WO2022127820A1 - 病原样抗原疫苗及其制备方法 - Google Patents

病原样抗原疫苗及其制备方法 Download PDF

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WO2022127820A1
WO2022127820A1 PCT/CN2021/138312 CN2021138312W WO2022127820A1 WO 2022127820 A1 WO2022127820 A1 WO 2022127820A1 CN 2021138312 W CN2021138312 W CN 2021138312W WO 2022127820 A1 WO2022127820 A1 WO 2022127820A1
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antigen
virus
pathogen
particle
fusion protein
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French (fr)
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侯百东
华兆琳
郭畅
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榕森生物科技(北京)有限公司
中国科学院生物物理研究所
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Definitions

  • the present invention relates to vaccine and immunotherapy technology. Specifically, it relates to a pathogen-like antigen (PLA) vaccine, a preparation method thereof, and the use of the vaccine to prevent or treat related diseases.
  • PLA pathogen-like antigen
  • Subunit vaccines based on purified protein have the advantages of clear composition, good safety, and easy production, and are the development direction of modern vaccine technology.
  • protein subunit vaccines have also been found to have weak immunogenicity, so it is difficult to produce high-level and durable immune protection.
  • the common method to improve the immunogenicity of subunit protein antigens is to add adjuvants.
  • Adjuvants can enhance antigen-specific immune responses by stimulating innate immune signaling.
  • overactive innate immune signaling may have unacceptable inflammatory side effects. This limits the use of adjuvants to enhance the immunogenicity of protein subunit vaccines.
  • VLPs virus-like particles
  • HBsAg hepatitis B virus surface antigen
  • the immunogenicity of these antigens will also be improved, which may be related to the increased antigen density.
  • this approach is limited to proteins that can self-assemble into VLPs.
  • the effect of VLP alone in enhancing immunogenicity is still limited, and adjuvants are still needed to achieve better effects.
  • a class of VLPs (including Q ⁇ , MS2, AP205, etc.) derived from E.
  • coli phages were found to be highly immunogenic (Paul Pumpens et al., The True Story and Advantages of RNA Phage Capsids as Nanotools, Intervirology, 2016.11, 59:74-110).
  • Thomas M. Kundig et al. Thomas M. Kundig et al., Der p 1 peptide on virus-like particles is safe and highly immunogenic in healthy adults, J Allergy Clin Immunol, 2006, VOL.117, NO.6 found that the antigen After being linked to the surface of Q ⁇ -VLP by genetic engineering or chemical cross-linking, the specific immune response to the antigen can be significantly enhanced.
  • AP205 protein (hereinafter referred to as AP205) is the major capsid protein of the newly identified AP205 RNA phage.
  • AP205 self-assembles into VLP particles, each VLP contains 180 AP205 capsid protein molecules. Both the N-terminus and C-terminus of AP205 can be linked to the target protein.
  • the SpyTag(ST)/SpyCatcher(SC) system is derived from the CnaB2 domain, which can spontaneously form stable isopeptide bonds under various conditions, so it can be used to solve the problems of protein subunit fusion expression and chemical coupling.
  • SpyCatcher(SC) system is derived from the CnaB2 domain, which can spontaneously form stable isopeptide bonds under various conditions, so it can be used to solve the problems of protein subunit fusion expression and chemical coupling.
  • due to the obvious mismatch between antigenic protein and VLP it is easy to lead to VLP aggregation, causing precipitation, which seriously affects the efficacy
  • the present invention aims to solve the above-mentioned defects and deficiencies in the prior art.
  • PHA soluble pathogen-like antigen
  • a virus-like particle which is self-assembled from a first fusion protein, the first fusion protein comprises a viral capsid protein or a variant thereof at the N-terminus, and a SpyTag at the C-terminus, (2) a second fusion protein, the The second fusion protein comprises the antigen or variant thereof and SpyCatcher;
  • virus-like particle also encapsulates nucleic acid within it, and wherein the antigen or variant thereof is displayed on the surface of the virus-like particle by covalent linkage between SpyCatcher and SpyTag.
  • the soluble pathogen-like antigen complex according to the present invention wherein the nucleic acid encapsulated in the virus-like particle is the nucleic acid from the host bacterium used to express the virus-like particle, which is encapsulated by the virus-like particle during its self-assembly,
  • the host bacterium is E. coli
  • the nucleic acid is RNA.
  • the soluble pathogen-like antigen complex according to the present invention wherein the capsid protein is from Escherichia coli Q ⁇ , MS2, or AP205.
  • the soluble pathogen-like antigen complex according to the present invention wherein the capsid protein is from Escherichia coli phage AP205.
  • the soluble pathogen-like antigen complex according to the present invention wherein the antigen is selected from the RBD sequence of SARS-CoV2 virus S protein, African swine fever virus antigen eP22, influenza virus antigen M2E, autoantigen myelin oligodendrocyte glycoprotein MOG.
  • the soluble pathogen-like antigen complex according to the present invention wherein the sequence of the phage AP205 capsid protein has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% consistency.
  • the sequence of the phage AP205 capsid protein is SEQ ID NO: 1.
  • the soluble pathogen-like antigen complex according to the present invention wherein the sequence of the SpyTag has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with SEQ ID NO: 3 %, 99% or 100% identity, the sequence of SpyCatcher is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO:4 % or 100% consistency.
  • the sequence of the SpyTag is SEQ ID NO: 3
  • the sequence of the SpyCatcher is SEQ ID NO: 4.
  • the soluble pathogen-like antigen complex according to the present invention wherein an isopeptide bond is formed between Asp at position 7 of SpyTag sequence SEQ ID NO:3 and Lys at position 31 of SpyCatcher sequence SEQ ID NO:4.
  • the soluble pathogen-like antigen complex according to the present invention, wherein in the first fusion protein, the phage capsid protein or its variant is linked to SpyTag through a first linking peptide, and in the second fusion protein, the antigen or its variant is linked to SpyCatcher Linked via a second linker peptide.
  • the sequence of the first connecting peptide is SEQ ID NO: 5
  • the sequence of the second connecting peptide is SEQ ID NO: 6.
  • the second fusion protein is linked to the virus-like particle at a ratio of less than or equal to 1:1, preferably at a ratio of 1:1, 1:1.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12 ratio connections to ensure Solubility and immunogenicity of pathogen-like antigen complexes, the ratios calculated as the ratio of SpyCatcher on the second fusion protein to SpyTag on the virus-like particle.
  • the soluble pathogen-like antigen complex according to the present invention is formulated as a vaccine composition together with a pharmaceutically acceptable carrier and/or excipient.
  • Another aspect of the present invention provides a method for preparing a soluble pathogen-like antigen complex, comprising purifying virus-like particles at a pH value in the range of pH 4.0-9.0, preferably 5.5-8.5.
  • Yet another aspect of the present invention provides a method for increasing the solubility of a pathogen-like antigen complex, comprising the steps of: (1) preparing a virus-like particle and a second fusion protein as defined above; and (2) preparing the second fusion protein When the fusion protein is linked to the virus-like particle, the linking ratio of the second fusion protein to the virus-like particle is reduced to obtain a soluble pathogen-like antigen complex.
  • the method for improving the solubility of a pathogen-like antigen complex wherein the antigen is selected from the RBD sequence of the S protein of SARS-CoV2 virus, the African swine fever virus antigen eP22, the influenza virus antigen M2E, or the self-antigen myelin oligodendrocytes Glial glycoprotein MOG.
  • Yet another aspect of the invention relates to the prevention and/or treatment of SARS-CoV2 virus, influenza virus, or African swine fever virus or diseases associated with the self-antigen myelin oligodendrocyte glycoprotein MOG in a subject in need thereof
  • a method of comprising administering to said subject a prophylactically and/or therapeutically effective amount of a soluble pathogen-like antigen complex or vaccine composition of the present invention.
  • the method for preparing a pathogen-like antigen vaccine of the present invention avoids the degradation of nucleic acid in the process of separating and purifying the PLA vaccine, so that the immunogenicity of the vaccine can be effectively improved without additional adjuvant, and at the same time, additional adjuvant addition is reduced or avoided. resulting in an excessive inflammatory response.
  • Figure 1 Whole bacterial lysis before and after induction of AP205 fusion protein expression, wherein Figure 1A is SC-AP205 and Figure 1B is AP205-SC.
  • Figure 2 Nucleic acid gel images of SC-AP205 and AP205-SC after centrifugation on sucrose pads, where 1 is AP205-ST, 2 is SC-AP205, and 3 is AP205-SC.
  • Figure 3 The gel image of the AP205 fusion protein after cesium chloride density gradient centrifugation and the collected protein gels, wherein Figure 3A is SC-AP205, and Figure 3B is AP205-SC.
  • Figure 4 Whole bacterial lysis before and after induction of AP205-ST.
  • Figure 5 Stratified protein gel image of AP205-ST cesium chloride density gradient centrifugation.
  • Figure 7 Degradation of AP205-RBD (SC at the C-terminus).
  • Figure 8 Stability of SC-RBD ligated products to AP205-ST.
  • Figure 9 The effect of modified AP205 on the solubility of the ligation product, 1 is the wild-type AP205, 2 is the AP205 after the modification of the present invention, Figure 9A and Figure 9B are SDS-PAGE and nucleic acid gel images, respectively.
  • Figure 10 The effect of adjusting the antigen ratio on the solubility of the ligation product, Figure 10A, Figure 10B and Figure 10C are SDS-PAGE, nucleic acid gel, Coomassie R-250 plots, respectively.
  • Figure 11 The connection of African swine fever antigen eP22 to AP205-ST, Figure 11A, Figure 11B and Figure 11C are SDS-PAGE, nucleic acid gel, Coomassie R-250 images, respectively.
  • Figure 12 The connection of influenza virus antigen M2E to AP205-ST, Figure 12A, Figure 12B and Figure 12C are SDS-PAGE, nucleic acid gel, Coomassie R-250 images, respectively.
  • Figure 13 The connection of autoantigen MOG to AP205-ST, Figure 13A, Figure 13B and Figure 13C are SDS-PAGE, nucleic acid gel, Coomassie R-250 maps, respectively.
  • Figure 14 Influence of VLP purification conditions on the presence or absence of RNA inside
  • Figure 14A and Figure 14B are SDS-PAGE and nucleic acid gel images, respectively.
  • Figure 15 Changes in nucleic acids within VLPs under different pH gradients.
  • FIG 16 Production of anti-RBD IgG antibodies in mice immunized with the PLA-SARS-CoV2 vaccine (primary immunization).
  • FIG 17 Production of anti-RBD IgG antibodies in mice immunized with the PLA-SARS-CoV2 vaccine (secondary immunization).
  • Figure 18 Changes in RBD IgG-type antibody titers after primary immunization and re-immunization with PLA-SARS-CoV2 vaccine.
  • Figure 19 Compared with the traditional vaccine with adjuvant, the vaccine complex constructed by the VLP of the present invention and several other antigens significantly enhanced the ability to induce antibody production (Figure 19A, Figure 19B and Figure 19C, respectively, using the African swine fever virus antigen eP22 , influenza virus antigen M2E and autoantigen myelin oligodendrocyte glycoprotein MOG).
  • Figure 20 The production of neutralizing antibodies in mice immunized with the PLA-SARS-CoV2 vaccine.
  • Figure 21 Anti-RBD IgG antibodies produced in cynomolgus monkeys immunized with the PLA-SARS-CoV2 vaccine.
  • Figure 22 Neutralizing antibody production in cynomolgus monkeys immunized with PLA-SARS-CoV2 vaccine.
  • Figure 23 Lung viral loads in macaques immunized with PLA-SARS-CoV2 vaccine.
  • the SpyCatcher sequence can be located at the N-terminus or C-terminus of the antigen sequence, the fusion protein formed when it is located at the N-terminus of the antigen is relatively more stable.
  • the fusion protein is connected with the virus-like particle carrying the SpyTag sequence. Also more stable (Example 2).
  • RNA nucleic acid in VLP is another key factor for the effect of unadjuvanted PLA vaccine.
  • PLA vaccine although some conditions do not affect the stability of the protein, it may degrade the RNA.
  • PHA soluble pathogen-like antigen
  • a virus-like particle which is self-assembled from a first fusion protein, the first fusion protein comprises a viral capsid protein at the N-terminus or a variant thereof and a SpyTag at its C-terminus, (2) a second fusion protein, the The second fusion protein comprises the antigen or variant thereof and SpyCatcher;
  • virus-like particle also encapsulates nucleic acid inside it, and wherein the virus-like particle and the second fusion protein are covalently linked via the SpyCatcher and SpyTag to display the antigen or variant thereof on the virus-like particle s surface.
  • the soluble pathogen-like antigen complex according to the present invention wherein the nucleic acid encapsulated in the virus-like particle is a nucleic acid from a host bacterium for expressing the virus-like particle, preferably the host bacterium is Escherichia coli, preferably the nucleic acid is RNA.
  • the soluble pathogen-like antigen complex according to the present invention wherein the capsid protein is from Escherichia coli Q ⁇ , MS2, or AP205.
  • the soluble pathogen-like antigen complex according to the present invention wherein the capsid protein is from Escherichia coli phage AP205.
  • the soluble pathogen-like antigen complex according to the present invention wherein the antigen is selected from the RBD sequence of SARS-CoV2 virus S protein, African swine fever virus antigen eP22, influenza virus antigen M2E, autoantigen myelin oligodendrocyte glycoprotein MOG.
  • the soluble pathogen-like antigen complex according to the present invention wherein the sequence of the phage AP205 capsid protein has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% consistency.
  • the sequence of the phage AP205 capsid protein is SEQ ID NO: 1.
  • the soluble pathogen-like antigen complex according to the present invention wherein the sequence of the SpyTag has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with SEQ ID NO: 3 %, 99% or 100% identity, the sequence of SpyCatcher is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with SEQ ID NO:4 % or 100% consistency.
  • the sequence of the SpyTag is SEQ ID NO: 3
  • the sequence of the SpyCatcher is SEQ ID NO: 4.
  • the soluble pathogen-like antigen complex according to the present invention wherein an isopeptide bond is formed between Asp at position 7 of SpyTag sequence SEQ ID NO:3 and Lys at position 31 of SpyCatcher sequence SEQ ID NO:4.
