WO2020063370A4 - 免疫组合物及其制备方法与应用 - Google Patents

免疫组合物及其制备方法与应用 Download PDF

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WO2020063370A4
WO2020063370A4 PCT/CN2019/105716 CN2019105716W WO2020063370A4 WO 2020063370 A4 WO2020063370 A4 WO 2020063370A4 CN 2019105716 W CN2019105716 W CN 2019105716W WO 2020063370 A4 WO2020063370 A4 WO 2020063370A4
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flagellin
protein
vector
seq
composition according
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PCT/CN2019/105716
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WO2020063370A3 (zh
WO2020063370A2 (zh
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慕婷
赵萍
徐龙
肖杨
朱利安·琼·菲利普
吴月
谢亮
陈雪婷
刘奇
谢铎源
庄再成
克莱因·米歇尔
杜林森
吴克
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武汉博沃生物科技有限公司
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Priority to AU2019349036A priority Critical patent/AU2019349036A1/en
Priority to EP19866309.8A priority patent/EP3868399A4/en
Priority to CA3114658A priority patent/CA3114658A1/en
Priority to CN201980063806.6A priority patent/CN113164586B/zh
Publication of WO2020063370A2 publication Critical patent/WO2020063370A2/zh
Publication of WO2020063370A3 publication Critical patent/WO2020063370A3/zh
Publication of WO2020063370A4 publication Critical patent/WO2020063370A4/zh
Priority to US17/216,619 priority patent/US20210290759A1/en

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Definitions

  • the invention relates to a recombinant VZVgE glycoprotein and a fusion protein with its own adjuvant function, a recombinant carrier, a preparation method, an immune composition, and a preparation method and application thereof.
  • VZV Varicella-zoster virus
  • Herpesviridae alpha herpesvirus subfamily It is a double-stranded DNA virus with a diameter of 150 to 200 nm. Morphology is composed of nucleic acid, protein The concentric structure formed by the shell and the envelope, the surface is a symmetric regular icosahedron composed of 162 shell particles. VZV is a global pathogen with characteristics of skin and nerve addiction. Primary infections in children can cause chickenpox. Chickenpox is a highly contagious disease spread through skin contact or respiratory droplets. It is characterized by a disseminated blister-like rash on the face and trunk, accompanied by itching and fever.
  • VZV primary infections occasionally have visceral complications, such as encephalitis, hepatitis, pancreatitis, or pneumonia, which can be serious life-threatening complications, especially in unvaccinated children and adults, and immunosuppressed people.
  • VZV can be latent in the cranial nerve and dorsal root ganglion for life. Decades later, VZV can still be reactivated, causing painful herpes zoster (HZ) disease or other serious neurological complications or ocular complications, and can lead to post-healing neuralgia of the failure herpes zoster—most Common chronic complications of herpes zoster.
  • HZ herpes zoster
  • Herpes zoster HZ is also a common complication in organ transplant patients. Diseases caused by VZV and their related sequelae (such as postherpetic neuralgia) have gradually become a major disease burden and an important public health problem, and urgently need more medical attention.
  • Antiviral drugs such as acyclovir, valacyclovir, famciclovir, etc., although helpful for the recovery of varicella and HZ patients, cannot prevent VZV infection.
  • the administration of virus-specific immunoglobulin after exposure to VZV is also limited to suspend or reduce disease burden.
  • anti-VZV membrane antigen antibody titers ⁇ 1/64 are considered to be related to disease protection, and anti-gE glycoprotein antibodies have also been found to be related to long-term protection.
  • the live attenuated varicella vaccine was first developed by the Takahashi research group in Japan in 1974. They isolated a strain of VZV from a 3-year-old child with chickenpox named Oka, which was attenuated by serial passage of human embryonic fibroblasts, guinea pig fibroblasts and human diploid fibroblasts. This live attenuated vaccine is called Oka vaccine (vOka).
  • Oka vaccine vOka
  • the Oka vaccine is currently included in routine immunization programs in multiple countries. In general, the Oka vaccine is very safe. Even in partially immunocompromised children and children with human immunodeficiency virus infection, no serious adverse reactions have occurred, and it also shows good immune protection effects.
  • the persistence of the immune protection induced by the Oka vaccine is not long enough, and some individuals cannot achieve effective protection after continuous vaccination. And for adolescents, the immunization effect of the Oka vaccine is lower than that for children aged 1-12, so for preschool children, the vaccine needs to be vaccinated twice. All varicella vaccines currently on the market are live attenuated vaccines. Although there are rare serious side effects, there are reports of serious rashes, lung or liver infections, meningitis, convulsions, pneumonia or systemic vaccine strains after vaccination, especially It is among immunocompromised children.
  • herpes zoster vaccines include Merck's Zostavax and GSK's Shingrix.
  • Zostavax is a concentrated version of the Oka vaccine, which was approved by the US FDA in 2006. Its effectiveness decreases with the age of the vaccinated person. It is not recommended for people over the age of 60. It has been proven that it can provide 50% protection in about five years, and its effectiveness gradually decreases 5-8 years after vaccination. And the protective power is no longer statistically significant after 8 years of vaccination (Morrison VA, et al., Clin Infect Dis, 60:900-909, 2015).
  • GSK's Shingrix uses genetic recombination technology to express varicella zoster virus glycoprotein E in Chinese hamster ovary cells.
  • Shingrix protects herpes zoster by 90%, reduces the risk of neuralgia after herpes zoster, and is Zostavax's preferred alternative.
  • the adjuvant used by Shingrix is AS01 from GSK, which has side effects.
  • VZV vaccines that are safe, have low side effects, no latent risk, and post-herpetic neuralgia complication risk, as well as lower prices, but no progress has been made.
  • the new vaccine should not only cause a strong humoral response to neutralize the virus, but also induce extensive cellular immunity to control the disease.
  • the open reading frame (ORF) of the VZV genome encodes a total of 8 glycoproteins: glycoprotein E (gE), gB, gH, gI, gC, gL, gK and gM.
  • the gE glycoprotein is encoded by the ORF68 gene and belongs to the type I membrane protein. It is a glycoprotein necessary for the generation of infectious virus particles. It is also the most abundant and immunogenic glycoprotein in the viral envelope, which exists on the surface of the virus particles. And the cytoplasm of VZV-infected cells exists in different glycosylation forms at different stages of virus maturation.
  • VZV antibodies are mainly directed against gE, gB and gH.
  • Specific anti-gE monoclonal antibodies can neutralize VZV and mediate antibody-dependent cytotoxicity (ADCC).
  • gE is also the main target of cellular immunity, which can control diseases and destroy cells infected with viruses. These characteristics make gE an ideal immunogen for the development of safe and effective broad-spectrum vaccines.
  • Inactive human vaccines usually consist of one or more immunogens, and immune adjuvants can be added to the formulation to enhance its effectiveness. Only a limited number of immune adjuvants are currently available for human use, such as aluminum salts, mineral oil, plant or bacterial extracts. Immune adjuvants have different enhancing properties and can cause various adverse side effects. With the deepening of the understanding of the immune response regulation mechanism, people have discovered Toll-like expressions expressed on the surface of sentinel cells (such as dendritic cells and macrophages) of the immune system and expressed on lymphocytes, which jointly regulate innate immunity and adaptive immunity. Receptors (Toll-like receptors, TLRs). TLR recognizes the conserved microbial-associated molecular pattern (MAMP). Agonists trigger TLR to produce multiple pleiotropic immune mediators, such as cytokines and chemokines, which participate in pre-inflammatory responses, stimulate innate immunity, and thus act as an immune adjuvant.
  • MAMP conserved microbial-associated
  • Toll-like receptor 5 is a transmembrane receptor that specifically recognizes bacterial flagellin protein.
  • Flagellin protein is the main structural protein of gram-negative bacteria flagella. Flagellin induces the activation of TLR5, initiates innate immunity, induces the activation of monocytes-macrophages, and epithelial cells, and releases proinflammatory factors such as IL-1, IL-8, and TNF- ⁇ . Therefore, flagellin protein is a powerful systemic and mucosal immune adjuvant. It consists of four domains D0, D1, D2 and D3, of which the domains D0 and D1 are highly conserved in Proteobacteria.
  • flagellin The interaction between the N-terminal D0-D1 helix of the flagellin protein and the C-terminal D1-D0 helix forms a stalk-like core structure, which is essential for the binding and activation of TLR5.
  • the D2 and D3 domains of flagellin vary greatly between different bacteria, have strong immunogenicity but are not functionally necessary. Deleting the D2-D3 domain does not impair TLR5 activation and can minimize the useless anti-flagellin antibody response.
  • flagellin must be combined with the target immunogen to produce the best immune adjuvant effect. This can be achieved by constructing a fusion protein with its own adjuvant effect, where the immunogen is covalently linked to flagellin or its functional fragment that retains TLR5 binding activity, and thus retains its inherent immunostimulatory properties.
  • the present invention is to overcome the above-mentioned shortcomings of the marketed vaccines and to improve the adverse reactions.
  • Two different methods are used to develop new immune compositions.
  • One is to produce strong neutralizing antibodies and CD4+ T cell responses with lower side effects.
  • the present invention provides an immune composition comprising an antigen based on varicella-zoster virus glycoprotein E (abbreviated as gE), which can be used to prevent or treat varicella-zoster virus (VZV) infection.
  • gE varicella-zoster virus glycoprotein E
  • VZV varicella-zoster virus
  • the gE-based immunogen comprises at least: (i) gE extracellular region or fragment thereof, or its corresponding encoding nucleic acid molecule; (ii) gE-based fusion protein, or its encoding nucleic acid molecule; (iii ) GE-based recombinant vector; or (iv) a combination of two or more of the above.
  • the gE-based fusion protein comprises at least: gE extracellular region or a fragment thereof covalently coupled to a bacterial flagellin protein or fragment thereof having an adjuvant effect, wherein the bacterial flagellin protein or fragment thereof has TLR- 5 agonistic activity.
  • VZVgE and flagellin can be found in publicly available databases such as GenBank (GB), SwissPro (sp), EMBL, etc.
  • Representative database entries for gE include but are not limited to: GBAQT34120.1, AAG32558.1, ABE03086.1, etc., the sequence represented by the registration number is incorporated by reference into the present invention.
  • GE glycoprotein is a membrane protein whose structure contains signal peptide, extracellular domain, transmembrane domain and intracellular domain.
  • the extracellular region is exposed on the surface of bacteria and is a target recognized by the immune system. Therefore, it should be understood that the gE mentioned in the present invention contains at least its extracellular region or fragments thereof, and if necessary, can further contain other structural fragments such as transmembrane and/or intracellular regions on the basis of retaining certain antigen activity of gE. According to common sense, those skilled in the art can determine each structural fragment of gE, and the fragment of the extracellular region of gE can be understood as a fragment that retains certain autoimmunity of gE.
  • the extracellular region of gE has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the amino acid sequence shown in SEQ ID NO. % Or 100% homology.
  • Bacterial flagellin protein is the main flagella component of many Gram-negative bacteria (such as E. coli or Salmonella, etc.), and its primary amino acid sequence differs in composition and size with different bacterial species.
  • the conserved N-terminal D0-D1 and C-terminal D1-D0 domains interact to form a functional stem-like structure, which is necessary for TLR5 binding and signal transmission.
  • the "hypervariable" D2 and D3 regions in the middle are non-essential domains for TLR-5 signal transduction, but the "hypervariable” D2 and D3 domains in the molecule are not required for TLR5 signal transduction and have very Strong immunogenicity and induce adverse reactions, so this part of the area can be deleted without affecting TLR5 binding activity.
  • the bacterial flagellin protein in the present invention may be the original or modified flagellin protein. Such modifications include but are not limited to mutations, substitutions (such as conservative substitutions of functionally similar amino acids), additions, deletions, or truncations, etc., but a certain degree of TLR-5 binding capacity should be retained to activate innate immunity. It should be understood that the bacterial flagellin protein or fragments thereof in the present invention should not cause significant proinflammatory side effects.
  • the published patents US2011110962A1 and/or US2011230643A1 describe the immunity of flagellin proteins and certain modifications thereof, and are introduced into the present invention.
  • the N-terminus of flagellin protein refers to its N-terminal D0-D1 region
  • the C-terminus of flagellin protein refers to its C-terminal D1-D0 region.
  • the gE-based fusion protein comprises at least: the N-terminal region of flagellin protein, the C-terminal region of flagellin protein, and the extracellular region of gE or a fragment thereof.
  • the gE-based fusion protein may also contain flagellin protein or other fragments of gE
  • the gE extracellular region or fragment thereof is located at the N-terminus or C-terminus of the gE-based fusion protein; or inserted between the N-terminus and the C-terminus of the flagellin protein.
  • the gE-based fusion protein is selected from any of the following fusion forms:
  • Fusion form 1 Flagellin N-terminal region-Flagellin C-terminal region-gE extracellular region or fragments thereof;
  • Fusion form 2 gE extracellular region or its fragment-flagellin N-terminal region-flagellin C-terminal region;
  • Fusion form 3 Flagellin N-terminal region-gE extracellular region or its fragment-Flagellin C-terminal region;
  • the N-terminal region or C-terminal region of the flagellin protein can be directly or through a linker connected to the gE extracellular region or a fragment thereof;
  • the N-terminal region of the flagellin protein can be directly or through a linker connected to the C-terminal region of the flagellin protein.
  • the linkers include genetically engineered peptide chains (such as amino acids linked by 1-20 peptide bonds) and non-peptide chemical linkers (such as alkyl linkers or polyethylene glycol groups, where the alkyl linkers can also be non-sterically hindered Groups such as halogen, CN, NH2, etc.). It should be understood that the selected linker does not interfere with the biological activity of the fusion protein.
  • the linker is an amino acid connected by 1-20 peptide bonds, such as linker I or linker II; linker I is shown in SEQ ID NO: 4; linker II is shown in SEQ ID NO: 7.
  • SEQ ID: NO: 4 SPGISGGGGGILDSMG
  • SEQ ID: NO: 7 GGGGSGGGGSGGGGS
  • the N-terminal region or the C-terminal region of the flagellin protein is connected to the extracellular region of gE or a fragment thereof via linker II, respectively.
  • the N-terminal region of the flagellin protein is connected to the C-terminal region of the flagellin protein via a linker I
  • the flagellin protein is derived from Salmonella, such as Salmonella enterica (Salmonella subsp.enterica serovar typhimurium (S.typhimurium)) or Salmonella enteritidis (Salmonella subsp.enterica serovar typhi (S.typhi) ), the Salmonella typhimurium includes but not limited to strain LT2; Salmonella enterica includes but not limited to strain Ty2.
  • the amino acid sequence of the flagellin protein is shown in SEQ ID NO: 3 (derived from strain LT2) or SEQ ID NO: 29 (derived from strain Ty2).
  • methionine is the first amino acid at the N-terminus of the natural flagellin molecule
  • the N-terminus of the flagellin protein begins with the second amino acid (Ala) of the natural sequence.
  • the N-terminal region of the flagellin protein of Salmonella typhimurium according to the present invention generally starts from the second alanine (Alanine) in SEQ ID NO: 3 and ends at any amino acid of amino acids 137-176 ;
  • the C-terminal region generally starts at amino acids 392-406 and ends at amino acid 495.
  • the N-terminal region of the flagellin protein is at least 95% homologous (eg, 97%, 98%, or 99%) to the amino acid regions 2 to 176 in SEQ ID NO:3 Amino acid sequence; the C-terminal region is at least 95% homologous (e.g., 97%, 98%, or 99% homology) to the amino acid region from 392 to 495 in SEQ ID NO:3 Amino acid sequence.
  • amino acid sequence of the N-terminal region of the flagellin protein is shown in the sequence table SEQ ID NO: 5: the amino acid sequence of the C-terminal region of the flagellin protein is shown in the sequence table SEQ ID NO: 6 shown
  • the N-terminal region of the Tymon flagellin protein of Salmonella enterica of the present invention generally starts from the second alanine (Alanine) of SEQ ID NO: 29 and ends at any amino acid of amino acids 180-200;
  • the C-terminal region starts at any amino acid at positions 278-400 and ends at amino acid 506.
  • the N-terminal region of Ty2 flagellin protein is 2-180 of SEQ ID NO: 29, and the C-terminal region is position 400-506; or the N-terminal region of Ty2 flagellin protein is SEQ ID NO: 29, 2-220, the C-terminal region is 320-506; or the N-terminal region of Ty2 flagellin protein is 1-190 of SEQ ID NO: 29, and the C-terminal region is 278-506.
  • the N-terminal region of the flagellin protein is at least 95% (e.g., 97%, 98%, or 99% identical to the amino acid region from 2 to 180 in SEQ ID NO: 29 Amino acid sequence of homology; the C-terminal conserved region is at least 95% (e.g., 97%, 98%, or 99% homologous) homologous to the amino acid regions 400 to 506 in SEQ ID NO:29 Amino acid sequence.
  • amino acid sequence of the N-terminal conserved region is shown in SEQ ID NO: 30; the amino acid sequence of the C-terminal conserved region is shown in SEQ ID NO: 31 in the Sequence Listing.
  • the amino acid sequence of the gE-based fusion protein is shown in any one of SEQ ID NO: 8-10 and SEQ ID NO: 32-34.
  • the nucleic acid molecule described in the present invention is generally a nucleic acid molecule optimized according to an expression system, including but not limited to: DNA, RNA, mRNA, ssDNA or cDNA.
  • the nucleic acid molecule can be operatively linked to an expression control sequence including, but not limited to, promoters, enhancers, transcription terminators, start codons (such as ATG), splicing signals and termination of introns Codons, etc., in which the use of in vitro and in vivo conditional expression control elements may be considered.
  • additional gene fragments can be added, such as, but not limited to, human cytomegalovirus early enhancer, Kozak consensus sequence, leader sequence, Woodchuck hepatitis virus post-transcriptional regulatory element, encoding glycosylation receptor sequence Nucleic acid sequences, or extraneous proteins such as markers or cleavage sites, etc.
  • the extraneous proteins that can be added include those used to optimize gene expression, information stability, protein yield, secretion and purification.
  • Various gene cloning and construction methods well known to the skilled person, as well as host cell expression systems can be used.
  • the protein-encoding DNA sequence disclosed in the present invention can be expressed in prokaryotic and eukaryotic host cells.
  • a nucleic acid leader sequence may be further added at the 5′ end of the nucleic acid molecule to promote protein secretion, and the nucleic acid leader sequence includes but is not limited to the Japanese encephalitis virus (JEV) prM protein gene leader Sequence or the mouse Ig ⁇ light chain gene leader sequence; and/or the 5′ end of the nucleic acid molecule can also be added with Kozak sequence to enhance translation efficiency; and/or the 3′ end of the nucleic acid molecule can also be added with polyadelynation (polyA)
  • JEV Japanese encephalitis virus
  • polyA polyadelynation
  • the sequence increases the stability of the nucleic acid molecule; the polyA sequence includes but is not limited to SV40polyA.
  • the JEV signal peptide gene sequence is shown in SEQ ID NO: 14.
  • the Ig ⁇ signal peptide gene sequence is shown in SEQ ID NO: 15.
  • the Kozak sequence gene sequence is shown in SEQ ID NO:16.
  • the SV40polyA gene sequence is shown in SEQ ID NO:17.
  • the signal peptide encoded by the nucleic acid leader sequence is hydrolytically cleaved during the intracellular processing of the natural protein.
  • the nucleic acid molecule encoding the gE extracellular region or a fragment thereof is shown in any one of SEQ ID NO: 2, 18-19.
  • the nucleic acid molecule encoding the gE-based fusion protein is shown in any one of SEQ ID NO: 11-13 and SEQ ID NO: 20-26.
  • the gene sequence encoding the gE-based fusion protein shown in SEQ ID NO: 8 is shown in SEQ ID NO: 11, any one of 20-21: encoding the gE-based fusion protein shown in SEQ ID NO: 9
  • the nucleic acid molecule sequence is shown in SEQ ID NO: 12, 22-23: the nucleic acid molecule sequence encoding the gE-based fusion protein shown in SEQ ID NO: 10 is shown in SEQ ID NO: 13, 33-34. Show.
  • the nucleic acid molecule sequence encoding the fusion protein based on gE shown in SEQ ID NO: 10 is shown in any one of SEQ ID NO: 13, 33-34.
  • the nucleic acid molecule sequence encoding the gE-based fusion protein shown in SEQ ID NO: 34 is shown in SEQ ID NO: 26.
  • the recombinant vector based on gE of the present invention carries the nucleic acid molecule as described above. It should be understood that the gE-based recombinant vector may carry the gene encoding the gE extracellular region or fragment thereof as described above, or the gene carrying the fusion protein of gE as described above.
  • the vector may be an expression vector, a cloning vector or a transfer vector, including but not limited to: a viral vector, a DNA vector or an mRNA vector, and the like.
  • viral vectors include but are not limited to: adenovirus vectors, adenovirus-related virus vectors, pox virus vectors, vesicular stomatitis virus vectors, bovine parainfluenza virus vectors, human parainfluenza virus vectors, Newcastle disease virus vectors, Sendai virus vectors, Measles virus vector, attenuated RSV vector, paramyxovirus vector, A virus vector (such as Venezuelan equine encephalitis virus vector, Semliki Forest virus vector, Sindby virus vector), baculovirus vector, rabies virus vector, small Ribonucleic acid viruses, lentiviral vectors, herpes virus vectors, or plant-derived viruses are used for expression in plant expression systems.
  • a virus vector such as Venezuelan equine encephalitis virus vector, Semliki Forest virus vector, Sindby virus vector
  • baculovirus vector such as Venezuelan equine encephalitis virus vector, Semliki Forest virus vector, Sindby
  • the adenovirus vector is a human-derived adenovirus vector (such as type 5 adenovirus vector Ad5), chimpanzee-derived adenovirus vector (such as ChAd68), gorilla adenovirus vector, or other human-applicable adenovirus vector .
  • a human-derived adenovirus vector such as type 5 adenovirus vector Ad5
  • chimpanzee-derived adenovirus vector such as ChAd68
  • gorilla adenovirus vector gorilla adenovirus vector
  • other human-applicable adenovirus vector such as type 5 adenovirus vector Ad5
  • the recombinant adenovirus vector is a replication-defective recombinant adenovirus vector
  • the replication-defective type may be deletion or functional deletion of the E1 region of the adenovirus genome to form a replication-deficient adenovirus, or E3
  • the region is further deleted or functionally deleted; or both E1 and E3 regions are deleted or functionally deleted; all E1 functionally deleted vectors are replication-defective vectors.
  • the functional loss generally refers to the loss of the original function of E1 due to mutations, deletions, or increased sites, which in turn affects adenovirus replication. Therefore, these viruses can only replicate in mammalian cells that supplement the expression of E1 protein, such as HEK293 and PER.C6 cells, whose genomes are modified to express the E1 gene.
  • the remaining adenovirus genome of the replication-defective recombinant adenovirus vector of the present invention can be the original adenovirus genome (that is, it can be understood that except for the deletion or functional deletion of the E1 region, or the deletion or functional deletion of both E1 and E3, the remaining genomes are not There are further modifications, such as the pAd5-CMV/V5-Dest vector purchased from Thermo Fisher Scientific) or the adenovirus genome that can be further modified.
  • the modification refers to the replacement, mutation and other modifications of the original adenovirus genome, for example
  • the E4 region of the replication-deficient chimpanzee adenovirus itself is replaced with the human adenovirus E4 region to improve the performance of the vector.
  • the gE-based recombinant vector When the gE-based recombinant vector carries a nucleic acid molecule encoding the extracellular region of gE or a fragment thereof as described above (for example, the nucleic acid molecule shown in SEQ ID NO: 2, 18-19), it is called a recombinant gland. Viral vector A, in other words, the gE is expressed in a non-fused form.
  • the recombinant adenovirus vector A is constructed by homologous recombination.
  • the backbone plasmid used to construct the recombinant adenovirus vector A is pAd5-CMV/V5-DEST.
  • the shuttle plasmid used to construct the recombinant adenovirus vector A is pDONR221.
  • the host cell lines used to construct the recombinant adenovirus vector A include but are not limited to HEK293 or PER.C6 cell lines.
  • the recombinant adenovirus vector A is constructed by the following method: homologous recombination of the recombinant shuttle plasmid pDONR221-gE gene-PolyA sequenced correctly and the viral backbone plasmid pAd5-CMV/V5-DEST, Transform the recombinant mixture into E. coli TOP10 competent cells, screen and sequence the correct adenovirus vector pAd5-CMV-gE gene-PolyA, linearize the adenovirus vector pAd5-CMV-gE gene-PolyA, and transfect HEK293 or PER C6 cells are packaged to obtain the recombinant adenovirus vector A.
