HSV-1 type nanoparticle vaccine as well as preparation method and application thereof

Abstract

The invention relates to an HSV-1 type nanoparticle vaccine and a preparation method and application thereof.The nanoparticle comprises HSV-1 PreF gB mutant protein and ferritin, and after assembling, a plurality of ferritin-PreF gB nanoparticles with the PreF gB mutant protein displayed on the surface in a centralized mode are obtained. After PreF gB is mutated, the stability and the effective immunogenicity of gB protein are enhanced, the immunogenicity is further enhanced through display on the surfaces of the nanoparticles, the cellular immune mechanism of an organism can be effectively stimulated, and the preF gB becomes an effective vaccine for preventing HSV infection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an HSV-1 type nanoparticle vaccine and a preparation method and application thereof. Background Art

[0002] Herpes simplex virus (HSV) is a common infectious virus that can cause herpes lesions on the skin and mucous membranes. Herpes simplex virus infection is very common in the population. According to data, its infection rate is as high as over 80%, which indicates that most people may have been or are currently infected with herpes simplex virus. The incidence of herpes simplex virus is quite high and is a public health issue worthy of attention.

[0003] HSV is mainly divided into two types: HSV-1 and HSV-2. HSV-1 is usually transmitted through oral-to-oral contact. It mainly causes herpes simplex in non-genital areas such as the oral mucosa, face, lips, and corneal conjunctiva.

[0004] The gB protein is an important structural protein of the herpes simplex virus. It is located on the viral envelope and plays a key role when the virus enters the host cell. It participates in the fusion process between the virus and the host cell membrane. The pre-fusion (Pre-F) state is a conformational state of the gB protein on the viral envelope. The gB protein in the pre-fusion state (Pre-F gB) plays a key role when the virus enters the host cell. It binds to the receptors on the host cell membrane, triggering a series of conformational changes, which ultimately leads to the fusion of the viral envelope with the host cell membrane. This fusion process enables the viral genetic material to enter the host cell, thereby initiating the viral infection and replication process. The fused gB protein is very stable, but before fusion, the gB protein is not stable.

[0005] At present, the prevention and treatment of herpes simplex virus mainly focus on avoiding contact transmission, antiviral, symptomatic treatment, etc., and there is still a lack of effective prevention or treatment drugs. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides an HSV-1 type nanoparticle vaccine and a preparation method and application thereof. Specifically,

[0007] In a first aspect of the present invention, a Pre-F gB mutant protein is provided, wherein the mutant protein is obtained by mutation of the HSV-1 wild-type Pre-F gB protein, wherein the mutation comprises substitution, deletion and / or insertion, and wherein the mutation comprises mutation at positions 161, 209, 240, 352, 516, 531, 541 and / or 607.

[0008] Preferably, the above mutation sites include a combination of the following sites:

[0009] (1), 161st, 209th, 352nd and 541st;

[0010] (2), Nos. 240, 516, 531 and 607;

[0011] (3) Nos. 240, 352, 531 and 541; or

[0012] (4), 161st, 209th, 531st and 607th.

[0013] Preferably, the mutation includes replacement, and the HSV-1 wild-type Pre-F gB protein is subjected to at least one mutation in the following 1)-8) to obtain a Pre-F gB mutant protein:

[0014] 1) The alanine (A) at position 161 of the amino acid sequence of the Pre-F gB protein was mutated to cysteine ​​(C), i.e. A161C.

[0015] 2) The serine (S) at position 209 of the amino acid sequence of the Pre-F gB protein was mutated to phenylalanine (F), i.e. S209F.

[0016] 3) The alanine (A) at position 240 of the amino acid sequence of the Pre-F gB protein was mutated to cysteine ​​(C), i.e. A240C.

[0017] 4) The valine (V) at position 352 of the amino acid sequence of the Pre-F gB protein was mutated to leucine (L), i.e., V352L.

[0018] 5) The histidine (H) at position 516 of the amino acid sequence of the Pre-F gB protein was mutated to proline (P), i.e. H516P.

[0019] 6) The leucine (L) at position 531 of the amino acid sequence of the Pre-F gB protein was mutated to glutamic acid (E), i.e., L531E.

[0020] 7) The glutamic acid (E) at position 541 of the amino acid sequence of the Pre-F gB protein was mutated to cysteine ​​(C), i.e., E541C.

[0021] 8) The glutamic acid (E) at position 607 of the amino acid sequence of the Pre-F gB protein was mutated to cysteine ​​(C), i.e., E607C.

[0022] More preferably, the mutation comprises:

[0023] (1), A161C, S209F, V352L and E541C;

[0024] (2), A240C, H516P, L531E and E607C;

[0025] (3), A240C, V352L, L531E, and E541C; or,

[0026] (4), A161C, S209F, L531E and E607C,

[0027] Further preferably, the amino acid sequence of the wild-type Pre-F gB protein of HSV-1 is shown in GenBank Seq ID: P06437.2, positions 1 to 730. This fragment contains the main extracellular region of the gB protein, and deletes the transmembrane region and intracellular region after position 730.

[0028] It is understandable that the wild-type Pre-F gB protein amino acids of different subtypes or strains of viruses are not necessarily the same, and there may be substitution, insertion or deletion mutations. The above wild-type sequence can be used for reference, but it can also be a wild-type sequence with other different structures, and its mutation site corresponds to any site defined above. The correspondence is understood as a corresponding relationship based on amino acid structure and / or function analysis.

[0029] In a specific embodiment, the amino acid sequence of the mutant protein comprises:

[0030] (A1) any one of SEQ ID No. 1-4;

[0031] (A2) a protein having the same function as (A1) obtained by replacing and / or deleting and / or adding one or more amino acid residues;

[0032] (A3) A protein having 80% or more identity with any of (A1) to (A2) and having the same function.

[0033] More preferably, the (A2) or (A3) retains any of the mutations in (1) to (4) above.

