An HSV-2 nanoparticle vaccine, its preparation method and application

Abstract

This invention relates to an HSV-2 nanoparticle vaccine, its preparation method, and its application. The nanoparticles comprise HSV-2 PreF gB mutant protein and ferritin. After assembly, multiple PreF gB mutant proteins are concentrated and displayed on the surface, forming ferritin-PreF gB nanoparticles. The mutation of PreF gB enhances the stability and effective immunogenicity of the gB protein, and its display on the nanoparticle surface further enhances immunogenicity, effectively stimulating the body's cellular immune mechanism, thus becoming an effective vaccine for preventing HSV infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an HSV-2 type nanoparticle vaccine, its preparation method, and its application. Background Technology

[0002] Herpes simplex virus (HSV) is a common infectious virus that causes herpetic lesions on the skin and mucous membranes. HSV infection is very common in the population; data shows an infection rate of over 80%, indicating that most people have likely been or are currently infected with HSV. The high incidence of HSV is a significant public health concern.

[0003] HSV is mainly divided into two types: HSV-2 and HSV-2. HSV-2 primarily infects the genitals and surrounding areas, such as the vulva and labia. After infection, patients may develop small blisters or ulcers around the genitals. These rashes or ulcers are usually clustered and accompanied by significant pain or itching. In addition, HSV-2 can occasionally infect the area around the lips or nose, but this is relatively rare.

[0004] The gB protein is an important structural protein of herpes simplex virus (HSV). Located on the viral envelope, it plays a crucial role in viral entry into host cells, participating 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, and this pre-fusion state plays a key role in viral entry into the host cell. It binds to receptors on the host cell membrane, triggering a series of conformational changes that ultimately lead to the fusion of the viral envelope with the host cell membrane. This fusion process allows the viral genetic material to enter the host cell, initiating viral infection and replication. The fused gB protein is very stable, but before fusion, it is unstable.

[0005] Currently, the prevention and treatment of herpes simplex virus mainly involve avoiding sexual contact, antiviral therapy, and symptomatic treatment, but there is still a lack of effective preventive or therapeutic drugs. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides an HSV-2 nanoparticle vaccine, its preparation method, and its application. Specifically,

[0007] In a first aspect, the present invention provides a Pre-F gB mutant protein, the mutant protein being obtained by mutation of HSV-2 wild-type Pre-F gB protein, the mutation including substitution, deletion and / or insertion, the mutation including mutations at positions 204, 235, 217, 283, 513, 528, 604 and / or 611.

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

[0009] (1) The 235th, 513th, 528th and 611th positions;

[0010] (2) The 204th, 235th, 513th and 604th positions;

[0011] (3) The 204th, 217th, 283rd, and 611th positions; or,

[0012] (4) The 235th, 513th, 528th and 604th positions.

[0013] Preferably, the mutation includes substitution, wherein the wild-type HSV-2 Pre-F gB protein is mutated by at least one of the following 1)-8) to obtain the Pre-F gB mutant protein:

[0014] 1) Mutate serine (S) at position 204 of the Pre-F gB protein amino acid sequence to phenylalanine (F), i.e., S204F.

[0015] 2) Mutate alanine (A) at position 217 of the Pre-F gB protein amino acid sequence to cysteine ​​(C), i.e., A217C.

[0016] 3) Mutate alanine (A) at position 235 of the Pre-F gB protein amino acid sequence to cysteine ​​(C), i.e., A235C.

[0017] 4) Mutate valine (V) at position 283 of the Pre-F gB protein amino acid sequence to leucine (L), i.e., V283L.

[0018] 5) Mutate histidine (H) at position 513 of the Pre-F gB protein amino acid sequence to proline (P), i.e., H513P.

[0019] 6) Mutate leucine (L) at position 528 of the Pre-F gB protein amino acid sequence to glutamic acid (E), i.e., L528E.

[0020] 7) Mutate the glutamic acid (E) at position 604 of the Pre-F gB protein amino acid sequence to cysteine ​​(C), i.e., E604C.

[0021] 8) Mutate the glutamic acid (E) at position 611 of the Pre-F gB protein amino acid sequence to cysteine ​​(C), i.e., E611C.

[0022] More preferably, the mutation includes:

[0023] (1), A235C, H513P, L528E, E611C;

[0024] (2), S204F, A235C, H513P, E604C;

[0025] (3) S204F, A217C, V283L, E611C; or,

[0026] (4), A235C, H513P, L528E, E604C.

[0027] Further preferably, the amino acid sequence of the HSV-2 wild-type Pre-F gB protein is shown in GenBank Sequence ID: P08666.2, and the present invention includes positions 1-727 therein. This fragment contains the main extracellular region of the gB protein, with the transmembrane and intracellular regions deleted after position 727.

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

[0029] In one specific embodiment, the amino acid sequence of the mutant protein includes

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

[0031] (A2) A protein with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of (A1);

[0032] (A3) and any one of (A1)-(A2) have more than 80% identity and the same function.

[0033] More preferably, (A2) or (A3) retains any of the mutations in (1)-(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 comprising any of the Pre-FgB mutant proteins described above and ferritin.