  • This technique is well known to those skilled in the art.
  • the gE-based recombinant vector carries a nucleic acid molecule encoding a gE-based fusion protein as described above (for example, the nucleic acid molecule shown in SEQ ID NO: 11-13, 20-26), it is called a recombinant adenovirus Carrier B.
  • the recombinant adenovirus vector B is constructed by homologous recombination.
  • the viral backbone plasmid used to construct the recombinant adenovirus vector B is pAd5-CMV/V5-DEST.
  • the shuttle plasmid used to construct the recombinant adenovirus vector B is pDONR221.
  • the host cell lines used to construct the recombinant adenovirus vector B include but are not limited to HEK293 or PER.C6 cell lines.
  • the recombinant adenovirus B is constructed by the following method: transforming the recombinant shuttle plasmid pDONR221-gE-flagellin fusion protein gene-PolyA sequenced correctly and the viral backbone plasmid pAd5-CMV/V5-DEST Perform homologous recombination, transform the recombination mixture into E.
  • coli TOP10 competent cells screen and sequence the correct adenovirus vector pAd5-CMV-gE-flagellin fusion protein gene-PolyA, and convert the adenovirus vector pAd5-CMV-fusion protein gene- After linearizing PolyA, transfect HEK293 or PER.C6 cells for packaging to obtain the recombinant adenovirus vector B.
  • This technique is well known to those skilled in the art.
  • the immunological composition of the present invention as described above may further include one or more other components, such as a pharmaceutically acceptable carrier, and/or adjuvant, and/or immunostimulatory molecules, and the like.
  • the adjuvants include, but are not limited to: aluminum salt (such as aluminum hydroxide or aluminum phosphate) oil-in-water emulsion or water-in-oil emulsion, MF-59, TLR agonist (such as monophosphoryl lipid A (MPL) or Its analogs, or CpG oligonucleotides), Quil A or its QS21 component, chitosan, or a combination of two or more thereof.
  • the adjuvant has the purpose of enhancing body fluid and/or cell response.
  • Immunostimulatory molecules may include but are not limited to Escherichia coli heat-resistant enterotoxin LT, cholera toxin CT or its analogs, etc.; cytokines or chemokines; antibodies or fragments thereof, which are directed against specific cell surface differentiation antigens or Receptors involved in immune response, and can enhance humoral and cellular immune responses.
  • the medically acceptable carrier may be a carrier conventionally used in the art, and generally depends on the administration method of the drug.
  • dosage forms for parenteral administration generally include medically and physiologically acceptable injectable fluids, including but not limited to water, physiological saline, balanced salt solution, glycerin, or other carbohydrates as carriers.
  • the immune composition may also contain a small amount of non-toxic auxiliary substances, such as emulsifiers, pH buffers, stabilizers or preservatives. Sterile solutions are prepared by sterile filtration or other methods known in the art.
  • the pH value of the solution is generally between 3.0 and 9.0, preferably between pH 5.0 and 7.5.
  • the preparations can be stored in liquid form or lyophilized form, and can be provided in a single dose or in multiple-dose sealed containers.
  • the immune composition of the present invention can also be delivered using carrier systems (including but not limited to liposomes, microspheres, micelle systems, immunostimulatory complexes (ISCOMS) and nanoparticles), the nanoparticles including Ferritin, cystin, thiooxygenase reductase (SOR), and luminamide synthase-nanoparticles.
  • carrier systems including but not limited to liposomes, microspheres, micelle systems, immunostimulatory complexes (ISCOMS) and nanoparticles
  • the nanoparticles including Ferritin, cystin, thiooxygenase reductase (SOR), and luminamide synthase-nanoparticles.
  • the immune composition of the present invention as described above can be administered by a delivery system well known to those skilled in the art, including subcutaneous, intramuscular, intradermal, or intranasal routes.
  • the nucleic acid-based immune composition of the present invention can also be administered by the gene gun method, and the recombinant protein immunogen can be administered by a needle-free delivery system.
  • the immune composition of the present invention as described above can be used to prevent and/or treat varicella-zoster infection.
  • the immune composition can be used to vaccinate infants, children, adolescents, adults or the elderly against varicella infections or the elderly to vaccinate against herpes zoster infections.
  • the immune composition can be used to treat Neuralgia after herpes zoster and/or herpes zoster.
  • infants are between 0-12 months old, children are 1-12 years old, teenagers are 12-18 years old, adults over 18 years old are adults, and people over 50 years old are elderly. It should be understood that the division of age is not limited to the above description, and the immune composition can be used to immunize people of appropriate ages against varicella or herpes zoster infections.
  • Another aspect of the present invention provides the use of the immune composition as described above for the preparation of a medicament for preventing and/or treating varicella-zoster virus infection; further, for the preparation of varicella vaccine and/or herpes zoster Application in vaccine; or the immune composition can be used to prepare medicine for treating herpes zoster and/or postherpetic neuralgia.
  • the present invention further provides a combination vaccine comprising the immune composition as described above and one or more other vaccines.
  • the antigen components of the combined vaccine do not interfere with each other, or a synergistic effect can be further achieved.
  • Non-interference generally refers to maintaining the stability of the immunogen and compatibility between the immune components, and there is no competition between antigens or the risk of serious adverse reactions.
  • each antigen component in the combination vaccine should have the same or similar subject population and immunization program.
  • the other vaccines that can be combined include but are not limited to: mumps, measles and rubella vaccines.
  • the gE-based fusion protein, the corresponding nucleic acid molecule, and the gE-based recombinant vector of the present invention as described above can be used to prevent and/or treat infections caused by varicella-zoster, specifically for infants, children, Adolescents, adults or the elderly are vaccinated against varicella infections or the elderly are vaccinated against herpes zoster infections.
  • One aspect of the present invention provides the above-mentioned gE-based fusion protein, the nucleic acid molecule, and the gE-based fusion protein recombinant vector in the preparation of a medicament for preventing and/or treating varicella-zoster virus infection Application; further, for the preparation of varicella vaccine and/or herpes zoster vaccine.
  • the immune composition can be used for preparing a medicine for treating herpes zoster and/or neuralgia after herpes zoster.
  • Vaccination may involve single or multiple injections at intervals of one or more months at doses ranging from 1 ⁇ g to 100 ⁇ g of recombinant protein or adenovirus vectors of 10 10 to 10 12 virus particles (VP).
  • the specific dosage will be determined in clinical trials and depends on the route of administration and target population. If necessary, booster immunization can be given every year.
  • the priming-boosting immunization program includes administering a first immune composition (primary vaccine) to the subject, and then administering a second immune composition (boosting vaccine) to induce the optimal immune response.
  • a first immune composition primary vaccine
  • boosting vaccine second immune composition
  • the immune composition administered for the primary immunization and booster immunization may be the same or different and the respective amounts may be different.
  • the gE extracellular region or its fragment, gE-based fusion protein, nucleic acid molecule and gE-based recombinant vector can be used for primary immunization or booster immunization, respectively.
  • the present invention provides the following initial immunity-boosting immunization procedures: (1) The above-mentioned gE-based recombinant vector can be used for the initial immunization, and the gE extracellular region or its fragment or gE-based fusion protein can be used for boosting immunization; or (2) The gE extracellular region or its fragment or gE-based fusion protein can be used for primary immunization, and gE-based recombinant vector can be used for boosting immunization.
  • the combination of priming and boosting immunization programs includes but is not limited to the above expressions.
  • priming can be performed using gE-based adenovirus vectors, and then boosting immunization can be performed using different vectors (such as poxvirus vectors, etc.) derived from expressing the same gene, or in turn, priming can be based on
  • the gE heterologous vector can be understood as other vectors than adenovirus vector
  • the gE-based adenovirus vector of the present invention is used for boosting immunization.
  • two types of adenovirus vectors expressing the same or different gE genes based on different types or different species can also be used in combination in priming-boosting immunization procedures.
  • the dosage depends on the immune components, administration route, target population and other factors. Clinical trial personnel will determine the appropriate dosage and effective immunization schedule for each immune component based on their knowledge. A single administration is sufficient or requires multiple administrations using single and/or combined immunogens.
  • Yet another aspect of the present invention provides an isolated host cell comprising a gE-based gene as described above (such as a nucleic acid molecule encoding a gE extracellular region or fragment thereof, or a nucleic acid molecule encoding a gE-based fusion protein).
  • the host cells include but are not limited to: E. coli, Bacillus subtilis, Salmonella, Saccharomyces cerevisiae, Pichia pastoris, insect cells, HEK293 cells, PER.C6 cells, Vero cells, CHO cells, W38 cells, BHK Cells or COS cells.
  • the present invention provides a method for preparing the gE extracellular region or fragment thereof as shown above, or the gE-based fusion protein as described above, which can be specifically expressed by a prokaryotic expression system or a eukaryotic expression system.
  • the gE extracellular region or a fragment thereof is prepared with or without a covalent binding protein tag conducive to purification;
  • the gE-based fusion protein is prepared with or without a covalent binding protein tag conducive to purification;
  • the covalently bound protein tags include but are not limited to polyhistidine tags (His tags).
  • the prokaryotic expression system includes but is not limited to the E. coli expression system.
  • the E. coli used is BL21(DE3)
  • the prokaryotic expression vector may contain but not limited to the T7 promoter, preferably the expression vector is pET28a.
  • the amino acid sequence of the gE extracellular region is shown in SEQ ID NO: 35
  • the gene sequence of the gE extracellular region is shown in SEQ ID NO: 36
  • the amino acid sequence of the gE-based fusion protein is SEQ ID NO: 37-39
  • the gene sequence of the gE-based fusion protein is shown in SEQ ID NO: 37-39.
  • the prokaryotic expression may include the steps of: transforming the gene carrying the gE extracellular region or fragment thereof or the pET28a expression vector carrying the gE-based fusion protein gene into E. coli BL21 (DE3), cultured on LB medium supplemented with kanamycin (50 ⁇ g/ml) coated on agar plates. Pick single clones and inoculate them into LB liquid medium containing kanamycin. When cultured at 37°C until OD 600 reaches 0.6, add 0.1 ⁇ 1mM IPTG and induce expression at 16 ⁇ 37°C.
  • the harvested cells were broken by ultrasonic or high-pressure homogenizer, and the inclusion bodies (IB) were harvested by centrifugation, and the inclusion bodies were washed several times with brine containing detergent.
  • the inclusions were resuspended and dissolved in buffer containing 6M guanidine hydrochloride or 8M urea (20 mM Tris, 5 mM imidazole, 500 mM NaCl, pH 8.0).
  • the eukaryotic expression system includes but is not limited to yeast expression system, mammalian cell expression system, or recombinant virus (such as human, animal or plant recombinant virus expression system, such as baculovirus, adenovirus, lentivirus or poxvirus) Expression system, or plant expression system.
  • mammalian cell lines for expression include but are not limited to 293 cells or PER.C6 cell lines, Chinese hamster ovary CHO cell lines, insect cell lines such as SF9 cells, Vero cells, or transgenic animal or plant cell lines.
  • Recombinant proteins can be expressed by transient expression, stable cell line expression or recombinant viral vector expression.
  • Cell culture media are available from commercial sources, and suitable conditions for culturing cells are well known, and those skilled in the art can easily select the culture media and the culture conditions of the host cells to express the immunogen of interest. Suitable media may or may not contain serum.
  • the eukaryotic expression includes the steps of: fusing the gE-based recombinant vector (preferably a recombinant adenovirus vector A encoding the gE protein and the gE-flagellin as described above) Protein recombinant adenovirus vector B) Infect a 90% confluent host cell with a certain MOI value (in some embodiments, the host cell includes but is not limited to Vero or CHO and other cells), four to five days after infection, harvest The supernatant is cultured, and the corresponding protein is obtained after purification of the harvested supernatant.
  • the MOI value may be 10-500, and more preferably the MOI value may be 100-200.
  • Purification steps include hydrophobic chromatography followed by ion exchange chromatography and/or size exclusion chromatography purification; wherein, hydrophobic fillers include but are not limited to: Phenyl, Octyl or butyl related fillers; ion exchange fillers include but are not limited to: QsephraseFF, DEAE or Source 30Q; among them, size exclusion chromatography packing includes but not limited to Sephadex G200, G100 or G75.
  • the purification process is first performed by hydrophobic chromatography and then by ion exchange chromatography.
  • the hydrophobic filler is Capto Phenyl Impress, and the ion exchange filler is Source30Q.
  • the present invention also provides a recombinant adenovirus vector pAd5-CMV-gE gene-PolyA as described above.
  • the gE gene is a nucleic acid sequence as shown in SEQ ID NO: 2, 18-19.
  • the present invention also provides a recombinant adenovirus vector Ad5-CMV-gE-flagellin fusion gene-PolyA as described above, the gE-flagellin fusion gene has any of SEQ ID NO: 11-13, 20-26 The nucleic acid sequence shown.
  • Another aspect of the present invention also provides a modified flagellin protein, the N-terminal region of the flagellin protein is at least 95% (e.g., 96%, 97) of the amino acid region 2 to 176 in SEQ ID NO: 3 %, 98% or 99% homology) homologous amino acid sequence; flagellin protein C-terminal region is at least 95% (for example: 96%, 96%, 97%, 98% or 99% homology) an amino acid sequence of homology; the N-terminal region of the flagellin protein is directly or through a linker connected to the C-terminal region of the flagellin protein.
  • the N-terminal region of the flagellin protein is at least 95% (e.g., 96%, 97) of the amino acid region 2 to 176 in SEQ ID NO: 3 %, 98% or 99% homology) homologous amino acid sequence
  • flagellin protein C-terminal region is at least 95% (for example: 96%, 96%, 97%, 98% or
  • the linker may be an amino acid linked by 1-20 peptide bonds, such as having the amino acid sequence shown in SEQ ID NO:4.
  • amino acid sequence of the N-terminal region of the flagellin protein is shown in the sequence table SEQ ID NO: 5: the amino acid sequence of the C-terminal region is shown in the sequence table SEQ ID NO: 6.
  • the modified flagellin protein has the amino acid sequence shown in SEQ ID NO:27.
  • the invention also provides a nucleic acid sequence which can encode the amino acid sequence shown in SEQ ID NO:27.
  • the nucleic acid sequence is shown in SEQ ID NO:28.
  • the present invention also provides the application of the modified flagellin protein as an immune adjuvant.
  • the fusion protein formed when it is coupled with gE or its fragments has inherent adjuvant properties. Therefore, the fusion protein based on gE may be Recombinant adenovirus vectors expressing gE-flagellin fusion protein (such as recombinant adenovirus vector B) can be directly used to prepare vaccines for immunizing hosts (human or animal) to induce and/or enhance the immune response to VZV against acute or Potential VZV infection.
  • the invention discloses a method for efficiently expressing gE or gE-flagellin fusion immunogen through a prokaryotic expression system or a recombinant adenovirus system.
  • the prepared gE, gE flagellin fusion protein and recombinant adenovirus vector can stimulate immunity
  • the host produces high levels of antibody titers and good cellular immunity and can be developed as a new generation and improved VZV vaccine.
  • prevention refers to the inhibition of the overall development of an infection or disease in a subject at risk of disease (eg, VZV infection).
  • Treatment refers to a therapeutic intervention to improve the signs or symptoms of a disease or pathological state after it begins to develop.
  • improvement refers to any observable beneficial therapeutic effect, such as delayed clinical symptoms of the disease, reduced symptoms of the disease, slowed progression of the disease, improved overall health of the subject, or other specific indicators of special diseases recognized in the field .
  • Preventive treatment is treatment of subjects who do not have symptoms of the disease or only early symptoms, with the aim of reducing the risk of developing the disease.
  • Adenovirus type 5 (Ad5): A double-stranded DNA virus that belongs to the adenoviridae family and mainly causes respiratory infections in humans.
  • E1 gene products (including E1A and E1B) are involved in virus replication.
  • Most E3 proteins are involved in regulating the immune response of infected cells. You can delete the E1 region to make the virus lose its replication ability, and then insert the heterologous transgene into the deleted E1 and E3 regions, using the virus as a vector to achieve immunity or gene therapy purposes.
  • Adjuvant A substance that enhances the host's immune response to an immunogen or vaccine.
  • Antibodies Blood proteins produced by specific plasma cells play a major role in the humoral adaptive immune response against foreign molecules or pathogens. Antibodies recognize specific sites on the homologous immunogen, thereby neutralizing or eliminating these antigens.
  • ADCC Antibody-dependent cellular toxicity
  • Dendritic cells are responsible for the initiation of non-specific innate immunity and immunogen-specific adaptive immunity. Lymphocyte subsets are responsible for CD4+ helper T cells including pro-inflammatory response, helper antibody production, and cytotoxic CD8+ T cells that kill infected targets.
  • Conditional gene expression refers to the ability to arbitrarily activate or inhibit the expression of specific genes or gene products.
  • CpG oligonucleotides are short single-stranded synthetic DNA molecules containing cytosine deoxynucleotides and guanine triphosphate deoxynucleotides.
  • the CpG motif is a pathogen-associated molecular model, so it has immunoadjuvant properties as a TLR9 agonist.
  • Extracellular domain A domain in which membrane proteins extend into the extracellular space.
  • Membrane proteins consist of an extracellular domain (extracellular domain), a transmembrane segment and an intracytoplasmic tail.
  • Enhancer A DNA sequence that can increase the transcription level of genes located near the coding sequence.
  • Flagellin a polymeric protein that is the main component of flagella in Gram-negative bacteria and determines the specificity of flagella in causing an immune response. Flagellin is an effective immunomodulator.
  • Fusion protein A protein produced by the combination of two or more genes that originally encoded an isolated protein.
  • Homologous recombination The exchange of genetic material between two strands of DNA containing long stretches of similar base sequences. Homologous recombination naturally exists in eukaryotes, bacteria and certain viruses, and is a powerful tool for genetic engineering.
  • Host cell A cell containing foreign molecules, viruses, or microorganisms.
  • Immunogen A substance or organism that can cause immunity after entering the host, including humoral (antibody) and cellular reactions.
  • Immune composition A composition that induces immunity.
  • Immunostimulatory molecules Molecules that can stimulate or enhance the immune response.
  • Innate immunity A natural mechanism of defense by the sentinel cells of the immune system (such as dendritic cells and macrophages). This immunization was not caused by previous sensitization to the immunogen, such as infection or vaccination. Since innate immunity is not stimulated by specific immunogens, innate immunity is usually immediate, non-specific and memoryless, which is completely different from acquired immunity with immunogen specificity and memory.
  • ISCOM Immunostimulating Complex
  • Kozak sequence A nucleic acid sequence present on the mRNA of a eukaryote, usually (gcc)gccRccAUGG. The Kozak sequence plays an important role in initiating the translation process.
  • Leader sequence The nucleotide sequence at the 5'end of the messenger RNA (and DNA) located upstream of the translation initiation codon.
  • Liposomes tiny spheres that wrap water droplets of phospholipid molecules, especially artificially formed liposomes that transport vaccines, drugs, or other substances into tissues.
  • Nanoparticles Microparticles smaller than 100 nanometers can not only improve the stability and immunogenicity of vaccines, but also effectively deliver and slow release.
  • Packaging cell line The recombinant vector is transfected into the packaging cell line to supplement the viral genes missing from the recombinant viral vector, thereby generating a recombinant virus containing the transgene.
  • Polyadenylation sequence (polyA tail): Adding multiple adenosine monophosphates to messenger RNA is part of the pre-translation messenger RNA (mRNA) maturation process.
  • Promoter A site in a DNA molecule where RNA polymerase and a transcription factor combine to initiate the transcription of mRNA by a specific gene.
  • Replication-defective vector Refers to the fact that a critical part of the viral genome has been deleted, making the viral vector unable to replicate.
  • Shuttle plasmid A plasmid that can reproduce in two different host species.
  • Signal peptide A short peptide (5-30 amino acids in length) that exists at the N-terminus of most newly synthesized proteins and eventually enters the secretory pathway.
  • Start codon is the first codon of the messenger RNA (mRNA) transcript translated by the ribosome. In eukaryotes, the start codon always encodes methionine, while in prokaryotes, the start codon always encodes modified methionine (fMet). The most common start codon is AUG.
  • the SV40polyA sequence is a terminator sequence, indicating the end of a transcription unit.
  • a protein tag is a peptide sequence that a gene is grafted onto a recombinant protein, especially to facilitate purification.
  • a polyhistidine tag is bound to a nickel column so that the protein can be purified by affinity chromatography.
  • a protein tag is a peptide sequence fused to a recombinant protein, especially to facilitate its purification.
  • a polyhistidine tag is combined with a Ni 2+ column, enabling protein purification by affinity chromatography.
  • T cell subpopulation A subset of lymphocytes with specific immune functions to the immune response.
  • CD4+ helper T cells are indispensable for antibody production. It also participates in the pro-inflammatory response by releasing soluble immunostimulatory mediators such as cytokines and chemokines.
  • Type 1 helper T cells (Th1) are the cells necessary for the host to resist intracellular viruses and bacterial pathogens and produce interferon gamma (IFN- ⁇ ).
  • Type 2 helper T cells (Th2) play an important role in the host's resistance to extracellular pathogens and secrete IL-4.
  • Cytotoxic CD8+ T cells are a subset of lymphocytes responsible for killing infected cells and secreting IFN- ⁇ .
  • TLR agonist an agent that can activate immune cells by interacting with homologous TLR receptors, thereby promoting and coordinating the initiation of innate immunity and adaptive immunity.
  • TLRs Toll-like receptors
  • Transcription terminator A portion of the nucleic acid sequence that marks the end of a gene or operon in genomic DNA during transcription.
  • Transfection The process of introducing nucleic acids into mammalian cells. There are many different methods and techniques, including lipofection and chemical and physical methods, such as electroporation.
  • Transformation Insert foreign plasmids or ligation products into bacteria such as E. coli.
  • Viral vectors tools commonly used by molecular biologists to deliver genetic material into cells. This process can be carried out in living organisms (in vivo) or cell culture (in vitro). Viruses have evolved specialized molecular mechanisms to efficiently transport their genomes, enabling them to transfer genes and other genetic material within the cells they infect.
  • Figure 1 Schematic diagram of the three-dimensional structure of modified flagellin protein interacting with Toll-like receptors using Phyre2 software. (Reference: Phyre2 web portal for protein modeling, prediction and analysis. Kelley LA et al., Nature Protocols 10, 845-858, 2015)
  • Figure 2 Schematic diagram of three-dimensional computer simulation of the interaction of gE-flagellin fusion protein with Toll-like receptors.
  • the computer predictive immunogen design method is as follows: first, the Phyre2 webpage is used to generate the enveloped glycoprotein E model of varicella-zoster virus (strain Dumas; UniProtKB P09259), and then the signal peptide secretion sequence and transmembrane region of VZVgE are removed from the protein model And the intracellular region, and then select the flagellin protein sequence of Salmonella typhimurium (strain) LT2; UniProtKB P06179 based on the information in the database PDB ID's 3v47 and 3a5x (Yoon S-il et al., Science, 335: 859-864, 2012).
  • FIG. 3 The naming abbreviation of recombinant adenovirus vector carrying gE and gE-flagellin fusion genes and the legend of their corresponding inserted genes.
  • Js represents the Japanese encephalitis virus (JEV) prM leader peptide gene sequence.
  • Ig ⁇ refers to the mouse IgG ⁇ light chain leader peptide gene sequence.
  • FIG. 4A Western Bloting (WB) detection of recombinant adenovirus 1: rAd5-ACF (Js); 2: rAd5-ACF-SV40 (Js) 3: rAd5-ANF (Js); 4: rAd5-ANF-SV40 (Js) ; 5: rAd5-gE(Js); 6: rAd5-gE-SV40(Js)-expression of foreign genes in Vero cell supernatant after infection.
  • the primary antibody used in FIG. 4A is a mouse anti-VZV monoclonal antibody; the primary antibody used in FIG. 4B is a rabbit anti-flagellin D0, D1 polyclonal antibody.
  • M protein molecular weight markers.
  • FIG. 1 Western Bloting and SDS-PAGE analysis of foreign gene expression in 293A cell supernatant (S) and cell lysate (L) after recombinant adenovirus infection.
  • 5A Mouse anti-VZV gE monoclonal antibody as primary antibody, WB test result;
  • 5B Rabbit anti-flagellin D0, D1 antiserum as primary antibody, WB test result; 5C.
  • gE represents rAd5 -gE-SV40 (Js) infected HEK293 cell supernatant (S) and cell lysate (L);
  • ANF stands for rAd5-ANF-SV40 (Js) infected HEK293 cell supernatant (S) and cell lysate ( L);
  • ACF stands for rAd5-ACF-SV40 (Js) infected HEK293 cell supernatant (S) and cell lysate (L);
  • ASF stands for rAd5 ASF (Js)-infected HEK293 cell supernatant (S) and Cell lysate (L).