[0034] More preferably, the mutant protein may further include a signal peptide and / or a tag protein.

[0035] In a second aspect, the present invention provides a fusion protein, wherein the fusion protein comprises any one of the above-mentioned Pre-FgB mutant proteins and ferritin.

[0036] Preferably, the ferritin comprises ferritin or a mutant thereof.

[0037] More preferably, the ferritin mutant is a protein obtained by subjecting the wild-type ferritin amino acid sequence to at least one of the following mutations b1) to b3):

[0038] b1) mutating the asparagine (N) at position 15 of the wild-type ferritin amino acid sequence to glutamine (Q);

[0039] b2) mutating the serine (S) at position 96 of the wild-type ferritin amino acid sequence to asparagine (N);

[0040] b3) The tyrosine (Y) at position 119 of the wild-type ferritin amino acid sequence was mutated to arginine (R).

[0041] It is understandable that the wild-type ferritin amino acids of different subtypes or strains of viruses are not necessarily the same, and there may be mutations of substitution, insertion or deletion. The mutation sites of various wild-type sequences correspond to the above-mentioned sites, and the correspondence is understood to be a corresponding relationship based on amino acid structure and / or functional analysis.

[0042] In a specific embodiment, the amino acid sequence of ferritin comprises:

[0043] (B1) protein represented by SEQ ID No.5;

[0044] (B2) a protein having the same function as (B1) obtained by replacing and / or deleting and / or adding one or more amino acid residues;

[0045] (B3) A protein having 80% or more identity with any of (B1) to (B2) and having the same function.

[0046] Preferably, (B2) and (B3) retain the mutations of ferritin relative to wild-type ferritin, i.e., the mutations b1) to b3) above.

[0047] Preferably, the fusion protein further comprises a linker. More preferably, the linker may be SGSGGGSG (SEQ ID No. 6).

[0048] The connecting peptide can also be a variant of the above sequence, which has a connecting function without affecting the protein function at both ends, for example: GSGGGGSG (SEQ ID No: 11), GS,

[0049] SGGGSGGGGSGGGGSAGSGGGGSGGGGSAGSGGGSGGGGSGGGGSAGSGGGSGGGGSGGGGSAGSGGGS GGGGSGGGGSA (SEQ ID No: 12), SGSGSG (SEQ ID No: 13), or, GGGGSGGGGSGGGG (SEQ ID No: 14), etc.

[0050] More preferably, the fusion protein includes Pre-F gB mutant protein, linker and ferritin from N-terminus to C-terminus.

[0051] Preferably, the fusion protein may further include a signal peptide and / or a tag protein.

[0052] The above-mentioned tag protein refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0053] Any of the above mentioned mutant proteins or fusion proteins can be artificially synthesized, or the encoding gene thereof can be synthesized first and then obtained by biological expression.

[0054] Herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and searching to calculate the identity of an amino acid sequence, the value (%) of identity can then be obtained.

[0055] Herein, the 80% or greater identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0056] In a third aspect, the present invention provides a biomaterial, comprising:

[0057] 1. A nucleic acid molecule encoding any one of the Pre-F gB mutant proteins or fusion proteins described above;

[0058] II. an expression cassette or vector comprising the nucleic acid molecule described in I; or

[0059] III. A host cell, which contains the nucleic acid molecule described in I or the expression cassette or vector described in II.

[0060] The mutant protein or fusion protein refers to the above definition of the present invention.

[0061] The nucleic acid molecule may be DNA, such as recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA.

[0062] Preferably, the nucleic acid molecule comprises:

[0063] (I-1), a DNA molecule shown at position 61 to 2709 or the full length of any sequence of SEQ ID No. 7 to 10;

[0064] (I-2) or (I-1) which are complementary or degenerate sequences;

[0065] (I-3) A DNA molecule having 75% or more identity with the DNA molecule defined in (I-1) or (I-2) and encoding the mutant protein or fusion protein.

[0066] Those skilled in the art can easily mutate the nucleotide sequence encoding the above-mentioned protein or fusion protein of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or higher identity with the nucleotide sequence encoding the above-mentioned mutant protein or fusion protein are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the above-mentioned mutant protein or fusion protein and have the same function.

[0067] The identity refers to the sequence similarity with the natural nucleic acid sequence. "Identity" includes nucleotide sequences with 75% or more, 80% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of the protein composed of the coding amino acid sequence shown in the present invention. The identity can be evaluated by the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0068] The aforementioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0069] In the above biological material, the expression cassette refers to a DNA capable of expressing the above protein or fusion protein in a host cell, and the DNA may include not only a promoter for initiating transcription of the gene sequence encoding the above protein or fusion protein, but also a terminator for terminating transcription of the gene sequence encoding the above protein or fusion protein. Furthermore, the expression cassette may also include an enhancer sequence.

[0070] The vector described herein refers to a vector that can carry exogenous DNA or target gene into host cells for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmid, phage (such as lambda phage or M13 filamentous phage, etc.), cosmid (i.e., cosmid), Ti plasmid, viral vector (such as retrovirus (including lentivirus), adenovirus, adeno-associated virus, etc.). In one or more embodiments of the present invention, the vector is a pUC57 vector and / or a pKS001 vector.

[0071] Microorganisms described herein may be bacteria, fungi, actinomycetes, protozoa, algae or viruses. Wherein, the bacteria may be from Escherichia sp., Erwinia sp., Agrobacterium sp., Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., Bacillus sp., etc., but not limited thereto, for example, the bacteria may be Escherichia coli, Bacillus subtilis or Bacillus pumilus. In one or more embodiments of the present invention, the microorganism is a TOP10 competent cell.