  • the soluble pathogen-like antigen complex according to the present invention, wherein in the first fusion protein, the phage capsid protein or its variant is linked to SpyTag through a first linking peptide, and in the second fusion protein, the antigen or its variant is linked to SpyCatcher Linked via a second linker peptide.
  • the sequence of the first connecting peptide is SEQ ID NO: 5
  • the sequence of the second connecting peptide is SEQ ID NO: 6.
  • the second fusion protein is linked to the virus-like particle at a ratio of less than or equal to 1:1, preferably at a ratio of 1:1, 1:1.5, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12 ratio connections to ensure Solubility and immunogenicity of pathogen-like antigen complexes, the ratios calculated as the ratio of SpyCatcher on the second fusion protein to SpyTag on the virus-like particle.
  • the present invention also relates to nucleic acid sequences encoding the first and second fusion proteins and vectors comprising the nucleic acid sequences.
  • nucleic acid sequences or nucleic acid molecules or vectors described herein can be codon optimized.
  • nucleic acid sequences or nucleic acid molecules or vectors described herein may be degenerate versions thereof.
  • the soluble pathogen-like antigen complex according to the present invention is formulated as a vaccine composition together with a pharmaceutically acceptable carrier and/or excipient.
  • Another aspect of the present invention provides a method for preparing a soluble pathogen-like antigen complex, comprising purifying virus-like particles at a pH value in the range of pH 4.0-9.0, preferably 5.5-8.5.
  • Yet another aspect of the present invention provides a method for improving the solubility of a pathogen-like antigen complex, comprising the steps of: (1) preparing the second fusion protein and the virus-like particle as defined in any one of the foregoing; (2) ) when the second fusion protein is linked to the virus-like particle, reducing the linking ratio of the second fusion protein to the virus-like particle to obtain a soluble pathogen-like antigen complex.
  • the method for improving the solubility of a pathogen-like antigen complex wherein the antigen is selected from the RBD sequence of the S protein of SARS-CoV2 virus, the African swine fever virus antigen eP22, the influenza virus antigen M2E, or the self-antigen myelin oligodendrocytes cytoplasmic glycoprotein MOG.
  • Yet another aspect of the present invention relates to the prevention and/or treatment of diseases associated with SARS-CoV2 virus, influenza virus, or African swine fever virus infection or with the self-antigen myelin oligodendrocyte glycoprotein MOG in a subject in need thereof
  • a method for a related disease comprising administering to said subject a prophylactically and/or therapeutically effective amount of a soluble pathogen-like antigen complex or vaccine composition of the present invention.
  • the associated disease may be caused by SARS-COV-2 virus and/or mutants thereof.
  • the associated disease may be COVID-19.
  • fusion protein refers to a genetically engineered protein encoded by a nucleotide sequence formed by two or more complete or partial genes or series of nucleic acids joined together. Alternatively, fusion proteins can be made by combining two or more heterologous peptides.
  • linker peptide refers to one or more (eg, about 2-10) amino acid residues between two adjacent motifs, regions or domains of a polypeptide such as between an antigenic peptide or between an antigenic peptide and an adjacent peptide encoded by a multiple translation leader sequence, or between an antigenic peptide and a spacer or cleavage site.
  • the linker peptide can be derived from the construct design of the fusion protein (eg, amino acid residues resulting from the use of restriction enzyme sites in the construction of the nucleic acid molecule encoding the fusion protein).
  • variant refers to a protein or nucleic acid molecule whose sequence is similar but not identical to the reference sequence, wherein the activity of the variant protein (or protein encoded by the variant nucleic acid molecule) is not significantly altered.
  • variations in sequence can be naturally occurring variations or can be engineered using genetic engineering techniques known to those skilled in the art. Examples of such techniques can be found in Sambrook J, Fritsch EF, Maniatis T et al., in Molecular Cloning--A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, pp. 9.31-9.57), or Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • any type of change in amino acid or nucleic acid sequence is permissible as long as the activity of the resulting variant protein or polynucleotide is not significantly altered.
  • variations include, but are not limited to, deletions, insertions, substitutions, and combinations thereof.
  • amino acids can be divided into charged amino acids, uncharged amino acids, polar uncharged amino acids, and hydrophobic amino acids.
  • protein variants containing substitutions may be those protein variants in which amino acids are substituted with amino acids from the same group. Such substitutions are referred to as "conservative" substitutions.
  • antigen refers to a polypeptide that can stimulate a cell to mount an immune response.
  • virus-like particles are particles assembled from one or more viral structural proteins, which have an external structure and antigenicity similar to viral particles, but do not contain viral genes .
  • vaccine and “vaccine composition” used in the present invention refer to a pharmaceutical composition containing corresponding virus antigens, and the pharmaceutical composition can induce, stimulate or enhance the immune response of a subject against the corresponding virus.
  • nucleic acid or “nucleic acid molecule” as used herein refers to, for example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, fragments produced by the polymerase chain reaction (PCR) or by in vitro translation Any of , and fragments produced by any one or more of ligation, cleavage, endonuclease action, or exonuclease action.
  • the nucleic acids of the present disclosure are generated by PCR.
  • Nucleic acids can be composed of monomers that are naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (eg, naturally occurring nucleotides) the ⁇ -enantiomeric form) or a combination thereof.
  • Modified nucleotides may have modifications in or instead of sugar moieties, or pyrimidine or purine base moieties, or pyrimidine or purine base moieties.
  • construct refers to any polynucleotide containing a recombinant nucleic acid.
  • the construct can be present in a vector (eg, bacterial vector, viral vector), or can be integrated into the genome.
  • a "vector” is a nucleic acid molecule capable of transporting another nucleic acid.
  • a vector can be, for example, a plasmid, cosmid, virus, RNA vector or linear or circular DNA or RNA molecule, which can include chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic acids.
  • Exemplary vectors are those capable of autonomously replicating (episomal vectors) and/or expressing the nucleic acids to which they are linked (expression vectors).
  • the terms "signal peptide” and “leader sequence” are used interchangeably herein and refer to an amino acid sequence that can be linked to the amino terminus of the proteins set forth herein.
  • the signal peptide/leader sequence usually directs the localization of the protein.
  • the signal peptide/leader sequence used herein preferably facilitates the secretion of the protein from the cell in which it is produced.
  • the signal peptide/leader sequence is often cleaved from the rest of the protein (often referred to as the mature protein) after secretion from the cell.
  • the signal peptide/leader sequence is attached to the N-terminus of the protein and is about 9 to 200 nucleotides in length (3 to 60 nucleic acids).
  • the signal peptide used in the present invention can be the signal peptide sequence of the SARS-COV-2 virus S protein or the signal peptide sequence from other eukaryotic/viral proteins.
  • expression vector refers to a DNA construct containing a nucleic acid molecule operably linked to suitable control sequences that enable expression of the nucleic acid molecule in a suitable host.
  • control sequences include promoters for effecting transcription, optional operator sequences for controlling such transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control the termination of transcription and translation.
  • the vector may be a plasmid, phage particle, virus, or simply a potential genomic insert.
  • Viral vectors can be DNA (eg, adenovirus or vaccinia virus) or RNA based, including oncolytic viral vectors (eg, VSV), replicable or non-replicable. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases, can integrate into the genome itself.
  • plasmid "expression plasmid"
  • vector are often used interchangeably.
  • expression refers to the process of producing a polypeptide based on the nucleic acid sequence of a gene.
  • the process includes transcription and translation.
  • Translation can start with an unconventional start codon, such as a CUG codon, or translation can start with several start codons (standard AUG and unconventional) to produce more protein than mRNA produced (on a per mole basis). quantity).
  • the term "introduced” as used herein refers to “transfection” or “transformation” or “transduction” and includes the integration of nucleic acid sequences into eukaryotic or prokaryotic cells Mention, wherein the nucleic acid sequence can be integrated into the genome of the cell (eg, chromosomal, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (eg, transfected mRNA).
  • host refers to any organism or cell thereof, whether eukaryotic or prokaryotic, into which a construct of the invention may be introduced, in particular a host in which RNA silencing occurs.
  • host includes E. coli such as E. coli.
  • the term "host” is used to refer to eukaryotes, including unicellular eukaryotes such as yeast and fungi, and multicellular eukaryotes such as animals, non-limiting examples including invertebrates (eg, insects, coelenterates, echinoderms, nematodes etc.); eukaryotic parasites (eg, malaria parasites such as Plasmodium falciparum, worms, etc.); vertebrates (eg, fish, amphibians, reptiles, birds, mammals); and mammals (eg, rodents, primates such as humans and non-human primates).
  • the term "host cell” appropriately encompasses cells of such eukaryotes as well as cell lines derived from such eukaryotes.
  • adjuvant refers to a natural or synthetic substance that participates in the immune response to a hapten or antigen by enhancing the activity of macrophages to promote the response of T cells or B cells in the body.
  • prophylaxis and/or treatment refers to inhibiting the replication, spread or colonization of the corresponding virus in a host, as well as alleviating the symptoms of a virus-infected disease or disorder.
  • the treatment is considered therapeutic if there is a reduction in viral load, a reduction in symptoms, and/or an increase in food intake and/or growth.
  • the term "therapeutically effective amount (or dose)" or “effective amount (or dose)” of a compound or composition means sufficient to cause one or more symptoms of the disease being treated in a statistically significant manner amount of improved compound. The precise amount depends on numerous factors, eg, the activity of the composition, the method of delivery employed, the immunostimulatory capacity of the composition, the intended patient and patient considerations, etc., and can be readily determined by one of ordinary skill in the art.
  • a therapeutic effect can include, directly or indirectly, the alleviation of one or more symptoms of a disease, and a therapeutic effect can also include, directly or indirectly, the stimulation of a cellular immune response.
  • pharmaceutically acceptable carrier includes any carrier that does not by itself induce the production of antibodies detrimental to the individual receiving the pharmaceutical composition. Suitable carriers are usually large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, amino acid polymers, amino acid copolymers, lipid aggregates (eg, oil droplets or liposomes), and the like. These pharmaceutically acceptable carriers are well known to those of ordinary skill in the art.
  • subject can be any organism capable of developing a cellular immune response, such as a human, pet, livestock, display animal, zoo specimen or other animal.
  • a subject can be a human, non-human primate, dog, cat, rabbit, rat, mouse, guinea pig, horse, cow, sheep, goat, pig, and the like.
  • Subjects in need of administration of a therapeutic agent as described herein include subjects who have been infected with SARS-COV-2 virus or even have developed a disease associated with viral infection, or are at risk of SARS-COV-2 virus infection.
  • subject in need refers to a subject at high risk for or suffering from a disease, disorder or condition for which the compounds provided herein or Its composition treats or improves.
  • the subject in need is a human.
  • the desired outcome is a safe product capable of inducing durable protective immunity with minimal side effects, and compared to other strategies (eg, intact live or attenuated pathogens), Inexpensive production minimizes or eliminates contraindications that are otherwise (usually) associated with the use of intact or attenuated viral immunization compositions.
  • Inexpensive production minimizes or eliminates contraindications that are otherwise (usually) associated with the use of intact or attenuated viral immunization compositions.
  • the ability to respond rapidly to infectious disease emergencies is one benefit of effective application of the embodiments disclosed herein, whether in the context of biodefense or immunotherapy or technology.
  • the pathogen-like antigen vaccines of the invention can be administered, for example, by intramuscular injection, subcutaneously, intranasally, transmucosally, intravenously, or by intradermal or subcutaneous administration.
  • the soluble pathogen-like antigen (PLA) vaccine of the present invention comprises four structural elements: 1) a phage VLP or other nanoparticle-based antigen display chassis; 2) a TLR stimulator, such as nucleic acid, such as RNA, carried inside the chassis particle, preferably from The expression host; 3) the Spycather/Spytag sequence used to link the chassis particle to the antigen; and 4) the antigen to be displayed.
  • the necessary conditions for the fusion protein to be used as an unadjuvanted protein engineering vaccine include: the fusion protein has sufficient structural stability, is soluble, and will not aggregate or precipitate; at the same time, the chassis TLR stimulators such as nucleic acids encapsulated inside the particles are not eliminated by degradation.
  • the chassis TLR stimulators such as nucleic acids encapsulated inside the particles are not eliminated by degradation.
  • Example 1 Influence of SpyCatcher (SC) and SpyTag (ST) connection with AP205 on VLP self-assembly
  • amino acid sequence of SC is SEQ ID NO: 4, and the amino acid sequence of AP205 (non-wild type) after the transformation is SEQ ID NO: 1, and the two are connected by connecting sequence SEQ ID NO: 5.
  • a 393bp full-length cDNA (SEQ ID NO: 9) fragment encoding AP205 was artificially synthesized, and a BamHI restriction site was added to its 5' end and a GSGGSG connection, an AgeI restriction site, and a stop codon were added to the 3' end. TAA, KpnI restriction sites.
  • the synthesized AP205 cDNA fragment (1 ⁇ g) and pET21 plasmid (1 ⁇ g) were digested with BamHI (Takara 1010A) and KpnI endonuclease (Takara 1068A), respectively, at 37°C for 2 hours.
  • the digested cDNA fragment and the pET21a plasmid fragment were then separated by agarose gel electrophoresis.
  • the isolated cDNA fragment and pET21a plasmid fragment were purified separately using a small amount of DNA product purification kit (Zhuangmeng Biotechnology ZP201-3).
  • the purified cDNA fragment was further subjected to a DNA ligation reaction with the pET21a plasmid fragment to construct a pET21a plasmid (referred to as pET21a-AP205 plasmid) containing the cDNA fragment.
  • the ligase was T4 DNA ligase (Takara 2011A), and the ligation buffer was T4 DNA Ligase Buffer (Takara 2011A).
  • the ratio of the pET21a plasmid fragment to the AP205 cDNA fragment in the ligation reaction was about 1:3, and the total DNA was about 200 ng, 22 °C connection for 2 hours.
  • the pET21a-AP205 plasmid was transformed into the expression host as follows: 15 ⁇ l of the ligation reaction solution was added to 150 ⁇ l of XLI-Blue competent E. coli (full gold CD401-02) at 42° C. for 1 minute.
  • PCR upstream primer (SEQ ID NO: 13): acgggatccATGTCGTACTACCATCACCATC
  • downstream primer SEQ ID NO: 14
  • the PCR program is 194°C for 5 minutes 294°C for 30 seconds 358°C for 30 seconds 472°C for 1 minute, 234 cycle 30 Second, 572°C for 5 minutes, 64°C hold) to artificially synthesize a full-length 276bp cDNA (SEQ ID NO: 10) fragment encoding SC, and add BamHI restriction sites at both the 5' and 3' ends.