  • Figure 6A WB identification results of purified recombinant adenovirus (using rabbit anti-Ad5 polyclonal antibody as the primary antibody).
  • M molecular weight Marekers; lane 1: purified rAd5-gE-SV40 (Js) virus; lane 2: purified rAd5-ANF-SV40 (Js) virus; lane 3: purified rAd5-ACF-SV40 (Js) )virus.
  • Lane 4 purified rAd5-ASF (Js) virus.
  • Lane 5 purified rAd5-SE (Ig ⁇ ) virus. 6B.
  • TEM Transmission electron microscope
  • 6C Anion exchange-high performance liquid chromatography (agilent 1260) analysis of purified rAd5-gE-SV40(Js) virus. Load 40 ⁇ l of purified virus sample onto a column (4.8) equilibrated with 90% mobile phase A (20 mM Tris, pH 8.0) and 10% mobile phase B (20 mM Tris, 1 M NaCl, pH 8.0) x 250mm Sepax SAX-NP5 anion exchange column Sepax, China).
  • FIG. 7 Prokaryotic expression of his-tagged recombinant gE and gE-flagellin fusion protein naming abbreviations and their corresponding inserted gene legends.
  • FIG. 8A SDS-PAGE and Western Bloting detection of recombinant gE and recombinant gE-flagellin fusion protein expressed in purified E. coli.
  • 8B Results of SDS-PAGE; 8B. Results of using mouse anti-VZV-gE monoclonal antibody as primary antibody WB; 8C. Results of using rabbit anti-flagellin D0, D1 antiserum as primary antibody WB results.
  • M protein molecular weight markers; lane 1: purified gE protein; lane 2: purified ENF protein; lane 3: purified ESF protein; lane 4: purified ECF protein.
  • Figure 9 SDS-PAGE and Western Bloting detection of recombinant gE and recombinant gE-flagellin fusion protein expressed in purified Vero cells.
  • 9A SDS-PAGE test result;
  • 9B Use mouse anti-VZV-gE monoclonal antibody as the primary antibody WB result;
  • 9C Use rabbit anti-flagellin D0, D1 antiserum as the primary antibody WB result.
  • M protein molecular weight markers; lane 1: purified gE protein; lane 2: purified ANF protein; lane 3: purified ASF protein; lane 4: purified ACF protein.
  • FIG. 10 Detection of VZV-gE specific antibodies in the serum of mice immunized with recombinant adenovirus.
  • Various recombinant adenoviruses (10 9 TCID 50 /dose) or commercial varicella vaccine (700 pfu/dose) were immunized into C57BL/6 mice by intramuscular injection, with a total of two doses, and the immunization interval was 30 days. Serum was collected on days 12, 26, and 42 after the first immunization, and gE-specific antibody titers were detected using an enzyme-linked immunosorbent assay (ELISA method) as described in the materials and methods.
  • the results of the gE-specific antibody reaction are expressed as geometric mean titers (GMT), with a 95% upper and lower confidence interval. *** p ⁇ 0.001 (ANOVA/Bonferroni one-way analysis of variance).
  • FIG 11. Analysis of antibody-mediated neutralizing VZV virus infection activity in the serum of mice immunized with recombinant adenovirus.
  • Various recombinant adenoviruses (10 9 TCID 50 /dose) or commercial varicella vaccine (700 pfu/dose) were immunized into C57BL/6 mice by intramuscular injection, with a total of two doses, and the immunization interval was 30 days. Thirty days after the second immunization, mouse sera were collected and the titer of VZV specific neutralizing antibody was detected. The average value of the multiwell determination is taken to indicate the neutralizing antibody titer. Calculate the dilution factor that can reduce the number of plaques by 50%, and take the reciprocal to indicate the neutralizing antibody titer. ** p ⁇ 0.01, *** p ⁇ 0.001 (ANOVA/Bonferroni one-way analysis of variance).
  • FIG. 12 Flow cytometry analysis of gE-specific CD4+ and CD8+ T cell responses induced by recombinant adenovirus.
  • Spleen cells were collected 36 days after the second immunization, and the splenocytes were stimulated with 15 overlapping polypeptide mixtures (2 ⁇ g/peptide) covering the entire gE extracellular region.
  • intracellular factor staining (ICS) flow cytometry analysis was performed with fluorescently labeled anti-IFN- ⁇ antibody. The results were expressed in terms of the percentage of CD4+ and CD8+ T cells expressing IFN- ⁇ , with an upper and lower confidence interval of 95%. **p ⁇ 0.01, ****p ⁇ 0.0001 (ANOVA/Bonferroni one-way analysis of variance).
  • the negative control was unstimulated spleen cells, and the positive control was PMA (50ng).
  • FIG. 13 Elispot analysis of recombinant adenovirus-induced T cells producing IFN- ⁇ and IL-4. 36 days after the second immunization, spleen cells were collected, 15 spectacular polypeptide mixtures (2 ⁇ g/peptide) covering the entire extracellular region of gE were used to stimulate the spleen cells, and the number of T cells producing IFN- ⁇ and IL-4 was analyzed. The results are expressed as the average of the number of spots/ 5 ⁇ 105. * p ⁇ 0.05, ** p ⁇ 0.01, **** p ⁇ 0.0001 (ANOVA/Bonferroni one-way analysis of variance). The negative control was the unstimulated empty vector adenovirus group spleen cells, and the positive control was PMA (50ng).
  • FIG. 14 gE-specific antibody titers induced by gE and gE-flagellin fusion proteins.
  • C57BL/6 mice were immunized with gE protein (5 ⁇ g/dose) or gE-flagellin fusion protein (8 ⁇ g/dose) with or without MF59 adjuvant (50 ⁇ l/dose), immunized with two doses, and the immunization interval was 14 days. 14 days after the second dose of immunization, immune sera were collected and the gE-specific antibody titer was detected by ELISA. *** p ⁇ 0.001, **** p ⁇ 0.0001 (ANOVA/Bonferroni one-way analysis of variance).
  • FIG. 15 Antibody-mediated VZV infection activity neutralizing titer in gE or gE-flagellin fusion protein (with or without MF59 adjuvant) immune serum.
  • C57BL/6 mice were immunized with gE protein (5 ⁇ g/dose) or gE-flagellin fusion protein (8 ⁇ g/dose) with or without MF59 adjuvant (50 ⁇ l/dose) or commercial varicella vaccine.
  • the immunization interval is 14 days. 14 days after the second dose of immunization, immune sera were collected, and the sera of each group of mice were combined two by two to detect VZV specific neutralizing antibody titers. The average value of the multiwell determination is taken to indicate the neutralizing antibody titer. Calculate the dilution factor that can reduce the number of plaques by 50%, and take the reciprocal to indicate the neutralizing antibody titer.
  • * p ⁇ 0.05 ANOVA/Bonferroni one-way analysis of variance).
  • FIG. 16 Elispot analysis of gE and gE-flagellin fusion proteins (with or without MF59 adjuvant) induced T cells producing IFN- ⁇ and IL-4.
  • Spleen cells were stimulated with 15 overlapping polypeptide mixtures (2 ⁇ g/peptide) covering the entire extracellular region of gE and the number of T cells producing IFN- ⁇ and IL-4 was analyzed. The results are expressed as the average of the number of spots/ 5 ⁇ 105.
  • * p ⁇ 0.05, ** p ⁇ 0.01 ANOVA/Bonferroni one-way analysis of variance).
  • the negative control was spleen cells immunized in the saline group, and the positive control was PMA (50ng).
  • Mouse anti-VZV-gE monoclonal antibody was purchased from Merck (USA), rabbit anti-flagellin D0-D1 antibody was prepared by immunizing rabbits. Three synthetic peptides from flagellin D0 and D1 domains (see Table 1.) were combined with carrier protein (CCH, Thermo Fisher Scientific). Immune process: first dose, 0.4 mg conjugate with complete Freund's adjuvant, intramuscular injection, second and third dose, with 0.2 mg conjugate containing incomplete Freund's adjuvant, intramuscular injection; finally with 0.1 mg conjugate Venous impact; immunize rabbits separately.
  • Rabbit Anti-Ad5 monoclonal antibody was purchased from Abcam (UK). Cell culture flasks and pipettes were purchased from Corning Corporation (USA).
  • Endotoxin-free flagellin protein was purchased from Alpha Diagnostics (USA); IL-8 and TNF- ⁇ content ELISA kits and Elispot kits were purchased from Daktronics; guinea pig complement serum used in neutralizing antibody detection experiments was purchased from BD Corporation (United States). All antibodies used in flow cytometry were purchased from Thermo Fisher.
  • the commercial live attenuated varicella vaccine is produced by Changchun Qijian (China) or Changchun Baike Company (China).
  • Adenovirus titer-TCID 50 method is :
  • a flask (T-75 flask) of 293 cells containing 90% confluence grown in DMEM medium containing 10% FBS was taken. The day before the measurement, after washing with PBS, 1x TypLE was added for digestion for 2 mins, and DMEM medium containing 2% FBS was added to terminate the digestion, and the cells were resuspended in the same medium and counted.
  • the 96-well plate was placed in a CO 2 incubator at 37°C for 10 days, and then observed under an inverted microscope to determine and record the effect of cytopathic effect (CPE) in each column.
  • CPE cytopathic effect
  • the criterion for judgment is that as long as a small number of cells develop CPE, they are positive.
  • the virus titer was calculated according to the Karber method. ( G., Archiv f experiment Pathol u Pharmakol, 162:480-483, 1931).
  • THP-1 cells that expressed TLR5 receptor in the logarithmic growth phase and grown in RPM-1640 containing 10% FBS centrifuge at 125g for 5mins, discard the supernatant, and resuspend the cells in RPMI-1640 medium containing 10% FBS , Adjust the cell concentration to 1x10 7 cells/ml, inoculate in 96-well cell culture plate, 100 ⁇ l/well.
  • the positive control was diluted with RPMI-1640 medium solution containing 10% FBS to a final concentration of 2.5 ⁇ g/ml (endotoxin-free flagellin protein).
  • the purified gE-flagellin fusion protein with endotoxin content ⁇ 5EU/ml was diluted to equimolar concentration (5 ⁇ g/ml) with the same medium, and the purified gE protein was used as a negative control.
  • the diluted samples, endotoxin-free flagellin or gE were added to 96-well plates, 100 ⁇ l/well. Place the 96-well cell culture plate in a CO 2 incubator at 37°C for 12 to 24 hours. After the cultivation, the cells in each well were sucked out, centrifuged at 2000g for 10mins, and the cell supernatant was collected.
  • the activity of TLR5 was detected by detecting the content of IL-8 and TNF- ⁇ cytokines in the culture supernatant, and the cytokine content was operated according to the instructions of the IL-8 and TNF- ⁇ cytokine Elisa detection kit.
  • Enzyme-linked immunoassay to detect anti-gE antibody titer in serum :
  • the purified prokaryotic expression gE protein was diluted to 1 ⁇ g/ml with sterile sodium carbonate buffer (8.4 g/L NaHCO 3 , 3.5 g/L Na 2 CO 3 , pH 9.6), and 100 ⁇ l/well was added to 96-well microplate , Coated overnight at 4°C. The next day, the microplate was removed, the liquid in the well was discarded, and the plate was washed 3 times with PBST (PBS solution containing 0.1% Tween 20). Add blocking solution (PBST solution containing 10% skim milk powder) to each well and block at 37°C for 1 hour.
  • PBST PBS solution containing 0.1% Tween 20
  • the blocking solution was discarded, the immunized mouse serum was serially diluted with the blocking solution, and the blocking solution was set as a blank control.
  • the diluted serum was added to a 96-well plate at 100 ⁇ l per well, and three dilution wells of each diluted serum were made and incubated at 37°C for 1 hour.
  • 100 ⁇ l peroxidase (HRP)-labeled goat anti-mouse IgG antibody diluted 1:1000 was added to each well and incubated at 37°C for 1 hour.
  • TMB substrate (3,3',5,5'-tetramethylbenzidine, KPL, USA) was added.
  • 0.2M sulfuric acid was added to stop the reaction. The absorbance was measured with a microplate reader at a wavelength of 450 nm and a reference wavelength of 620.
  • Antibody-mediated neutralization titer determination procedure for neutralizing VZV virus infection activity is as follows: VZV virus is diluted with VZV dilution to 2 ⁇ 10 3 PFU/ml (phosphate buffered salt (PBS), sucrose 5%, glutamic acid) 1%, fetal bovine serum (FBS) 10%, pH 7.1). 150 ⁇ l of virus and 150 ⁇ l of serially diluted heat-inactivated serum and 5 ⁇ l of guinea pig complement were incubated at 37° C. for 1 hour. The virus serum mixture after incubation was added to a 24-well plate (100ul/well) overgrown with MRC-5 monolayer cells.
  • PBS phosphate buffered salt
  • FBS fetal bovine serum
  • Two dilution wells were made for each dilution and incubated at 37°C for 2 hours. After 2 hours, 2 ml of virus maintenance solution (MEM containing 2% FBS) was added. After 7 days, the medium was removed, the cells were fixed, stained with Coomassie blue solution (Coomassie blue 0.5%, methanol 45%, acetic acid 10%) for 10 minutes, and the plate was washed with distilled water and several spots were counted. Two duplicate wells were tested for each dilution. The reciprocal of the serum dilution that reduces the number of plaques by 50% is the neutralizing antibody titer.
  • the mouse spleen was aseptically removed and transferred to a cell strainer placed in a single well of a 6-well plate, 3 ml of culture medium (RPMI-1640, containing 5% FBS) was added, and the spleen cells were milled to release a 200-mesh cell screen. Filter the spleen. The cells were collected in a 15ml test tube and centrifuged at 350 ⁇ g at 4°C for 5min.
  • gE-specific cellular immunity was detected by Elispot of interferon- ⁇ (IFN- ⁇ ) and IL-4, using a mixture of 15 overlapping polypeptides covering the entire extracellular region of gE as a stimulus.
  • Elispot plates Dakco pre-coated with IFN- ⁇ or IL-4 antibodies were added to each well, and 200 ⁇ l of RPMI-1640 medium was added, and they were withdrawn after standing at room temperature for 10 minutes. Splenocytes adjusted to a final concentration of 2 ⁇ 8x 10 6 cells / ml. 100 ⁇ l of spleen cell suspension was mixed with polypeptide (each peptide concentration was 2 ⁇ g/ml), and three replicate wells were made for each sample.
  • AttB1-JEV-F GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGCCGCCGCCATGGGAAAACGGTCC
  • AttB2-SV40-R GGGGACCACTTTGTACAAGAAAGCTGGGTCAGACATGATAAGATACATTGATGAG
  • AttB2-GE-R GGGGACCACTTTGTACAAGAAAGCTGGGTCTTATTATTATCTGATCAGGGGGCTAG
  • AttB2-hOACF-R GGGGACCACTTTGTACAAGAAAGCTGGGTCTTATTATTACCTCAGCAGGCTCAG
  • AttB2-hOANF-R GGGGACCACTTTGTACAAGAAAGCTGGGTCTTATTATTATCTAATCAGAGGGCTAG
  • F represents the forward primer and R represents the reverse primer;
  • R represents the reverse primer;
  • the forward primers used to amplify the gE and gE-flagellin genes with or without SV40polyA are the same, namely AttB1-JEV -F;
  • the reverse primers used to amplify the SV40polyA-containing gE and gE-flagellin genes are AttB2-SV40-R.
  • the gene fragments shown in 1.1 were subjected to gene synthesis, and each target gene fragment synthesized by the gene was amplified with high-fidelity DNA polymerase (for amplification primer sequences, see Table 2. in 1.1.2). After PCR amplification, the PCR products were detected by 1% agarose gel electrophoresis, and the target DNA fragments were respectively recovered by DNA gel recovery kit.
  • PCR cycle conditions first step: 95°C, 2mins; second step, 95°C, 15s , 55 °C, 15 s, 72 °C, 1min 30s, a total of 30 cycles, the third step, 72 °C, 5mins.
  • the recovered DNA fragments of interest and pDONR221 plasmid were used for BP recombination (Thermo Fisher Scientific, Cat 11789020), the recombination mixture was transformed into E. coli TOP10 competent cells and coated with Kana-resistant solid LB plates. Extract the plasmid and send it for sequencing.
  • the prepared TOP10/pDONR221-Js-ASF-SV40plyA, TOP10/pDONR221-Js-ACF-SV40plyA and TOP10/pDONR221-Js-ANF-SV40plyA were deposited in the Chinese Typical Culture Collection Center on September 10, 2019 ( CCTCC) for deposit, the deposit numbers are: CCTCC M 2019707, CCTCC M 2019708 and CCTCC M 2019709.
  • the recombinant pDONR221 plasmid sequenced correctly and the target plasmid pAd5-CMV/V5-DEST were used for LR recombination (Thermo Fisher Scientific, Cat 11791020).
  • the recombinant mixture was transformed into E. coli TOP10 competent cells and plated with solid LB plates containing Ampicillin (Amp, 100 ⁇ g/ml) resistance.
  • pAd5-CMV plasmids which may contain different pAd5-CMV plasmids, and carry these pAd5-CMV plasmids (referred to as pAd5-) with or without SV40 polyA fusion gE or gE-flagellin fusion genes. CMV (VZV)).
  • the picked colonies were cultured in LB medium containing Amp resistance. Extract the plasmid and sequence.
  • VZV pAd5-CMV
  • the prepared TOP10/pAd5-Js-gE-SV40plyA was deposited on September 10, 2019 at the China Type Culture Collection (CCTCC), and the deposit number is CCTCC M 2019710.
  • the plasmids obtained in 1.2.3 were digested with PacI restriction enzyme (NEB, USA) at 37°C for 3h.
  • the digestion system was as follows: pAd5-CMV (VZV) plasmid: 10 ⁇ g, 10*NEB CutSmart buffer: 5 ⁇ l , PacI enzyme: 5 ⁇ l, add ddH 2 O to a final volume of 50 ⁇ l.
  • the PCR product recovery kit is used to recover the digested DNA fragments. And quantify the recovered DNA fragments with a micro nucleic acid quantifier.
  • PacI-linearized pAd5-CMV (VZV) plasmids were transfected into HEK293 cells in 6-well plates with 60-70% confluence, respectively. 2h before transfection, the medium was replaced with antibiotic-free medium, and the DNA/liposome complex was added. Five hours after transfection, the medium was replaced with DMEM medium containing 10% FBS and 1% double antibody.
  • Viral RNA/DNA extraction kit for the initial virus amplification preservation solution, according to the operating instructions, extract viral genomic DNA, PCR amplify the extracted viral genomic DNA, and identify the VZV gE or gE inserted into the recombinant adenovirus vector -Flagellin fusion gene.
  • Primer T7-F/V5-CR
  • PCR conditions 1 ⁇ l of viral DNA, 0.5 ⁇ l of forward and reverse primers, 5 ⁇ l 2 ⁇ PrimerSTAR mix, ddH 2 O 3 ⁇ l
  • cycling conditions first step: 95°C, 2min; second Step, 95 °C, 15 s, 45 °C, 15 s, 72 °C, 1min 30 s total 30 cycles; the third step, 72 °C, 5 mins.
  • the PCR products are electrophoresed on a 1% agarose gel, and the target band is recovered by gel cutting and sent to a sequencing company for sequencing.
  • the gE protein and gE-flagellin fusion protein can be specifically recognized by mouse anti-VZV gE monoclonal antibody. And gE-flagellin fusion protein can be specifically recognized by anti-flagellin polyclonal antibody.
  • HEK293 cells with 90% confluence were inoculated with different recombinant adenoviruses according to MOI 0.01 ⁇ 1, and the cells were continuously cultured in a 37°C, 5% CO 2 incubator until more than 70% of the cells became round and shed
  • the cells were scraped off with cells, 2265g, centrifuged for ten minutes, and the supernatant and cell pellet were harvested separately.
  • the cell pellet was resuspended in PBS, and placed in a refrigerator at -80°C for repeated freezing and thawing three times, 2265g, and the supernatant was harvested by centrifugation for ten minutes, which was used for the next purification.
  • the genes shown in 2.1 were inserted into NcoI and XhoI and inserted into the pET28a vector after the same enzyme digestion. After ligation and transformation, pick a single clone and inoculate LB medium containing kanamycin (50 ⁇ g/ml) resistance overnight. After cultivation, the plasmid was extracted and sent to a sequencing company for sequencing. The pET28a-gE, pET28a-ENF, pET28a-ECF, pET28a-ESF expression plasmids were obtained.
  • the correctly sequenced pET28a-gE, pET28a-ENF, pET28a-ECF, pET28a-ESF plasmids were transformed into BL21 (DE3) competent cells, and the single clones were picked and inoculated into LB medium containing kanamycin resistance, 37°C, 200rpm Incubate overnight. The next day, the strains were transferred to fresh LB medium containing kanamycin resistance. Incubate at 37°C and 200 rpm for 4 hours. When the OD 600 reaches 0.6 to 0.8, add 0.1 to 1 mM IPTG to induce expression. The expression temperature is 16 to 37°C to induce 4 to 16 hours. Harvest the cells for further purification.
  • inclusion bodies After crushing the collected microbial cells with a high-pressure homogenizer, centrifuge at 2,265xg for 10 minutes to collect inclusion bodies.
  • the inclusion bodies were washed with physiological saline containing detergents 3 to 4 times, and then added with 20 mM Tris, 5 mM imidazole, 500 mM NaCl, pH 8.0 buffer containing 6 M guanidine hydrochloride or 8 M urea for dissolution.
  • the cleaned nickel column was equilibrated with 5 column volumes (CV) with equilibration solution A (20mM Tris, 8M Urea, 5mM imidazole, 500mM NaCl, pH 8.0). Load the dissolved inclusion body onto the nickel column.
  • the eluent B solution is 20mM Tris, 8M Urea , 500mM, imidazole, 500mM NaCl, pH 8.0. Collect each elution peak separately.
  • Dialysis renaturation The purified inclusion bodies (dissolved in 8M Urea) were gradually dialyzed into a PBS solution containing 6M, 4M, 2M Urea using a dialysis bag. Change the dialysate every 2h. Finally, the purified inclusion body protein was slowly dialyzed into PBS solution.
  • On-column renaturation After the inclusion body has been loaded, the column is rinsed with 5CV of equilibration solution A; use a linear gradient of 20CV ⁇ 40CV to 100% renaturation solution B for on-column renaturation.
  • the renaturation solution B is: 20mM Tris+ 2M Urea+5mM imidazole+500mM NaCl+0.1mMGSGS/1mMGSH, pH: 8.0.
  • the column 5CV is rinsed with buffer C (20 mM Tris, 2M Urea, 5 mM imidazole, 500 mM NaCl, pH 8.0).
  • buffer C (20 mM Tris, 2M Urea, 5 mM imidazole, 500 mM NaCl, pH 8.0).
  • a 20CV linear gradient to 100% eluent D was used for elution.
  • the eluent D was: 20mM Tris + 2M Urea + 5mM imidazole + 500mM NaCl, pH: 8.0. Collect each elution peak separately. The collected elution peak was dialyzed into PBS solution using a dialysis bag.
  • the purified gE protein has a molecular weight of about 58Kd and the gE-flagellin fusion protein is about 90Kd. Except for the ECF protein, the purity of other proteins after purification is more than 80%.
  • Each protein can be specifically recognized by the mouse anti-gE monoclonal antibody, and the gE-flagellin fusion protein can be specifically recognized by the rabbit anti-flagellin D0-D1 antiserum.
  • the protein concentration was detected by the BCA method.
  • the yield of gE protein was 15 mg-20 mg/L, and the yield of gE-flagellin fusion protein was 8-15 mg/L. Due to the contamination of the remaining lipopolysaccharide after purification (LPS, an adjuvant that interferes with flagellin activity determination) and the degradation of some proteins, the immunogen from E. coli and the eukaryotic system (recombinant adenovirus vector) were not compared The corresponding protein expressed immunogenicity gap. However, one of ordinary skill in the art should be able to optimize yield, prevent or minimize protein hydrolytic degradation, and significantly reduce residual LPS content. The protein of prokaryotic expression is not further optimized in the present invention because the present invention uses the adenovirus eukaryotic expression system to obtain a complete, high-yield and LPS-free recombinant protein.
  • the equilibration buffer is 10 mM PBS + 500 mM (NH 4 ) 2 SO 4 , pH: 7.5.
  • solution B is 10mM PBS, pH: 7.5. The elution peak at 100% B was collected.
  • the purified gE and gE flagellin fusion protein were analyzed by SDS-PAGE (see Figure 9A).
  • the purity of the purified gE protein was above 95%, and the purity of the purified gE-flagellin fusion protein was above 85%.