[0072] The host cell (also referred to as recipient cell) described herein can be a plant cell or an animal cell. The host cell can be understood to refer not only to a specific recipient cell, but also to the progeny of such a cell, and due to natural, accidental or intentional mutations and / or changes, the progeny may not necessarily be completely identical to the original parent cell, but is still included in the scope of the host cell. Suitable host cells are known in the art, wherein: the plant cell may be Arabidopsis thaliana, tobacco (Nicotiana tabacum), corn (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum) and the like, but are not limited thereto; the animal cell may be a mammalian cell (e.g., Chinese hamster ovary cell (CHO cell), African green monkey kidney cell (Vero cell), baby hamster kidney cell (BHK cell), mouse breast cancer cell (C127 cell), human embryonic kidney cell (HEK293 cell), human HeLa cell, fibroblast, bone marrow cell line, T cell or NK cell, etc.), avian cell (e.g., chicken or duck cell), amphibian cell (e.g., African clawed frog (Xenopus laevis) cell or giant salamander (Andrias davidianus) cell), fish cell (e.g., grass carp, carp, rainbow trout or catfish cell), insect cell (e.g., Sf21 cell or Sf-9 cell), etc., but are not limited thereto. In one or more embodiments of the present invention, the host cell is a CHO-K1Q cell.

[0073] In a fourth aspect, the present invention provides a method for preparing any one of the above-mentioned Pre-F gB mutant proteins or fusion proteins, the preparation method comprising:

[0074] 1) constructing a recombinant expression vector containing a nucleic acid molecule encoding the mutant protein or fusion protein;

[0075] 2) introducing the recombinant expression vector into a host cell to obtain a recombinant cell;

[0076] 3) Cultivating the recombinant cells, and obtaining the mutant protein or fusion protein by separation and / or purification.

[0077] The above-mentioned nucleic acid molecule, vector, and host cell are as defined in the third aspect.

[0078] In a specific embodiment, the preparation method comprises the following steps: introducing a nucleic acid molecule encoding the mutant protein or fusion protein into CHO K1Q cells to obtain recombinant cells; and culturing the recombinant cells to obtain the mutant protein or fusion protein.

[0079] Furthermore, the nucleic acid molecule encoding the mutant protein or fusion protein is introduced into CHOK1Q cells via a recombinant plasmid.

[0080] The recombinant plasmid is a plasmid obtained by inserting the nucleic acid molecule of the mutant protein or fusion protein into a vector plasmid.

[0081] In a specific embodiment of the present invention, the vector plasmid is pKS001 vector plasmid.

[0082] In a fifth aspect, the present invention provides an application of the above-mentioned mutant protein, fusion protein or biomaterial, characterized in that the application comprises:

[0083] (1) Use in the preparation of products for preventing and / or treating diseases caused by HSV virus infection;

[0084] (2) Application in the preparation of products for inducing immune response to HSV virus antigens;

[0085] (3) Use in the prevention and / or treatment of diseases caused by HSV virus infection;

[0086] (4) Application in inducing immune response to HSV virus antigens.

[0087] The product described herein may be a reagent or a drug, such as a diagnostic reagent or a vaccine.

[0088] Preferably, the preparation further comprises screening, for example, using the above mutant protein or fusion protein as a target to screen candidate preventive or therapeutic drugs or diagnostic agents.

[0089] Preferably, the infectious diseases caused by the HSV virus include infectious diseases caused by HSV-1 and / or HSV-2 viruses, more preferably HSV-1.

[0090] More preferably, the infection sites of the infectious disease include the mouth, pharynx, nose, eyes and reproductive tract, skin, digestive tract, respiratory tract, central nervous system and the like.

[0091] In a sixth aspect, the present invention further provides a drug, which comprises any one of the above-mentioned Pre-F gB mutant proteins, fusion proteins or biological materials.

[0092] Preferably, the drug is a vaccine, and more preferably, the vaccine further comprises an adjuvant and / or a vaccine delivery system.

[0093] More preferably, the adjuvant may be a substance that can stimulate the body to produce a stronger humoral and / or cellular immune response to the antigen inoculated with it. The adjuvant described herein may be known to those skilled in the art, including but not limited to: plant adjuvants (such as alkylamines, phenolic components, quinine, saponin, sesquiterpenes, proteins, polypeptides, polysaccharides, glycolipids, phytohemagglutinins, etc.), bacterial adjuvants (such as cholera toxin, Escherichia coli heat-labile toxin, bacterial lipopolysaccharides, etc.), aluminum adjuvants and other inorganic adjuvants (such as calcium adjuvants), cytokines and nucleic acid adjuvants (such as monocyte colony stimulating factor, leukocyte factor IL-1, IL-2, IL-4, IL-5, IL-6, IFN-γ, CpG motifs, nucleic acid vectors, etc.), emulsion adjuvants (such as Freund's adjuvant). The adjuvant may be a pharmaceutically acceptable adjuvant.

[0094] It is well known to those skilled in the art that in order to enhance the immunogenicity of antigenic proteins, in addition to adding compounds with immunopotentiating effects as adjuvants, the gene combination can be adjusted to allow them to be expressed in a granular structure; or they can be aggregated in vitro and encapsulated in liposomes or capsule microspheres.

[0095] Preferably, the vaccine further comprises a vaccine delivery system.

[0096] The vaccine delivery system can be a type of substance that can carry antigenic substances to the body's immune system, store and exert its antigenic effect therein for a long time. The vaccine delivery system described herein can be an aluminum salt gel adjuvant vaccine delivery system, an emulsion adjuvant vaccine delivery system, a liposome adjuvant vaccine delivery system or a nano adjuvant vaccine delivery system.

[0097] Furthermore, the medicine or vaccine also includes one or more pharmaceutically acceptable carriers.

[0098] The pharmaceutically acceptable carrier may be a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, an adsorption carrier, a surfactant or a lubricant but is not limited thereto.

[0099] The vaccine for preventing infection of the present invention can be an intramuscular liquid injection, an intravenous liquid injection, an intranasal liquid injection, an intradermal liquid injection or a subcutaneous liquid injection.