  • the synthesized SC cDNA fragment (1 ⁇ g) and pET21a-AP205 plasmid (1 ⁇ g) were digested with BamHI endonuclease (Takara 1010A) at 37°C for 2 hours, respectively.
  • the digested cDNA fragment and the pET21a-AP205 plasmid fragment were then separated by agarose gel electrophoresis.
  • the isolated cDNA fragment and pET21a-AP205 plasmid fragment were purified separately using a small amount of DNA product purification kit (Zhuangmeng Biotechnology ZP201-3).
  • the purified cDNA fragment was further subjected to a DNA ligation reaction with the pET21a-AP205 plasmid fragment to construct a pET21a-AP205 plasmid (referred to as pET21a-SC-AP205 plasmid) containing the cDNA fragment.
  • the ligase was T4 DNA ligase (Takara 2011A), and the ligation buffer was T4 DNA Ligase Buffer (Takara 2011A).
  • the ratio of pET21a-AP205 plasmid fragment to SC cDNA fragment in the ligation reaction was about 1:3, and the total DNA was about 200ng. Connect for 2 hours at 22°C.
  • the pET21a-SC-AP205 plasmid was transformed into the expression host as follows: 15 ⁇ l of the ligation reaction was added to 150 ⁇ l of XLI-Blue competent E. coli (full gold CD401-02), 42°C for 1 minute. Pipette 150 ⁇ l to plate on ampicillin-resistant LB plates and incubate at 37°C for 14-16 hours. A single colony was taken on the plate, and plasmid DNA was extracted with a plasmid purification kit (full-type gold EM101-02) and verified by enzyme digestion, confirming that the pET21a-SC-AP205 plasmid was successfully constructed.
  • a plasmid purification kit full-type gold EM101-02
  • the fusion protein AP205-SC expression plasmid was prepared by the same method as above (1), except that by PCR (upstream primer: acgaccggtATGTCGTACTACCATCACCATC (SEQ ID NO: 15), downstream primer: cccaccggtAATATGAGCGTCACCTTTAGTTGC (SEQ ID NO: 16),
  • the PCR program was 194°C for 5 minutes 294°C for 30 seconds 358°C for 30 seconds 472°C for 1 minute, 234 for 30 cycles, 572°C for 5 minutes 64°C hold) to artificially synthesize a full-length SC cDNA fragment of 276 bp, at its 5' and 3' AgeI restriction sites are added to the ends.
  • the synthesized SC cDNA fragment (1 ⁇ g) and pET21a-AP205 plasmid (1 ⁇ g) were digested with AgeI endonuclease (NEB R0552V) and ligated to construct pET21a-AP205-SC plasmid, respectively.
  • the fusion protein AP205-ST expression plasmid was constructed by the same method as above (1), except that the coding DNA sequence (gcccacatcgtgatggtggacgcctacaagccgacgaag) of ST was synthesized through the following process (the encoded amino acid sequence is SEQ ID NO:4):
  • PCR by annealing (5 ⁇ l 200 ⁇ M Primer-F, 5 ⁇ l 200 ⁇ M Primer-R, 2 ⁇ l 10x Annealing Buffer (100 mM Tris 8.0, 1 M NaCl, 10 mM EDTA), 8 ⁇ l dH 2 O.
  • Set PCR program to 99°C for 3 min, 99-20°C
  • the DNA sequence encoding ST was obtained by decreasing the temperature by 0.5 °C every 30 seconds, and finally keeping it at 4 °C.
  • the pET21a-AP205 plasmid (1 ⁇ g) was digested with AgeI endonuclease (NEB R0552V) at 37°C for 2 hours.
  • the digested pET21a-AP205 plasmid fragment was then separated on agarose gel electrophoresis.
  • the isolated pET21a-AP205 plasmid fragment was purified using a small amount of DNA product purification kit (Zhuangmeng Biotechnology ZP201-3).
  • the ST DNA fragment obtained by PCR was further subjected to DNA ligation reaction with the purified pET21a-AP205 plasmid fragment to construct a pET21a-AP205 plasmid (referred to as pET21a-AP205-ST plasmid) containing the DNA fragment.
  • the ligase was T4 DNA ligase (Takara 2011A), and the ligation buffer was T4 DNA Ligase Buffer (Takara 2011A).
  • the ratio of pET21a-AP205 plasmid fragment to ST DNA fragment in the ligation reaction was about 1:3, and the total DNA was about 200ng , 22 °C connection for 2 hours.
  • the pET21a-AP205-ST plasmid was transformed into the expression host as follows: 15 ⁇ l of the ligation reaction solution was added to 150 ⁇ l of XLI-Blue competent E. coli (full gold CD401-02) at 42°C for 1 minute.
  • Fusion protein expression The BL21 (DE3) competent Escherichia coli (CD601-02) transformed with the plasmid constructed above was verified by sequencing, and single clones were picked and placed in ampicillin-resistant LB medium for overnight shaking at 37°C, 220rpm. The next day, the culture was expanded. When the OD value of the logarithmic growth phase was 0.6-0.9, the inducer IPTG (Yisheng Bio 10902ES08) with a final concentration of 0.1 mM was added to induce the expression of the fusion protein, and the bacteria were harvested after 5 hours of induction.
  • the harvested E. coli was centrifuged (6000 rpm for 10 minutes) to obtain a cell pellet.
  • the pellet was resuspended in 20 mM pH 7.5 Tris. Sonication to break the bacteria to obtain the lysed supernatant, centrifuge twice (5000rpm for 10 minutes, 20000g for 30 minutes) to remove insoluble impurities such as cell debris, and then centrifuge through a 30% sucrose pad to precipitate granular proteins (in a 12 ml centrifuge tube, add 2 ml 30% sucrose on top of which 10 ml lysate supernatant was added, 33000 rpm for 3.5 hours), resuspended in 1 ml PBS (KCl 2.6 mM, KH 2 PO 4 1.47 mM, NaCl 136 mM, Na 2 HPO 4 .12H 2 O 8 mM) Granular protein, and then pass through a cesium chloride density gradient (in a 5 ml ultra-centrifuge
  • the expression plasmids of SC-AP205, AP205-SC and AP205-ST can express the corresponding fusion proteins well.
  • VLPs successfully self-assembled from AP205-ST were obtained in subsequent purification (see Figures 2 and 4).
  • Cesium chloride density gradient centrifugation showed obvious target fusion protein bands from layer 14 to layer 20 in the collected protein gel image (see Figure 5), and the corresponding layer was dialyzed to PBS to obtain purified, assembled from AP205-ST well-dispersed, non-aggregated VLPs (AP205-ST VLPs).
  • 50-60 mg of VLP can be obtained per liter of bacteria.
  • a 1068bp full-length cDNA encoding RBD-SC (SEQ ID NO: 11) was artificially synthesized, and the Kozak sequence GCCACC and KpnI restriction site for regulating protein expression were added at the 5' end and XhoI restriction was added at the 3' end. site.
  • the synthesized RBD-SC cDNA fragment (1 ⁇ g) and pCEP4 plasmid (1 ⁇ g) were digested with KpnI and XhoI endonucleases (Takara), respectively, at 37°C for 2 hours. The digested cDNA fragment and the pCEP4 plasmid fragment were then separated by agarose gel electrophoresis.
  • the isolated cDNA fragment and pCEP4 plasmid fragment were purified using a small amount of DNA product purification kit (Zhuangmeng Biotechnology ZP201-3).
  • the purified cDNA fragment was further subjected to a DNA ligation reaction with the pCEP4 plasmid fragment to construct a pCEP4 plasmid (referred to as pCEP4-RBD-SC plasmid) containing the cDNA fragment.
  • the ligase was T4 DNA ligase (Takara 2011A), and the ligation buffer was T4 DNA Ligase Buffer (Takara 2011A).
  • the ratio of the pCEP4 plasmid fragment to the RBD-SC cDNA fragment in the ligation reaction was about 1:3, and the total DNA was about 200 ng , 22 °C connection for 2 hours.
  • the pCEP4-RBD-SC plasmid was transformed into the expression host as follows: 15 ⁇ l of the ligation reaction solution was added to 150 ⁇ l of XLI-Blue competent E. coli (full gold CD401-02) at 42° C. for 1 minute. Pipette 150 ⁇ l to plate on ampicillin-resistant LB plates and incubate at 37°C for 14-16 hours.
  • plasmid DNA was extracted with a plasmid purification kit (full-type gold EM101-02) and verified by enzyme digestion to confirm the successful construction of the pCEP4-RBD-SC plasmid.
  • the pCEP4-RBD-SC plasmid was extracted from the host bacteria using an endotoxin-free large-scale kit (Tiangen Bio DP117).
  • the extracted pCEP4-RBD-SC plasmid was transfected into 293F cell line (Life technologies) using PEI reagent (polyscience 23966-1). Configure the transfection mixture: 1 300 micrograms of plasmid plus 15 ml of SMM 293-TII medium (Sino biological M293TII), 2 1.5 ml of PEI plus 15 ml of SMM 293-TII medium, mix the two, and let stand at room temperature for 2 minutes.
  • Two-step centrifugation 500g for 10 minutes, 8000rpm for 30 minutes
  • removed insoluble impurities such as cell debris
  • the supernatant was passed through a 0.2 ⁇ m filter to further remove insoluble impurities.
  • the resulting fusion protein, RBD-SC had poor stability and was severely degraded when placed at 4°C for three days (so the situation after 3 days is not shown in Figure 6) (see Figure 6).
  • the ratio of RBD-SC to AP205-ST VLP is 1:10 (a VLP is self-assembled from 180 AP205-ST sequences, that is, there are 180 STs, the ratio refers to the SC on the RBD and the ST on the VLP to which it is to be connected.
  • the ratio was incubated in PBS buffer for 1 hour at 4°C, whereby Asp at position 7 of ST amino acid sequence and Lys at position 31 of SC amino acid sequence spontaneously formed an isopeptide covalent bond, allowing RBD-SC to pass through Covalently coupled to AP205-ST VLP. This reaction process does not require any special enzymes and buffer systems.
  • the fusion protein SC-RBD expression plasmid was constructed by the same method as the above (1), and the SC-RBD and AP205-ST VLP were connected by the same method as the above (2).
  • the fusion protein SC-RBD sequence is SEQ ID NO:8.
  • the full-length 1059bp SC-RBD cDNA (SEQ ID NO: 12) fragment was artificially synthesized, and the Kozak sequence GCCACC and HindIII restriction sites for regulating protein expression were added to the 5' end and the XhoI restriction site was added to the 3' end. .
  • the synthesized SC-RBD cDNA fragment (1 ⁇ g) and pCEP4 plasmid (1 ⁇ g) were digested with HindIII and XhoI endonucleases (Takara), respectively, at 37°C for 2 hours.
  • the obtained fusion protein SC-RBD has significantly improved stability.
  • the ligation ratio of SC-RBD to AP205-ST VLP was 1:10 at 4°C
  • the ligation product AP205-ST VLP/SC-RBD was stable for 5 days, and a small amount of antigen shedding did not appear until the 7th day. phenomenon, and at 14 days the vast majority were still intact ligation products (see Figure 8). It can be seen that the stability of AP205-ST VLP/SC-RBD is significantly better than that of AP205-ST VLP/RBD-SC, which is completely degraded on the 9th day under the same conditions.
  • the inventors used the modified AP205 capsid protein sequence used in the present invention (that is, in the wild-type (WT) AP205 capsid protein sequence N Five amino acids MEFGS are added at the end, unless otherwise stated, AP205 and the corresponding VLP and vaccine products used in this paper are all prepared using the modified AP205) and the unmodified WT AP205 capsid protein sequence A series of comparative experiments were conducted .
  • WT AP205-ST VLPs were obtained in the same manner as described above. Then it was ligated with fusion protein SC-RBD in the same way as the aforementioned ligation method to obtain the corresponding ligation product. After reducing and denaturing the ligation product, run SDS-PAGE to show the covalent connection between the antigen and VLP (Figure 9A); and run nucleic acid gel electrophoresis ( Figure 9B) to detect the solubility of the ligation product.
  • the specific measurement process and conditions are: The loading volume was 10 ⁇ g PLA or ligation product, 1% nucleic acid gel, 90 volts, 20 minutes.
  • antigens tested include African swine fever virus antigen eP22 (SEQ ID NO:24), influenza virus antigen M2E (SEQ ID NO:25) and autoantigen myelin oligodendrocyte glycoprotein MOG (SEQ ID NO:26)
  • SEQ ID NO:24 African swine fever virus antigen eP22
  • influenza virus antigen M2E SEQ ID NO:25
  • autoantigen myelin oligodendrocyte glycoprotein MOG SEQ ID NO:26
  • the inventors studied the effect of the ratio of antigen to VLP on the solubility of the ligation product PLA, in order to further improve the solubility of the ligation product by adjusting the ratio.
  • the inventor used SC-RBD and AP205-ST VLP, and tested the ratios of 1:2, 1:4, 1:5, 1:6, 1:7, 1:8, 1:10 respectively, and the test method was the same as the embodiment 3.
  • Figure 10A shows that ligation products were successfully obtained at these ratios
  • Figure 10B shows that when the ratio of antigen to VLP is high (1:2, 1:4, 1:5)), significant deposition is seen in the nucleic acid gel wells, and There is obvious precipitation visible to the naked eye, indicating that there is PLA aggregation at this time, and reducing the connection ratio of antigen and VLP, such as the connection ratio of 1:6, 1:7, 1:8, 1:10, basically no visible precipitation, and no visible precipitation.
  • Proteins are deposited in nucleic acid gel wells
  • Figure 10C is the result of protein staining on agarose gel, showing the accompanying movement of RNA and AP205 protein in electrophoresis.