  • the protein content after purification by BCA detection shows that the yield of gE protein expressed by this method can reach 100 mg/L, and the yield of gE-flagellin fusion protein is 50-80 mg/L.
  • the recombinant protein prepared in the present invention is soluble in an aqueous solution with a concentration ranging from 100 ⁇ g to 5 mg/ml, such as phosphate buffer (pH 7.0-7.5) or 4 mM acetate buffer (pH 5.4) aqueous solution.
  • phosphate buffer pH 7.0-7.5
  • 4 mM acetate buffer pH 5.4
  • the purified protein was analyzed by WB (see Figures 9B and 9C). Both the gE protein and the gE-flagellin fusion protein can be specifically recognized by the mouse anti-gE monoclonal antibody. Only gE-flagellin fusion protein can be specifically recognized by rabbit anti-flagellin D0-D1 antiserum, but not gE protein.
  • TLR-5 activity analysis shows that ANF, ACF, and ASF fusion proteins can induce THP-1 cells to secrete higher concentrations of IL-8 in a dose-dependent manner by activating THP-1 and TLR-5 receptors. And TNF- ⁇ factor.
  • the gE protein prepared and purified by the same method cannot induce the secretion of TLR-5 active cytokines. It shows that these three gE-flagellin fusion proteins have the specific activity of flagellin protein through TLR-5.
  • the flagellin activity of ASF is basically the same as that of commercial flagellin protein.
  • mice All animal experiments were conducted in accordance with the protocol approved by the Hubei Provincial Food and Drug Safety Evaluation Center and the Animal Protection and Utilization Committee (IACUC). Thirty-six female C57BL/6 mice without special pathogens (SPF grade) weighing 12 to 16g were raised in the Hubei Provincial Food and Drug Safety Evaluation Center. After the inspection and quarantine, the mice were randomly divided into 6 groups according to body weight, and were intramuscularly inoculated with 10 9 TCID 50 /dose recombinant adenovirus A, recombinant adenovirus B or 700 pfu commercial VZV vaccine (Changchun) on day 1 and day 28, respectively. Qi Jian Biotechnology Co., Ltd., China). Table 4 summarizes the grouping details. Blood was collected from the orbital venous plexus on days 0, 12, 42, and 56 respectively.
  • IACUC Animal Protection and Utilization Committee
  • Serum anti-gE IgG antibody The anti-gE IgG antibody titer in the serum after immunization was detected by ELISA. The test results are shown in Figure 10 and Table 5. The empty vector control mice were not detected at 12, 42, 56 days after immunization. The antibody titer increased. In the remaining groups at 109 doses, antibody titers increased significantly 12 days after immunization. After the second dose of booster immunization, antibody titer levels further increased. The antibody levels of different recombinant adenoviruses carrying the gE-flagellin fusion protein on the 12th day after immunization were significantly higher than those of the rAd5-gE group and the commercially available varicella vaccine group.
  • the antibody levels of the recombinant adenovirus groups were significantly different from those of the commercial vaccine group (p ⁇ 0.001).
  • Serum neutralizing antibody titers As shown in Figure 11, at a dose of 109 , each recombinant adenovirus group induced higher levels of neutralizing antibody 56 days after the first dose of immunization.
  • the levels of neutralizing antibodies induced by rAd5-ACF group were significantly different from those of other recombinant adenovirus groups and commercially available VZV vaccines (p ⁇ 0.001). Although there were no significant differences among the remaining groups, the level of induced neutralizing antibodies was comparable to that of commercially available live attenuated vaccines.
  • the levels of neutralizing antibodies induced by the rAd5-ANF and rAd5-SE groups were not statistically different from the rAd5-gE group, but they were more consistent and uniform.
  • Detection of cellular immunity The results of intracellular cytokine staining are shown in Figure 12. After immunizing C57BL/6 mice with recombinant adenovirus for 8 weeks, VZVgE-specific CD4+ T cell immunity can be detected in the rAd5-gE group and rAd5-SE group. The percentage of IFN- ⁇ positive cells in CD4+ and CD8+ T cells of both was significantly higher than that of empty adenovirus control group (P ⁇ 0.01 or P ⁇ 0.0001). As shown in Figure 13, the results of IFN- ⁇ Elispot detection are shown in Figure 13 further confirming the results of intracellular cytokine staining.
  • the number of IFN- ⁇ and IL-4 spots in spleen cells of rAd5-gE group and rAd5-SE group were significantly different from those of other experimental groups (P ⁇ 0.01 or P ⁇ 0.0001).
  • the rAd5-gE group was also significantly different from the commercially available vaccine group (P ⁇ 0.05). This indicates that the rAd5-gE and rAd5-SE groups can induce strong CD4+Th1 and Th2 cell immune responses, and can also induce strong CD8+T cytotoxic cell immune responses.
  • mice All animal experiments were conducted in accordance with the protocol approved by the Hubei Provincial Food and Drug Safety Evaluation Center and the Animal Protection and Utilization Committee (IACUC). 60 female C57BL/6 mice without special pathogens (SPF grade), weighing 12-16g, were raised in the Hubei Food and Drug Safety Evaluation Center. After the inspection and quarantine, the mice were randomly divided into 10 groups according to body weight, and were intramuscularly inoculated with and without MF59 (50 ⁇ l/dose) adjuvant gE protein (5 ⁇ g/dose) or with and without MF59 on day 1 and 14 respectively.
  • IACUC Animal Protection and Utilization Committee
  • GE-flagellin fusion protein (8 ⁇ g/dose) containing MF59 adjuvant or 700 pfu commercial VZV vaccine (Changchun Qijian Biotechnology Co., Ltd., China). Table 6 summarizes the grouping details. Blood was collected from the orbital venous plexus on days 0 and 28, respectively.
  • Quantity Negative control group Saline / Intramuscular injection, 0.1ml/piece 6 gE gE 5 Intramuscular injection, 0.1ml/piece 6 ANF ANF 8 Intramuscular injection, 0.1ml/piece 6 ACF ACF 8 Intramuscular injection, 0.1ml/piece 6 ASF ASF 8 Intramuscular injection, 0.1ml/piece 6 gE+MF59 gE+MF59 5+50 ⁇ l Intramuscular injection, 0.1ml/piece 6 ANF+MF59 ANF+MF59 8+50 ⁇ l Intramuscular injection, 0.1ml/piece 6 ACF+MF59 ACF+MF59 8+50 ⁇ l Intramuscular injection, 0.1ml/piece 6 ASF+MF59 ASF+MF59 8+50 ⁇ l Intramuscular injection, 0.1ml/piece 6 ASF+MF59 ASF+MF59 8+50 ⁇ l Intramuscular injection, 0.1ml/piece 6 Positive vaccine LicensedVZV 700PFU Intramuscular injection
  • Serum anti-gE IgG antibody titer On the 28th day after immunization, that is, on the 14th day after the second dose of immunization, the serum of mice was tested by ELISA for gE-specific antibody titer. The results are shown in Figure 14 and Table 7.
  • the gE-specific antibody titer of ACF group was significantly increased and had statistical difference compared with saline group and gE group.
  • the gE-specific antibody titers of the ANF and ASF groups also increased significantly, which was statistically different from the saline group; and the gE-specific antibody titers were also higher than the gE group.
  • Serum neutralizing antibody titer As shown in Figure 15, 14 days after the second dose of immunization, the level of neutralizing antibody induced by the ACF group containing MF59 adjuvant was significantly higher than that induced by the gE protein group containing MF59 adjuvant. Antibody levels. Although the other two gE-flagellin fusion proteins containing MF59 adjuvant showed no significant difference in neutralizing antibody levels compared to the gE protein group containing MF59 adjuvant, the gE-flagellin fusion protein group containing MF59 adjuvant induced And the antibody level is still higher than the MF59 adjuvant gE protein group.
  • the level of neutralizing antibody induced by the gE-flagellin fusion protein group containing MF59 adjuvant is comparable to that induced by the commercially available live attenuated varicella live vaccine.
  • the neutralizing antibody responses induced by the ASF and ACF groups containing MF59 adjuvant were more consistent and uniform than those induced by the commercial vaccine group.
  • the present invention discloses a method of preparing and implementing new immune components that can be used to prevent vaccines against VZV infection and induce a wide range of protective humoral and cellular immunity.
  • the immune component selects the VZV-gE glycoprotein as the immunogen because the gE protein is the most abundant and most immunogenic protein in the VZV virus.
  • the immune component of the present invention includes an adjuvant-containing recombinant VZV-gE protein and a gE-flagelin fusion protein with inherent adjuvant properties.
  • the immune component can be prepared by expression in a prokaryotic or eukaryotic expression system, or in a replication-deficient adenovirus vector expressing gE or gE-flagellin protein.
  • the part of the flagellin protein covalently linked to the gE protein through genetic engineering has been shown to bind and activate TLR5, thereby triggering innate immunity.
  • This fusion protein may not require further adjuvants in human vaccines, thereby reducing the risk of adverse reactions caused by adjuvants.
  • all immune components have a high degree of immunogenicity and can induce strong gE-specific antibodies and functional neutralizing antibodies in vitro related to protection; at the same time, the immune components can also be induced in bands CD4+Th1 and Th2 T cell immunity plays an important role in herpes prevention and rehabilitation.
  • the gE-flagellin fusion protein with its own adjuvant effect is more immunogenic than its corresponding gE protein whether it is directly purified or delivered via an adenovirus vector. If necessary, conventional adjuvants with much lower AS01 reactivity in Shingrix can be used to significantly improve the immunogenicity of the purified protein.
  • the non-replicating adenovirus vector expressing gE or gE-flagellin fusion protein can not only induce good gE-specific antibodies, VZV neutralization reaction and CD4+ T cell response, but also induce the body to produce CD8+ T cell immunity, which can Further destroy the cells infected by VZV.
  • Almost all immune components in the present invention are more immunogenic than commercially available live attenuated varicella vaccine.
  • the various immune components described in the present invention can be used as part of a priming-boost immunization strategy to enhance and expand VZV-specific immunity.
  • the various immune components can also be mixed with other immunogens and used in combination vaccines. These immune components are safer than commercially available live attenuated varicella vaccines because they are not contagious, do not cause accidental serious adverse events that may be related to use, and most importantly, do not expose vaccine recipients to Significant risk of developing herpes zoster and neuralgia.
  • the invention also discloses a method for expressing and preparing gE and gE flagellin protein fusion protein in a prokaryotic system, which can reduce the production cost of the vaccine.
  • the adenovirus vector disclosed in the present invention can also be developed as a single immunization vaccine, thereby reducing the frequency of immunization.
  • the immune components provided by the present invention can be used to produce safer, more effective and possibly cheaper new vaccines for the prevention and control of varicella and herpes zoster. It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, but should not be construed as limiting the present invention. Those skilled in the art can make further improvements and adjustments to the above content of the present invention, which belong to the protection scope of the present invention.