[0100] In a seventh aspect, the present invention also provides a method for producing an immune response, which may include administering any of the above vaccines to a subject.

[0101] In the above method, after the vaccine is administered to the subject, an immune response against HSV can be induced in the subject. The immune response can be a cellular immune response, a humoral immune response, or a cellular immune response and a humoral immune response.

[0102] The cellular immune response may include a B cell immune response and a T cell immune response.

[0103] The subject described herein can be a human or a non-human animal.

[0104] Furthermore, the non-human animal may be a non-human mammal.

[0105] The non-human mammal may be any one of mice, rats, guinea pigs, hamsters, pigs, dogs, sheep, monkeys, rabbits, cats, cows, and horses, but is not limited thereto.

[0106] The subjects described herein include, but are not limited to, healthy subjects, symptomatic infected subjects, asymptomatic infected subjects, or recovered subjects (subjects who have recovered after HSV infection).

[0107] The administration described herein includes, but is not limited to, intramuscular injection, subcutaneous injection, intradermal injection, intravenous injection, arterial injection, intraperitoneal injection, microneedle injection, mucosal administration, oral administration, oral and nasal spraying, or aerosol inhalation.

[0108] In an eighth aspect, the present invention also provides a method for preventing and / or treating infectious diseases caused by HSV, which may include administering the drug or vaccine to a subject.

[0109] Preferably, the infectious diseases caused by the HSV virus include infectious diseases caused by HSV-1 and / or HSV-2 viruses, more preferably HSV-1.

[0110] More preferably, the infection sites of the infectious disease include the mouth, pharynx, nose, eyes and reproductive tract, skin, digestive tract, respiratory tract, central nervous system and the like.

[0111] In the above method, after the drug is administered to a subject, an immune response against HSV can be induced in the subject. The immune response can be a cellular immune response, a humoral immune response, or a cellular immune response and a humoral immune response.

[0112] The cellular immune response may include a B cell immune response and a T cell immune response.

[0113] The subject described herein can be a human or a non-human animal.

[0114] Furthermore, the non-human animal may be a non-human mammal.

[0115] The non-human mammal may be any one of mice, rats, guinea pigs, hamsters, pigs, dogs, sheep, monkeys, rabbits, cats, cows, and horses, but is not limited thereto.

[0116] The subjects described herein include, but are not limited to, healthy subjects, symptomatic infected subjects, asymptomatic infected subjects, or recovered subjects (subjects who have recovered after infection).

[0117] The administration described herein includes, but is not limited to, intramuscular injection, subcutaneous injection, intradermal injection, intravenous injection, arterial injection, intraperitoneal injection, microneedle injection, mucosal administration, oral administration, oral and nasal spraying, or aerosol inhalation.

[0118] It should be noted that any form of numbering in the present invention, such as I, II, III, A, B, a, b, etc., is only used to distinguish each other and does not indicate the order of time or space, unless otherwise specified.

[0119] In summary, compared with the prior art, the present invention has the following beneficial effects:

[0120] The present invention provides a method for preparing a vaccine by centrally displaying a mutated herpes simplex virus type 1 prefusion (Pre-F) gB protein fragment. The present invention enhances the stability and effective immunogenicity of the gB protein through mutation, and further enhances the immunogenicity by displaying it on the surface of nanoparticles.

[0121] The present invention solves the problem of poor stability of wild antigens, so that after entering the body, it can induce simple herpes simplex virus gB antibodies with good binding activity, thereby giving the body corresponding immune protection. The present invention can obtain a good immune effect at a low dose by fusing the mutated HSV antigen with nanoparticles for expression, and can effectively stimulate the body's cellular immune mechanism. BRIEF DESCRIPTION OF THE DRAWINGS

[0122] Figure 1 Schematic diagram of the structure of the pKS001 vector.

[0123] Figure 2 These are the eluted products after purification of fusion proteins A, B, C, and D. The left figure corresponds to the eluted product after purification of fusion protein A, and the right figure corresponds to the eluted product after purification of fusion proteins B, C, and D.

[0124] Figure 3 Electron micrographs of fusion proteins A, B, C, and D.

[0125] Figure 4 It is the antibody titer produced after mice are immunized with fusion proteins A, B, C, D, and E. DETAILED DESCRIPTION

[0126] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0127] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0128] Example 1 Design, Preparation and Purification of Ferritin-HSV-1pre-F gB Mutant Protein Fusion Protein

[0129] 1. Design of ferritin-HSV-1pre-F gB mutant protein fusion protein

[0130] The HSV-1 pre-F gB related sequence is mutated and designed to obtain the pre-F gB mutant protein, and it is fused with the ferritin related sequence to form a subunit of ferritin-pre-F gB-mutant protein (ferritin-gB for short), and then the self-assembly property of ferritin is used to form nanoparticles with good Pre-F gB antigen display effect. The specific steps are as follows:

[0131] 1. Design of pre-F gB mutant protein

[0132] Objective: To design a stable HSV-1pre-F gB mutant protein that can induce highly efficient binding antibodies.

[0133] Methods: pre-F gB mutant protein design

[0134] The wild-type pre-F gB was mutated. The wild-type pre-F gB can be found in GenBank Seq ID: P06437.2, position 1-730. The sequence of the wild-type gB is:

[0135] MHQGAPSWGRRWFVVWALLGLTLGVLVASAAPTSPGTPGVAAATQAANGGPATPAPPPLGAAPTGDPKPKKNKKPKNPTPPRPAGDNATVAAGHATLREHLRDIKAENTDANFYVCPPPTGATVVQFEQPRRCPTRPEGQNYTEGIAVVFKENIAPYKFKATMYYKDVTVSQVWFGHRYSQFMGIFEDRAPVPFEEVIDKINAKGVCRSTAKYVRNNLETTAFHRDDHETDMELKPANAATRTSRGWHTTDLKYNPSRVEAFHRYGTTVNCIVEEVDARSVYPYDEFVLATGDFVYMSPFYGYREGSHTEHTTYAADRFKQVDGFYARDLTTKARATAPTTRNLLTTPKFTVAWDWVPKRPSVCTMTKWQEVDEMLRSEYGGSFRFSSDAISTTFTTNLTEYPLSRVDLGDCIGKDARDAMDRIFARRYNATHIKVGQPQYYQANGGFLIAYQPLLSNTLAELYVREHLREQSRKPPNPTPPPPGASANASVERIKTTSSIEFARLQFTYNHIQRHVNDMLGRVAIAWCELQNHELTLWNEARKLNPNAIASVTVGRRVSARMLGDVMAVSTCVPVAADNVIVQNSMRISSRPGACYSRPLVSFRYEDQGPLVEGQLGENNELRLTRDAIEPCTVGHRRYFTFGGGYVYFEEYAYSHQLSRADITTVSTFIDLNITMLEDHEFVPLEVYTRHEIKDSGLLDYTEVQRRNQLHDLRFADIDTVIHADANAA(SEQ ID No:15)

[0136] The gene sequence after fusion with ferritin is:

[0137]

[0138] In order to display and stabilize the required epitopes and destroy or mask the unnecessary epitopes, the wild-type Pre-FgB protein of HSV-1 is subjected to at least one mutation in the following 1)-8) to obtain a Pre-F gB mutant protein:

[0139] 1) The alanine (A) at position 161 of the amino acid sequence of the Pre-F gB protein was mutated to cysteine ​​(C).

[0140] 2) The serine (S) at position 209 of the amino acid sequence of the Pre-F gB protein was mutated to phenylalanine (F).

[0141] 3) The alanine (A) at position 240 of the amino acid sequence of the Pre-F gB protein was mutated to cysteine ​​(C).

[0142] 4) The valine (V) at position 352 of the amino acid sequence of the Pre-F gB protein was mutated to leucine (L).

[0143] 5) The histidine (H) at position 516 of the amino acid sequence of the Pre-F gB protein was mutated to proline (P).

[0144] 6) The leucine (L) at position 531 of the amino acid sequence of the Pre-F gB protein was mutated to glutamic acid (E).

[0145] 7) The glutamic acid (E) at position 541 of the amino acid sequence of the Pre-F gB protein was mutated to cysteine ​​(C).

[0146] 8) The glutamic acid (E) at position 607 of the amino acid sequence of the Pre-F gB protein was mutated to cysteine ​​(C).

[0147] In a preferred embodiment, mutations are performed on some sites to stabilize the pre-fusion (Pre-F) gB conformation, wherein the mutation sites of mutant proteins A, B, C, and D (corresponding to SEQ ID No. 1-4, respectively) are shown in Table 1:

[0148] Table 1: Mutation sites of pre-F gB mutant proteins

[0149] sequence Mutation site 1 A161C, S209F, V352L, E541C 2 A240C, H516P, L531E, E607C 3 A240C, V352L, L531E, E541C 4 A161C, S209F, L531E, E607C

[0150] Its specific structure is as follows:

[0151] SEQ ID No.1(A):

[0152] MHQGAPSWGRRWFVVWALLGLTLGVLVASAAPTSPGTPGVAAATQAANGGPATPAPPPLGAAPTGDPKPKKNKKPKNPTPPRPAGDNATVAAGHATLREHLRDIKAENTDANFYVCPPPTGATVVQFEQPRRCPTRPEGQNYTEGIAVVFKENIAPYKFKCTMYYKDVTVSQVWFGHRYSQFMGIFEDREPVPFEEVIDKINAKGVCRFTAKYVRNNLETTAFHRDDHETDMELKPANAATRTSRGWHTTDLKYNPSRVEAFHRYGTTVNCIVEEVDARSVYPYDEFVLATGDFVYMSPFYGYREGSHTEHTTYAADRFKQVDGFYARDLTTKARATAPTTRNLLTTPKFTLAWDWVPKRPSVCTMTKWQEVDEMLRSEYGGSFRFSSDAISTTFTTNLTEYPLSRVDLGDCIGKDARDAMDRIFARRYNATHIKVGQPQYYQANGGFLIAYQPLLSNTLAELYVREHLREQSRKPPNPTPPPPGASANASVERIKTTSSIEFARLQFTYNHIQRLVNDMLGRVAIAWCEHQNHELTLWNCARKLNPNAIASVTVGRRVSARMLGDVMAVSTCVPVAADNVIVQNSMRISSRPGACYSRPLVSFRYEDQGPLVEGQLGENNELRLTRDAIEPCTVGHRRYFTFGGGYVYFEEYAYSHQLSRADITTVSTFIDLNITMLEDHEFVPLEVYTRHEIKDSGLLDYTEVQRRNQLHDLRFADIDTVIHADANAA。

[0153] SEQ ID No.2(B):

[0154] MHQGAPSWGRRWFVVWALLGLTLGVLVASAAPTSPGTPGVAAATQAANGGPATPAPPPLGAAPTGDPKPKKNKKPKNPTPPRPAGDNATVAAGHATLREHLRDIKAENTDANFYVCPPPTGATVVQFEQPRRCPTRPEGQNYTEGIAVVFKENIAPYKFKATMYYKDVTVSQVWFGHRYSQFMGIFEDRAPVPFEEVIDKINAKGVCRSTAKYVRNNLETTAFHRDDHETDMELKPANACTRTSRGWHTTDLKYNPSRVEAFHRYGTTVNCIVEEVDARSVYPYDEFVLATGDFVYMSPFYGYREGSHTEHTTYAADRFKQVDGFYARDLTTKARATAPTTRNLLTTPKFTVAWDWVPKRPSVCTMTKWQEVDEMLRSEYGGSFRFSSDAISTTFTTNLTEYPLSRVDLGDCIGKDARDAMDRIFARRYNATHIKVGQPQYYQANGGFLIAYQPLLSNTLAELYVREHLREQSRKPPNPTPPPPGASANASVERIKTTSSIEFARLQFTYNHIQRPVNDMLGRVAIAWCEEQNHELTLWNEARKLNPNAIASVTVGRRVSARMLGDVMAVSTCVPVAADNVIVQNSMRISSRPGACYSRPLVSFRYCDQGPLVEGQLGENNELRLTRDAIEPCTVGHRRYFTFGGGYVYFEEYAYSHQLSRADITTVSTFIDLNITMLEDHEFVPLEVYTRHEIKDSGLLDYTEVQRRNQLHDLRFADIDTVIHADANAA。