  • the inventors also tested African swine fever virus antigen eP22 (SEQ ID NO: 24), influenza virus antigen M2E (SEQ ID NO: 25) and autoantigen myelin oligodendrocyte glycoprotein MOG (SEQ ID NO: 25) using the same method. ID NO: 26) in the SC-antigen form of the fusion protein at different ratios to AP205-ST VLP ligation of the resulting product of the solubility. Combining the results of SDS-PAGE protein gel, nucleic acid gel and Coomassie R-250 protein staining, it was found that:
  • African swine fever virus antigen eP22 when the ligation ratio is as high as 1:2, there is no visible deposition (see Figure 11), so the suitable ligation ratio for African swine fever virus antigen eP22 can be determined as 1:1 to 1:5, For example 1:1, 1:2, 1:3, 1:4, 1:5;
  • connection ratio suitable for influenza virus antigen M2E when the connection ratio is as high as 1:1, there is no visible deposition (see Figure 12), so the connection ratio suitable for influenza virus antigen M2E can be determined to be 1:1 to 1:1.5;
  • the suitable ligation ratio for self-antigen MOG can be determined to be 1:4 to 1:10, such as 1:4 , 1:5, 1:6, 1:7, 1:8, 1:10.
  • connection ratios suitable for the formation of soluble ligation product PLA are different, that is, the type of antigen itself has an influence on the solubility of the ligation product, but the trend is the same, that is, with As the ligation ratio decreases, the solubility of the ligation product gradually increases.
  • Example 5 Influence of VLP purification conditions on the presence or absence of RNA inside it
  • the inventors When exploring the industrial purification process of VLP, the inventors found that the RNA inside the purified VLP disappeared when the pH of the ion exchange solution was 10.5 (see Figure 14), suggesting that the pH of the solution may affect the presence of RNA in VLP. Therefore, based on the VLP purification conditions described in Example 1, the inventors examined the effect of solution pH on the presence of RNA in VLPs.
  • the specific method is: adjust the pH of PBS with hydrochloric acid and NaOH respectively, then place 2.5 ⁇ g of purified VLP in a water bath at 37 °C for 2 hours, and then detect the effect of pH on the presence of RNA in VLP by agarose gel electrophoresis and EB staining .
  • RNA content inside the VLP was stable in the range of pH 4.5-8.5, and began to decrease at pH 9.5.
  • pH was 10.5 and above, the RNA inside the VLP was greatly reduced.
  • pH 11.0 the internal RNA could not be detected.
  • VLP RNA appeared outside, indicating that RNA is released from inside the VLP under such alkaline conditions (see Figure 15).
  • RNA inside the VLP of PLA plays a key role in the B cell-related immune activation mechanism of PLA (Sheng Hong et al., B Cells Are the Dominant Antigen-Presenting Cells that Activate Naive CD4+T Cells upon Immunization with a Virus-Derived Nanoparticle Antigen, Immunity, 2018.10, 49:1-14), testing found that when there is RNA inside the VLP of PLA, the RNA acts as a TLR stimulator, enabling PLA to rely on B cell-related immune mechanisms to function, and the immune effect is better than VLP PLA without RNA inside. Therefore, the inventor proposes that the purification process of VLP needs to be under suitable pH conditions, such as pH 4.0-9.0, and strong alkaline conditions above pH 10.5 should be avoided.
  • suitable pH conditions such as pH 4.0-9.0, and strong alkaline conditions above pH 10.5 should be avoided.
  • Example 6 The ability of PLA-SARS-CoV2 vaccine to induce anti-COVID-19 RBD antibodies
  • mice purchased from Speyford
  • RBD antigen mixed with aluminum adjuvant Al, purchased from Pierce
  • 12 mice 10 ⁇ g/mice
  • RBD antigen mixed with CpG1826 adjuvant sequence: tccatgacgttcctgacgtt
  • 4 mice 10 ⁇ g/piece (the dosage of CpG is 50 ⁇ g/piece)
  • S protein extracellular segment mixed with aluminum adjuvant 4 mice, 50 ⁇ g/piece
  • PLA-SARS-CoV2 that is, the vaccine complex formed by connecting the VLP formed after AP205 transformation and the SARS CoV2 RBD antigen, the same below
  • the blood was collected on the 14th day of the first immunization and recorded as the first immune serum, the second immunization was performed on the 21st day of the first immunization, and the blood was collected on the 7th day of the second immunization (ie, the first immunization was 28 days), which was recorded as the second immunization. serum.
  • Elisa detects RBD-specific antibody responses.
  • the amount of RBD antigen coating was 2 ⁇ g/ml, 50 ⁇ l/well, overnight at 4°C.
  • Serum was diluted by gradient (the initial dilution of serum was 1:1000, and then continued to be diluted by 5 times, making a total of 8 gradients), and incubated with RBD-coated Elisa 96-well plate at room temperature for 3 hours.
  • the secondary antibody IgG-HRP Bethyl Laboratories
  • the microplate reader reads the OD value of the corresponding well. Take the wells with unincubated serum as blank control, the average value of OD values of 4-8 blank control wells plus 10 times the standard deviation is the reference value, and the minimum dilution of serum greater than the reference value is recorded as the antibody titer.
  • mice can produce higher titers of RBD IgG antibodies after one immunization (see Figure 16 ); after re-immunization, the titers of RBD IgG antibodies can reach about 3 ⁇ 10 6 (see Figure 17 ).
  • the RBD IgG type of PLA-SARS-CoV2 vaccine after primary immunization or re-immunization Antibody titers were all increased by about 100-fold (see Figure 18).
  • the inventors further used the same method to detect the antibody response of the PLA vaccine constructed with influenza virus M2E antigen, African swine fever virus eP22 antigen and self-antigen MOG.
  • the results showed that: African swine fever virus eP22, influenza virus M2E antigen, and PLA vaccine constructed with self-antigen MOG could also induce a good antibody response. 14 days after immunization of C57BL/6 mice with PLA vaccines of several antigens, serum was collected to detect IgG-type antibody responses, and it was found that compared with the corresponding antigens plus adjuvant, PLA vaccines could induce a good level of specific IgG antibodies ( 19A, B, C, each point in the graph represents the serum antibody titer level of one mouse).
  • Example 7 The production of neutralizing antibodies in mice immunized with PLA-SARS-CoV2 vaccine
  • RBD antigen mixed with aluminum adjuvant RBD antigen mixed with CpG1826 adjuvant, S protein extracellular segment mixed with aluminum adjuvant, and PLA-SARS-CoV2
  • Serum dilution Dilute 3-fold in 300 ⁇ l of 2% DMEM medium 200 ⁇ l of serum at different dilutions were incubated with live virus (10 ⁇ l) at MOI 0.01 at 37°C for 1 h. 200 ⁇ l was used to infect VERO-E6 cells in a 48-well plate. After 1 h, the medium was changed, and the cells were cultured in 2% DMEM medium for 24 h.
  • Upstream primer CAATGGTTTAACAGGCACAGG (SEQ ID NO: 19); Downstream primer (5'-3'): CTCAAGTGTCTGTGGATCACG (SEQ ID NO: 20).