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Abstract

本发明提供了一种免疫组合物及其制备方法与应用。本发明通过原核表达体系或重组腺病毒体系高效表达VZV包膜gE糖蛋白及其gE鞭毛素融合蛋白。将制得的重组gE蛋白、gE鞭毛素融合蛋白及重组腺病毒载体或其组合物免疫小鼠后可刺激机体产生gE和VZV特异性抗体滴度及gE和VZV特异性的细胞免疫。

Description

免疫组合物及其制备方法与应用 技术领域
本发明涉及一种重组VZV gE糖蛋白及自身具有佐剂功能的融合蛋白、重组载体、制备方法、免疫组合物及其制备方法与应用。
背景技术
水痘-带状疱疹病毒(varicella-zoster virus，VZV)是疱疹病毒属(Herpesviridae)α疱疹病毒亚科成员，是一种直径150～200nm的双链DNA病毒，形态学上是由核酸内心、蛋白衣壳和包膜构成的同心圆状结构，表面由162个壳微粒组成的对称正二十面体。VZV是一种全球性病原体，有嗜皮肤和神经的特征。儿童原发性感染可导致水痘。水痘是一种通过皮肤接触或呼吸道飞沫传播的高传染性疾病，其特征是面部和躯干出现播散性水疱样皮疹，伴随瘙痒和发热。VZV原发性感染偶见内脏并发症，例如脑炎、肝炎、胰腺炎或肺炎等可能危及生命的严重并发症，尤其是在未接种疫苗的幼儿和成人以及免疫抑制人群中。初次感染后，VZV可终生潜伏于颅神经和背根神经节。数十年后，VZV仍可被重新激活，引起疼痛性带状疱疹(HZ)疾病或其他严重的神经系统并发症或眼部并发症，并可导致衰竭性带状疱疹愈后神经痛—最常见的带状疱疹慢性并发症。超过95％的50岁以上免疫力正常人群血清呈VZV抗体阳性，因而有罹患带状疱疹的风险。带状疱疹的终生发病风险在25％至30％之间，80岁后风险则上升到50％(Johnson RW et al.,Ther Adv Vaccines,3:109-120,2015)。在免疫受损的个体中，VZV感染的发生率和死亡率都很高。例如，在器官移植患者中，水痘可能是致命性的，伴随有严重的播散性皮损、脏器损伤和血管内凝血。带状疱疹(HZ)也是器官移植患者常见的并发症。VZV引起的疾病及其相关后遗症(如带状疱疹愈后神经痛)已逐渐成为重大的疾病负担及重要的公共卫生问题，并急需得到更多医疗方面的关注。
对于水痘和带状疱疹，主要是对症处理，无特效治疗方法。抗病毒药物，如阿昔洛韦、伐昔洛韦、泛昔洛韦等，虽有助于水痘及HZ患者康复，但不能预防VZV感染。而暴露VZV后给予病毒特异性免疫球蛋白，对于中止或减轻疾病负担也是有限的。研究发现，抗VZV膜抗原抗体滴度≥1/64被认为与疾病的保护相关，抗gE糖蛋白的抗体也被发现与长期保护有关。虽然感染可产生对病毒的终生免疫，但完整的细胞免疫对于从感染和复发性疾病中恢复至关重要，因为带状疱疹发生于随年龄增长T细胞免疫下降或免疫抑制的时候。CD4+T细胞增殖反应在带状疱疹预防和/或治疗中起到重要作用，不过迄今为止还未建立其与效力的相关性(Plotkin SA.，Clin Vaccine Immunol 17:1055-1065 2010)。
鉴于此，接种疫苗仍然是最有效和最可靠的预防和控制水痘和带状疱疹的手段。
水痘减毒活疫苗最早由日本的高桥(Takahashi)研究组于1974年研发成功。他们从1例3岁名叫Oka的水痘患儿体内分离出一株VZV，经人胚胎成纤维细胞、豚鼠成纤维细胞和人二倍体成纤维细胞连续传代后减毒。这一减毒活疫苗被称为Oka疫苗(vOka)。Oka疫苗目前已纳入多个国家的常规免疫计划。一般情况下，Oka疫苗十分安全，即使在免疫力部分损伤的儿童和人类免疫缺陷病毒感染儿童中也没有出现严重不良反应，还显示出良好的免疫保护效果。然而，Oka疫苗诱导的免疫保护的持久性不够长，并且一些个体在连续接种疫苗后不能达到有效的保护状态。且对青少年，Oka疫苗的免疫效果低于1～12岁的儿童，因此对于学龄前儿童，需要对该疫苗进行二次接种。目前市面上所有水痘疫苗均为减毒活疫苗，虽然罕见严重副作用，但也有报道反映接种疫苗后出现严重皮疹、肺部或肝脏感染、脑膜炎、惊厥、肺炎或全身性疫苗株严重感染，尤其是在免疫受损儿童中。
目前，带状疱疹疫苗有Merck的Zostavax以及GSK的Shingrix。Zostavax是浓缩版的Oka疫苗，于2006年获得美国FDA批准。它的有效性随着疫苗接种者的年龄下降，60岁以上人群不推荐使用，目前已被证明其能在大约五年内提供50％的保护，其效力在疫苗接种后5-8年逐步降低，且在疫苗接种8年后保护力不再具有统计学意义(Morrison VA,et al.,Clin Infect Dis,60:900-909,2015)。GSK的Shingrix采用基因重组技术，在中国仓鼠卵巢细胞中表达水痘带状疱疹病毒糖蛋白E，于2017年获得FDA批准，用于50岁及以上的人群。Shingrix对带状疱疹的保护率为90％，降低了带状疱疹愈后神经痛的风险，是Zostavax的首选替代品。但是，Shingrix使用的佐剂为GSK公司的AS01，具有副作用。
水痘减毒活疫苗给接种者带来多重风险，包括罕见但十分严重的并发症、传染给免疫受损个体及潜伏感染，更为重要地，30％接种者会遭受潜伏病毒被重新激活而导致带状疱疹发生。带状疱疹疫苗方面，中国国家药品监督局于2019年批准了Shingrix的进口注册申请，填补了国内带状疱疹疫苗的空白。但Shingrix具有副作用，国外售价大约在150美元/剂，而且目前只是针对于50岁及以上的人，由于副作用太强不能作为水痘疫苗用于儿童人群。因此，需进一步开发安全、副作用较低、无潜伏风险和带状疱疹愈后神经痛并发症风险以及更低价的改良VZV疫苗，但目前尚无进展。新型疫苗应既能引起强烈的体液反应来中和病毒，又可诱发广泛的细胞免疫以控制疾病。
VZV基因组的开放读码框架(openreadingframe，ORF)共编码8种糖蛋白：糖蛋白E(gE)、gB、gH、gI、gC、gL、gK和gM。其中gE糖蛋白由ORF68基因编码，属于I型膜蛋白，是生成感染性病毒颗粒必需的糖蛋白，也是病毒包膜中含量最丰富、免疫原性最强的糖蛋白，存在于病毒颗粒的表面及VZV感染细胞的胞质内，在病毒不同成熟阶段以不同的糖基化形式存在。在处于恢复期的水痘和带状疱疹患者血清中，VZV抗体主要针对gE、gB和gH。特异性抗gE单克隆抗体可中和VZV，介导抗体依赖性细胞毒性(ADCC)。gE还是细胞免疫的主要靶点，能控制疾病和破坏感染病毒的细胞。这些特性使得gE成为开发安全、有效广谱疫苗的理想免疫原。
非活性人用疫苗通常由一种或多种免疫原组成，制剂中加入可增强其效力的免疫佐剂。目前仅有有限数量的免疫佐剂可供人类使用，如铝盐、矿物油、植物或细菌提取物。免疫佐剂具有不同的增强特性，并可引起各种不良副作用。随着对免疫应答调节机制认识的不断深入，人们发现了表达于免疫系统前哨细胞(如树突状细胞和巨噬细胞)表面及表达于淋巴细胞、共同调节先天免疫和适应性免疫的Toll样受体(Toll-like receptors，TLRs)。TLR识别保守的微生物相关分子模式(MAMP)。激动剂触发TLR产生多种多效性免疫介质，如细胞因子和趋化因子，它们参与前炎症反应，刺激先天免疫，从而起到免疫佐剂的作用。
Toll样受体5(TLR5)是一种特异性识别细菌鞭毛素蛋白的跨膜受体。鞭毛素蛋白是革兰氏阴性菌鞭毛的主要结构蛋白。鞭毛素诱导激活TLR5，启动先天免疫，诱导单核-巨噬细胞、上皮细胞活化，并释放IL-1、IL-8和TNF-α等前炎症因子。因此，鞭毛素蛋白是一种强效的全身和粘膜免疫佐剂。它由四个结构域D0、D1、D2和D3组成，其中结构域D0和D1在变形菌门中高度保守。鞭毛素蛋白N端D0-D1的螺旋和C端D1-D0的螺旋之间的相互作用形成茎状核心结构，该结构对TLR5的结合和活化至关重要。与此相反，鞭毛素的D2和D3结构域在不同细菌之间差异很大，具有很强的免疫原性但在功能性上非必须。删除D2-D3结构域不会削弱TLR5的活化，且可最大程度降低无用的抗鞭毛素蛋白抗体应答。研究显示，鞭毛素必须与目标免疫原结合才能产生最佳的免疫佐剂效应。这可以通过构建一种自身具有佐剂效应的融合蛋白来实现，其中免疫原共价连接到鞭毛素或其保留TLR 5结合活性的功能片段上，并由此保留其固有免疫刺激属性。
发明内容
本发明是为了克服已上市疫苗的上述缺陷以及改善不良反应，采用两种的不同方法开发新型免疫组合物，一种是生产能够诱导强的中和抗体和CD4+T细胞反应，副作用更低的原性的重组gE蛋白或基于gE的融合免疫原；另一种是构建更加安全的复制缺陷型腺病毒载体来表达gE基因或gE鞭毛素融合蛋白基因，以引起中和抗体应答和更广泛的CD4+T细胞和CD8+T细胞免疫；从而获得了新的糖蛋白、融合蛋白、重组载体、制备方法、组合物，且可运用于抗VZV感染的新疫苗制备中。
本发明提供了一种免疫组合物，包含基于水痘带状疱疹病毒糖蛋白E(简称gE)的抗原，可用来预防或者治疗水痘带状疱疹病毒(VZV)感染。
在一些实施方案中，基于gE的免疫原至少包含：(i)gE胞外区或其片段，或者其相应的编码核酸分子；(ii)基于gE的融合蛋白，或者其编码核酸分子；(iii)基于gE的重组载体；或者(iv)上述两种或更多的组合。
进一步地，基于gE的融合蛋白至少包含：gE胞外区或其片段共价偶联至自身具有佐剂效应的细菌鞭毛素蛋白或其片段，其中所述细菌鞭毛素蛋白或其片段具有TLR-5激动活性。
VZV gE以及鞭毛素的氨基酸序列或核酸序列可以在公众可获得的如GenBank(GB)、SwissPro(sp)、EMBL等数据库中找到，gE代表性的数据库条目包括但不限于：GB AQT34120.1、AAG32558.1、ABE03086.1等，所述登记号代表的序列通过引用并入本发明。
gE糖蛋白是一种膜蛋白，其结构包含信号肽、胞外区、跨膜区和胞内区。胞外区暴露在细菌表面并是免疫系统识别的靶点。故应当理解为本发明中提及的gE至少包含其胞外区或其片段，如有需要可在保留gE一定的抗原活性基础上进一步包含其它结构片段如跨膜和/或胞内区。根据常识，本领域技术人员可以确定gE的各结构片段，gE胞外区片段可理解为保留一定的gE的自身免疫原性的片段。
应理解的是，在保留gE一定的免疫活性的情况下，可对其做一定微小的修饰，包括但不限于：突变、替换(如功能上类似的氨基酸的保守置换)、增加、缺失或截短等，仍视为本发明公开内容。
在一些实施方案中，gE胞外区具有与SEQ ID NO.1所示氨基酸序列至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同源性。
细菌鞭毛素蛋白是许多革兰氏阴性细菌(如大肠杆菌或沙门氏菌等)的主要鞭毛成分，其一级氨基酸序列在组成和大小上随着细菌种类的不同而不同。在不同的细菌中，保守的N端D0-D1区和C端D1-D0区的结构域相互作用形成了一个功能茎样结构，该结构是TLR5结合和信号传递必所必须的。中间“超变的”D2和D3区为TLR-5信号转导非必须的结构域，但分子中的“超可变”D2和D3结构域不是TLR 5信号转导所必需的，且具有很强的免疫原性并诱发不良反应，因此该部分区域可被删除且不会影响TLR5结合活性。本发明中的细菌鞭毛素蛋白可为原始的或经改造的鞭毛素蛋白。所述改造包括但不限于突变、替换(如功能上类似的氨基酸的保守置换)、增加、缺失或截短等，但应保留一定程度的TLR-5结合能力以激活天然免疫。应当理解为：本发明中的细菌鞭毛素蛋白或其片段不应引起显著的促炎性副作用。实际上，在公开的专利US2011110962A1和/或US2011230643A1中描述了鞭毛素蛋白的免疫性及其某些修饰，被引入到本发明中。在本发明中若无特殊说明，鞭毛素蛋白的N端是指其N端D0-D1区，鞭毛素蛋白的C端是指其C端D1-D0区。
在一些实施方案中，基于gE的融合蛋白至少包含：鞭毛素蛋白的N端区、鞭毛素蛋白的C端区，和gE胞外区或其片段。换言之，基于gE的融合蛋白还可包含鞭毛素蛋白或gE的其它片段
在一些具体的实施方案中，gE胞外区或其片段位于所述基于gE的融合蛋白的N端或C端；或者插入到鞭毛素蛋白N端和C端之间。
作为优选的实施方式：所述基于gE的融合蛋白选自如下任一融合形式：
融合形式1：鞭毛素蛋白N端区-鞭毛素蛋白C端区-gE胞外区或其片段；
融合形式2：gE胞外区或其片段-鞭毛素蛋白N端区-鞭毛素蛋白C端区；
融合形式3：鞭毛素蛋白N端区-gE胞外区或其片段-鞭毛素蛋白C端区；
其中，所述鞭毛素蛋白的N端区或C端区可直接或者通过连接体与gE胞外区或其片段相连；
所述鞭毛素蛋白N端区可直接或者通过连接体与鞭毛素蛋白C端区相连。
所述的连接体包括基因工程肽链(如1-20个肽键连接的氨基酸)和非肽化学接头(如烷基接头或聚乙二醇基团，其中烷基接头还可被非立体阻碍性的基团如卤素、CN、NH2等基团取代)。应当理解为所选择的连接体不会干扰所述融合蛋白的生物活性。
优选地，所述连接体为1-20个肽键连接的氨基酸，如连接体I或连接体II；连接体I如SEQ IDNO:4所示；连接体II如SEQ ID NO:7所示。
SEQ ID NO:4：SPGISGGGGGILDSMG
SEQ ID NO:7：GGGGSGGGGSGGGGS
在一些具体的实施方案中，所述鞭毛素蛋白的N端区或C端区分别通过连接体II与gE胞外区或其片段相连进行连接。
在一些具体的实施方案中，所述鞭毛素蛋白N端区通过连接体I与鞭毛素蛋白C端区相连
在一些实施方案中，所述的鞭毛素蛋白来自沙门氏菌，例如鼠伤寒沙门氏菌(Salmonella enterica subsp.enterica serovar typhimurium(S.typhimurium))或肠道沙门氏菌(Salmonella enterica subsp.enterica serovar typhi(S.typhi))，所述鼠伤寒沙门氏菌包括但不限于strain LT2；肠道沙门氏菌包括但不限strain Ty2。
在一些具体的实施方案中，所述鞭毛素蛋白的氨基酸序列如SEQ ID NO:3(源自strain LT2)或SEQ ID NO:29(源自strain Ty2)所示。虽然蛋氨酸是天然鞭毛素分子的N端第一位氨基酸，但本发明中鞭毛素蛋白的N端始于天然序列的第二氨基酸(Ala)。
本发明所述的鼠伤寒沙门氏菌LT2鞭毛素蛋白的N端区一般为起始于SEQ ID NO:3中的第2位丙氨酸(Alanine)，终止于137-176位氨基酸的任一位氨基酸；C端区一般为起始于第392-406任一位氨基酸，终止于495位氨基酸。
作为本发明一个具体的实施方式，所述鞭毛素蛋白的N端区为至少与SEQ ID NO:3中第2至176位氨基酸区域有95％同源性(例如97％,98％或99％的同源性)的氨基酸序列；C端区为至少与SEQ ID NO:3中第392至495位氨基酸区域有95％同源性(例如97％,98％或99％的同源性)的氨基酸序列。
在一个具体的实施方式中，所述鞭毛素蛋白的N端区的氨基酸序列如序列表SEQ ID NO:5所示：所述鞭毛素蛋白的C端区的氨基酸序列如序列表SEQ ID NO:6所示
本发明所述的肠道沙门氏菌Ty2鞭毛素蛋白的N端区一般为起始于SEQ ID NO:29的第2位丙氨酸(Alanine)，终止于180-200位氨基酸的任一位氨基酸；C端区起始于第278-400任一位氨基酸，终止于506位氨基酸。
在一些实施方式中，所述Ty2鞭毛素蛋白的N端区为SEQ ID NO:29的2-180，C端区为400-506位；或者Ty2鞭毛素蛋白的N端区为SEQ ID NO:29的2-220，C端区为320-506位；或者Ty2鞭毛素蛋白的N端区为SEQ ID NO:29的1-190，C端区为278-506位。
在本发明的一些具体的实施方式中，所述鞭毛素蛋白N端区为至少与SEQ ID NO:29中第2至180位氨基酸区域有95％(例如97％,98％或99％的同源性)同源性的氨基酸序列；C端保守区为至少与SEQ ID NO:29中第400至506位氨基酸区域有95％(例如97％,98％或99％的同源性)同源性的氨基酸序列。
在一个具体的实施方式中，所述的N端保守区的氨基酸序列如SEQ ID NO:30所示；所述的C端保守区的氨基酸序列如序列表SEQ ID NO:31所示。
本发明的一些具体的实施方式中，所述基于gE的融合蛋白的氨基酸序列如SEQ ID NO:8～10、SEQ ID NO:32-34任一所示。
本发明所述的核酸分子一般为根据表达系统优化后的核酸分子，包括但不限于：DNA，RNA，mRNA，ssDNA或cDNA。
所述核酸分子可通过操作与表达控制序列连接,所述表达控制序列包括但不限于：启动子、增强子、转录终止子、起始密码子(如ATG)、内含子的剪接信号和终止密码子等，其中可考虑使用体外和体内条件性表达控制元件。利用标准的分子生物学技术，可添加额外的基因片段，例如但不限于人巨细胞病毒的早期增强子、Kozak共识序列、先导序列、Woodchuck肝炎病毒转录后调控元件、编码糖基化受体序列的核酸序列、或无关蛋白如标记或切割位点等，所述可添加的无关蛋白包括用于优化基因表达、信息稳定性、蛋白质产量、分泌和纯化等。技术人员所熟知的多种基因的克隆和构建方法，以及宿主细胞的表达系统均可使用。本发明公开的编码蛋白质的DNA序列可以在原核和真核宿主细胞中表达。
在真核表达系统中，所述核酸分子5’端还可进一步添加核酸先导序列来促进蛋白分泌，所述核酸先导序列包括但不限于日本脑炎病毒(Japanese encephalitis virus，JEV)prM蛋白基因先导序列或小鼠Igκ轻链基因先导序列；和/或所述核酸分子的5’端还可添加Kozak序列以增强翻译效率；和/或所述核酸分子的3’端还可添加polyadelynation(polyA)序列增加核酸分子的稳定性；所述polyA序列包括但不限于SV40polyA。
优选地，JEV信号肽基因序列如SEQ ID NO:14所示。
优选地，Igκ信号肽基因序列如SEQ ID NO:15所示。
优选地，Kozak序列基因序列如SEQ ID NO:16所示。
优选地，SV40polyA基因序列如SEQ ID NO:17所示。
由核酸先导序列编码的信号肽在天然蛋白的细胞内的加工过程中被水解切割。
一些具体的实施方式中，编码所述gE胞外区或其片段的核酸分子如SEQ ID NO:2，18-19任一所示。
一些具体的实施方式中，编码所述基于gE的融合蛋白的核酸分子如SEQ ID NO:11-13、SEQ ID NO:20-26任一所示。
其中编码如SEQ ID NO:8所示的基于gE的融合蛋白的基因序列如SEQ ID NO:11，20-21任一所示：编码如SEQ ID NO:9所示的基于gE的融合蛋白的核酸分子序列如SEQ ID NO:12，22-23任一所示：编码如SEQ ID NO:10所示的基于gE的融合蛋白的核酸分子序列如SEQ ID NO:13，33-34任一所示。编码如SEQ ID NO:10所示的基于gE的融合蛋白的核酸分子序列如SEQ ID NO:13，33-34任一所示。编码如SEQ ID NO:34所示的基于gE的融合蛋白的核酸分子序列如SEQ ID NO:26所示。
本发明所述的基于gE的重组载体，携带如上所述的核酸分子。应当理解为所述的基于gE的重组载体可携带如上所述的gE胞外区或其片段的编码基因，或携带如上所述的gE的融合蛋白的基因。所述载体可以是表达载体、克隆载体或转移载体，包含但不限于：病毒载体、DNA载体或mRNA载体等。其中病毒载体包括但不限于：腺病毒载体、腺病毒相关病毒载体、痘病毒载体、水疱性口炎病毒载体、牛副流感病毒载体、人副流感病毒载体、新城疫病毒载体、仙台病毒载体、麻疹病毒载体、减毒RSV载体、副粘病毒载体、甲型病毒载体(如委内瑞拉马脑炎病毒载、塞姆利基森林病毒载体、辛德比病毒载体)、棒状病毒载体、狂犬病病毒载体、小核糖核酸病毒、慢病毒载体、疱疹病毒载体、或植物来源的病毒用于在植物表达系统中表达。
一些具体的实施方式中，所述腺病毒载体为人源腺病毒载体(如5型腺病毒载体Ad5)、黑猩猩源腺病毒载体(如ChAd68)、大猩猩腺病毒载体或其他人类适用的腺病毒载体。
一些具体的实施方式中，所述重组腺病毒载体为复制缺陷型重组腺病毒载体，所述复制缺陷型可为腺病毒基因组的E1区删除或功能性缺失从而形成复制缺陷型腺病毒，或E3区进一步删除或功能性缺失；或E1区和E3区中均删除或功能性缺失；所有E1功能性缺失的载体均为复制缺陷型载体。所述的功能性缺失一般指由于突变、缺失或增加位点等原因的导致的E1原有功能的缺失，进而影响腺病毒复制。因此，这些病毒只能在补充表达E1蛋白的哺乳动物细胞中复制，例如HEK293及PER.C6细胞，这些细胞的基因组被修饰以表达E1基因。
本发明的复制缺陷型重组腺病毒载体的其余腺病毒基因组可为腺病毒原始基因组(即可理解为除了E1区删除或功能性缺失，或E1和E3都删除或功能性缺失外，其余基因组未有进一步修饰，如购自Thermo Fisher Scientific公司的pAd5-CMV/V5-Dest载体)或可被进一步修饰的腺病毒基因组，所述的修饰指对腺病毒原始基因组的进行替换，突变等修饰，例如在特定的实施例中，复制缺陷型黑猩猩腺病毒(如ChAd68)本身的E4区被人5型腺病毒E4区取代以提高载体的性能。
当所述基于gE的重组载体携带如上所述的编码gE胞外区或其片段的核酸分子(例如SEQ ID NO:2,18-19任一所示的核酸分子)时，被称为重组腺病毒载体A，换言之，该gE为非融合形式表达。
优选地，所述的重组腺病毒载体A通过同源重组的方式进行构建。
优选地，构建所述的重组腺病毒载体A所用的骨架质粒为pAd5-CMV/V5-DEST。
优选地，构建所述的重组腺病毒载体A所用的穿梭质粒为pDONR221。
优选地，构建所述的重组腺病毒载体A所用的宿主细胞系包括但不限于HEK 293或PER.C6细胞系。
一些具体的实施方式中，所述的重组腺病毒载体A由下述方法构建：将测序正确的重组穿梭质粒pDONR221-gE基因-PolyA与病毒骨架质粒pAd5-CMV/V5-DEST进行同源重组，将重组混合物转化至大肠杆菌TOP10感受态细胞中，筛选测序正确的腺病毒载体pAd5-CMV-gE基因-PolyA，将腺病毒载体pAd5-CMV-gE基因-PolyA线性化后转染HEK 293或PER.C6细胞进行包装得到所述的重组腺病毒载体A。该技术是本领域技术人员熟知的。
当所述基于gE的重组载体携带如上所述的编码基于gE的融合蛋白的核酸分子时(例如SEQ ID NO:11-13,20-26任一所示的核酸分子)被称为重组腺病毒载体B。
优选地，所述的重组腺病毒载体B通过同源重组的方式进行构建。
优选地，构建所述的重组腺病毒载体B所用的病毒骨架质粒为pAd5-CMV/V5-DEST。
优选地，构建所述的重组腺病毒载体B所用的穿梭质粒为pDONR221。
优选地，构建所述的重组腺病毒载体B所用的宿主细胞系包括但不限于HEK 293或PER.C6细胞系。
一些具体的实施方式中，所述的重组腺病毒B由下述方法构建：将测序正确的重组穿梭质粒pDONR221-gE-鞭毛素融合蛋白基因-PolyA转化与病毒骨架质粒pAd5-CMV/V5-DEST进行同源重组，将重组混合物转化至大肠杆菌TOP10感受态细胞，筛选测序正确的腺病毒载体pAd5-CMV-gE-鞭毛素融合蛋白基因-PolyA，将腺病毒载体pAd5-CMV-融合蛋白基因-PolyA线性化后转染HEK 293或PER.C6细胞进行包装得到所述的重组腺病毒载体B。该技术是本领域技术人员熟知的。
本发明如上所述的免疫组合物还可以进一步包含一种或多种其他组分，例如药学可接受的载体，和/或佐剂，和/或免疫刺激分子等。所述的佐剂包括但不限于：铝盐(如氢氧化铝或磷酸铝)水包油乳液或油包水乳液、MF-59、TLR激动剂(如单磷酰脂质A(MPL)或其类似物，或CpG寡核苷酸)、Quil A或其QS21组分、壳聚糖、或其两种或多种的组合。所述的佐剂具有增强体液和/或细胞反应的用途。免疫刺激分子可包括但不限于大肠杆菌耐热肠毒素LT、霍乱毒素CT或其类似物等；细胞因子或趋化因子；抗体或其片段，该抗体或其片段针对特异性细胞表面分化抗原或参与免疫应答的受体，并可增强体液和细胞免疫反应。
医学上可接受的载体可为本领域的常规使用的载体，一般取决于药物的给药方式。例如胃肠外给药剂型等通常包含医学上和生理上可接受的可注射的流体，包括但不限于水、生理盐水、平衡盐溶液、甘油或其他碳水化合物等作载体。另外所述的免疫组合物中还可含有少量的无毒辅助物质，如乳化剂、pH缓冲液、稳定剂或防腐剂等。无菌溶液是通过无菌过滤或本领域已知的其他方法制备的。溶液的pH值一般在3.0～9.0之间，优先为pH5.0～7.5。制剂可以液体形式或冻干剂的形式保存，可以单剂量提供或者多剂量密封容器提供。本发明中的所述的免疫组合物也可以使用载体系统(包括但不限于脂质体、微球、胶束系统、免疫刺激复合物(ISCOMS)和纳米颗粒)来传递，所述纳米颗粒包括铁蛋白、包囊素、硫加氧酶还原酶(SOR)和鲁米嗪合成酶-纳米颗粒。
本发明如上所述的免疫组合物可通过本领域熟练人员所熟知的传递系统进行给药包括通过皮下、肌肉、皮内或鼻内等途径给药。本发明基于核酸的免疫组合物也可以通过基因枪法给药，重组蛋白免疫原可通过无针输送系统进行给药。
本发明如上所述的免疫组合物可用于预防和/或治疗水痘带状疱疹感染。具体地，所述的免疫组合物可用于婴儿、儿童、青少年、成年或老人接种免疫对抗水痘感染或老年人接种免疫对抗带状疱疹感染，另一方面，所述的免疫组合物可用于治疗带状疱疹和/或带状疱疹愈后神经痛。一般0-12月龄之间为婴儿，1-12岁为儿童，青少年为12-18岁，大于18岁为成年，50岁以上为老人。应当理解为年龄的划分并不绝限与上述描述，所述的免疫组合物可用于免疫适龄人群对抗水痘或带状疱疹感染。
本发明另一方面提供了如上所述的免疫组合物在用于制备预防和/或治疗水痘-带状疱疹病毒感染的药物中的应用；进一步地，为在制备水痘疫苗和/或带状疱疹疫苗中的应用；或所述的免疫组合物可用于制备治疗带状疱疹和/或带状疱疹愈后神经痛的药物。
本发明进一步还提供了一种联合疫苗，其包含如上所述的免疫组合物以及一种或多种其他疫苗。应当理解为，该联合疫苗各抗原组分间互不干扰，或可进一步达到协同作用。互不干扰一般指维持免疫原的稳定性及各免疫组分之间兼容性，且无抗原间的竞争，或严重不良反应的风险。另外，联合疫苗中的各抗原组分应当具有相同或相似的受试人群以及免疫程序。本发明中，所述的可联合的其他疫苗包含但不限于：流行性腮腺炎、麻疹和风疹疫苗。
本发明如上所述的基于gE的融合蛋白、相应的所述的核酸分子、所述的基于gE的重组载体可用于预防和/或治疗由水痘带状疱疹感染，具体地可用于婴儿、儿童、青少年、成年或老人接种免疫对抗水痘感染或老年人接种免疫对抗带状疱疹感染。
本发明一方面提供了如上所述的基于gE的融合蛋白、所述的核酸分子、所述的基于gE的融合蛋白重组载体在制备用于预防和/或治疗水痘-带状疱疹病毒感染的药物中的应用；进一步地，为在制备水痘疫苗和/或带状疱疹疫苗中的应用。所述的免疫组合物可用于制备治疗带状疱疹和/或带状疱疹愈后神经痛的药物。疫苗接种可能涉及在一个或多个月间隔时间内的单次或多次注射，剂量范围为在1μg至100μg的重组蛋白或10 10到10 12个病毒颗粒(VP)的腺病毒载体。具体的使用剂量将在临床试验中确定，并取决于给药途径和目标人群。如果需要，每年可给予加强免疫。