[0155] SEQ ID No.3(C):

[0156] MHQGAPSWGRRWFVVWALLGLTLGVLVASAAPTSPGTPGVAAATQAANGGPATPAPPPLGAAPTGDPKPKKNKKPKNPTPPRPAGDNATVAAGHATLREHLRDIKAENTDANFYVCPPPTGATVVQFEQPRRCPTRPEGQNYTEGIAVVFKENIAPYKFKATMYYKDVTVSQVWFGHRYSQFMGIFEDRAPVPFEEVIDKINAKGVCRSTAKYVRNNLETTAFHRDDHETDMELKPANACTRTSRGWHTTDLKYNPSRVEAFHRYGTTVNCIVEEVDARSVYPYDEFVLATGDFVYMSPFYGYREGSHTEHTTYAADRFKQVDGFYARDLTTKARATAPTTRNLLTTPKFTLAWDWVPKRPSVCTMTKWQEVDEMLRSEYGGSFRFSSDAISTTFTTNLTEYPLSRVDLGDCIGKDARDAMDRIFARRYNATHIKVGQPQYYQANGGFLIAYQPLLSNTLAELYVREHLREQSRKPPNPTPPPPGASANASVERIKTTSSIEFARLQFTYNHIQRLVNDMLGRVAIAWCEEQNHELTLWNCARKLNPNAIASVTVGRRVSARMLGDVMAVSTCVPVAADNVIVQNSMRISSRPGACYSRPLVSFRYEDQGPLVEGQLGENNELRLTRDAIEPCTVGHRRYFTFGGGYVYFEEYAYSHQLSRADITTVSTFIDLNITMLEDHEFVPLEVYTRHEIKDSGLLDYTEVQRRNQLHDLRFADIDTVIHADANAA。

[0157] SEQ ID No.4(D):

[0158] .

[0159] 2. Design of Nanoparticle Sequence

[0160] In order to improve the stability and integrity of the particles, ferritin or its mutant is linked to the above-mentioned Pre-F gB mutant protein to form a fusion protein.

[0161] Reference may be made to the definition and description of ferritin or its mutants and the method for preparing its nanoparticles in CN115850396A, which is incorporated herein by reference in its entirety as a part of the present application.

[0162] In a preferred embodiment, the ferritin sequence is:

[0163] SEQ ID No.5:

[0164] DIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPE HKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLA DQYVKGIAKSRK.

[0165] 2. Preparation of Ferritin-gB Fusion Protein

[0166] 1. Ferritin-gB gene fusion design

[0167] The Pre-F gB mutant protein and ferritin were fused via a linker (SGSGGGSG, SEQ ID No. 6) to prepare a ferritin-gB fusion protein, which included the Pre-F gB mutant protein, the linker and ferritin in sequence from the N-terminus to the C-terminus.

[0168] The ferritin-gB fusion proteins are represented by A-NP, B-NP, C-NP, and D-NP, respectively (hereinafter also referred to as fusion proteins A, B, C, and D, respectively), and their encoding gene sequences are shown in SEQ ID No.7-SEQ ID No.10, respectively.

[0169] SEQ ID No.7:

[0170]

[0171] SEQ ID No.8:

[0172]

[0173] SEQ ID No.9:

[0174]

[0175] SEQ ID No.10:

[0176]

[0177] 2) Construction of recombinant plasmid

[0178] The above four sequences were synthesized into the pKS001 vector plasmid (Zhongshan Kangtian Shenghe Biotechnology Co., Ltd., catalog number A14101) between Hind III and Not I by gene synthesis (synthesized, prepared and sequenced by Nanjing GenScript Biotechnology Co., Ltd.). Figure 1 shown.

[0179] 2. Expression of ferritin-gB fusion protein

[0180] The recombinant plasmids A, B, C, D, and E comprising SEQ ID Nos: 7-10 and 16 were electroporated and expressed in CHO K1Q cells (Kangsheng Biopharmaceutical Co., Ltd., catalog number A14101), and cell lines with high expression were screened.

[0181] The electroporation was performed using the EBXP-F1 electroporator from Suzhou Yida Biotechnology Co., Ltd. according to the voltage and other conditions recommended by the device (180 V, 4 times, 2000 μs pulse time, 611 ms interval, 1X10 7 Cell clone screening was carried out in the form of minipool. After the cells and plasmids were mixed and electroporated and plated, positive clones were screened by ELISA, and the culture was expanded from 96-well plates to 24-well plates, 6-well plates, T25 square bottles, and 125 shake flasks. Finally, the highest expression strain was selected through fed-batch culture in 125 shake flasks.

[0182] 3. Purification of Ferritin-gB Fusion Protein

[0183] The culture supernatant of the expressing cell line was purified by Capto Lentil Lectin (Cytiva, Catalog No.: 17548902), Q Sepharose FF (Cytiva, Catalog No.: 17051060), Capto Core 400 (Cytiva, Catalog No.: 17372402), and Superose 6prep grade (Cytiva, Catalog No.: 10321079).