  • the ordinate is the neutralizing antibody titer (ID50 titer) detected by ELISA, showing that the level of neutralizing antibody induced by the PLA-SARS-CoV2 of the present invention was 100 times higher than that of other mixed adjuvant conventional vaccines (see Figure 20).
  • Example 8 Anti-RBD IgG antibodies produced by PLA-SARS-CoV2 vaccine immunized macaques
  • Figure 21 shows anti-RBD IgG antibody levels in serum 14 days after primary immunization (1st) and 7 days after re-immunization (2nd) (the ordinate is the antibody titer detected by ELISA), indicating that anti-RBD IgG in serum at the time of primary immunization
  • the antibody level was about 100 times higher than that of the PBS control, and the anti-RBD IgG antibody level after re-immunization was more than 1000 times higher than that of the PBS control.
  • Example 9 PLA-SARS-CoV2 vaccine immunized rhesus monkeys to produce neutralizing antibodies
  • the inventors further tested the PLA-SARS-CoV2 vaccine of the present invention to induce the production of neutralizing antibodies in rhesus monkeys.
  • the immunization process and conditions were the same as in Example 8 above.
  • the detection method of serum neutralizing antibody titer is the same as that in Example 7 above.
  • Figure 22 shows that the level of 2019-nCoV neutralizing antibodies in the serum after 7 days of re-immunization (2nd) was increased by several tens of times relative to the PBS control.
  • Example 10 Pulmonary viral load after immunization of macaques with PLA-SARS-CoV2 vaccine
  • the inventors next tested the viral load in the lungs of macaques immunized with the PLA-SARS-CoV2 vaccine.
  • the process of immunizing cynomolgus monkeys with PLA-SARS-CoV2 vaccine is the same as that in Example 8.
  • the virus used in the experiment was 107 new coronavirus strains (provided by the Guangdong Provincial Center for Disease Control and Prevention, China).
  • the virus strains were expanded by Vero-E6 cell line, and the half of the tissue culture infectious dose was determined by the Reed-Muench method.
  • Virus challenge tests were performed 10 days after re-immunization.
  • the challenge route was a combination of intranasal (0.4 mL/nostril) and intratracheal (1.2 mL, fiberoptic bronchoscopy), with a total virus titer of 1 ⁇ 10 7 TCID 50 mL, diluted with sterile 0.9% saline.
  • the results showed that the rhesus monkey lung virus almost completely disappeared after immunization with the PLA-SARS-CoV2 vaccine of the present invention (Fig. 23, the vertical axis of the graph shows the logarithm of the number of virus copies per microgram of RNA). Moreover, according to the results of the specific antibodies and neutralizing antibodies induced by the PLA-SARS-CoV2 vaccine of the present invention in the foregoing examples, it can be reasonably inferred that the vaccine of the present invention is also effective in the challenge test compared to other traditional vaccines that require additional adjuvants. will have significantly better results.

Abstract

本申请涉及一种病原样抗原(PLA)复合物及其制备方法和应用，该病原样抗原复合物由结构经改造的大肠杆菌噬菌体病毒样颗粒(VLP)及展示在其上的抗原构成，病毒样颗粒内部包裹有核酸。

Description

病原样抗原疫苗及其制备方法 技术领域
本发明涉及疫苗与免疫治疗技术。具体地，涉及病原样抗原(pathogen-like antigen，PLA)疫苗及其制备方法，以及该疫苗预防或治疗相关疾病的用途。
背景技术
基于纯化蛋白的亚单位疫苗具有组成成分明确、安全性好、便于生产等方面的优点，是现代疫苗技术发展的方向。但是，在应用中，蛋白亚单位疫苗也被发现存在免疫原性弱的问题，因此难以产生高水平、持久的免疫保护。目前，提高亚单位蛋白抗原免疫原性的常用方法是添加佐剂。佐剂能够通过刺激天然免疫信号来增强抗原特异性免疫应答。但是，过度活化的天然免疫信号又可能产生不可接受的炎症副作用。这就限制了使用佐剂增强蛋白亚单位疫苗免疫原性的效果。
在亚单位蛋白疫苗中，某些特殊的重组蛋白可以组装成病毒样颗粒(Virus-like particle,VLP)，如乙肝病毒表面抗原(HBsAg)的VLP抗原。这些抗原的免疫原性也会得到提高，其原理可能与提高了抗原密度有关。但是，这种方法仅限于能自组装成VLP的蛋白。并且，单独的VLP增强免疫原性的作用依然有限，仍然需要使用佐剂才能达到较好的效果。来自于大肠杆菌噬菌体的一类VLP(包括Qβ、MS2、AP205等)被发现具有很强的免疫原性(Paul Pumpens et al.,The True Story and Advantages of RNA Phage Capsids as Nanotools,Intervirology，2016.11,59:74-110)。Thomas M.Kundig等人(Thomas M.Kundig et al.,Der p 1 peptide on virus-like particles is safeand highly immunogenic in healthy adults,J Allergy Clin Immunol,2006,VOL.117,NO.6)发现将抗原通过基因工程方法或者化学交联方式连接到Qβ-VLP表面后，可以明显增强对抗原的特异性免疫应答。但是，这种方式的缺点是：1)基因工程融合表达抗原的种类有限(抗原融合表达后不能组装成VLP)；2)化学偶联的方式不适合规模化生产，而且抗原在VLP表面的取向不可控，影响免疫效力。
AP205蛋白(以下简称AP205)是最近新鉴定的AP205 RNA噬菌体的主要衣壳蛋白。在体外，AP205可自行组装成VLP颗粒，每个VLP含有180个AP205衣壳蛋白分子。AP205的N端和C端都可以连接目的蛋白。SpyTag(ST)/SpyCatcher(SC)体系源于 CnaB2结构域，其在各种条件下均能自发形成稳定的异肽键，因此可用于解决蛋白亚单位融合表达与化学偶联的问题。但是，我们发现，由于抗原蛋白与VLP会存在明显的不匹配而容易导致VLP聚集，引发沉淀，严重影响疫苗的效力。
本发明致力于解决现有技术中存在的上述缺陷和不足。
发明内容
为了解决前述现有技术的不足，我们对病原样抗原(Pathogen Like Antigen，PLA)蛋白工程疫苗进行了一系列的探索和改进。
本发明的一个方面提供一种可溶性病原样抗原(PLA)复合物，其包含：
(1)病毒样颗粒，其由第一融合蛋白自组装而成，该第一融合蛋白包含位于N端的病毒衣壳蛋白或其变体，以及C端的SpyTag，(2)第二融合蛋白，该第二融合蛋白包含抗原或其变体和SpyCatcher；
其中所述病毒样颗粒还在其内部包裹核酸，且其中所述抗原或其变体通过在SpyCatcher和SpyTag之间的共价连接而展示于病毒样颗粒的表面。
根据本发明的可溶性病原样抗原复合物，其中所述病毒样颗粒内包裹的核酸为所述病毒样颗粒在其自组装时包裹的、来自用于表达所述病毒样颗粒的宿主细菌的核酸，优选所述宿主细菌为大肠杆菌，优选所述核酸是RNA。
根据本发明的可溶性病原样抗原复合物，其中所述衣壳蛋白来自大肠杆菌噬菌体Qβ、MS2、或AP205。
根据本发明的可溶性病原样抗原复合物，其中所述衣壳蛋白来自大肠杆菌噬菌体AP205。
根据本发明的可溶性病原样抗原复合物，其中所述抗原选自SARS-CoV2病毒S蛋白的RBD序列、非洲猪瘟病毒抗原eP22、流感病毒抗原M2E、自身抗原髓鞘少突胶质细胞糖蛋白MOG。
根据本发明的可溶性病原样抗原复合物，其中所述噬菌体AP205衣壳蛋白的序列与SEQ ID NO：1具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的一致性。
根据本发明的可溶性病原样抗原复合物，其中所述噬菌体AP205衣壳蛋白的序列为 SEQ ID NO：1。
根据本发明的可溶性病原样抗原复合物，其中所述SpyTag的序列与SEQ ID NO：3具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的一致性，SpyCatcher的序列与SEQ ID NO：4具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的一致性。
根据本发明的可溶性病原样抗原复合物，其中所述SpyTag的序列为SEQ ID NO：3，SpyCatcher的序列为SEQ ID NO：4。
根据本发明的可溶性病原样抗原复合物，其中在SpyTag序列SEQ ID NO：3的第7位的Asp和SpyCatcher序列SEQ ID NO：4的第31位Lys之间形成异肽键。
根据本发明的可溶性病原样抗原复合物，其中在第一融合蛋白中，噬菌体衣壳蛋白或其变体与SpyTag通过第一连接肽连接，在第二融合蛋白中，抗原或其变体与SpyCatcher通过第二连接肽连接。
根据本发明的可溶性病原样抗原复合物，其中所述第一连接肽的序列为SEQ ID NO：5，所述第二连接肽的序列为SEQ ID NO：6。
根据本发明的可溶性病原样抗原复合物，其中根据不同的抗原，所述第二融合蛋白与所述病毒样颗粒以小于或等于1:1的比率连接，优选以1:1、1:1.5、1:2、1:3、1:4、1:5、1:6、1:7、1:8、1:9、1:10、1:11、1:12的比率连接，以此确保病原样抗原复合物的可溶性和免疫原性，所述比率以第二融合蛋白上的SpyCatcher与病毒样颗粒上的SpyTag之比计算。
根据本发明的可溶性病原样抗原复合物，其与药学上可接受的载体和/或赋形剂一起被配制为疫苗组合物。
本发明的另一个方面提供一种制备可溶性病原样抗原复合物的方法，包括在pH4.0～9.0，优选5.5～8.5范围内的pH值条件下纯化病毒样颗粒。
本发明的又一个方面提供一种提高病原样抗原复合物的可溶性的方法，包括如下步骤：(1)制备前述定义的病毒样颗粒和第二融合蛋白；和(2)在将所述第二融合蛋白与所述病毒样颗粒连接时，降低所述第二融合蛋白与所述病毒样颗粒的连接比率，以获得可溶性病原样抗原复合物。
根据本发明的提高病原样抗原复合物的可溶性的方法，其中所述抗原选自SARS-CoV2病毒S蛋白的RBD序列、非洲猪瘟病毒抗原eP22、流感病毒抗原M2E、或 自身抗原髓鞘少突胶质细胞糖蛋白MOG。
本发明的再一个方面涉及在有需要的受试者中预防和/或治疗SARS-CoV2病毒、流感病毒、或非洲猪瘟病毒或与自身抗原髓鞘少突胶质细胞糖蛋白MOG相关的疾病的方法，其包括向所述受试者施用预防和/或治疗有效量的本发明的可溶性病原样抗原复合物或疫苗组合物。
本发明具有以下优点：
1)有效避免了由于抗原蛋白与接头之间、或者抗原蛋白与VLP之间适配性差所导致的颗粒聚集或沉淀现象发生，便利了疫苗的生产，确保疫苗效力的稳定。
3)本发明制备病原样抗原疫苗的方法避免了在分离纯化PLA疫苗过程中核酸的降解，使得无需额外添加佐剂就能有效提高疫苗的免疫原性，同时也减少或者避免了额外添加佐剂所导致的过度炎症反应。
附图说明
图1：AP205融合蛋白诱导表达前后的全细菌裂解，其中图1A是SC-AP205，图1B是AP205-SC。
图2：SC-AP205及AP205-SC蔗糖垫离心后核酸胶图，其中1为AP205-ST，2为SC-AP205，3为AP205-SC。
图3：AP205融合蛋白氯化铯密度梯度离心后分层收样蛋白胶图，其中图3A是SC-AP205，图3B是AP205-SC。
图4：AP205-ST诱导前后的全细菌裂解。
图5：AP205-ST氯化铯密度梯度离心后分层收样蛋白胶图。
图6：RBD-SC的降解。
图7：AP205-RBD(SC位于C端)的降解。
图8：SC-RBD与AP205-ST连接产物的稳定性。
图9：改造AP205对连接产物可溶性的影响，①为野生型AP205，②为本发明改造后的AP205，图9A和图9B分别为SDS-PAGE和核酸胶图。
图10：调整抗原比率对连接产物可溶性的影响，图10A、图10B和图10C分别 为SDS-PAGE、核酸胶、Coomassie R-250图。
图11：非洲猪瘟抗原eP22与AP205-ST的连接，图11A、图11B和图11C分别为SDS-PAGE、核酸胶、Coomassie R-250图。
图12：流感病毒抗原M2E与AP205-ST的连接，图12A、图12B和图12C分别为SDS-PAGE、核酸胶、Coomassie R-250图。