初免-强化免疫程序包括向受试者施用第一种免疫组合物(初免疫苗)，然后施用第二种免疫组合物(加强疫苗)以诱导最佳的免疫反应。本领域技术人员应当了解初次免疫与加强免疫之间的合适时间间隔。初次免疫和加强免疫施用的免疫组合物可以相同或者不同且各自的数量可能不同。本发明中：所述的gE胞外区或其片段、基于gE的融合蛋白、核酸分子和基于gE的重组载体可分别用于初次免疫或加强免疫。例如，本发明提供了如下初免-强化免疫程序：(1)可以使用上述基于gE的重组载体做初次免疫，gE胞外区或其片段或基于gE的融合蛋白进行加强免疫；或者(2)可以使用上述gE胞外区或其片段或基于gE的融合蛋白做初次免疫，基于gE的重组载体进行加强免疫。所述的初免-加强免疫程序的组合包括但不限于上述的表述。例如，初免可以用基于gE的腺病毒载体来执行，然后用来自于表达相同基因的如上所述的不同载体(如痘病毒载体等)来执行加强免疫，或者反过来，初免可以用基于gE的异源载体(可理解为除了腺病毒载体之外的其它载体)来实现，并用本发明的所述的基于gE的腺病毒载体进行加强免疫。此外，两种表达相同或不同的基于gE基因的不同类型或不同物种的腺病毒载体也可以在初免-强化免疫程序中联合使用。
使用剂量取决于免疫组分、给药途径、目标人群和其他因素。临床试验人员将根据他们的知识确定每种免疫组分的适当剂量和有效的免疫方案。单次给药即足够或需要采用单个和/或联合免疫原进行多次给药。
本发明又一方面提供了一种分离的宿主细胞其包含如上所述的基于gE的基因(如gE胞外区或其片段的编码核酸分子，或基于gE的融合蛋白的编码核酸分子)。所述的宿主细胞包含但不限于：大肠杆菌、枯草芽孢杆菌、沙门氏菌、酿酒酵母、巴斯德毕赤酵母、昆虫细胞、HEK293细胞、PER.C6细胞、Vero细胞、CHO细胞、W38细胞、BHK细胞或COS细胞胞。
本发明一方面提供了一种制备如上所示的gE胞外区或其片段，或如上所述的基于gE的融合蛋白的方法，具体地可通过原核表达系统或真核表达系统表达。所述gE胞外区或其片段被制备成含或者不含有利于纯化的共价结合蛋白标签；所述基于gE的融合蛋白被制备成含或者不含有利于纯化的共价结合蛋白标签；所述的共价结合蛋白标签包含但不限于多聚组氨酸标签(His标签)。
所述原核表达系统包括但不限于大肠杆菌表达系统。一些具体实施方式中，所用大肠杆菌为BL21(DE3)，所述原核表达载体可含有但不限于T7启动子，优选地该表达载体为pET28a。优选地，所述gE胞外区的氨基酸序列如SEQ ID NO:35所示，所述gE胞外区的基因序列如SEQ ID NO:36所示；所述基于gE的融合蛋白的氨基酸序列如SEQ ID NO:37-39所示；所述基于gE的融合蛋白的基因序列如SEQ ID NO:37-39所示。
作为本发明一具体的实施方式，所述原核表达可包括以下步骤：将携带所述gE胞外区或其片段的基因或携带所述基于gE的融合蛋白基因的pET28a表达载体转化至大肠杆菌BL21(DE3)，涂布在琼脂平板上的添有加卡那霉素(50μg/ml)的LB培养基培养。挑取单克隆接种至含有卡那霉素的LB液体培养基中，37℃培养至OD 600达到0.6时，加入0.1～1mM IPTG，16～37℃诱导表达。超声或高压均匀仪破碎收获的菌体，离心收获包涵体(IB)，用含去污剂的盐水洗涤包涵体数次。将包涵体重悬并用含有6M盐酸胍或8M尿素的缓冲液中(20mM Tris,5mM咪唑,500mM NaCl,pH 8.0)进行溶解。将溶解后的包涵体上样至Ni柱中，清洗Ni柱用5-10个柱体积(20mM Tris，8M尿素，5-50mM咪唑，500mM NaCl，pH 8.0)，并用适当浓度的咪唑(20mM Tris,8M尿素,500mM咪唑,500mM NaCl,pH 8.0)洗脱蛋白质。蛋白复性可在柱上或纯化后进行。
所述真核表达系统包括但不限于酵母表达系统，哺乳动物细胞表达系统，或重组病毒(如人、动物或植物重组病毒表达系统，又如杆状病毒、腺病毒、慢病毒或痘病毒)表达系统，或植物表达系统。优选地，用于表达的哺乳动物细胞系包括但不限于293细胞或PER.C6细胞系，中国仓鼠卵巢CHO细胞系，昆虫细胞系如SF9细胞，Vero细胞，或转基因动物或植物细胞系。重组蛋白可通过瞬时表达、稳转细胞系表达或重组病毒载体表达。细胞培养基可从商业来源获得，培养细胞的适当条件是众所周知的，本领域技术人员能够很容易地选择培养基和宿主细胞的培养条件来表达目的免疫原。合适的培养基可能含有或者不含血清。
一些具体的实施方式中，所述真核表达包括以下步骤：将如上所述基于gE的重组载体(优选地为编码所述的gE蛋白的重组腺病毒载体A和编码所述gE-鞭毛素融合蛋白的重组腺病毒载体B)以一定MOI值感染90％汇合点的宿主细胞(在一些实施例中，所述的宿主细胞包括但不限于Vero或CHO等细胞)，感染四到五天后，收获培养上清，将收获上清纯化后得到相应蛋白。其中：MOI值可以为10～500，更加优选地MOI值可以为100～200。纯化步骤包括疏水层析后经离子交换层析和/或分子排阻层析纯化；其中，疏水填料包括但不限于：Phenyl，Octyl或butyl相关填料；离子交换填料包括但不限于：Qsephrase FF，DEAE或Source 30Q；其中，分子排阻色谱填料包括但不限于Sephadex G200，G100或G75。作为优选的实施方式，所述的纯化过程是先经疏水层析，后经离子交换层析，优选地，所述疏水填料为Capto Phenyl Impress，所述离子交换填料是Source30Q。
本发明还提供了如上所述的重组腺病毒载体pAd5-CMV-gE基因-PolyA，gE基因为如SEQ ID NO:2,18-19任一所示的核酸序列。
本发明还提供了一种如上所述的重组腺病毒载体Ad5-CMV-gE-鞭毛素融合基因-PolyA，gE-鞭毛素融合基因具有如SEQ ID NO:11-13,20-26任一所示的核酸序列。
本发明另一方面还提供了一种改造的鞭毛素蛋白，所述鞭毛素蛋白N端区为至少与SEQ ID NO:3中第2至176位氨基酸区域有95％(例如：96％、97％、98％或99％的同源性)同源性的氨基酸序列；鞭毛素蛋白C端区为至少与SEQ ID NO:3中第392至495位氨基酸区域有95％(例如：96％、97％、98％或99％的同源性)同源性的氨基酸序列；所述鞭毛素蛋白N端区直接或者通过连接体与鞭毛素蛋白C端区相连。
所述连接体可为1-20个肽键连接的氨基酸，如具有如SEQ ID NO:4所示的氨基酸序列。
在一些实施方式中，所述鞭毛素蛋白的N端区的氨基酸序列如序列表SEQ ID NO:5所示：C端区的氨基酸序列如序列表SEQ ID NO:6所示。
在本发明的一些实施方式中，所述改造的鞭毛素蛋白具有如SEQ ID NO:27所示的氨基酸序列。
本发明还提供了一种核酸序列，其能编码如SEQ ID NO:27所示的氨基酸序列。优选地，所述的核酸序列如SEQ ID NO:28所示。
本发明还提供了所述的改造的鞭毛素蛋白作为免疫佐剂的应用，当其与gE或其片段偶联时形成的融合蛋白具有内在的佐剂特性，因此，基于gE的融合蛋白或可表达gE-鞭毛素融合蛋白的重组腺病毒载体(如重组腺病毒载体B)可直接用于制备用于免疫宿主(人或动物)诱导和/或增强对VZV的免疫应答的疫苗，对抗急性或者潜在VZV感染。
本发明公开了通过原核表达系统或重组腺病毒系统高效表达gE或gE-鞭毛素融合免疫原的方法，通过实验数据可知，制得的gE、gE鞭毛素融合蛋白及重组腺病毒载体可刺激免疫宿主产生高水平抗体滴度和良好的细胞免疫，可开发为新一代和改良的VZV疫苗。
术语
预防或治疗疾病：“预防”是指在有疾病风险的受试者(如VZV感染)中抑制感染或疾病的全面发展。“治疗”是指在疾病或病理状态开始发展之后，改善其体征或症状的治疗干预。术语“改善”是指任何可观察的有益治疗效果，如延迟出现疾病的临床症状、疾病症状减少、病情发展减缓、受试者的整体健康改善，或领域内中公认的特殊疾病的其他特定指标。“预防性”治疗是对没有出现疾病症状或仅出现早期症状的受试者进行的治疗，目的是降低发生病状的风险。
5型腺病毒(Ad5)：一种双链DNA病毒，属于腺病毒科，主要引起人类呼吸道感染。E1基因产物(包括E1A和E1B)参与病毒的复制。大多数E3蛋白参与调节感染细胞的免疫应答。可以通过删除E1区域使该病毒失去复制能力，然后将异源转基因插入到删除的E1和E3区域，使病毒作为载体，实现免疫或基因治疗目的。
佐剂：增强宿主对免疫原或疫苗的免疫反应的物质。
抗体：由特异性浆细胞产生的血液蛋白，在抵抗外来分子或病原体的体液适应性免疫反应中起主要作用。抗体识别同源免疫原上的特定位点，从而中和或消除这些抗原。
抗体依赖性细胞毒性(antibody-dependent cellular cytoxicity，ADCC)：一种免疫防御机制，通过这种机制，免疫系统的效应细胞主动裂解膜表面抗原已被特异性抗体结合的靶细胞。
细胞免疫：涉及激活哨兵树突状细胞和响应免疫原的淋巴细胞亚群的免疫反应。树突状细胞负责非特异性先天免疫以及免疫原特异性适应性免疫的启动。淋巴细胞亚群负责包括前炎症反应、辅助抗体产生的CD4+辅助性T细胞，以及杀死受感染靶点的细胞毒性CD8+T细胞。
条件性基因表达：指任意激活或抑制特定基因或基因产物表达的能力。
CpG寡核苷酸：CpG寡脱氧核苷酸是短单链合成DNA分子，含有胞嘧啶三磷酸脱氧核苷酸和鸟嘌呤三磷酸脱氧核苷酸。CpG基序是病原体相关分子模式，因此作为TLR9激动剂，具有免疫佐剂性质。
胞外域：膜蛋白延伸到细胞外空间的结构域。膜蛋白由胞外结构域(胞外域)、跨膜段和胞质内尾部组成。
增强子：一种DNA序列，能提高位于编码序列附近的基因的转录水平。
鞭毛素：一种聚合蛋白，是革兰氏阴性细菌鞭毛的主要成分，决定鞭毛在引起免疫反应方面的特异性。鞭毛素是一种有效的免疫调节剂。
融合蛋白：最初编码分离蛋白的两个或多个基因结合而产生的蛋白。
同源重组：含有长段相似碱基序列的两股DNA之间的遗传物质交换。同源重组自然存在于真核生物、细菌及某些病毒中，是基因工程的有力工具。
宿主细胞：含有外来分子、病毒或微生物的细胞。
免疫原：一种物质或有机体，进入宿主后能引起免疫，包括体液(抗体)和细胞反应。
免疫组合物：一种能诱导免疫的组合物。
免疫刺激分子：能刺激或增强免疫反应的分子。
先天免疫：由免疫系统的前哨细胞(如树突状细胞和巨噬细胞)进行防御的自然机制。这一免疫并非是由先前对免疫原的致敏，如感染或接种疫苗而引发。由于先天免疫不受特异性免疫原的刺激，因此先天免疫通常是即时的、非特异性和无记忆的，完全不同于具有免疫原特异性和记忆性的获得性免疫。
ISCOM：免疫刺激复合物(ISCOM)是胆固醇、磷脂和槲皮皂苷在特定的化学计量比下混合时自发形成的球形笼状结构。ISCOM显示出免疫佐剂的特性，可用于疫苗以增强其免疫应答。
Kozak序列：存在于真核生物mRNA上的核酸序列，通常为(gcc)gccRccAUGG。Kozak序列在翻译过程的启动中起着重要作用。
前导序列：信使RNA(和DNA)5'端的核苷酸序列，位于翻译起始密码子上游。
脂质体：包裹水滴的磷脂分子的微小球体，尤指人工形成的将疫苗、药物或其他物质输送到组织中的脂质体。
纳米颗粒：小于100纳米的微粒，不仅可以提高疫苗的稳定性和免疫原性，而且可以有效地递送和缓释。
中和：通过与病原体的特异性抗体相互作用而导致病原体感染性的丧失。
包装细胞系：将重组载体转染到包装细胞系中，以补充重组病毒载体中缺失的病毒基因，从而产生含有转基因的重组病毒。
多聚腺苷酸化序列(polyA tail)：在信使RNA上加入多个单磷酸腺苷，是翻译前信使RNA(mRNA)成熟过程的一部分。启动子：DNA分子中的一个位点，RNA聚合酶和转录因子在此位点结合，启动特定基因对mRNA的转录。
复制缺陷型载体：指病毒基因组的关键部分已经被删除，使得病毒载体不能复制。
穿梭质粒：一种能在两种不同宿主物种中繁殖的质粒。
信号肽：一种短肽(长度5-30个氨基酸)，存在于大多数新合成蛋白质的N端，并最终进入分泌途径。
起始密码子：起始密码子是由核糖体翻译的信使RNA(mRNA)转录物的第一个密码子。在真核生物中，起始密码子始终编码蛋氨酸，而在原核生物中，起始密码子始终编码修饰的蛋氨酸(fMet)。最常见的起始密码子是AUG。
SV40polyA：SV40polyA序列是一个终止子序列，表示一个转录单元的结束。
标签：蛋白标签是基因接枝到重组蛋白上的肽序列，尤其是为了便于纯化。例如，多组氨酸标签结合到镍柱，从而可利用亲和层析纯化蛋白。
Tag：蛋白质标签是基因融合至重组蛋白质上的肽序列，特别是为了促进其纯化。例如，多聚组氨酸标签与Ni 2+柱结合，从而能够通过亲和层析纯化蛋白质。
T细胞亚群：对免疫应答具有特异性免疫功能的淋巴细胞亚群。CD4+辅助性T细胞对于抗体产生不可或缺。它还通过释放可溶性免疫刺激介质，如细胞因子和趋化因子，参与前炎症反应。1型辅助性T细胞(Th1)是宿主抵抗细胞内病毒和细菌病原体所必需的细胞并产生干扰素γ(IFN-γ)。2型辅助性T细胞(Th2)在宿主抵抗细胞外病原体中起重要作用并分泌IL-4。细胞毒性CD8+T细胞是负责杀死受感染细胞并分泌IFN-γ的淋巴细胞的一个亚群。
TLR激动剂：能够通过与同源TLR受体相互作用来激活免疫细胞的试剂，从而促进和协调先天免疫和适应性免疫的启动。
Toll样受体(Toll-like receptors，TLRs)：是一类在先天免疫系统中起关键作用的蛋白，是单一、跨膜、非催化的受体，通常表达在前哨细胞(如巨噬细胞和树突状细胞)上，识别从微生物衍生的结构保守分子。
转录终止子：在转录过程中标记基因组DNA中基因或操纵子末端的核酸序列的一部分。
转染：将核酸导入哺乳动物细胞的过程。有很多不同的方法和技术，包括脂质转染及化学和物理方法，如电穿孔。
转化：将外源质粒或连接产物插入大肠杆菌等细菌中。
病毒载体：分子生物学家常用来将遗传物质输送到细胞中的工具。这一过程可在活的有机体(体内)或细胞培养(体外)中进行。病毒已经进化出专门的分子机制来有效地运输其基因组，能够在其感染的细胞内传递基因和其他遗传物质。
附图说明
图1.使用Phyre2软件模拟计算的修饰后鞭毛素蛋白与Toll样受体相互作用三维结构示意图。(参考文献:Phyre2 web portal for protein modeling,prediction and analysis.Kelley LA et al.,Nature Protocols 10,845-858,2015)
图2.gE-鞭毛素融合蛋白与Toll样受体相互作用计算机模拟三维结构示意图。计算机预测免疫原设计方法如下：首先使用Phyre2网页生成水痘带状疱疹病毒(strain Dumas；UniProtKB P09259)的包膜糖蛋白E模型，然后从蛋白模型中去除VZV gE的信号肽分泌序列、跨膜区以及胞内区，然后根据数据库PDB ID’s 3v47 and 3a5x(Yoon S-il et al.,Science,335:859-864,2012)中的信息，选择鼠伤寒沙门氏菌鞭毛素蛋白序列(strain LT2；UniProtKB P06179)的边界。在设计VZV gE和鞭毛素蛋白的融合蛋白时，根据融合的位置(N-端，C-端或gE蛋白插入中间)分别设计了不同长度的GGGGS连接体，以最小化空间位阻。图2A.修饰的鞭毛素蛋白融合至gE蛋白的N端(ANF)；图2B.修饰的鞭毛素蛋白融合至gE蛋白的C端(ACF)；图2C.将ge蛋白取代鞭毛素蛋白的D2和D3结构域插入其高变区(ASF)。
图3.携带gE及gE-鞭毛素融合基因的重组腺病毒载体命名简称及其对应插入基因图例。“Js”代表乙型脑炎病毒(JEV)prM先导肽基因序列。“Igκ”是指小鼠IgGκ轻链先导肽基因序列。
图4.Western Bloting(WB)检测重组腺病毒1:rAd5-ACF(Js)；2:rAd5-ACF-SV40(Js)3:rAd5-ANF(Js)；4:rAd5-ANF-SV40(Js)；5:rAd5-gE(Js)；6:rAd5-gE-SV40(Js)-感染后Vero细胞上清中外源基因的表达。图4A使用的一抗为小鼠anti-VZV gE单克隆抗体；图4B使用的一抗为兔anti-鞭毛素D0,D1多克隆抗体。M,蛋白分子量markers。
图5.Western Bloting及SDS-PAGE分析重组腺病毒感染后的293A细胞上清(S)及细胞裂解液(L)中外源基因表达结果。5A.鼠抗VZV gE单克隆抗体作为一抗，WB检测结果；5B.兔抗鞭毛素D0,D1抗血清作 为一抗，WB检测结果；5C.SDS-PAGE检测结果；图中，gE代表rAd5-gE-SV40(Js)感染的HEK293细胞上清液(S)和细胞裂解液(L)；ANF代表rAd5-ANF-SV40(Js)感染的HEK293细胞上清液(S)和细胞裂解液(L)；ACF代表rAd5-ACF-SV40(Js)感染的HEK293细胞上清液(S)和细胞裂解液(L)；ASF代表rAd5 ASF(Js)-感染的HEK293细胞上清液(S)和细胞裂解液(L)。
图6.纯化后的重组腺病毒检测。图6A,纯化后的重组腺病毒WB鉴定结果(以兔抗Ad5多克隆抗体作为一抗)。M:分子量Marekers；泳道1：纯化后的rAd5-gE-SV40(Js)病毒；泳道2:纯化后的rAd5-ANF-SV40(Js)病毒；泳道3：纯化后的rAd5-ACF-SV40(Js)病毒。泳道4：纯化后的rAd5-ASF(Js)病毒。泳道5：纯化后的rAd5-SE(Igκ)病毒。6B.透射电镜(TEM)分析10 10TCID 50/ml样品中的病毒颗粒检测结果。6C.阴离子交换-高效液相色谱法(agilent 1260)分析纯化后的rAd5-gE-SV40(Js)病毒。将40μl纯化后的病毒样品上样至用90％的流动相A(20mM Tris，pH值8.0)和10％的流动相B(20mM Tris，1M NaCl，pH值8.0)平衡好的柱子上(4.8x 250mm Sepax SAX-NP5阴离子交换柱Sepax，中国)。上样结束后，线性梯度(10-60％流动相B)洗脱柱子8分钟，然后用60％的流动相B冲洗柱子4分钟，之后再用另一线性梯度(60-100％流动相B)洗脱柱子4分钟。最后，用平衡缓冲液洗洗4分钟。
图7.原核表达的含His标签的重组gE及gE-鞭毛素融合蛋白命名简称及其对应插入基因图例。
图8.SDS-PAGE及Western Bloting检测纯化后的大肠杆菌表达的重组gE及重组gE-鞭毛素融合蛋白。8A.SDS-PAGE结果；8B.使用小鼠抗VZV-gE单克隆抗体作为一抗WB结果；8C.使用兔抗鞭毛素D0,D1抗血清作为一抗WB结果。M:蛋白分子量Markers；泳道1：纯化后的gE蛋白；泳道2：纯化后的ENF蛋白；泳道3：纯化后的ESF蛋白；泳道4：纯化后的ECF蛋白。
图9.SDS-PAGE及Western Bloting检测纯化后的Vero细胞表达的重组gE及重组gE-鞭毛素融合蛋白。9A.SDS-PAGE检测结果；9B.使用小鼠抗VZV-gE单克隆抗体作为一抗WB结果；9C.使用兔抗鞭毛素D0,D1抗血清作为一抗WB结果。M:蛋白分子量Markers；泳道1：纯化后的gE蛋白；泳道2：纯化后的ANF蛋白；泳道3：纯化后的ASF蛋白；泳道4：纯化后的ACF蛋白。
图10.重组腺病毒免疫的小鼠血清中的VZV-gE特异性抗体检测。将各种重组腺病毒(10 9TCID 50/剂)或商品化水痘疫苗(700pfu/剂)通过肌肉注射免疫C57BL/6小鼠，共免疫两剂，免疫间隔30天。在第一次免疫后的第12天、26天和42天收集血清，并按照材料和方法中的描述，使用酶联免疫吸附试验(ELISA法)检测gE特异性抗体滴度。gE特异性抗体反应的结果几何平均滴度(GMT)表示，上下置信区间95％。 ***p<0.001(ANOVA/Bonferroni单因素方差分析法)。
图11.重组腺病毒免疫的小鼠血清中，抗体介导的中和VZV病毒感染活性的分析。将各种重组腺病毒(10 9TCID 50/剂)或商品化水痘疫苗(700pfu/剂)通过肌肉注射免疫C57BL/6小鼠，共免疫两剂，免疫间隔30天。第二针免疫后30天，收集小鼠血清，并检测VZV特异性中和抗体滴度。取复孔测定的平均值表示中和抗体滴度。计算能够使空斑数减少50％的稀释倍数，取其倒数，表示中和抗体效价。 **p<0.01, ***p<0.001(ANOVA/Bonferroni单因素方差分析法)。
图12.流式细胞术分析重组腺病毒诱导的gE特异性CD4+和CD8+T细胞反应。第二次免疫后36天收集脾细胞，用覆盖整个gE胞外区的15个重叠多肽混合物(2μg/肽)刺激脾细胞。按照CD3+/CD4+和CD3+/CD8+T细胞双阳画门，用荧光标记的抗IFN-γ抗体进行胞内因子染色法(ICS)流式细胞术分析。结果按照表达IFN-γ的CD4+和CD8+T细胞的百分比表示，上下置信区间95％。**p<0.01，****p<0.0001(ANOVA/Bonferroni单因素方差分析法)。阴性对照为未刺激的脾细胞，阳性对照为PMA(50ng)。
图13.Elispot分析重组腺病毒诱导的产生IFN-γ及IL-4的T细胞。第二次免疫后36天收集脾细胞，用覆盖整个gE胞外区的15个重叠多肽混合物(2μg/肽)刺激脾细胞并分析产生IFN-γ及IL-4的T细胞数量。结果以斑点数/5x 10 5的平均值表示。 *p<0.05, **p<0.01, ****p<0.0001(ANOVA/Bonferroni单因素方差分析法)。阴性对照为未刺激的空载体腺病毒组脾细胞，阳性对照为PMA(50ng)。
图14.gE及gE-鞭毛素融合蛋白诱导的gE特异性抗体滴度。将含或不含MF59佐剂(50μl/剂)的gE蛋白(5μg/剂)或gE-鞭毛素融合蛋白(8μg/剂)免疫C57BL/6小鼠，共免疫两剂，免疫间隔14天。第二剂免疫后14天，收集免疫血清，ELISA法检测gE-特异性抗体滴度。 ***p<0.001, ****p<0.0001(ANOVA/Bonferroni单因素方差分析法)。
图15.gE或gE-鞭毛素融合蛋白(含或不含MF59佐剂)免疫血清中，抗体介导的VZV感染活性中和效价。将含或不含MF59佐剂(50μl/剂)的gE蛋白(5μg/剂)或gE-鞭毛素融合蛋白(8μg/剂)，或商品化的水痘疫苗免疫C57BL/6小鼠，共免疫两剂，免疫间隔14天。第二剂免疫后14天，收集免疫血清，将每组小鼠的血清两两合并后检测VZV特异性中和抗体滴度。取复孔测定的平均值表示中和抗体滴度。计算能够使空斑数减少50％的稀释倍数，取其倒数，表示中和抗体效价。 *p<0.05(ANOVA/Bonferroni单因素方差分析法)。
图16.Elispot分析gE及gE-鞭毛素融合蛋白(含或不含MF59佐剂)诱导的产生IFN-γ及IL-4的T细胞。用覆盖整个gE胞外区的15个重叠多肽混合物(2μg/肽)刺激脾细胞并分析产生IFN-γ及IL-4的T细胞数量。结果以斑点数/5x 10 5的平均值表示。 *p<0.05, **p<0.01(ANOVA/Bonferroni单因素方差分析法)。阴性对照为盐水组免疫的脾细胞，阳性对照为PMA(50ng)。
实施例
实施例中使用的材料和方法：
动物及细胞：
6-8周的无特定病原体(SPF级)C57BL/6雌性小鼠购自湖北省疾病预防控制中心。所有的动物研究在GLP(Good Laboratory)实验室条件下开展，并按照“动物福利伦理审查实验室动物指南”处理动物。人胚胎肾细胞HEK293购自(Thermo Fisher Scientific，美国)，使用含10％胎牛血清(FBS)的DMEM培养。THP-1细胞购自ATCC，使用含10％FBS及1％青霉素/链霉素双抗的RPMI-1640培养基培养(Gibco美国)。
试剂：
所有基因片段均有上海生工合成(上海，中国)，引物由武汉擎科生物公司合成(武汉，中国)；pDONR221，pAd5-CMV/V5-Dest载体，Gateway BP重组，LR重组酶，大肠杆菌TOP10感受态，lip2000转染试剂，均购自Thermo Fisher Scientific公司(美国)。pET28a表达质粒购自Novagen(美国)。质粒提取试剂盒，胶回收试剂盒购自Axygen公司(美国)。Mouse anti-VZV-gE单抗购自Merck公司(美国)，rabbit anti-鞭毛素D0-D1抗体通过免疫兔子制备。将三条合成的来自鞭毛素D0和D1结构域多肽(见表1.)与载体蛋白(CCH,Thermo Fisher Scientific.美国)结合。免疫过程：第一剂，0.4mg结合物含完全弗氏佐剂，肌肉注射，第二剂及第三剂，用0.2mg结合物含不完全弗氏佐剂，肌肉注射；最后用0.1mg结合物，静脉冲击；分别免疫兔子。Rabbit anti-Ad5单抗购自Abcam公司(英国)。细胞培养瓶及移液管购自Corning公司(美国)。不含内毒素的鞭毛素蛋白购自Alpha Diagnostic公司(美国)；IL-8及TNF-α含量ELISA试剂盒及Elispot试剂盒购自达科为公司；中和抗体检测实验所用豚鼠补体血清购自BD公司(美国)。流式所用抗体均购自Thermo fisher公司。商品化的水痘减毒活疫苗为长春祁健(中国)或长春百克公司(中国)生产。
表1.鞭毛素蛋白D0-D1多肽序列
序号 多肽序列
1 LNKSQSALGTAIERLSSGLRINSAKDDAAC
2 NNLQRVRELAVQSANSTNC
3 LTSARSRIEDSDYATEVSNM
PCR及琼脂糖电泳：
向含1μl上下游引物的管子中,分别加入2x PCR预混溶液25μl，DNA模板50-100ng，补加ddH2O至50μl,循环条件：第一步，95℃，2min；第二步：95℃，15s，45℃～55℃，15s，72℃，1min30s共30个循环；第三步：72℃5min。PCR结束后将PCR产物加入上样缓冲液并进行1％琼脂糖凝胶电泳，电泳条件180V,20～30min，紫外检测PCR结果。
SDS-PAGE及Westernblotting：
向80μl样品中加入20μl 5倍浓缩的上样缓冲液，煮沸5mins。将煮沸后的样品进行10％SDS-PAGE进行电泳(100V，20mins然后160V，1小时20mins)。电泳结束后，湿转法将蛋白转至PVDF膜上(Merck，美国)，用含5％脱脂奶粉的PBST溶液(含0.05％Tween 20的PBS溶液)4℃封闭过夜；PBST洗膜两次后加入鼠抗VZV-gE蛋白单克隆抗体(1:5000稀释，Millipore)或兔抗anti-鞭毛素抗血清(1:10000稀释)或兔抗-Ad5多克隆抗体(1:10000稀释)，37℃孵育1小时；PBST洗膜两次后加入辣根过氧化物酶(HRP)标记的羊抗鼠IgG(1:5 000稀释，碧云天)或HRP标记的羊抗兔IgG(1:5 000稀释，碧云天)，37℃孵育1小时；PBST洗膜两次后，使用Western Blotting ECL显色液处理后，化学发光法显色。
腺病毒滴度-TCID 50法 ：
取内含90％汇合度生长于含10％FBS的DMEM培养基的293细胞一瓶(T-75瓶)。在测定前一天，用PBS清洗后，加入1x TypLE消化2mins，加入含2％FBS的DMEM培养基终止消化，将细胞用相同培养基重悬后计数。调整细胞浓度至1.0～2.0x10 5细胞/mL，每孔100μl细胞，接种96孔板，将96孔板置于37℃，5％CO 2培养箱16～20小时；将待测病毒液及参考品分别用DMEM+2％FBS培养基进行十倍连续稀释(从10-1稀释至10-10)。将稀释后的病毒液分别加入1～10列，每孔100μl，每个病毒稀释度重复8孔。第11和12列加入100μl DMEM+2％FBS培养基做阴性对照。将96孔板置于CO 2培养箱37℃培养10天，然后在倒置显微镜下观察，判断并记录每列细胞病变效应(CPE)情况。判断的标准是只要有少量细胞发生CPE即为阳性。最后按照Karber法计算病毒滴度。(
Figure PCTCN2019105716-appb-000001
G.,Archiv f experiment Pathol u Pharmakol,162:480-483,1931).