[0184] The specific purification steps are as follows:

[0185] The selected cell supernatant culture fluid was centrifuged at 8000r / min for 20 minutes, filtered using a 0.45μm filter membrane (Jin Teng, catalog number: JTSF 025013 / 014), and the solution was about 100mL, and the balance solution was added to 200mL. The QFF column was equilibrated with the balance solution, and the sample was loaded with the A1 pump at a flow rate of 1.5mL / min. After the loading was completed, it was rinsed with the balance solution until the absorbance value returned to the value before loading and stabilized. The eluent (20mM Tris, 0.5M NaCl, pH8.5) was gradient eluted at a flow rate of 2mL / min, 0-100% B, 50min. The elution peak was collected. The supernatant was concentrated 5-10 times, passed through a Superose 6prep grade column at a flow rate of 1mL / min, and the sample of the absorption peak was collected. The purity was confirmed to be in line with expectations by SDS-PAGE.

[0186] For example, the reducing SDS-PAGE image of the purified fusion protein product was stained using 10% protein gel.

[0187] Results Figure 2 :

[0188] In the left figure, lane 1 is a molecular weight marker (Solarbio, catalog number PR1910), and lane 2 is the eluted product after purification of fusion protein A;

[0189] In the right figure, lane 1 from the left is a molecular weight marker (Solarbio, catalog number PR1910), and lane 2 from the left is the eluted product after purification of fusion proteins B, C, and D.

[0190] Example 3: Analysis of Nanoparticle Morphology of Ferritin-gB Fusion Protein

[0191] The purified products of fusion proteins A, B, C, and D prepared in Example 2 were photographed under an electron microscope. The specific operation is as follows:

[0192] Using PELCO easiGlow TM The copper grid with carbon film was discharged at 15 mA for 90 seconds, 3 μL of sample was dripped onto the grid, and excess liquid was removed with filter paper after 40 seconds. Finally, 3 μL of uranyl formate dye solution (0.75%) was taken with a pipette, dyed for 20 seconds, and dried. The grid was left for 2 minutes to dry.

[0193] The grids were observed using a Talos L120C electron microscope (Thermo Fisher Scientific, USA) with an operating voltage of 120 kV. Images were collected using a Ceta camera.

[0194] The results are as follows Figure 3 As shown, the results show that the purified product samples of fusion proteins A, B, C, and D have clear nanoparticle morphology and uniform protein morphology.

[0195] Example 4: Study on the immunogenicity of ferritin-gB fusion protein

[0196] 1. ELISA titer of antibodies in serum

[0197] Objective: To verify the effectiveness of this nanoparticle vaccine protein in animal experiments

[0198] Methods: 6-8 week old Balb / c mice (purchased from, catalog number) were selected and injected with 1 μg of the purified fusion proteins from the above four groups in each group of 10 mice. After two thigh muscle injections on days 0 and 21, mouse serum was collected on day 28 for ELISA analysis.

[0199] ELISA analysis used HSV 1 wild-type gB Protein (self-made, sequence source GenBank Seq ID: P06437.2, as shown in SEQ ID No. 15) as the coating protein, mouse serum as the primary antibody, and gradient dilution according to 100 times, 200 times, 400 times, 800 times, 1600 times, 3200 times, 6400 times, 12800 times, 25600 times, 51200 times, 102400 times, and 204800 times. The secondary antibody was mouse secondary antibody (Cell Signaling Technology, catalog number: 7076S), and the signal was read using an ELISA reader (Shanghai Kehua, catalog number: RD-SH-012). The titer test results of mouse serum after two immunizations in ELISA are shown in the following figure. Figure 4 (In the figure, fusion proteins A, B, C, D, and E are represented by A, B, C, D, and E, respectively), E corresponds to nanoparticles of the fusion protein formed by wild-type pre-F gB and ferritin.

[0200] The results showed that fusion proteins A and B had the highest ELISA titers, with mean values ​​of 32,000 and 35,840, respectively, with no significant difference between the two. Fusion proteins C and D had ELISA titers of 6,400 and 2,720, respectively, which were significantly different from fusion proteins A and B, but significantly higher than fusion protein E (mean value of 400).

[0201] 2. Mouse serum microfluorescence neutralization experiment

[0202] The virus titer of HSV virus type 1 strain (ATCC, VR-1789) cultured in EMEM medium with 10% bovine serum using Vero cells is 1.25E+06pfu / ml. 8 portions of mouse sera from each group were selected and diluted with PBS. Starting from 40-fold dilution, the serum was diluted to 29160 times according to a 3-fold dilution gradient, mixed with an equal volume of 400pfu virus solution, placed at 37°C for 1 hour, and 200ul per well was spread on the Vero cell plate. Three replicate wells were set for each mouse serum, and the serum was cultured at 37°C for 2 days to observe the cytopathic condition. For specific methods, please refer to the literature Development of a microneutralization assay for HSV-2.

[0203] The results are shown in Table 2.

[0204] Table 2: Results of mouse serum microfluorescence neutralization experiment

[0205] serial number Group Mean neutralization titer A Fusion Protein A 7317 B Fusion protein B 9857 C Fusion Protein C 3512 D Fusion protein D 1886 E Fusion protein E 227

[0206] The results show that:

[0207] The neutralizing protective antibody titers of fusion proteins A and B reached 7317 and 9857, respectively, with no significant difference between the two.

[0208] The neutralizing protective antibody titers of fusion proteins C and D reached 3512 and 1886, with no significant difference between the two, but there were significant differences with fusion proteins A and B.

[0209] A, B, C, and D in the present invention are significantly higher than 227 of the wild-type fusion protein E.

[0210] Example 5: Stability test of ferritin-gB fusion protein

[0211] In order to verify the stability of the ferritin-gB fusion protein prepared by the present invention, each group of purified ferritin-gB fusion proteins was subjected to a physical stability (physical environmental challenge) test, and the specific steps are as follows:

[0212] Five portions of 40 μg / μl ferritin-gB fusion protein solution and gradiently diluted ferritin-gB fusion protein solution were placed in pH 7.4 (25°C), pH 3.8 (25°C), pH 10 (25°C), 50°C (pH 7.4), and 70°C (pH 7.4) environments for 1 hour, and ELISA analysis was performed using the same method as cell clone screening.