图13：自身抗原MOG与AP205-ST的连接，图13A、图13B和图13C分别为SDS-PAGE、核酸胶、Coomassie R-250图。
图14：VLP纯化条件对其内部的RNA的存在与否的影响，图14A和图14B分别为SDS-PAGE和核酸胶图。
图15：不同pH梯度下VLP内部核酸的变化。
图16：PLA-SARS-CoV2疫苗免疫小鼠产生抗RBD IgG抗体的情况(初次免疫)。
图17：PLA-SARS-CoV2疫苗免疫小鼠产生抗RBD IgG抗体的情况(二次免疫)。
图18：PLA-SARS-CoV2疫苗初次免疫和再次免疫后产生的RBD IgG型抗体滴度的变化。
图19：本发明的VLP与其它几种抗原构建的疫苗复合物相对于添加佐剂的传统疫苗明显增强了诱导产生抗体的能力(图19A、图19B和图19C分别使用非洲猪瘟病毒抗原eP22、流感病毒抗原M2E和自身抗原髓鞘少突胶质细胞糖蛋白MOG)。
图20：PLA-SARS-CoV2疫苗免疫小鼠产生中和抗体的情况。
图21：PLA-SARS-CoV2疫苗免疫猕猴产生抗RBD IgG抗体的情况。
图22：PLA-SARS-CoV2疫苗免疫猕猴产生中和抗体的情况。
图23：PLA-SARS-CoV2疫苗免疫猕猴后的肺部病毒载量。
具体实施方式
为解决抗原蛋白与VLP不匹配所导致的VLP聚集和沉淀，从而影响疫苗的稳定性和免疫效力的问题，本发明人进行了大量的研究。
本发明人偶然发现，对组装成病毒样颗粒的噬菌体AP205衣壳蛋白序列(以下简称AP205序列)的改造能够明显改善连接所得疫苗产物的可溶性(实施例3)；底 盘颗粒与抗原连接时两者的比率也会影响连接产物的可溶性(实施例4)。本发明人还出乎意料地发现，AP205序列与SpyCatcher序列构建的融合蛋白无法正常组装为病毒样颗粒，只有将SpyTag构建于AP205序列C端形成的融合蛋白才能够正常组装(实施例1)。而SpyCatcher序列虽然可以位于抗原序列的N端或C端，但其位于抗原的N端时所形成的融合蛋白相对更为稳定，该融合蛋白与携带SpyTag序列的病毒样颗粒连接而成的连接产物也更为稳定(实施例2)。
病原样抗原复合物的聚集或沉淀将直接影响疫苗调动B细胞专职抗原提呈功能的作用，严重影响疫苗的生产和效力的稳定，因此，保证抗原与VLP连接后所得颗粒的可溶性是PLA疫苗发挥应有作用的一个关键因素。
VLP内的RNA核酸是无佐剂PLA疫苗发挥作用的另一个关键因素，在分离纯化PLA疫苗过程中，某些条件虽然不影响蛋白的稳定性，却可能使RNA降解。本发明人研究发现，VLP纯化过程中溶液的pH值过高会破坏VLP中的RNA甚至将其完全降解，适当的pH值能够确保RNA成分在VLP内部的留存。
本发明的一个方面提供一种可溶性病原样抗原(PLA)复合物，其包含：
(1)病毒样颗粒，其由第一融合蛋白自组装而成，该第一融合蛋白包含位于N端的病毒衣壳蛋白或其变体和其C端的SpyTag，(2)第二融合蛋白，该第二融合蛋白包含抗原或其变体和SpyCatcher；
其中所述病毒样颗粒还在其内部包裹核酸，和其中所述病毒样颗粒和所述第二融合蛋白通过所述SpyCatcher和SpyTag共价连接而使所述抗原或其变体展示于病毒样颗粒的表面。
根据本发明的可溶性病原样抗原复合物，其中所述病毒样颗粒内包裹的核酸为来自用于表达所述病毒样颗粒的宿主细菌的核酸，优选所述宿主细菌为大肠杆菌，优选所述核酸是RNA。
根据本发明的可溶性病原样抗原复合物，其中所述衣壳蛋白来自大肠杆菌噬菌体Qβ、MS2、或AP205。
根据本发明的可溶性病原样抗原复合物，其中所述衣壳蛋白来自大肠杆菌噬菌体AP205。
根据本发明的可溶性病原样抗原复合物，其中所述抗原选自SARS-CoV2病毒S蛋白的 RBD序列、非洲猪瘟病毒抗原eP22、流感病毒抗原M2E、自身抗原髓鞘少突胶质细胞糖蛋白MOG。
根据本发明的可溶性病原样抗原复合物，其中所述噬菌体AP205衣壳蛋白的序列与SEQ ID NO：1具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的一致性。
根据本发明的可溶性病原样抗原复合物，其中所述噬菌体AP205衣壳蛋白的序列为SEQ ID NO：1。
根据本发明的可溶性病原样抗原复合物，其中所述SpyTag的序列与SEQ ID NO：3具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的一致性，SpyCatcher的序列与SEQ ID NO：4具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的一致性。
根据本发明的可溶性病原样抗原复合物，其中所述SpyTag的序列为SEQ ID NO：3，SpyCatcher的序列为SEQ ID NO：4。
根据本发明的可溶性病原样抗原复合物，其中在SpyTag序列SEQ ID NO：3的第7位的Asp和SpyCatcher序列SEQ ID NO：4的第31位Lys之间形成异肽键。
根据本发明的可溶性病原样抗原复合物，其中在第一融合蛋白中，噬菌体衣壳蛋白或其变体与SpyTag通过第一连接肽连接，在第二融合蛋白中，抗原或其变体与SpyCatcher通过第二连接肽连接。
根据本发明的可溶性病原样抗原复合物，其中所述第一连接肽的序列为SEQ ID NO：5，所述第二连接肽的序列为SEQ ID NO：6。
根据本发明的可溶性病原样抗原复合物，其中根据不同的抗原，所述第二融合蛋白与所述病毒样颗粒以小于或等于1:1的比率连接，优选以1:1、1:1.5、1:2、1:3、1:4、1:5、1:6、1:7、1:8、1:9、1:10、1:11、1:12的比率连接，以此确保病原样抗原复合物的可溶性和免疫原性，所述比率以第二融合蛋白上的SpyCatcher与病毒样颗粒上的SpyTag之比计算。
本发明还涉及编码所述第一融合蛋白和第二融合蛋白的核酸序列以及包含该核酸序列的载体。
在一些实施方式中，本文所述的核酸序列或核酸分子或载体可以被密码子优化。
在一些实施方式中，本文所述的核酸序列或核酸分子或载体可以是其简并版本。
根据本发明的可溶性病原样抗原复合物，其与药学上可接受的载体和/或赋形剂一起被配制为疫苗组合物。
本发明的另一个方面提供一种制备可溶性病原样抗原复合物的方法，包括在pH 4.0～9.0，优选5.5～8.5范围内的pH值条件下纯化病毒样颗粒。
本发明的又一个方面提供一种提高病原样抗原复合物的可溶性的方法，包括如下步骤：(1)制备前述任一项中定义的所述第二融合蛋白与所述病毒样颗粒；(2)在将所述第二融合蛋白与所述病毒样颗粒连接时，降低第二融合蛋白与所述病毒样颗粒的连接比率，以获得可溶性病原样抗原复合物。
根据本发明提高病原样抗原复合物的可溶性的方法，其中所述抗原选自SARS-CoV2病毒S蛋白的RBD序列、非洲猪瘟病毒抗原eP22、流感病毒抗原M2E、或自身抗原髓鞘少突胶质细胞糖蛋白MOG。
本发明的再一个方面涉及在有需要的受试者中预防和/或治疗SARS-CoV2病毒、流感病毒、或非洲猪瘟病毒感染相关疾病或与自身抗原髓鞘少突胶质细胞糖蛋白MOG相关的疾病的方法，其包括向所述受试者施用预防和/或治疗有效量的本发明的可溶性病原样抗原复合物或疫苗组合物。
在一些实施方式中，所述相关疾病可以是由SARS-COV-2病毒和/或其突变体引起。
在一些实施方式中，所述相关疾病可以是COVID-19。
本文中所用术语“融合蛋白”是指基因工程改造的蛋白质，其由两个或更多个结合在一起的完整或部分基因或一系列核酸形成的核苷酸序列所编码。可替代地，可通过结合两个或更多个异源肽来制造融合蛋白。
本文中使用的术语“连接肽”或“连接序列”表示这样的一个或多个(例如，约2-10个)氨基酸残基：其在多肽的两个邻近的基序、区域或结构域之间，诸如在抗原性肽之间或在抗原性肽和由多重翻译前导序列编码的邻近肽之间，或在抗原性肽和间隔区或切割位点之间。连接肽可以源自融合蛋白的构建体设计(例如，在构建编码融合蛋白的核酸分子的过程中由限制性酶位点的使用引起的氨基酸残基)。
本文所述术语“变体”是指蛋白质或核酸分子，其序列与参考序列相似但不相同， 其中变体蛋白(或由变体核酸分子编码的蛋白质)的活性没有明显改变。序列上的这些变异可以是天然发生的变异，或者可以通过使用本领域技术人员已知的遗传工程技术进行工程化改造。此类技术的实例可见于Sambrook J,Fritsch E F,Maniatis T et al.,in MolecularCloning--A Laboratory Manual,2nd Edition,Cold Spring Harbor Laboratory Press,1989,pp.9.31-9.57),或Current Protocols in Molecular Biology,John Wiley&Sons,N.Y.(1989),6.3.1-6.3.6。关于变体，氨基酸或核酸序列的任何类型的改变都是允许的，只要所得的变体蛋白或多核苷酸的活性没有显著改变。此类变异的实例包括但不限于缺失、插入、取代及其组合。根据其性质，氨基酸可以分为带电荷的氨基酸、不带电荷的氨基酸、极性的不带电荷的氨基酸和疏水性氨基酸。因此，含有取代的蛋白质变体可以是其中氨基酸被来自相同组的氨基酸取代的那些蛋白质变体。此类取代被称为“保守”取代。
本文中使用的术语“抗原”或其变体表示可以刺激细胞产生免疫应答的多肽。
本文中使用的术语“病毒样颗粒(virus-like particles，VLPs)”是由一种或多种病毒结构蛋白组装成的颗粒，具有与病毒颗粒相似的外部结构和抗原性，但不含病毒基因。
本发明所用术语“疫苗”、“疫苗组合物”指含有相应病毒抗原的药物组合物，该药物组合物可诱发、刺激或增强受试者针对相应病毒的免疫反应。
本文中使用的术语“核酸”或“核酸分子”表示，例如通过聚合酶链式反应(PCR)或通过体外翻译产生的脱氧核糖核酸(DNA)、核糖核酸(RNA)、寡核苷酸、片段中的任一种，和通过连接、切割、内切核酸酶作用或外切核酸酶作用中的任意一种或多种产生的片段。在某些实施方式中，本发明内容的核酸通过PCR产生。核酸可以由单体组成，所述单体是天然存在的核苷酸(诸如脱氧核糖核苷酸和核糖核苷酸)、天然存在的核苷酸的类似物(例如，天然存在的核苷酸的α-对映异构形式)或它们的组合。修饰的核苷酸可以具有在糖部分、或嘧啶或嘌呤碱基部分中或替代糖部分、或嘧啶或嘌呤碱基部分的修饰。
本文中使用的术语“构建体”表示含有重组核酸的任何多核苷酸。构建体可以存在于载体(例如，细菌载体、病毒载体)中，或可以整合进基因组中。“载体”是能够运输另一种核酸的核酸分子。载体可以是，例如，质粒、粘粒、病毒、RNA载体或线性或圆形DNA或RNA分子，其可以包括染色体、非染色体、半合成的或合成的核酸。 示例性的载体是能够自主复制(附加型载体)和/或表达它们所连接的核酸(表达载体)的那些载体。
本文中使用的术语“信号肽”和“前导序列”在本文可互换使用并且是指可以连接在本文阐述的蛋白质的氨基末端的氨基酸序列。信号肽/前导序列通常指导蛋白质的定位。本文所用的信号肽/前导序列优选地促进蛋白质从产生其的细胞中分泌。信号肽/前导序列常常在从细胞分泌后从蛋白质的其余部分(通常称为成熟蛋白质)切割下来。信号肽/前导序列连接在所述蛋白质的N端，长度为约9至200个核苷酸(3至60个核酸)。本发明使用的信号肽可以是SARS-COV-2病毒S蛋白的信号肽序列或来自其它真核/病毒蛋白的信号肽序列。
本文中使用的术语“表达载体”表示含有与合适的控制序列可操作地连接的核酸分子的DNA构建体，所述控制序列能够实现所述核酸分子在合适的宿主中的表达。这样的控制序列包括用于实现转录的启动子、任选的用于控制这样的转录的操纵基因序列、编码合适的mRNA核糖体结合位点的序列、和控制转录和翻译的终止的序列。所述载体可以是质粒、噬菌体颗粒、病毒，或简单地是潜在基因组插入物。病毒载体可以是基于DNA(例如，腺病毒或痘苗病毒)或RNA的，包括溶瘤病毒载体(例如，VSV)，能复制的或不能复制的。一旦转化进合适的宿主中，所述载体可以独立于宿主基因组而复制和起作用，或在某些情况下，可以整合进基因组本身中。在本说明书中，“质粒”、“表达质粒”、和“载体”经常互换使用。
本文中使用的术语“表达”表示基于基因的核酸序列而生产多肽的过程。所述过程包括转录和翻译。翻译可以开始于非常规起始密码子，诸如CUG密码子，或翻译可以开始于几种起始密码子(标准的AUG和非常规的)以产生比产生的mRNA更多的蛋白(基于每摩尔量)。
在将核酸序列插入细胞中的背景下，本文中使用的术语“引入”是指“转染”或“转化”或“转导”，且包括对核酸序列向真核或原核细胞中的整合的提及，其中所述核酸序列可以整合进细胞的基因组(例如，染色体、质粒、质体或线粒体的DNA)中，转化成自主复制子，或短暂地表达(例如，转染的mRNA)。
用于在细胞中表达外源或异源核酸的重组方法是本领域众所周知的。这样的方法可以参见，例如，Sambrook等人，Molecular Cloning：A Laboratory Manual，第三版，ColdSpring Harbor Laboratory，New York(2001)；和Ausubel等人，Current Protocols  inMolecular Biology，John Wiley and Sons，Baltimore，MD(1999)。对编码融合抗原蛋白的核酸分子的遗传修饰可以给从它的天然存在状态改变的重组或非天然细胞赋予生化或代谢能力。
本文中使用的术语“宿主”指可以向其引入本发明构建体的任何生物或其细胞，无论真核或原核，特别地，其中出现RNA沉默的宿主。在具体的实施方案中，“宿主”包括大肠杆菌如E.coli。术语“宿主”在指真核生物，包括单细胞真核生物如酵母和真菌以及多细胞真核生物如动物，非限制性例子包括无脊椎动物(例如，昆虫、腔肠动物、棘皮动物、线虫等)；真核寄生体(例如，疟疾寄生体，如恶性疟原虫(Plasmodium falciparum)、蠕虫等)；脊椎动物(例如，鱼类、两栖类、爬行类、鸟、哺乳动物)；和哺乳动物(例如，啮齿类、灵长类如人类和非人灵长类)。因此，术语“宿主细胞”适当地涵盖这类真核生物的细胞以及衍生自这类真核生物的细胞系。
本文中使用的术语“佐剂”指通过增强巨噬细胞活性促进机体T细胞或B细胞的反应，参与半抗原或抗原免疫应答的天然的或合成的物质。
本文中使用的术语“预防和/或治疗”指抑制相应病毒的复制、传播或防止其在宿主体内定居，以及减轻病毒感染的疾病或病症的症状。若病毒荷载量减少、病症减轻和/或摄食量和/或生长增加，那么就可以认为所述治疗达到了治疗效果。
本文中使用的术语化合物或组合物的“治疗有效量(或剂量)”或“有效量(或剂量)”表示，足以以统计上显著的方式导致正在治疗的疾病的一种或多种征状的改善的化合物的量。精确量取决于众多因素，例如，组合物的活性、采用的递送的方法、组合物的免疫刺激能力、预期的患者和患者考虑因素等，且可以由本领域普通技术人员容易地确定。治疗效果可以直接地或间接地包括疾病的一种或多种征状的减轻，治疗效果还可以直接地或间接地包括细胞免疫应答的刺激。
本文中使用的术语“药学上可接受的载体”包括本身不诱导产生对接受药物组合物的个体有害的抗体的任何载体。合适的载体通常是大的、代谢缓慢的大分子，如蛋白质、多糖、聚乳酸、聚乙醇酸、氨基酸聚合物、氨基酸共聚物、脂质凝集物(如油滴或脂质体)等。这些药学上可接受的载体是本领域普通技术人员所熟知的。
本文中使用的术语“受试者”可以是能够发生细胞免疫应答的任何生物体，诸如人类、宠物、家畜、展示动物、动物园样本或其它动物。例如，受试者可以是人、非人灵长类动物、狗、猫、兔、大鼠、小鼠、豚鼠、马、牛、绵羊、山羊、猪等。需要 施用如本文中所述的治疗剂的受试者包括已被SARS-COV-2病毒感染甚至已经出现病毒感染相关疾病，或者处于SARS-COV-2病毒感染风险中的受试者。
本文中使用的术语“有需要的受试者”表示处于疾病、障碍或病症的高危中或遭受疾病、障碍或病症的受试者，所述疾病、障碍或病症适合用本文所提供的化合物或其组合物治疗或改善。在某些实施方式中，有需要的受试者是人。
对于包含如本文中所述的病原样抗原复合物，期望的结果是能够以最小副作用诱导持久保护性免疫的安全产品，并且与其它策略(例如，完整活的或减毒的病原体)相比，廉价地生产，使已经在其它方面(通常)与完整的或减毒的病毒免疫组合物的应用相关联的禁忌最小化或消除。对传染性疾病紧急事件(天然爆发、大范围流行或生物恐怖主义)快速响应的能力是本文公开的实施方式的有效应用的一个益处，无论在生物防御还是免疫疗法或技术的背景下。
