苷酸残基均硫代修饰。
  15. 根据权利要13或14所述的带状疱疹疫苗，其特征在于：
    所述带状疱疹疫苗中，所述抗原、所述AL(OH)3和所述CpG佐剂质量比为2.5-30：10-200：1-10。
  16. 根据权利要求13-15中任一所述的带状疱疹疫苗，其特征在于：
    所述带状疱疹疫苗中，所述抗原、所述AL(OH)3和所述CpG佐剂质量比为7.5：50：5。
  17. 权利要求11-16任一中的所述CpG佐剂。
  18. 权利要求11-16任一中的所述CpG佐剂和权利要求13-16任一中的所述铝佐剂在如下任一种的应用：
    1)制备带状疱疹疫苗中的应用；
    2)提高带状疱疹疫苗或抗原的免疫原性。
  19. 一种制备权利要求11-16任一所述的带状疱疹疫苗的方法，包括如下步骤：将权利要求11-16任一中的所述抗原和所述佐剂混匀，得到带状疱疹疫苗。
  20. 权利要求1-3或12-16任一中的所述的融合蛋白，权利要求13-16任一中的所述铝佐剂，和权利要求11-16任一中的所述CpG佐剂在如下任一中的应用：
    1)制备预防或治疗带状疱疹的产品；
    2)制备中和带状疱疹病毒的产品；
    3)制备诱导机体对带状疱疹病毒产生体液免疫和/或细胞免疫的产品；
    4)制备带状疱疹病毒抗体；
    或，权利要求1-3或12-16任一中的所述的融合蛋白或其在如下任一中的应用：
    1)作为带状疱疹疫苗抗原；
    2)制备预防或治疗带状疱疹的产品；
    3)制备中和带状疱疹病毒的产品；
    4)制备诱导机体对带状疱疹病毒产生体液免疫和/或细胞免疫的产品；
    5)制备带状疱疹病毒抗体。
  21. 一种寡核苷酸，所述寡核苷酸中全部核苷酸均为硫代修饰；
    所述寡核苷酸的核苷酸序列为以下SEQ ID NO:11所示的核苷酸序列：
    TGACTX1X2X3CGTTTTAX4CGX5X6AGACTGA；
    其中：
    X1为G或A；
    X2为G或A；
    X3为A或T；
    X4为G或A；
    X5为T或C；并且
    X6为T或C。
  22. 权利要求21所述的寡核苷酸，其为选自以下序列的核苷酸序列：
    TGACTGAACGTTTTAACGTCAGACTGA(SEQ ID NO:5)；
    TGACTGAACGTTTTAGCGCTAGACTGA(SEQ ID NO:6)；
    TGACTAGTCGTTTTAACGTCAGACTGA(SEQ ID NO:7)；
    TGACTAGTCGTTTTAGCGCTAGACTGA(SEQ ID NO:8)；
    TGACTGTGAACGTTCGAGATGA(SEQ ID NO:9)。
  23. 一种CpG佐剂，其包含权利要求21或22所述寡核苷酸。
  24. 权利要求23所述的CpG佐剂，其还包含选自矿物盐类佐剂、油乳剂佐剂、靶向于模式识别受体的微生物和植物提取物以及衍生物类佐剂、微粒抗原递呈系统佐剂或细胞因子类佐剂中的一种或多种。
  25. 权利要求24所述的佐剂，其中所述矿物盐类佐剂是铝佐剂或镁佐剂中的一种或多种；所述油乳剂佐剂是皂角苷类佐剂、水包油类和油包水类乳剂中的一种或多种；所述靶向于模式识别受体的微生物和植物提取物以及衍生物类佐剂是磷酰脂A佐剂、CpG佐剂中的一种或多种；微粒抗原递呈系统佐剂是聚乙丙交酯(PLG)、脂质体中的一种或多种；细胞因子类佐剂是细胞因子IL-1、IL-2、IL-12中的一种或多种。
  26. 权利要求24或25所述的佐剂，其中权利要求21或22所述寡核苷酸作为有效成分。
  27. 疫苗，其包含权利要求23-26中任一项所述CpG佐剂。
  28. 权利要求27所述的疫苗，其中所述疫苗还包含抗原。
  29. 权利要求28所述的疫苗，其中每剂疫苗中所述寡核苷酸和所述铝佐剂的质量比为1:8-1:100。
  30. 权利要求21或22所述寡核苷酸、权利要求23-26中任一项所述CpG佐剂在制备疫苗，或者提高抗原或疫苗的免疫原性中的用途，优选的，所述疫苗用于治疗和/或预防带状疱疹病毒或冠状病毒引起的疾病或病症，更优选的，所述冠状病毒包括新型冠状病毒。
  31. 一种治疗或预防疾病或病状的方法，所述方法包括向受试者施用权利要求8、11-16、27-29任一所述的疫苗或权利要求7所述的药物组合物，优选的，所述疾病或病状包括带状疱疹病毒或冠状病毒引起的疾病或病症，更优选的，所述冠状病毒包括新型冠状病毒。
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