[0213] The results are shown in Table 3, which show that the antigen binding activity of each group of fusion proteins after different pH treatments is maintained at more than 65% of the original untreated protein, and the antigen binding activity after temperature treatment, even at an extreme temperature of 70°C, can be maintained at more than 48% of the original untreated protein. This shows that the ferritin-gB fusion proteins prepared by the present invention have sufficient physical stability. The mutant fusion proteins A, B, C, and D are superior to the wild type (E) after pH and temperature treatment.

[0214] Table 3. ELISA signal intensity retention percentage after physical environment challenge

[0215]

[0216] Note: The results in the table are the average values ​​of 4 dilution gradients of 10, 100, 1000 and 10000 times.

[0217] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0218] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0219] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A Pre-F gB mutant protein, characterized in that The mutant protein is obtained by mutation of HSV-1 wild-type Pre-F gB protein, wherein the mutation includes replacement, deletion and / or insertion, and the mutation includes mutation at positions 161, 209, 240, 352, 516, 531, 541 and / or 607.

2. The mutant protein according to claim 1, characterized in that The mutation includes replacement, and the HSV-1 wild-type Pre-F gB protein is subjected to at least one of the following mutations 1)-8) to obtain a Pre-F gB mutant protein: 1) The alanine (A) at position 161 of the amino acid sequence of the Pre-F gB protein was mutated to cysteine ​​(C), i.e. A161C. 2) The serine (S) at position 209 of the amino acid sequence of the Pre-F gB protein was mutated to phenylalanine (F), i.e. S209F. 3) The alanine (A) at position 240 of the amino acid sequence of the Pre-F gB protein was mutated to cysteine ​​(C), i.e. A240C. 4) The valine (V) at position 352 of the amino acid sequence of the Pre-F gB protein was mutated to leucine (L), i.e., V352L. 5) The histidine (H) at position 516 of the amino acid sequence of the Pre-F gB protein was mutated to proline (P), i.e. H516P. 6) The leucine (L) at position 531 of the amino acid sequence of the Pre-F gB protein was mutated to glutamic acid (E), i.e., L531E. 7) The glutamic acid (E) at position 541 of the amino acid sequence of the Pre-F gB protein was mutated to cysteine ​​(C), i.e., E541C. 8) The glutamic acid (E) at position 607 of the amino acid sequence of the Pre-F gB protein was mutated to cysteine ​​(C), i.e., E607C.

3. The mutant protein according to any one of claims 1-2, characterized in that The mutations include: (1), A161C, S209F, V352L and E541C; (2), A240C, H516P, L531E and E607C; (3), A240C, V352L, L531E, and E541C; or, (4), A161C, S209F, L531E and E607C, Preferably, the amino acid sequence of the mutant protein comprises: (A1) any one of SEQ ID No. 1-4; (A2) a protein having the same function as (A1) obtained by replacing and / or deleting and / or adding one or more amino acid residues; (A3) a protein having 80% or more identity with any of (A1) to (A2) and having the same function, More preferably, the mutant protein may further include a signal peptide and / or a tag protein.

4. A fusion protein, characterized in that The fusion protein comprises the Pre-F gB mutant protein according to any one of claims 1 to 3 and ferritin.

5. The fusion protein according to claim 4, characterized in that The ferritin protein includes ferritin or a mutant thereof, Preferably, the amino acid sequence of the ferritin comprises: (B1) protein represented by SEQ ID No.5; (B2) a protein having the same function as (B1) obtained by replacing and / or deleting and / or adding one or more amino acid residues; (B3) a protein having 80% or more identity with any of (B1) to (B2) and having the same function, More preferably, the fusion protein further comprises a connecting peptide, a tag protein and / or a signal peptide.

6. A biomaterial, characterized in that The biological material comprises: I. A nucleic acid molecule encoding the Pre-F gB mutant protein according to any one of claims 1 to 3 or the fusion protein according to any one of claims 4 to 5; II. an expression cassette or vector comprising the nucleic acid molecule described in I; or III. A host cell, which contains the nucleic acid molecule described in I or the expression cassette or vector described in II.

7. The biomaterial according to claim 6, characterized in that The nucleic acid molecule comprises: (I-1), a DNA molecule shown at position 61 to 2709 or the full length of any sequence of SEQ ID No. 7 to 10; (I-2) or (I-1) which are complementary or degenerate sequences; (I-3) A DNA molecule having 75% or more identity with the DNA molecule defined in (I-1) or (I-2) and encoding the mutant protein or fusion protein.

8. A method for preparing the Pre-F gB mutant protein according to any one of claims 1 to 3 or the fusion protein according to any one of claims 4 to 5, characterized in that: The preparation method comprises: 1) constructing a recombinant expression vector containing a nucleic acid molecule encoding the mutant protein or fusion protein; 2) introducing the recombinant expression vector into a host cell to obtain a recombinant cell; 3) Cultivating the recombinant cells, and obtaining the mutant protein or fusion protein by separation and / or purification.

9. Use of the Pre-F gB mutant protein according to any one of claims 1 to 3, the fusion protein according to claims 4 to 5, or the biomaterial according to any one of claims 6 to 7, characterized in that: The applications include: (1) Use in the preparation of products for preventing and / or treating diseases caused by HSV virus infection; (2) Application in the preparation of products for inducing immune response to HSV virus antigens; (3) Use in the prevention and / or treatment of diseases caused by HSV virus infection; (4) Application in inducing immune response to HSV virus antigens.

10. A drug, characterized in that The drug comprises the Pre-F gB mutant protein according to any one of claims 1-3, the fusion protein according to claims 4-5, or the biomaterial according to any one of claims 6-7. Preferably, the drug is a vaccine. More preferably, the vaccine further comprises an adjuvant and / or a vaccine delivery system.

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