本发明的病原样抗原疫苗可以通过例如肌肉内注射、皮下地、鼻内地、经粘膜呈递、静脉内地或通过真皮内或皮下施用。
以下将对有关病原样抗原疫苗发明通过具体实施的方式，对其进行举例说明。但应当理解这些实施例不以任何形式限制本发明的范围。
实施例
本发明的可溶性病原样抗原(PLA)疫苗包含四个结构要素：1)基于噬菌体VLP或者其他纳米颗粒的抗原展示底盘；2)底盘颗粒内部携带的TLR刺激剂，例如核酸，如RNA，优选来自表达宿主；3)用于连接底盘颗粒与抗原的Spycather/Spytag序列；和4)待展示的抗原。
对于包含上述四个结构要素的融合蛋白，其能够作为一种无佐剂蛋白质工程疫苗的必要条件包括：该融合蛋白具备足够的结构稳定性，是可溶性的，不会聚集或沉淀；同时，底盘颗粒内部包裹的TLR刺激剂如核酸未被降解消除。本发明人发现，多种因素影响作为蛋白质工程疫苗的融合蛋白的稳定性、可溶性。
实施例1：SpyCatcher(SC)和SpyTag(ST)与AP205的连接方式对VLP自组装的影响
(1)融合蛋白SC-AP205(SC位于AP205的N端)表达质粒的构建
SC的氨基酸序列为SEQ ID NO:4，改造后的AP205(非野生型)的氨基酸序列为SEQ  ID NO:1，两者通过连接序列SEQ ID NO:5连接。
I、构建AP205表达载体
人工合成全长393bp的编码AP205的cDNA(SEQ ID NO:9)片段，并在其5’端加上BamHI酶切位点和3’端加上GSGGSG连接、AgeI酶切位点、终止密码子TAA、KpnI酶切位点。用BamHI(Takara 1010A)和KpnI内切酶(Takara 1068A)分别对合成的AP205 cDNA片段(1μg)和pET21质粒(1μg)置37℃消化2小时。然后在琼脂糖凝胶电泳分离消化的cDNA片段和pET21a质粒片段。使用小量DNA产物纯化试剂盒(庄盟生物ZP201-3)分别将分离的cDNA片段和pET21a质粒片段纯化。进一步将纯化的cDNA片段与pET21a质粒片段进行DNA连接反应以构建含有该cDNA片段的pET21a质粒(称为pET21a-AP205质粒)。连接酶为T4 DNA连接酶(Takara 2011A)，连接缓冲液为T4 DNA Ligase Buffer(Takara 2011A)，连接反应中pET21a质粒片段与AP205 cDNA片段的比率约为1：3，总DNA约为200ng，22℃连接2小时。如下将pET21a-AP205质粒转化到表达宿主中：将15μl的连接反应液加入到150μl的XLI-Blue感受态大肠杆菌(全式金CD401-02)，42℃，1分钟。吸取150μl在氨苄抗性LB平板上涂板，37℃培养14-16小时。在平板上取单一菌落，用质粒纯化试剂盒(全式金EM101-02)提取质粒DNA并进行酶切验证，确认成功构建pET21a-AP205质粒。
II、构建SC-AP205表达载体
通过PCR(上游引物(SEQ ID NO：13)：acgggatccATGTCGTACTACCATCACCATC，下游引物(SEQ ID NO：14)：cccggatccactgccgctacctccAATATGAGCGTCACCTTTAGTTGC，PCR程序为①94℃5分钟②94℃30秒③58℃30秒④72℃1分钟，②③④循环30次，⑤72℃5分钟⑥4℃保持)人工合成全长276bp的编码SC的cDNA(SEQ ID NO：10)片段，并在其5’和3’端均加上BamHI酶切位点。用BamHI内切酶(Takara 1010A)分别对合成的SC cDNA片段(1μg)和pET21a-AP205质粒(1μg)置37℃消化2小时。然后通过琼脂糖凝胶电泳分离消化的cDNA片段和pET21a-AP205质粒片段。使用小量DNA产物纯化试剂盒(庄盟生物ZP201-3)分别将分离的cDNA片段和pET21a-AP205质粒片段纯化。进一步将纯化的cDNA片段与pET21a-AP205质粒片段进行DNA连接反应以构建含有该cDNA片段的pET21a-AP205质粒(称为pET21a-SC-AP205质粒)。连接酶为T4DNA连接酶(Takara 2011A)，连接缓冲液为T4 DNA Ligase Buffer(Takara 2011A)，连接反应中pET21a-AP205质粒片段与SC cDNA片段的比率约为1：3，总DNA约为200ng，22℃连接2小时。如下将pET21a-SC-AP205质粒转化到表达宿主中：将15μl的连接反应液加入到150μl的XLI-Blue 感受态大肠杆菌(全式金CD401-02)，42℃，1分钟。吸取150μl在氨苄抗性LB平板上涂板，37℃培养14-16小时。在平板上取单一菌落，用质粒纯化试剂盒(全式金EM101-02)提取质粒DNA并进行酶切验证，确认成功构建pET21a-SC-AP205质粒。
(2)融合蛋白AP205-SC(SC位于AP205的C端)表达质粒的构建
用与上述(1)相同的方法制备融合蛋白AP205-SC表达质粒，不同之处在于通过PCR(上游引物：acgaccggtATGTCGTACTACCATCACCATC(SEQ ID NO：15),下游引物为：cccaccggtAATATGAGCGTCACCTTTAGTTGC(SEQ ID NO：16)，PCR程序为①94℃5分钟②94℃30秒③58℃30秒④72℃1分钟，②③④循环30次，⑤72℃5分钟⑥4℃保持)人工合成全长276bp的SC cDNA片段，在其5’和3’端均加上AgeI酶切位点。用AgeI内切酶(NEB R0552V)分别对合成的SC cDNA片段(1μg)和pET21a-AP205质粒(1μg)进行消化并连接以构建pET21a-AP205-SC质粒。
(3)融合蛋白AP205-ST(ST位于AP205序列的C端)表达质粒的构建
用与上述(1)相同的方法构建融合蛋白AP205-ST表达质粒，不同之处在于通过以下过程合成ST的编码DNA序列(gcccacatcgtgatggtggacgcctacaagccgacgaag)(所编码的氨基酸序列为SEQ ID NO:4)：
人工合成引物：
F:ccggtggtagcggcgcccacatcgtgatggtggacgcctacaagccgacgaaga(SEQ ID NO：17)
R：ccggtcttcgtcggcttgtaggcgtccaccatcacgatgtgggcgccgctacca(SEQ ID NO：18)
通过退火PCR(5μl 200μM引物-F，5μl 200μM引物-R，2μl 10x退火缓冲液(100mM Tris 8.0,1M NaCl,10mM EDTA)，8μl dH 2O。设置PCR程序为99℃ 3min,99-20℃每30秒温度下降0.5℃,最后保持在4℃)的方式得到编码ST的DNA序列。用AgeI内切酶(NEB R0552V)对pET21a-AP205质粒(1μg)置37℃消化2小时。然后在琼脂糖凝胶电泳分离消化的pET21a-AP205质粒片段。使用小量DNA产物纯化试剂盒(庄盟生物ZP201-3)将分离pET21a-AP205质粒片段纯化。进一步将PCR获得的ST DNA片段与纯化的pET21a-AP205质粒片段进行DNA连接反应以构建含有该DNA片段的pET21a-AP205质粒(称为pET21a-AP205-ST质粒)。连接酶为T4 DNA连接酶(Takara 2011A)，连接缓冲液为T4 DNA Ligase Buffer(Takara 2011A)，连接反应中pET21a-AP205质粒片段与ST DNA片段的比率约为1：3，总DNA约为200ng，22℃连接2小时。如下将pET21a-AP205-ST质粒转化到表达 宿主中：将15μl的连接反应液加入到150μl的XLI-Blue感受态大肠杆菌(全式金CD401-02)，42℃，1分钟。吸取150μl在氨苄抗性LB平板上涂板，37℃培养14-16小时。用质粒纯化试剂盒(全式金EM101-02)提取质粒DNA并进行酶切验证，确认成功构建pET21a-AP205-ST质粒。
(4)融合蛋白表达和自组装成VLP、纯化VLP
融合蛋白表达：采用测序验证正确的、转化有前文构建的质粒的BL21(DE3)感受态大肠杆菌(CD601-02)，挑取单克隆到氨苄抗性LB培养基中过夜摇菌，37℃、220rpm。第二天扩大培养，在对数生长期OD值为0.6-0.9时加入终浓度为0.1mM的诱导剂IPTG(翊圣生物10902ES08)以诱导表达融合蛋白，诱导5小时后收菌。
纯化：将收获的大肠杆菌离心(6000rpm 10分钟)得到细胞沉淀。使用20mM pH 7.5Tris重悬沉淀。超声破碎菌体得到裂解上清，两次离心(5000rpm 10分钟，20000g 30分钟)除去细胞碎片等不溶性杂质，再通过30％蔗糖垫离心沉淀颗粒性蛋白(在12毫升离心管中，底部加2毫升30％蔗糖，其上加10ml裂解液上清，33000rpm 3.5小时)，用1毫升PBS(KCl 2.6mM，KH 2PO 4 1.47mM，NaCl 136mM，Na 2HPO 4.12H 2O 8mM)重悬颗粒性蛋白，再通过氯化铯密度梯度(在5毫升超离管中，依次加2毫升50％氯化铯，2毫升24％氯化铯，最后加1毫升样品)离心将杂蛋白与目的蛋白分离(200000g，22小时)。分层收样跑蛋白胶确认目的融合蛋白所在位置，取相应层蛋白透析至PBS保存。
(5)结果
SC-AP205、AP205-SC和AP205-ST表达质粒均可很好的表达相应融合蛋白。
SC-AP205和AP205-SC表达质粒诱导前后的全细菌裂解液对比蛋白胶图如图1，显示目的融合蛋白得以表达。但在纯化SC-AP205和AP205-SC时，蔗糖垫离心后离心管底部沉淀难以被重悬，说明SC-AP205和AP205-SC的自组装存在问题，无法得到良好分散的、非聚集的VLP(参见图2)。氯化铯密度梯度离心分层取样，13层之前肉眼可见浑浊，14层及以后澄清，但从蛋白胶条带上看，无法将目的蛋白与杂蛋白分开，而且产量非常少(参见图3)。
对于AP205-ST表达质粒，在后续纯化中则得到了从AP205-ST成功自组装的VLP(参见图2、4)。氯化铯密度梯度离心分层收样蛋白胶图中在14层到20层可见明显的目的融合蛋白条带(参见图5)，取相应层透析至PBS即得到纯化的、从AP205-ST组装的、良好分散的、非聚集的VLP(AP205-ST VLP)。在实验室阶段每升菌可得到50-60mg VLP。
实施例2：SpyCatcher(SC)与抗原的连接方式对PLA稳定性的影响
(1)融合蛋白RBD-SC(SC位于RBD的C端)表达质粒的构建
人工合成全长1068bp的编码RBD-SC的cDNA(SEQ ID NO：11)片段，并在其5’端加调控蛋白表达的Kozak序列GCCACC及KpnI酶切位点和3’端加上XhoI酶切位点。用KpnI和XhoI内切酶(Takara)分别对合成的RBD-SC cDNA片段(1μg)和pCEP4质粒(1μg)置37℃消化2小时。然后在琼脂糖凝胶电泳分离消化的cDNA片段和pCEP4质粒片段。使用小量DNA产物纯化试剂盒(庄盟生物ZP201-3)分别将分离的cDNA片段和pCEP4质粒片段纯化。进一步将纯化的cDNA片段与pCEP4质粒片段进行DNA连接反应以构建含有该cDNA片段的pCEP4质粒(称为pCEP4-RBD-SC质粒)。连接酶为T4 DNA连接酶(Takara 2011A)，连接缓冲液为T4 DNA Ligase Buffer(Takara 2011A)，连接反应中pCEP4质粒片段与RBD-SC cDNA片段的比率约为1：3，总DNA约为200ng，22℃连接2小时。如下将pCEP4-RBD-SC质粒转化到表达宿主中：将15μl的连接反应液加入到150μl的XLI-Blue感受态大肠杆菌(全式金CD401-02)，42℃，1分钟。吸取150μl在氨苄抗性LB平板上涂板，37℃培养14-16小时。在平板上取单一菌落，用质粒纯化试剂盒(全式金EM101-02)提取质粒DNA并进行酶切验证，确认成功构建pCEP4-RBD-SC质粒。
使用无内毒素大提试剂盒(天根生物DP117)从宿主细菌提取pCEP4-RBD-SC质粒。使用PEI试剂(polyscience 23966-1)将提取的pCEP4-RBD-SC质粒转染到293F细胞系(Life technologies)中。配置转染混合物：①300微克质粒加15毫升SMM 293-TII培养基(Sino biological M293TII)，②1.5毫升PEI加15毫升SMM 293-TII培养基，两者混匀后室温静置2分钟。将转染混合物①②充分混匀室温静置15分钟，之后将转染混合物加入300毫升细胞密度为2x10 5细胞/毫升的细胞液中，混匀后置于37℃，5％二氧化碳，125rpm摇床培养。中间每两天添加7毫升补料SMS 293-SUPI(Sino biological M293-SUPI)，第七天时收细胞。
两步离心(500g 10分钟，8000rpm 30分钟)除去细胞碎片等不溶性杂质，取上清过0.2μm滤膜进一步除去不溶性杂质。使用Ni-NTA预装重力柱(BBI C600791-0005)纯化所表达的目标蛋白，步骤如下：
a、平衡：先用50毫升超纯水洗，然后用50毫升结合缓冲液(5mM咪唑，500mM氯化钠，20mM Tris，10％甘油，pH7.9)；
b、上样：将细胞上清通过镍柱，重复三次上样；
c、洗脱：先用50毫升洗涤缓冲液(30mM咪唑，500mM氯化钠，20mM Tris，10％甘油，pH7.9)洗去杂蛋白。再用50毫升洗脱缓冲液(250mM咪唑，500mM氯化钠，20mM Tris，10％甘油，pH7.9)洗脱收取目标蛋白。将目标蛋白液浓缩至5毫升左右再透析至PBS保存。
所得融合蛋白RBD-SC稳定性差，4℃放置三天时严重降解(因此在图6中没有再显示3天以后的情况)(参见图6)。
(2)RBD-SC与AP205-ST VLP的连接
RBD-SC与AP205-ST VLP以1：10的比率(一个VLP由180条AP205-ST序列自组装而成，即有180个ST，该比率指RBD上的SC与其所要连接的VLP上的ST的比率，下同)在PBS缓冲液中4℃孵育1小时，由此ST氨基酸序列第7位的Asp和SC氨基酸序列的第31位的Lys自发形成异肽共价键，使RBD-SC通过共价键偶联到AP205-ST VLP上。这个反应过程不需要任何特殊的酶和缓冲液体系。
由此得到的连接产物AP205-ST VLP/RBD-SC在4℃放置9天时完全降解(参见图7)。
(3)融合蛋白SC-RBD(SC位于RBD的N端)表达质粒的构建以及SC-RBD与AP205-ST VLP的连接
用与上述(1)相同的方法构建融合蛋白SC-RBD表达质粒，并用与上述(2)相同的方法连接SC-RBD与AP205-ST VLP。融合蛋白SC-RBD序列为SEQ ID NO:8。人工合成全长1059bp的SC-RBD cDNA(SEQ ID NO：12)片段，并在其5’端加调控蛋白表达的Kozak序列GCCACC及HindIII酶切位点和3’端加上XhoI酶切位点。用HindIII和XhoI内切酶(Takara)分别对合成的SC-RBD cDNA片段(1μg)和pCEP4质粒(1μg)置37℃消化2小时。
所得融合蛋白SC-RBD与前述融合蛋白RBD-SC相比稳定性明显提高。而且，同样在4℃下在采用SC-RBD与AP205-ST VLP的连接比率为1:10时，连接产物AP205-ST VLP/SC-RBD在5天内稳定，直到第7天时才出现少量抗原脱落现象，并且在14天时绝大部分仍为完整连接产物(参见图8)。可见AP205-ST VLP/SC-RBD的稳定性明显优于在相同条件下在第9天即完全降解的AP205-ST VLP/RBD-SC。
实施例3：AP205的序列对PLA可溶性的影响
为了研究AP205的序列对PLA可溶性和AP205-ST VLP承载外接抗原的能力的影响，发明人将本发明所用的改造后的AP205衣壳蛋白序列(即在野生型(WT)AP205衣壳蛋白序列N末端添加了五个氨基酸MEFGS，除非另有说明，本文所用AP205及相应的VLP和疫苗产物均使用该改造后的AP205制得)与未改造的WT AP205衣壳蛋白序列作了一系列的对比试验。