TLR-5活性检测：
取对数生长期表达TLR5受体且生长于含10％FBS的RPM-1640培养中的THP-1细胞，125g离心5mins，弃上清，用含10％FBS的RPMI-1640培养基重悬细胞，调整细胞浓度至1x10 7cells/ml，接种于96孔细胞培养板中，100μl/孔。用含10％FBS的RPMI-1640培养基溶液稀释阳性对照至终浓度2.5μg/ml(不含内毒素的鞭毛素蛋白)。用相同的培养基将纯化后内毒素含量<5EU/ml的gE-鞭毛素融合蛋白稀释至等摩尔浓度(5μg/ml)，纯化后的gE蛋白作为阴性对照。将稀释后的样品，无内毒素-鞭毛素或gE分别加入96孔板中，100μl/孔。将96孔细胞培养板放入CO 2培养箱中37℃培养12～24小时。培养结束后，将各孔中的细胞吸出，2000g离心10mins，收集细胞上清液。通过检测培养上清中IL-8，TNF-α细胞因子的含量来检测TLR5的活性，细胞因子含量按照IL-8,TNF-α细胞因子Elisa检测试剂盒说明书进行操作。
酶联免疫反应(ELISA)检测血清中anti-gE抗体滴度：
将纯化后的原核表达gE蛋白用无菌碳酸钠缓冲液(8.4g/L NaHCO 3，3.5g/L Na 2CO 3，pH 9.6)稀释至1μg/ml，100μl/孔加入96孔酶标板，4℃包被过夜。次日，取出酶标板，弃去孔内液体，用PBST(含0.1％Tween 20的PBS溶液)洗板3次。每孔加入封闭液(含10％脱脂奶粉的PBST溶液)37℃封闭1小时。封闭结束后，弃封闭液，将免疫后的小鼠血清用封闭液进行系列梯度稀释，并设置封闭液作为空白对照。将稀释后的血清以100μl每孔加入96孔板，每个稀释度血清做三个复孔，37℃孵育1小时。用PBST洗板三次，之后每孔加入100μl 1:1000稀释的过氧化物酶(HRP)标记的羊抗小鼠IgG抗体，37℃孵育1小时。用PBST洗板3次后，加入TMB底物(3,3',5,5'-四甲基联苯胺，KPL,美国)。加入0.2M硫酸终止反应。用酶标仪在波长450nm，参比波长620处测定吸光度。
中和抗体检测：
抗体介导的中和VZV病毒感染活性中和效价测定步骤如下：将VZV病毒用VZV稀释液稀释至2×10 3PFU/ml(磷酸盐缓冲盐(PBS)、蔗糖5％、谷氨酸1％、胎牛血清(FBS)10％、pH 7.1)。将150μl病毒与150μl连续稀释的热灭活血清及5μl豚鼠补体在37℃孵育1小时。取孵育后的病毒血清混合物加入长满MRC-5单层细胞的24孔板(100ul/孔)中，每个稀释度做两个复孔，37℃孵育2小时。2小时后加入2ml病毒维持液(含2％FBS的MEM)。7天后，去除培养基，固定细胞，用考马斯蓝溶液(考马斯蓝0.5％，甲醇45％，乙酸10％)染色10分钟，用蒸馏水洗涤平板，数斑。每个稀释度检测两个复孔。取使空斑数减少50％的血清稀释度的倒数，即为中和抗体效价。
小鼠脾细胞分离：
将小鼠脾脏无菌取出并转移到放置在6孔板的单个孔中的细胞过滤器中，添加3ml培养基(RPMI-1640，含5％FBS)，研磨释放脾细胞，200目细胞筛网过滤脾脏。将细胞收集于15ml试管中，350×g、4℃下离心5min。丢弃上清液，将细胞沉淀重悬后加入2ml红细胞(RBC)裂解缓冲液(Thermo Fisher Scientific)室温裂解10mins，加入6ml RPMI-1640培养基终止裂解红细胞，离心(4℃，350g，5mins)。弃上清，加入10ml RPMI-1640培养基重悬细胞，离心(4℃，350g，5min)。弃上清，加5ml RPMI-1640+10％FBS重悬细胞。取重悬后的脾细胞悬液进行计数后待用。
Elispot检测：
gE-特异性细胞免疫通过干扰素-γ(IFN-γ)和IL-4的Elispot来检测，使用覆盖整个gE胞外区的15个重叠多肽混合物作为刺激物。将预包被有IFN-γ或IL-4抗体的Elispot板(达科为)，每孔加入200μl RPMI-1640培养基，室温静置10分钟后将其扣出。调整脾细胞终浓度至2～8x 10 6细胞/ml。将100μl脾细胞悬液与多肽混合(每条肽的浓度为2μg/ml)，每个样品做三个复孔。将ELISpot板置于37℃培养箱培养36～72小时。培养结束后，按照Elispot板子说明书进行斑点显色操作(具体操作流程见厂家说明书)。板子晾干后，使用酶联斑点成像系统进行斑点计数。计算每5x10 5个细胞中形成斑点的细胞数(SFC)。培养基背景水平通常<15SFC/5x10 5个细胞。
胞内细胞因子染色：
在体外用覆盖整个gE蛋白胞外区(含11个重叠氨基酸的15肽)的多肽混合物(2μg/ml)37℃刺激脾细胞2小时，刺激结束后加入Brefeldin A(3μg/ml)及离子霉素(1μg/ml)37℃孵育过夜。收获各孔细胞至EP管，350g离心5分钟，弃上清。使用50μl含2％Fc抗体及1％FBS的PBS溶液重悬细胞，4℃孵育10分钟。再加入50μl含anti-CD3Alex fluor 700、anti-CD4-FITC和anti-CD8-PE-Cy7(BD Biosciences,1：100稀释)的抗体混合液，避光4℃孵育30分钟。用FACS洗涤液洗涤细胞1次后，加入200μl固定液，避光室温孵育25分钟。固定结束后，再加入1.5ml稀释好的破膜剂洗涤细胞，350g离心细胞悬液5分钟，弃上清。使用破膜剂稀释IFN-γ-APC，IL-2-PerCp-Cy5.5 and IL-4-PE抗体，将稀释后的抗体混合物加入细胞悬液，避光室温孵育30分钟。使用CytoFLEX S流式细胞仪(Beckman)及Flow Jo软件分析CD3+/CD4+阳性和CD3+/CD8+阳性T细胞亚群。
实施例1
重组腺病毒的构建，鉴定，扩增及纯化
1.1试验设计
1.1.1根据计算机计算及模拟结果设计用于gE鞭毛素融合蛋白的linker序列，鞭毛素蛋白与TLR5受体结合计算机模拟图见图1，设计后的gE鞭毛素融合蛋白与TLR5受体结合模拟结构图见图2，其中图2A为ANF、图2B为ACF、图2C为ASF。
1.1.2本研究使用引物见表2，插入基因与对应制备的重组腺病毒简称见图3.
表2.本研究使用引物信息
引物名称 序列
AttB1-JEV-F GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGCCGCCGCCATGGGAAAACGGTCC
AttB2-SV40-R GGGGACCACTTTGTACAAGAAAGCTGGGTCAGACATGATAAGATACATTGATGAG
AttB2-GE-R GGGGACCACTTTGTACAAGAAAGCTGGGTCTTATTATTATCTGATCAGGGGGCTAG
AttB2-hOACF-R GGGGACCACTTTGTACAAGAAAGCTGGGTCTTATTATTACCTCAGCAGGCTCAG
AttB2-hOANF-R GGGGACCACTTTGTACAAGAAAGCTGGGTCTTATTATTATCTAATCAGAGGGCTAG
注：1.引物名称中，F代表正向引物，R代表反向引物；2.扩增含或者不含SV40polyA的gE及gE-鞭毛素基因所使用的正向引物均相同，即AttB1-JEV-F；3.扩增含SV40polyA的gE及gE-鞭毛素基因所使用的反向引物均为AttB2-SV40-R。
1.2重组腺病毒构建
1.2.1 pDONR221转移载体构建
将1.1所示的基因片段进行基因合成，用高保真DNA聚合酶分别扩增基因合成的各个目的基因片段(扩增引物序列见1.1.2中表2.所示)。PCR扩增后，将1％琼脂糖凝胶电泳检测PCR产物，用DNA胶回收试剂盒分别回收目的DNA片段，PCR循环条件：第一步：95℃，2mins；第二步，95℃，15s，55℃，15s，72℃，1min 30s，共30个循环，第三步，72℃，5mins。根据厂家说明书，将回收后的目的DNA片段分别和pDONR221质粒做BP重组(Thermo Fisher Scientific，Cat 11789020)，将重组混合物转化大肠杆菌TOP10感受态细胞并涂布含Kana抗性的固体LB平板。提取质粒，并送测序。
其中，制备得到的TOP10/pDONR221-Js-ASF-SV40plyA,TOP10/pDONR221-Js-ACF-SV40plyA和TOP 10/pDONR221-Js-ANF-SV40plyA于2019年9月10日在中国典型培养物保藏中心(CCTCC)进行保藏，保藏编号分别为：CCTCC M 2019707，CCTCC M 2019708 and CCTCC M 2019709。
1.2.2重组腺病毒表达载体构建
按照厂家说明书操作，将测序正确的重组pDONR221质粒，分别和目的质粒pAd5-CMV/V5-DEST做LR重组(Thermo Fisher Scientific,Cat 11791020)。将重组混合物并转化大肠杆菌TOP10感受态细胞，并涂布含Ampicillin(Amp，100μg/ml)抗性的固体LB平板。次日，挑取不同的菌落，这些菌落可能含有不同的pAd5-CMV质粒，将这些分别携带含或者不含SV40 poly A的gE或gE-鞭毛素融合基因的pAd5-CMV质粒(称为pAd5-CMV(VZV))。将挑取的菌落在含Amp抗性的LB培养基中培养。提取质粒并测序。
1.2.3重组腺病毒质粒的制备
将测序正确的pAd5-CMV(VZV)质粒分别转化大肠杆菌TOP10感受态细胞，并涂布含Amp抗性的固体LB平板。次日，分别挑取单克隆接种于200ml含Amp的LB液体培养基，过夜培养后，质粒大抽试剂盒，分别提取大量的pAd5-CMV(VZV)质粒。
其中，制备得到的TOP10/pAd5-Js-gE-SV40plyA于2019年9月10日在中国典型培养物保藏中心(CCTCC)进行保藏，保藏编号为：CCTCC M 2019710。
1.2.4重组腺病毒载体线性化处理
将1.2.3中获得的质粒分别用PacI限制性内切酶(NEB,美国),37℃酶切3h，酶切体系如下：pAd5-CMV(VZV)质粒：10μg，10*NEB CutSmart buffer：5μl，PacI酶：5μl，加ddH 2O至终体积50μl。酶切完成后用PCR产物回收试剂盒，回收酶切后的DNA片段。并用微量核酸定量仪对回收后的DNA片段进行定量。
1.2.5重组腺病毒的包装
根据Lipofectamine2000转染试剂的使用说明，将Pac I线性化的pAd5-CMV(VZV)质粒分别转染汇合度为60-70％的6孔板中的HEK293细胞。转染前2h，将培养基更换成无抗生素培养基，加入DNA/脂质体复合物。转染后5小时，将培养基更换为含10％FBS和1％双抗的DMEM培养基。在倒置显微镜下隔日观察细胞病变，直至60％的HEK293细胞出现噬斑时收集细胞，在室温与-80℃超低温之间反复冻融3次，1200g离心5min，收集上清即获得rAd5-gE(Js),rAd5-gE-SV40(Js),rAd5-ANF(Js),rAd5-ANF-SV40(Js),rAd5-ACF(Js),rAd5-ACF-SV40(Js),rAd5-ASF(Js)and rAd5-SE-SV40(Igκ)重组腺病毒，置于-80℃冰箱保存。
1.3重组腺病毒目的基因表达鉴定
1.3.1 PCR鉴定
初次病毒扩增保存液用病毒RNA/DNA提取试剂盒(Takara,日本)，按照操作说明书，提取病毒基因组DNA，PCR扩增提取后的病毒基因组DNA,鉴定插入重组腺病毒载体的VZV gE或gE-鞭毛素融合基因。引物：T7-F/V5-C-R,PCR条件：病毒DNA 1μl，正反向引物各0.5μl，5μl 2×PrimerSTAR mix,ddH 2O 3μl,循环条件：第一步：95℃，2min；第二步，95℃，15s，45℃，15s，72℃，1min 30s共30个循环；第三步，72℃，5mins。PCR结束后将PCR产物经1％琼脂糖凝胶电泳后，切胶回收目的条带并送测序公司进行测序。
1.3.2重组腺病毒的VZV gE和gE-鞭毛素融合基因表达
将HEK293细胞或Vero细胞接种于6孔板(5x 10 5/孔)，待6孔板中汇合率为90％时，将P3代重组腺病毒以MOI0.2(HEK293细胞)和20(Vero细胞)接种至6孔板中，并设置正常细胞为阴性对照。37℃培养48小时后，用细胞刮将细胞挂下,离心后分别收集细胞和上清，上清标记为细胞培养上清，细胞沉淀中加入100μl哺乳动物细胞裂解液(碧云天，中国),冰上裂解后，3,500xg离心5mins取裂解上清标记为细胞裂解液。向80μl细胞培养上清和细胞裂解液中分别加入20μl 5×上样缓冲液，100℃煮沸5mins，SDS-PAGE及WB检测gE或gE-鞭毛素融合蛋白的表达。Vero细胞检测结果见图4，HEK293细胞检测结果见图5。由图4和图5可知，经腺病毒A或B感染后，在HEK 293及Vero细胞的上清中均能够成功的检测到gE蛋白或gE鞭毛素融合蛋白的表达，表达的gE蛋白分子量约在80Kd左右，gE-鞭毛素融合蛋白分子量在120Kd左右。该gE蛋白及gE-鞭毛素融合蛋白能够被mouse anti-VZV gE单克隆抗体特异性识别。且gE-鞭毛素融合蛋白能被anti-鞭毛素多抗特异性识别。
14重组腺病毒小规模扩增：
将汇合度90％的HEK293细胞，按照MOI 0.01～1分别接种不同的重组腺病毒，并将细胞置于37℃，5％CO 2培养箱持续培养，待70％以上细胞出现变圆，脱落时，将细胞用细胞刮刮下，2265g，离心十分钟，分别收获上清及细胞沉淀。将细胞沉淀用PBS重悬，并置于-80℃冰箱反复冻融三次，2265g，离心十分钟收获上清，用于下一步纯化。
1.5重组腺病毒纯化
预冷离心转子至4℃。在生物安全柜中，向离心管中缓慢加入12ml 1.4g/ml氯化铯(53g+87ml 10mM Tris-HCl，pH 7.9)，再非常轻缓地加入9ml 1.2g/ml氯化铯(26.8g+92ml 10mM Tris-HCl，pH 7.9)。之后在不连续梯度顶部加入13ml病毒保存液，平衡离心管，100000×g(SW28转子上为23000rpm)4℃离心120分钟。小心抽吸病毒带，将含病毒的溶液转移至无菌15ml离心管，加入等体积的10mM Tris HCl,pH 7.9。将20ml 1.35g/ml氯化铯加入离心管中。非常缓慢地顶部加入 15ml上步中稀释的病毒悬液。平衡离心管后，100000×g 4℃离心18小时。超速离心后，小心收集蓝白色病毒带。将病毒在10000道尔顿的纤维素酯膜(购自美国BD公司)中进行4℃透析至PBS溶液中，以去除氯化铯盐。透析后的病毒溶液加入10％甘油，分装后冻存于-80℃冰箱。
1.6重组腺病毒的检定及分析
Western blotting检测纯化后的重组腺病毒(见图6A)，WB使用抗体为兔Anti-Ad5多克隆抗体。图6A可见每种重组腺病毒均可被rabit anti-rAd5多克隆抗体特异性识别。将纯化后的重组病毒rAd5-gE复染(1％～2％的磷钨酸溶液，pH 6.8)之后进行电镜检测，经电镜观察可看到完整的病毒颗粒见图6B，阴离子-HPLC分析结果显示，纯化后的病毒纯度在95％以上见图6C。TCID 50检测结果显示纯化后的腺病毒滴度在10 10TCID 50/ml以上。
实施例2
gE蛋白及gE-鞭毛素融合蛋白在原核系统中的表达，纯化及检定
2.1基因及蛋白命名
插入基因及对应表达的gE及gE-鞭毛素融合蛋白的命名见图7。
2.2 pET28a表达载体的构建
将2.1所示个基因分别插入用NcoI和XhoI酶切后插入用相同酶切后的pET28a载体中，连接转化后，挑取单克隆接种含kanamycin(50μg/ml)抗性的LB培养基，过夜培养后提取质粒，送测序公司进行测序。获得pET28a-gE，pET28a-ENF，pET28a-ECF，pET28a-ESF表达质粒。
2.3 gE及gE鞭毛素融合蛋白的表达
将测序正确的pET28a-gE，pET28a-ENF，pET28a-ECF，pET28a-ESF质粒分别转化BL21(DE3)感受态细胞，挑取单克隆接种于含kanamycin抗性的LB培养基中，37℃，200rpm过夜培养。次日，将菌种转接至新鲜的含kanamycin抗性的LB培养基中。37℃，200rpm培养4h，待OD 600达到0.6～0.8时，加入0.1～1mM的IPTG进行诱导表达，表达温度16～37℃诱导4～16h，收获菌体，用于下一步纯化。
2.3 gE及gE鞭毛素融合蛋白的纯化及复性。
将收集的菌体，使用高压匀浆仪进行破碎后，2,265x g，离心10min，收集包涵体。将包涵体用含去污剂的生理盐水洗涤3～4次后加入含6M盐酸胍或8M尿素的20mM Tris,5mM imidazole,500mM NaCl,pH 8.0缓冲液进行溶解。将清洗后的镍柱用平衡液A(20mM Tris,8M Urea,5mM imidazole,500mM NaCl,pH 8.0)平衡5个柱体积(CV)。将溶解后的包涵体上样至镍柱，上样结束后，用平衡液冲洗5CV，然后用20CV线性梯度至100％洗脱液B进行洗脱，洗脱液B液为20mM Tris,8M Urea,500mM imidazole,500mM NaCl,pH 8.0。分别收集各个洗脱峰。
透析复性：将纯化的包涵体(溶解于8M Urea)用透析袋逐步透析至含6M、4M、2M Urea的PBS溶液中。每隔2h更换一次透析液。最后再将纯化后的包涵体蛋白缓慢透析至PBS溶液中。
柱上复性：在包涵体上样结束后，使用平衡液A冲洗柱子5CV；使用20CV～40CV线性梯度至100％复性液B，进行柱上复性，复性液B为：20mM Tris+2M Urea+5mM咪唑+500mM NaCl+0.1mM GSSG/1mM GSH，pH：8.0。复性结束后，使用缓冲液C(20mM Tris,2M Urea,5mM imidazole,500mM NaCl,pH 8.0)冲洗柱子5CV。用20CV线性梯度至100％洗脱液D进行洗脱，洗脱液D为：20mM Tris+2M Urea+5mM咪唑+500mM NaCl，pH：8.0。分别收集各个洗脱峰。将收集的洗脱峰使用透析袋，透析至PBS溶液中。
2.4 gE及gE鞭毛素融合蛋白的检定。
10％SDS-PAGE电泳及WB分析纯化后的蛋白，结果见图8。检测后可知纯化后的gE蛋白分子量在58Kd左右，gE-鞭毛素融合蛋白在90Kd左右，除ECF蛋白外，其它蛋白经纯化后纯度达80％以上。各蛋白能被鼠anti-gE单克隆抗体特异性识别，gE-鞭毛素融合蛋白能被兔anti-鞭毛素D0-D1抗血清特异性识别。经BCA法检测蛋白浓度可知，纯化后gE蛋白的产量在15mg～20mg/L，gE-鞭毛素融合蛋白的产量在8～15mg/L。由于纯化后残留的脂多糖污染(LPS，一种佐剂，会干扰鞭毛素活性测定)且部分蛋白存在降解，因此未比较来自大肠杆菌产生的免疫原与来真核系统(重组腺病毒载体)表达的相应蛋白免疫原性差距。然而，本领域普通技术人员应当能够优化产率、防止或最小化蛋白质水解降解并显著降低残余LPS含量。本发明中未对原核表达的蛋白进行进一步优化是因为本发明使用腺病毒真核表达系统获得了完整的、高产量且无LPS的重组蛋白。
实施例3
gE蛋白及gE-鞭毛素融合蛋白在真核细胞中的表达，纯化，检定及活性分析
3.1 gE蛋白及gE-鞭毛素融合蛋白在Vero细胞中的表达。
取汇合度为90％的Vero细胞一瓶(T-75瓶)，用PBS清洗两次后，分别感染包装获得的重组腺病毒A及重组腺病毒B，MOI 100～200，37摄氏度吸附1小时后，每瓶补加20ml的DMEM培养基。将培养瓶放入CO 2培养箱，37℃继续培养4～5天。之后收获培养上清，用于下一步纯化。
3.2 gE蛋白及gE-鞭毛素融合蛋白的纯化
取收获的gE或gE鞭毛素融合蛋白表达上清加入等体积的10mM PBS+1M(NH 4) 2SO 4，pH：7.5溶液，0.2μm滤膜过滤后上样至平衡好的Capto Phenyl Impress柱子中。平衡缓冲液为10mM PBS+500mM(NH 4) 2SO 4，pH：7.5。上样结束后继续用平衡液冲洗5CV；使用10CV的线性梯度洗脱至100％B液，B液为10mM PBS，pH：7.5。收集100％B时的洗脱峰。
将收集的洗脱峰上样至10mM PBS，pH 7.5平衡好的Source 30Q柱中，上样结束后使用平衡液冲洗5CV，使用10mM PB+250mM NaCl，pH 7.5进行阶梯洗脱，并收集纯化液即为最终纯化液。将纯化液(100μg～5mg/ml)添加10％甘油后，冻存于-80℃冰箱中。
3.3 gE蛋白及gE鞭毛素融合蛋白的检定及活性分析。
将纯化后的gE及gE鞭毛素融合蛋白经SDS-PAGE分析可知(见图9A)，纯化后的gE蛋白纯度在95％以上，纯化后的gE-鞭毛素融合蛋白纯度在85％以上，经BCA检测纯化后蛋白含量可知，该方法表达的gE蛋白产量可达到100mg/L,gE-鞭毛素融合蛋白的产量在50～80mg/L。本发明所制备的重组蛋白在浓度范围100μg至5mg/ml的水溶液中均为可溶状态，例如磷酸盐缓冲液(pH7.0-7.5)或4mM醋酸盐缓冲液(pH5.4)水溶液。掌握本领域知识的技术人员均熟悉蛋白质长时间稳定保存的方法。
纯化后的蛋白经WB分析可知(见图9B和9C)gE蛋白及gE-鞭毛素融合蛋白均能被鼠anti-gE单克隆抗体特异性识别。只有gE-鞭毛素融合蛋白能够被兔anti-鞭毛素D0-D1抗血清特异性识别，但不识别gE蛋白。
TLR-5活性分析(见表3)显示ANF，ACF，ASF三种融合蛋白均能通过激活THP-1 TLR-5受体诱导THP-1细胞以剂量依赖的方式分泌较高浓度的IL-8及TNF-α因子。但按照相同方法制备并纯化的gE蛋白则不能诱导TLR-5活性细胞因子的分泌。表明这三种gE-鞭毛素融合蛋白均具通过TLR-5特异性的发挥鞭毛素蛋白的功能活性。其中ASF的鞭毛素活性与商品化鞭毛素蛋白活性基本一致。
表3.TLR-5活性分析结果
Figure PCTCN2019105716-appb-000002
Figure PCTCN2019105716-appb-000003
实施例4
免疫原性测试
4.1重组腺病毒A及重组腺病毒B免疫原性检测
4.1.1动物免疫及样品收集。
所有动物实验均按照湖北省食品药品安全评价中心，动物保护与利用委员会(IACUC)批准的方案进行。36只无特殊病原体(SPF级)的雌性C57BL/6小鼠，体重12～16g饲养于湖北省食品药品安全评价中心。检验检疫结束后，小鼠按体重随机分为6组，分别于第1天和第28天肌肉接种10 9TCID 50/剂量的重组腺病毒A、重组腺病毒B或700pfu市售VZV疫苗(长春祁健生物技术有限公司，中国)。表4总结了分组细节。分别于第0、12、42和56天从眼眶静脉丛采血。
表4重组腺病毒免疫原性检测分组情况
Figure PCTCN2019105716-appb-000004
4.1.5不同腺病毒载体疫苗及市售水痘疫苗对小鼠的免疫原性对比
血清anti-gE IgG抗体：通过ELISA检测免疫后血清中anti-gE IgG抗体滴度，检测结果见图10及表5，空载体对照组小鼠在免疫后12、42、56天均未检测到抗体滴度上升。而其余各组在109剂量下，免疫后12天抗体滴度均明显提高，在第二剂加强免疫后，抗体滴度水平进一步升高。携带gE-鞭毛素融合蛋白的不同重组腺病毒组，在免疫后第12天抗体水平均显著高于rAd5-gE组及市售水痘疫苗组抗体滴度水平。在免疫后第56天，重组腺病毒各组抗体水平与商品化疫苗组相比抗体滴度均具有显著性差异(p<0.001)。各腺病毒载体候选疫苗组之间比较，仅有rAd5-gE组抗体滴度略低于rAd5-ANF组(P＝0.031)，其余各gE-鞭毛素融合腺病毒组之间抗体滴度无显著差别(P>0.05)。
表5.重组腺病毒诱导的gE-特异性IgG抗体滴度
Figure PCTCN2019105716-appb-000005