以前述相同方法获得WT AP205-ST VLP。然后将其与融合蛋白SC-RBD以与前述连接方法相同的方法连接在一起获得相应的连接产物。将连接产物还原变性后跑SDS-PAGE以显示抗原与VLP之间的共价连接(图9A)；并通过跑核酸胶电泳(图9B)来检测连接产物的可溶性，具体测量过程和条件为：上样量为10μg PLA或者连接产物，1％核酸胶，90伏，20分钟。
结果发现：SC-RBD与野生型和改造后的AP205-ST VLP在不同比率(1:6、1:8、1:10)均能很好地连接(参见图9A)；但与WT AP205-ST VLP形成的连接产物极易聚集，生成可见沉淀，在核酸胶孔中可见沉积，而改造后的AP205-ST VLP形成的连接产物的可溶性有明显改善，无明显的可见沉淀，特别是在1:8和1:10比率下(参见图9B，图中①为改造前的，②为改造后的)。测试了其它抗原包括非洲猪瘟病毒抗原eP22(SEQ ID NO:24)、流感病毒抗原M2E(SEQ ID NO:25)和自身抗原髓鞘少突胶质细胞糖蛋白MOG(SEQ ID NO:26)与改造前后的AP205-ST VLP连接所得连接产物的可溶性，改造后的AP205-ST VLP形成的连接产物相比WT AP205-ST VLP形成的连接产物可溶性均有改善。
实施例4：抗原与VLP的比率对PLA可溶性的影响
本发明人研究了抗原与VLP的比率对连接产物PLA的可溶性的影响，以期通过调整该比率来进一步改善连接产物的可溶性。发明人采用SC-RBD与AP205-ST VLP，分别测试了1:2、1:4、1:5、1:6、1:7、1:8、1:10的比率，测试方法同实施例3。图10A显示了在这些比率下均可成功获得连接产物；图10B表明当抗原与VLP的比率较高(1:2、1:4、1:5))时在核酸胶孔可见明显沉积，而且有明显肉眼可见沉淀生成，说明此时存在PLA聚集，而降低抗原与VLP的连接比率如连接比率在1:6、1:7、1:8、1:10时基本无肉眼可见沉淀，同时没有蛋白沉积在核酸胶孔；图10C是对琼脂糖凝胶的蛋白染色结果，显示了RNA与AP205蛋白在电泳中伴随运动的情况，同样可以看出，在高比率连接时，胶孔中出现EB荧光和蛋白染色，说明发生PLA聚集。可见，降低抗原与VLP的比率能够明显提高连接 产物的可溶性。
发明人还使用相同的方法分别测试了非洲猪瘟病毒抗原eP22(SEQ ID NO:24)、流感病毒抗原M2E(SEQ ID NO:25)和自身抗原髓鞘少突胶质细胞糖蛋白MOG(SEQ ID NO:26)的SC-抗原形式的融合蛋白以不同的比率与AP205-ST VLP连接所得产物的可溶性。将SDS-PAGE蛋白胶、核酸胶和Coomassie R-250蛋白染色结果相结合发现：
对于非洲猪瘟病毒抗原eP22，当连接比率高至1:2时已无可见沉积(参见图11)，因此适合于非洲猪瘟病毒抗原eP22的连接比率可以确定为1:1～1:5，例如1:1、1:2、1:3、1:4、1:5；
对于流感病毒抗原M2E，当连接比率高至1:1时已无可见沉积(参见图12)，因此适合于流感病毒抗原M2E的连接比率可以确定为1:1～1:1.5；
对于自身抗原MOG，当连接比率高至1:4时连接产物已无可见沉积(参见图13)，因此适合于自身抗原MOG的连接比率可以确定为1:4～1:10，例如1:4、1:5、1:6、1:7、1:8、1:10。
可见，不同抗原的SC-抗原形式与AP205-ST VLP连接时，适于形成可溶性连接产物PLA的连接比率不同，即抗原自身的类型对连接产物的可溶性存在影响，但趋势是相同的，即随着连接比率的降低，连接产物的可溶性逐步提高。
实施例5：VLP纯化条件对其内部的RNA的存在与否的影响
在对VLP的工业化纯化工艺进行探索时，发明人发现当离子交换溶液的pH为10.5时纯化的VLP内部的RNA消失(参见图14)，提示该溶液的酸碱度可能影响VLP内RNA的存在。因此，发明人在实施例1记载的VLP纯化条件的基础上，检测了溶液pH对VLP内部RNA的存在的影响。具体方法为：分别用盐酸和NaOH调整PBS的酸碱度，然后将2.5微克纯化的VLP放置37℃水浴2小时，然后通过琼脂糖凝胶电泳和EB染色来检测溶液酸碱度对VLP内RNA的存在的影响。
结果显示：在pH4.5-8.5的范围内VLP内部的RNA含量稳定，pH 9.5时开始减少，在pH 10.5及以上时VLP内部的RNA大幅减少，pH 11.0时已检测不到内部的RNA，VLP外出现RNA，表明RNA在这样的碱性条件下会从VLP内部释放出来(参见图15)。PLA的VLP内部的RNA对于PLA的B细胞相关免疫活化机理起到关键作用(Sheng Hong et al.,B Cells Are the Dominant Antigen-Presenting Cells that Activate  Naive CD4+T Cells upon Immunization with a Virus-Derived Nanoparticle Antigen，Immunity,2018.10,49:1-14)，检测发现，当PLA的VLP内部存在RNA时，所述RNA充当TLR刺激剂，使PLA能够依靠B细胞相关免疫机制发挥作用，免疫效果优于VLP内部不存在RNA的PLA。因此，发明人提出VLP的纯化工艺需要在合适的pH条件下，例如pH 4.0-9.0，应避免pH 10.5以上的强碱性条件。
实施例6：PLA-SARS-CoV2疫苗诱导抗新冠病毒RBD抗体的能力
将C57BL/6小鼠(购自斯贝福)分为四组：(1)RBD抗原混合铝佐剂(Alum，购自Pierce)，12只，10μg/只；(2)RBD抗原混合CpG1826佐剂(序列为tccatgacgttcctgacgtt)，4只，10μg/只(CpG用量为50μg/只)；(3)S蛋白胞外段混合铝佐剂，4只，50μg/只；(4)PLA-SARS-CoV2(即对AP205改造后形成的VLP与SARS CoV2 RBD抗原连接而成的疫苗复合物，下同)，21只，10μg/只。采用腹腔免疫方式。在第一次免疫14天时取血，记为一免血清，第一次免疫21天进行第二次免疫，第二次免疫7天(即第一次免疫28天)时采血，记为二免血清。
Elisa检测RBD特异性抗体反应。RBD抗原包被量为2μg/ml，50μl/孔，4℃过夜。血清进行梯度(血清初始稀释为1：1000，再继续做5倍稀释，共做8梯度)稀释，室温下与包被RBD的Elisa 96孔板孵育3小时。二抗IgG-HRP(Bethyl Laboratories)室温孵育1小时。显色，酶标仪读取相应孔OD值。取未孵育血清的孔作为空白对照，4-8个空白对照孔OD值的平均值加10倍标准偏差值为基准值，大于基准值的血清最低稀释度记为抗体滴度。
不同分组的具体测定值，纵坐标为OD读值，横坐标为血清稀释度的Log值。可以看出在免疫一次后小鼠就能产生较高滴度的RBD IgG抗体(参见图16)；再次免疫后RBD IgG抗体滴度可以达到3x10 6左右(参见图17)。与免疫RBD抗原混合铝佐剂、RBD抗原混合CpG佐剂、以及免疫新冠病毒S蛋白胞外段混合铝佐剂相比，PLA-SARS-CoV2疫苗无论初次免疫还是再次免疫后产生的RBD IgG型抗体滴度都能提高100倍左右(参见图18)。
本发明人进一步采用相同方法检测了流感病毒M2E抗原、非洲猪瘟病毒eP22抗原和自身抗原MOG构建的PLA疫苗的抗体应答。结果表明：非洲猪瘟病毒eP22，流感病毒M2E抗原，自身抗原MOG构建的PLA疫苗也都可引起很好的抗体应答反应。几种抗原的PLA疫苗免疫C57BL/6小鼠后14天后取血清检测IgG型抗体应答情 况，发现与相应抗原加佐剂的情况相比，PLA疫苗均可以诱导出很好的特异性IgG抗体水平(参见图19A、B、C，图中每个点代表一只小鼠的血清抗体滴度(titer)水平)。
实施例7：PLA-SARS-CoV2疫苗免疫小鼠产生中和抗体的情况
同样使用RBD抗原混合铝佐剂、RBD抗原混合CpG1826佐剂、S蛋白胞外段混合铝佐剂和PLA-SARS-CoV2，通过如下中和抗体检测方法比较了诱导中和抗体的情况：血清稀释于300μl 2％DMEM培养基，3倍稀释。200μl不同稀释度的血清与MOI 0.01活病毒(10μl)37℃共孵育1h。200μl感染48孔板VERO-E6细胞。1h后换液，细胞置2％DMEM培养基培养24h。用MiniBEST Viral RNA/DNA Extraction Kit(Takara)收取150μl上清提取RNA，用PrimeScriptTM RT reagent Kit with gDNA Eraser(Takara)反转录出cDNA。用标准曲线法测拷贝数(ABI 7500(Takara TB
Figure PCTCN2021138312-appb-000001
Premix Ex Taq II))，引物靶向S基因。
上游引物(5’-3’):CAATGGTTTAACAGGCACAGG(SEQ ID NO：19)；下游引物(5’-3’):CTCAAGTGTCTGTGGATCACG(SEQ ID NO：20)。
纵坐标是ELISA检测的中和抗体滴度(ID50 titer)，显示本发明的PLA-SARS-CoV2诱导中和抗体的水平超过其它混合佐剂的传统疫苗100倍(参见图20)。
实施例8：PLA-SARS-CoV2疫苗免疫猕猴产生抗RBD IgG抗体的情况
实验使用8只青年健康猕猴(雄性，年龄3-6岁,均来源于中国科学院昆明灵长类研究中心)。免疫试验分成两组(各4只)分别接受PLA-SARS-CoV2(20微克/只/次)或者生理盐水(PBS，对照组)肌肉注射两次(间隔3周)。在第一次注射后14天和第二次注射后7天分别采集血液分离血清。抗RBD IgG抗体滴度检测方法参考实施例6描述的ELISA方法。仅将二抗换成HRR标记的山羊抗猴IgG(购自Abcam，货号ab112767)。图21显示了初次免疫后14天(1st)和再次免疫(2nd)后7天后血清中的抗RBD IgG抗体水平(纵坐标是ELISA检测的抗体滴度)，表明初次免疫时血清中抗RBD IgG抗体水平是PBS对照的约100倍以上，再次免疫后的抗RBD IgG抗体水平相对于PBS对照提高1000倍以上。
实施例9：PLA-SARS-CoV2疫苗免疫猕猴产生中和抗体的情况
发明人进一步测试了本发明的PLA-SARS-CoV2疫苗诱导猕猴产生中和抗体的情况。免疫过程和条件同上述实施例8。血清中和抗体滴度检测方法同上述实施例7。图22显示了再次免疫(2nd)7天后血清中的新冠病毒中和抗体的水平相对于PBS对照提高数十倍。
实施例10：PLA-SARS-CoV2疫苗免疫猕猴后的肺部病毒载量
发明人接下来测试了PLA-SARS-CoV2疫苗免疫猕猴后肺部病毒载量情况。PLA-SARS-CoV2疫苗免疫猕猴的过程同实施例8。实验采用的病毒为新型冠状病毒107株(由中国广东省疾病预防控制中心提供)，病毒株经Vero-E6细胞株扩培，半数组织培养感染剂量测定方法为Reed-Muench法。
病毒攻毒试验是在再次免疫后10天进行。攻毒途径为组合方式，滴鼻(0.4mL/鼻孔)和气管内(1.2mL，纤维支气管镜)，总病毒滴度为1×10 7TCID50mL，使用无菌0.9％生理盐水稀释。
两组动物在7天后的肺部病毒载量(viral load)通过RT-PCR进行了检测。采用试剂盒(Roche德国)提取拭子和气管刷总RNA，组织样本RNA采用TRIzol试剂法提取(Thermo USA)。病毒RNA检测采用探针一步法实时定量PCR试剂盒检测(TOYOBO，Japan)。引物和探针分别为：上游引物5'-GGGGAACTTCTCCTGCTAGAAT-3'(SEQ ID NO：21)、下游引物5'-CAGACATTTTGCTCTCAAGCTG-3'(SEQ ID NO：22)和FAM-TTGCTGCTGCTTGACAGATT-TAMRA-3'(SEQ ID NO：23)。每个检测样本稀释参照中国计量科学研究院标准，最后计算每一个样本的拷贝数。
结果显示，使用本发明的PLA-SARS-CoV2疫苗免疫后猕猴肺部病毒几乎完全消失(图23，图纵坐标显示的是每微克RNA中的病毒拷贝数的对数值)。而且，根据前述实施例中本发明PLA-SARS-CoV2疫苗诱导特异性抗体和中和抗体的结果，可以合理推知本发明的疫苗相对于其它需额外添加佐剂的传统疫苗在攻毒试验中也将具有明显更优的效果。

Claims (15)

  1. 一种可溶性病原样抗原(PLA)复合物，其包含：
    (1)病毒样颗粒(VLP)，其由第一融合蛋白自组装而成，所述第一融合蛋白包含位于其N端的病毒衣壳蛋白或其变体和其C端的SpyTag和连接两者的第一连接肽，
    (2)第二融合蛋白，所述第二融合蛋白包含抗原或其变体和SpyCatcher和连接两者的第二连接肽，优选所述SpyCatcher在所述第二融合蛋白的N端；
    其中所述病毒样颗粒还在其内部包裹核酸，和
    其中所述病毒样颗粒和所述第二融合蛋白通过所述SpyCatcher和所述SpyTag共价连接而使所述抗原或其变体展示于所述病毒样颗粒的表面。
  2. 根据权利要求1所述的可溶性病原样抗原复合物，其中所述病毒样颗粒内包裹的所述核酸为所述病毒样颗粒在其自组装时包裹的、来自于表达所述病毒样颗粒的宿主细菌的核酸，优选所述宿主细菌为大肠杆菌，优选所述核酸是RNA。
  3. 根据权利要求1或2所述的可溶性病原样抗原复合物，其中所述衣壳蛋白来自大肠杆菌噬菌体Qβ、MS2或AP205，优选来自大肠杆菌噬菌体AP205。
  4. 根据前述权利要求中任一项所述的可溶性病原样抗原复合物，其中所述抗原选自SARS-CoV2病毒S蛋白的RBD序列、非洲猪瘟病毒抗原eP22、流感病毒抗原M2E和自身抗原髓鞘少突胶质细胞糖蛋白MOG。
  5. 根据前述权利要求中任一项所述的可溶性病原样抗原复合物，其中所述噬菌体AP205衣壳蛋白的序列与SEQ ID NO：1具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的一致性。
  6. 根据前述权利要求中任一项所述的可溶性病原样抗原复合物，其中所述SpyTag的序列与SEQ ID NO：3具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的一致性。
  7. 根据前述权利要求中任一项所述的可溶性病原样抗原复合物，其中所述SpyCatcher的序列与SEQ ID NO：4具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的一致性。
  8. 根据前述权利要求中任一项所述的可溶性病原样抗原复合物，其中在所述SpyTag的序列SEQ ID NO：3的第7位的Asp和所述SpyCatcher序列SEQ ID NO：4的第31位Lys之间形成异肽键。
  9. 根据前述权利要求中任一项所述的可溶性病原样抗原复合物，其中所述第一连接肽的序列为SEQ ID NO：5。
  10. 根据前述权利要求中任一项所述的可溶性病原样抗原复合物，其中所述第二连接肽的序列为SEQ ID NO：6。
  11. 根据前述权利要求中任一项所述的可溶性病原样抗原复合物，其中根据不同的抗原，所述第二融合蛋白与所述病毒样颗粒以小于或等于1:1的比率连接，优选以1:1、1:1.5、1:2、1:3、1:4、1:5、1:6、1:7、1:8、1:9、1:10、1:11、1:12的比率连接，以此确保病原样抗原复合物的可溶性和免疫原性，所述比率以第二融合蛋白上的SpyCatcher与病毒样颗粒上的SpyTag之比计算。
  12. 一种病原样抗原疫苗组合物，其包含前述权利要求中任一项所述的可溶性病原样抗原复合物和药学上可接受的载体和/或赋形剂。
  13. 一种制备权利要求1-11任一项所述的可溶性病原样抗原复合物的方法，其包括在4.0～9.0，优选5.5-8.5范围内的pH下纯化所述病毒样颗粒。
  14. 一种提高病原样抗原复合物的可溶性的方法，其包括：
    (1)制备权利要求1-13任一项中定义的病毒样颗粒和第二融合蛋白；和
    (2)在将所述第二融合蛋白与所述病毒样颗粒连接时，降低第二融合蛋白与所述病毒样颗粒的连接比率，以获得可溶性病原样抗原复合物。
  15. 一种在有需要的受试者中预防和/或治疗SARS-CoV2病毒、流感病毒、非洲猪瘟病毒或与自身抗原髓鞘少突胶质细胞糖蛋白MOG相关的疾病的方法，其包括向所述受试者施用预防和/或治疗有效量的权利要求1-11中任一项所述的可溶性病原样抗原复合物或权利要求12所述的病原样抗原疫苗组合物。
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