血清中和抗体滴度：如图11所示，10 9剂量条件下，在第一剂免疫后56天，各重组腺病毒组均诱导较高的中和抗体水平。而rAd5-ACF组诱导的中和抗体水平与其余各重组腺病毒组及市售VZV疫苗相比均有显著性差异(p<0.001)。其余各组虽无显著性差异，但诱导的中和抗体水平与市售的减毒活疫苗水平相当。rAd5-ANF及rAd5-SE组诱导的中和抗体水平与rAd5-gE组相比虽无统计学差异，但更为一致和均一。
细胞免疫水平检测：细胞内细胞因子染色结果见图12，重组腺病毒免疫C57BL/6小鼠8周后，rAd5-gE组及rAd5-SE组可检测到VZV gE特异性CD4+T细胞免疫。两者的CD4+及CD8+T细胞中IFN-γ阳性细胞百分比均显著高于腺病毒空载体对照组(P<0.01或P<0.0001)。如图13所示，IFN-γElispot检测结果见图13进一步证实了细胞内细胞因子染色的结果。rAd5-gE组与rAd5-SE组脾细胞IFN-γ和IL-4斑点数与其余各实验组相比均有显著性差异(P<0.01或P<0.0001)。rAd5-gE组与市售疫苗组相比也具有显著性差异(P<0.05)。说明rAd5-gE和rAd5-SE组可诱导强烈的CD4+Th1和Th2细胞免疫反应，同时亦可诱导较强的CD8+T细胞毒性细胞免疫反应。
4.2 gE鞭毛素融合蛋白免疫原性检测
4.2.1动物免疫及样品收集。
所有动物实验均按照湖北省食品药品安全评价中心，动物保护与利用委员会(IACUC)批准的方案进行。60只无特殊病原体(SPF级)的雌性C57BL/6小鼠，体重12～16g饲养于湖北省食品药品安全评价中心。检验检疫结束后，小鼠按体重随机分为10组，分别于第1天和第14天肌肉接种含和不含MF59(50μl/剂)佐剂的gE蛋白(5μg/剂)或含和不含MF59佐剂的gE-鞭毛素融合蛋白(8μg/剂)或700pfu市售VZV疫苗(长春祁健生物技术有限公司，中国)。表6总结了分组细节。分别于第0天和第28天从眼眶静脉丛采血。
表6.gE鞭毛素融合蛋白动物实验分组
组别 处理 剂量(μg/dose) 给药途径 数量
阴性对照组 生理盐水 / 肌肉注射，0.1ml/只 6
gE gE 5 肌肉注射，0.1ml/只 6
ANF ANF 8 肌肉注射，0.1ml/只 6
ACF ACF 8 肌肉注射，0.1ml/只 6
ASF ASF 8 肌肉注射，0.1ml/只 6
gE+MF59 gE+MF59 5+50μl 肌肉注射，0.1ml/只 6
ANF+MF59 ANF+MF59 8+50μl 肌肉注射，0.1ml/只 6
ACF+MF59 ACF+MF59 8+50μl 肌肉注射，0.1ml/只 6
ASF+MF59 ASF+MF59 8+50μl 肌肉注射，0.1ml/只 6
阳性疫苗 Licensed VZV 700PFU 肌肉注射，0.15ml/只 6
4.2.2含佐剂与不含佐剂的gE蛋白及gE-鞭毛素融合蛋白及市售水痘疫苗免疫原性对比。
血清anti-gE IgG抗体滴度：免疫后第28天，即第二剂免疫后第14天小鼠血清进行ELISA检测gE特异性抗体滴度,结果见图14及表7。ACF组gE特异性抗体滴度明显升高且与盐水组及gE组相比均具有统计学差异。ANF与ASF组gE特异性抗体滴度亦明显升高，与盐水组相比具有统计学差异；且gE特异性抗体滴度水平亦均高于gE组。该结果表明，在免疫后4周，所有不含MF59佐剂的的自身具有佐剂效应gE-鞭毛素融合蛋白诱导的抗体水平均显著高于对照组(p<0.0001)。在添加MF59佐剂后，抗体滴度进一步提高，表明该免疫组合物与佐剂联合具有增强体液免疫的作用，后期还可以考虑与其它能够诱导产生细胞免疫的佐剂联合使用。
表7.gE及gE-鞭毛素融合蛋白诱导的gE-特异性IgG抗体滴度
Figure PCTCN2019105716-appb-000006
血清中和抗体滴度：如图15所示，在第二剂免疫后14天，含MF59佐剂的ACF组诱导的中和抗体水平显著高于含MF59佐剂的gE蛋白组诱导的中和抗体水平。虽然其它两个含MF59佐剂的gE-鞭毛素融合蛋白与含MF59佐剂的gE蛋白组相比中和抗体水平无显著差异，但含MF59佐剂的gE-鞭毛素融合蛋白组诱导的中和抗体水平仍然高于MF59佐剂的gE蛋白组。且含MF59佐剂的gE-鞭毛素融合蛋白组诱导的中和抗体水平与市售减毒水痘活疫苗诱导的中和抗体水平相当。除此之外，含MF59佐剂的ASF和ACF组诱导的中和抗体反应比市售疫苗组诱导的中和抗体水平更为一致和均一。
细胞免疫：IFN-γ及IL-4Elispot检测结果见图16。含MF59佐剂的gE及ACF组脾细胞产生的IL-4斑点数有明显增加。与市售水痘减毒活疫苗相比具有显著性差异。
结论：目前仍需要开发一种更安全的改良型水痘和带状疱疹疫苗。市售的水痘减毒活疫苗仍然会给疫苗接种者带来罕见但非常严重的不良反应风险，尤其是对婴儿及免疫抑制人群，一旦出现这些反应，即需要紧急医疗处理。此外，市售的水痘减毒活疫苗还存在传染免疫受损个体的风险。三分之一接种了减毒活疫苗的受试者日后将有患带状疱疹的风险，他们当中有五分之一的受试者将受到带状疱疹愈后衰弱慢性神经痛的影响。因而目前孕妇和免疫功能低下的人禁止使用水痘和带状疱疹减毒活疫苗。尽管含佐剂的亚单位带状疱疹疫苗Shingrix比减毒活疫苗更有效，但它不良反应更多，会引起更多的局部和全身不良反应(Tricco AC et al.，BMJ，363:k4029，2018)。
本发明公开了制备和实施新的免疫组分的方法，所述免疫组分可用于预防针对VZV感染的疫苗并诱导广泛的保护性体液和细胞免疫。所述免疫组分选取VZV-gE糖蛋白作为免疫原，是因为gE蛋白是VZV病毒中含量最丰富、免疫原性最强的一种蛋白。本发明所述的免疫组分包括含佐剂的重组VZV-gE蛋白和具有内在佐剂特性的gE-flagelin融合蛋白。所述免疫组分可在原核或真核表达系统中表达制备，亦可在表达gE或gE-鞭毛素蛋白的复制缺陷型腺病毒载体中制备。通过基因工程与gE蛋白共价连接的鞭毛素蛋白部分已被证明能够结合并激活TLR5，从而触发天然免疫。这种融合蛋白在人类疫苗中可能不需要进一步的佐剂，从而降低由佐剂引发的不良反应的风险。根据本发明，所有免疫组分都具有高度的免疫原性，能诱导很强的与保护作用相关的gE特异性抗体、体外功能性中和抗体；同时该免疫组分还可诱导出在带状疱疹预防及康复中起重要作用的CD4+Th1和Th2 T细胞免疫。具有自身佐剂效应的gE-鞭毛素融合蛋白无论是直接纯化后的或经腺病毒载体递送均比其相应的gE蛋白具有更高的免疫原性。如有需要，还可使用Shingrix中AS01反应性低得多的常规佐剂即可显著提高纯化蛋白的免疫原性。表达gE或gE-鞭毛素融合蛋白的非复制腺病毒载体不仅能诱导良好的gE特异性抗体、VZV中和反应和CD4+T细胞反应，而且还能诱导机体产生CD8+T细胞免疫，从而能够进一步破坏被VZV感染细胞。
本发明中几乎所有的免疫组分均比市售减毒水痘活疫苗的免疫原性更强。此外，本发明所述的各种免疫组分可被用作初免-加强免疫策略的一部分，以增强及扩大VZV特异性免疫。所述的各种免疫组分还可与其他免疫原混合用于联合疫苗中。这些免疫组分比市售的水痘减毒活疫苗更安全，因为它们不会传染，不会引起偶然的可能与使用相关的严重的不良事件，而且最重要的是不会使疫苗接种者暴露于罹患带状疱疹及愈后神经痛的重大风险。本发明还公开了gE和gE鞭毛素蛋白融合蛋白在原核系统中表达制备的方法，该方法可降低疫苗的生产成本。本发明公开的腺病毒载体亦可被开发为单次免疫的疫苗，从而减少免疫频次。
总之，本发明提供的免疫组分可用于生产更安全、有效和可能更便宜的预防和控制水痘和带状疱疹的新型疫苗。需要注意的是，上述实施例仅用于说明本发明的技术方案，但不应理解为对本发明的限制。本领域技术人员可以对本发明的上述内容进行进一步的改进和调整，属于本发明的保护范围。
Figure PCTCN2019105716-appb-000007
Figure PCTCN2019105716-appb-000008
Figure PCTCN2019105716-appb-000009
Figure PCTCN2019105716-appb-000010
Figure PCTCN2019105716-appb-000011
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Figure PCTCN2019105716-appb-000013
Figure PCTCN2019105716-appb-000014
Figure PCTCN2019105716-appb-000015
Figure PCTCN2019105716-appb-000016
Figure PCTCN2019105716-appb-000017
Figure PCTCN2019105716-appb-000018
Figure PCTCN2019105716-appb-000019
Figure PCTCN2019105716-appb-000020
Figure PCTCN2019105716-appb-000021
Figure PCTCN2019105716-appb-000022
Figure PCTCN2019105716-appb-000023
Figure PCTCN2019105716-appb-000024
Figure PCTCN2019105716-appb-000025
Figure PCTCN2019105716-appb-000026
Figure PCTCN2019105716-appb-000027
Figure PCTCN2019105716-appb-000028
Figure PCTCN2019105716-appb-000029
Figure PCTCN2019105716-appb-000030
Figure PCTCN2019105716-appb-000031
Figure PCTCN2019105716-appb-000032
Figure PCTCN2019105716-appb-000033
Figure PCTCN2019105716-appb-000034
Figure PCTCN2019105716-appb-000035
Figure PCTCN2019105716-appb-000036
Figure PCTCN2019105716-appb-000037
Figure PCTCN2019105716-appb-000038
Figure PCTCN2019105716-appb-000039

Claims (64)

  1. 一种免疫组合物，其特征在于，至少包含基于水痘带状疱疹病毒糖蛋白E(简称gE)所得的抗原。
  2. 如权利要求1所述的免疫组合物，其特征在于，基于gE的抗原至少包含：(i)gE胞外区或其片段，或者其编码核酸分子；(ii)基于gE的融合蛋白，或者其编码核酸分子；(iii)基于gE的重组载体；(iv)或者上述两种或更多的组合。
  3. 如权利要求2所述的免疫组合物，其特征在于，基于gE的融合蛋白包含：gE胞外区或其片段共价偶联至细菌鞭毛素蛋白或其片段，其中所述细菌鞭毛素蛋白或其片段作为TLR-5激动剂。
  4. 如权利要求1所述的免疫组合物，其特征在于，gE胞外区具有与SEQ ID NO.1所示氨基酸序列至少90％的同源性。
  5. 如权利要求1所述的免疫组合物，其特征在于，基于gE的融合蛋白至少包含：所述鞭毛素蛋白的N端D0-D1区、鞭毛素蛋白的C端D0-D1区，和gE胞外区或其片段。
  6. 如权利要求5所述的免疫组合物，其特征在于，gE胞外区或其片段位于所述融合蛋白的N端或C端；或者插入到所述鞭毛素蛋白N端和C端之间。
  7. 如权利要求2所述的免疫组合物，其特征在于，所述融合蛋白选自如下任一融合形式：
    融合形式1：鞭毛素蛋白N端区-鞭毛素蛋白C端区-gE胞外区或其片段；
    融合形式2：gE胞外区或其片段-鞭毛素蛋白N端区-鞭毛素蛋白C端区；
    融合形式3：鞭毛素蛋白N端区-gE胞外区或其片段-鞭毛素蛋白C端区；
    其中，所述鞭毛素蛋白的N端区或C端区分别直接或者通过连接体与gE胞外区或其片段相连；或，
    所述鞭毛素蛋白N端区直接或者通过连接体与鞭毛素蛋白C端区相连。
  8. 如权利要求7所述的免疫组合物，其特征在于，所述连接体为1-20个肽键连接的氨基酸。
  9. 如权利要求8所述的免疫组合物，其特征在于，所述连接体为连接体I或连接体II；连接体I如SEQ IDNO：4所示；连接体II如SEQ ID NO：7所示。
  10. 如权利要求9所述的免疫组合物，其特征在于，所述鞭毛素蛋白的N端区或C端区分别通过连接体II与gE胞外区或其片段进行连接。
  11. 如权利要求9所述的免疫组合物，其特征在于，所述鞭毛素蛋白N端区通过连接体I与鞭毛素蛋白C端区相连。
  12. 如权利要求3所述的免疫组合物，其特征在于，所述细菌鞭毛素蛋白来自沙门氏菌。
  13. 如权利要求12所述的免疫组合物，其特征在于，所述沙门氏菌为鼠伤寒沙门氏菌或肠道沙门氏菌。
  14. 如权利要求13所述的免疫组合物，其特征在于，所述鞭毛素蛋白的氨基酸序列如SEQ ID NO：3或SEQ ID NO：29所示。
  15. 如权利要求13所述的免疫组合物，其特征在于，所述鞭毛素蛋白N端区为至少与SEQ ID NO：3中第2至176位氨基酸区域有95％同源性的氨基酸序列；所述鞭毛素蛋白C端区为至少与SEQ ID NO：3中第392至495位氨基酸区域有95％同源性的氨基酸序列。
  16. 如权利要求13所述的免疫组合物，其特征在于，所述鞭毛素蛋白N端区的氨基酸序列如SEQ ID NO：5所示；所述鞭毛素蛋白C端区的氨基酸序列如SEQ ID NO：6所示。
  17. 如权利要求13所述的免疫组合物，其特征在于，所述鞭毛素蛋白N端区为至少与SEQ ID NO：29中第2至180位氨基酸区域有95％同源性的氨基酸序列；所述鞭毛素蛋白C端区为至少与SEQ ID NO：29中第400至506位氨基酸区域有95％同源性的氨基酸序列。
  18. 如权利要求13所述的免疫组合物，其特征在于，所述鞭毛素蛋白N端区的氨基酸序列如SEQ ID NO：30所示；所述鞭毛素蛋白C端区的氨基酸序列如序列表SEQ ID NO：31所示。
  19. 如权利要求2所述的免疫组合物，其特征在于，所述基于gE的融合蛋白的氨基酸序列如SEQ ID NO：8～10、SEQ ID NO：32-34任一所示。
  20. 如权利要求2所述的免疫组合物，其特征在于，编码所述gE胞外区或其片段的核酸分子如SEQ ID NO：2，18-19任一所示。
  21. 如权利要求2所述的免疫组合物，其特征在于，编码所述基于gE的融合蛋白的核酸分子如SEQ ID NO：11-13、SEQ ID NO：20-26任一所示。
  22. 如权利要求2所述的免疫组合物，其特征在于，所述基于gE的重组载体包含如权利要求2-21任一项中所描述的核酸分子。
  23. 如权利要求22所述的免疫组合物，其特征在于，所述载体为腺病毒载体、腺病毒相关病毒载体、痘病毒载体、水疱性口炎病毒载体、牛副流感病毒载体、人副流感病毒载体、新城疫病毒载体、仙台病毒载体、麻疹病毒载体、减毒RSV载体、副粘病毒载体、甲型病毒载体(如委内瑞拉马脑炎病毒载、塞姆利基森林病毒载体、辛德比病毒载体)、棒状病毒载体、狂犬病病毒载体、小核糖核酸病毒、慢病毒载体、疱疹病毒载体、或植物来源的病毒用于在植物表达系统中表达。
  24. 如权利要求23所述的免疫组合物，其特征在于，所述腺病毒载体为人源腺病毒载体、黑猩猩源腺病毒载体或大猩猩腺病毒载体。
  25. 如权利要求24所述的免疫组合物，其特征在于，所述人源腺病毒为5型腺病毒载体(Ad5)；所述黑猩猩源腺病毒载体为ChAd68。
  26. 如权利要求24所述的免疫组合物，其特征在于，所述腺病毒载体为复制缺陷型腺病毒载体。
  27. 如权利要求26所述的免疫组合物，其特征在于，所述腺病毒载体的E1区被删除或功能性缺失从而形成复制缺陷型载体；或E1区和E3区均被删除或功能性缺失。
  28. 如权利要求27所述的免疫组合物，其特征在于，所述黑猩猩源腺病毒载体自身的E4区进一步被人5型腺病毒相应的E4区取代以增强载体的功能。
  29. 如权利要求22-28任一项所述的免疫组合物，其特征在于，当所述基于gE的重组载体携带编码gE胞外区或其片段的核酸分子时被称为重组腺病毒载体A。
  30. 如权利要求29所述的免疫组合物，其特征在于，所述重组腺病毒载体A携带如SEQ ID NO：2，18-19任一所示的核酸分子。
  31. 如权利要求29所述的免疫组合物，其特征在于，构建所述重组腺病毒载体A所用的骨架质粒为pAd5-CMV/V5-DEST。
  32. 如权利要求29所述的免疫组合物，其特征在于，构建所述重组腺病毒载体A所用的穿梭质粒为pDONR221。
  33. 如权利要求29所述的免疫组合物，其特征在于，构建所述的重组腺病毒载体A所用的宿主细胞系包括但不限定于HEK293或PER.C6细胞系。
  34. 如权利要求29-33任一项所述的免疫组合物，其特征在于，所述的重组腺病毒载体A由下述方法构建：将测序正确的重组穿梭质粒pDONR221-gE基因-PolyA与病毒骨架质粒pAd5-CMV/V5-DEST进行同源重组，将重组混合物转化至大肠杆菌TOP10感受态细胞中，筛选测序正确的腺病毒载体pAd5-CMV-gE基因-PolyA，将腺病毒载体pAd5-CMV-gE基因-PolyA线性化后转染HEK 293或PER.C6细胞进行包装得到所述的重组腺病毒载体A。
  35. 如权利要求22-28任一项所述的免疫组合物，其特征在于，当所述基于gE的重组载体携带编码基于gE的融合蛋白的核酸分子时被称为重组腺病毒载体B。
  36. 如权利要求35所述的免疫组合物，其特征在于，所述重组腺病毒载体B携带如SEQ ID NO：11-13，20-26任一所示的核酸分子。
  37. 如权利要求35所述的免疫组合物，其特征在于，构建所述的重组腺病毒载体B所用的骨架质粒为pAd5-CMV/V5-DEST。
  38. 如权利要求35所述的免疫组合物，其特征在于，构建所述的重组腺病毒载体B所用的穿梭质粒为pDONR221。
  39. 如权利要求35所述的免疫组合物，其特征在于，构建所述的重组腺病毒载体B所用的宿主细胞系包括但不限于HEK 293或PER.C6细胞系。
  40. 如权利要求35-39任一项所述的免疫组合物，其特征在于，所述的重组腺病毒载体B由下述方法构建：将测序正确的重组穿梭质粒pDONR221-gE-鞭毛素融合蛋白基因-PolyA转化与病毒骨架质粒pAd5-CMV/V5-DEST进行同源重组，将重组混合物转化至大肠杆菌TOP10感受态细胞，筛选测序正确的腺病毒载体pAd5-CMV-gE-鞭毛素融合蛋白基因-PolyA，将腺病毒载体pAd5-CMV-gE-鞭毛素融合蛋白基因-PolyA线性化后转染HEK 293或PER.C6细胞进行包装得到所述的重组腺病毒载体B。
  41. 如权利要求1所述的免疫组合物，其特征在于，进一步包含药学上可接受的载体，和/或佐剂，和/或免疫刺激分子。
  42. 如权利要求41所述的免疫组合物，其特征在于，所述佐剂包含但不限于：铝盐、水包油乳液或油包水乳液、MF-59、Quil A或其QS21组分、TLR激动剂、壳聚糖、免疫刺激复合物(ISCOMs)或其两种或多种的组合。
  43. 如权利要求1-42任一项所述的免疫组合物被用于制备预防和/或治疗由水痘带状疱疹感染的药物组合物中的应用。
  44. 如权利要求43所述的应用，其特征在于，所述的免疫组合物用于制备水痘疫苗或带状疱疹疫苗，或用于制备治疗带状疱疹或其愈后神经痛的药物中的应用。
  45. 一种联合疫苗，其特征在于，至少包含如权利要求1-42任一项所述的免疫组合物和其他疫苗，所述其它疫苗包含但不限于：流行性腮腺炎、麻疹和风疹疫苗。
  46. 如权利要求1-21任一所述免疫组合物中所描述的基于gE的融合蛋白。
  47. 如权利要求1-21任一所述免疫组合物中所描述的核酸分子。
  48. 如权利要求22-40任一所述免疫组合物中所描述的基于gE的重组载体。
  49. 一种分离的宿主细胞，其特征在于，包含如权利要求47所述的核酸分子。
  50. 一种制备gE胞外区或其片段，或如权利要求46所述的基于gE的融合蛋白的方法。
  51. 如权利要求50所述的方法，其特征在于，通过原核表达系统或者真核表达系统来制备。
  52. 如权利要求50所述的方法，其特征在于，所述原核表达为大肠杆菌表达，大肠杆菌为BL21(DE3)，表达载体为pET28a；所述gE胞外区的氨基酸序列如SEQ ID NO：35所示，所述gE胞外区的基因序列如SEQ ID NO：36所示；所述基于gE的融合蛋白的氨基酸序列如SEQ ID NO：37-39所示；所述基于gE的融合蛋白的基因序列如SEQ ID NO：37-39所示。
  53. 如权利要求50所述的方法，其特征在于，其特征在于，所述gE胞外区或其片段通过将如权利要求29-34任一项中所描述的重组腺病毒载体A感染培养Vero细胞获得；所述基于gE的融合蛋白通过将如权利要求35-40任一项中所描述的重组腺病毒载体B感染培养Vero细胞获得。
  54. 一种初免-强化免疫方案，其特征在于，使用如权利要求48所述的基于gE的重组载体进行初次免疫，然后用gE胞外区或其片段或者如权利要求46所述的基于gE的融合蛋白进行加强免疫；或者相反地，用gE胞外区或其片段或者如权利要求46所述的基于gE的融合蛋白进行首次免疫，然后用如权利要求48所述的基于gE的重组载体进行加强免疫，其中，使用gE胞外区或其片段进行免疫时可添加如权利要求41-42中所描述的佐剂。
  55. 一种初免-强化免疫方案，其特征在于，用如权利要求23所述的基于gE的重组异源载体进行初次免疫，用如权利要求24-40任一项所述的基于gE的重组腺病毒载体进行加强免疫；或者相反地，用如权利要求24-40任一项所述的基于gE的重组腺病毒载体进行初次免疫，用如权利要求23所述的基于gE的重组异源载体进行加强免疫；其中，所述异源载体指非腺病毒载体。
  56. 一种初免-强化免疫方案，其特征在于，将如权利要求24-40任一项中所描述的两种不同类型或不同物种的基于gE的重组腺病毒载体分别用作初次免疫或加强免疫，所述重组腺病毒载体携带gE胞外区或基于gE的融合蛋白基因。
  57. 一种重组腺病毒载体pAd5-CMV-gE基因-PolyA，其特征在于，gE基因具有如SEQ ID NO：2，18-19任一所示的核酸序列。
  58. 一种重组腺病毒载体pAd5-CMV-gE-鞭毛素融合基因-PolyA，其特征在于，gE-鞭毛素融合基因具有如SEQ ID NO：11-13，20-26任一所示的核酸序列。
  59. 一种改造的鞭毛素蛋白，其特征在于，鞭毛素蛋白N端区为至少与SEQ ID NO：3中第2至176位氨基酸区域有95％同源性的氨基酸序列；鞭毛素蛋白C端区为至少与SEQ ID NO：3中第392至495位氨基酸区域有95％同源性的氨基酸序列；所述鞭毛素蛋白N端区直接或者通过连接体与鞭毛素蛋白C端区相连。
  60. 如权利要求59所述的改造的鞭毛素蛋白，其特征在于，所述连接体为1-20个肽键连接的氨基酸。
  61. 如权利要求60所述的改造的鞭毛素蛋白，其特征在于，所述连接体具有如SEQ ID NO：4所示的氨基酸序列。
  62. 如权利要求59所述的改造的鞭毛素蛋白，其特征在于，所述的N端区的氨基酸序列如SEQ ID NO：5所示；所述的C端区的氨基酸序列如序列表SEQ ID NO：6所示。
  63. 如权利要求59所述的改造的鞭毛素蛋白，其特征在于，所述的改造的鞭毛素蛋白具有如SEQ ID NO：27所示的氨基酸序列。
  64. 如权利要求59-63任一项所述的改造的鞭毛素蛋白作为免疫佐剂的应用。
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