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

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

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

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

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

[0041] It can be understood that the amino acid composition of wild-type ferritin may not be the same for different subtypes or strains of the virus. There may be mutations such as substitution, insertion or deletion. The mutation sites of various wild-type sequences correspond to the above-mentioned sites. The correspondence is understood to be a correspondence based on the amino acid structure and / or function analysis.

[0042] In one specific embodiment, the amino acid sequence of the ferritin includes:

[0043] (B1) The protein shown in SEQ ID No. 5;

[0044] (B2) A protein with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of (B1);

[0045] (B3) and any one of (B1)-(B2) have more than 80% identity and the same function.

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

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

[0048] The linker peptide can also be a variant of the above sequence, which has a linking function without affecting the function of the proteins at both ends, such as: GSGGGGGSG (SEQ ID No: 11), GS, SGGGSGGGGSGGGGSAGSGGGSGGGGSGGGSAGSGGGSGGGGSGGGSAGSGGGSGGGGSGGGSAGSGGGSGGGGSGGGSAGSGGGSGGGGSGGGSAGSGGGSGGGSGGGSSA (SEQ ID No: 12), SGSGSG (SEQ ID No: 13), or GGGGSGGGGSGGGG (SEQ ID No: 14), etc.

[0049] More preferably, the fusion protein comprises, from the N-terminus to the C-terminus, a Pre-F gB mutant protein, a linker, and ferritin.

[0050] Preferably, the fusion protein may further include a signal peptide and / or a tag protein.

[0051] The aforementioned tagged protein refers to a polypeptide or protein expressed by fusing it with a target protein using in vitro DNA recombination technology, in order to facilitate the expression, detection, tracing, and / or purification of the target protein. The tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0052] Any of the mutant or fusion proteins described above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0053] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, a search can be performed to calculate the identity of amino acid sequences, and then the identity value (%) can be obtained.

[0054] In this document, the 80% or more of identity can 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.

[0055] In a third aspect, the present invention provides a biomaterial comprising:

[0056] I. A nucleic acid molecule, wherein the nucleic acid molecule encodes any of the Pre-F gB mutant proteins or fusion proteins described above;

[0057] II. An expression cassette or vector, wherein the expression cassette or vector comprises the nucleic acid molecule described in I; or,

[0058] III. Host cell, wherein the host cell contains the nucleic acid molecule described in I or the expression cassette or vector described in II.

[0059] The mutant protein or fusion protein is defined as described above in this invention.

[0060] The nucleic acid molecule can be DNA, such as recombinant DNA; or it can be RNA, such as mRNA.

[0061] Preferably, the nucleic acid molecule comprises:

[0062] (I-1) DNA molecules containing any sequence of SEQ ID No. 7-10, positions 1-2181, or positions 1-2181 and 2206-2691, or the full length thereof;

[0063] Complementary or degenerate sequences of (I-2) and (I-1);

[0064] (I-3) A DNA molecule that has more than 75% identity with the DNA molecule defined in (I-1) or (I-2) and encodes the mutant or fusion protein.

[0065] Those skilled in the art can readily mutate the nucleotide sequences encoding the aforementioned proteins or fusion proteins using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that possess 75% or higher identity to the nucleotide sequences encoding the aforementioned mutant or fusion proteins, provided they encode the aforementioned mutant or fusion proteins and have the same function, are derived from and equivalent to the sequences of this invention.

[0066] The term "identity" refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher identity with the nucleotide sequence encoding the amino acid sequence shown in this invention. Identity can be evaluated visually or using 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.

[0067] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0068] In the aforementioned biological materials, the expression cassette refers to DNA capable of expressing the aforementioned protein or fusion protein in host cells. This DNA may include not only a promoter to initiate transcription of the gene sequence encoding the aforementioned protein or fusion protein, but also a terminator to terminate transcription of the gene sequence encoding the aforementioned protein or fusion protein. Furthermore, the expression cassette may also include an enhancer sequence.

[0069] The vectors described herein refer to vectors capable of delivering exogenous DNA or target genes into host cells for amplification and expression. These vectors can be cloning vectors or expression vectors, including but not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), Ti plasmids, and viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.). In one or more embodiments of this invention, the vector is the pUC57 vector and / or the pKS001 vector.

[0070] The microorganisms described herein may be bacteria, fungi, actinomycetes, protozoa, algae, or viruses. Specifically, the bacteria may originate from genera such as *Escherichia sp.*, *Erwinia sp.*, *Agrobacterium sp.*, *Flavobacterium sp.*, *Alcaligenes sp.*, *Pseudomonas sp.*, and *Bacillus sp.*, but are not limited to these. For example, the bacteria may be *Escherichia coli*, *Bacillus subtilis*, or *Bacillus pumilus*. In one or more embodiments of the present invention, the microorganisms are TOP10 competent cells.

[0071] The host cell (also called the recipient cell) described herein may be a plant cell or an animal cell. The term "host cell" can be understood not only to refer to a specific recipient cell, but also to the offspring of such a cell, which may not necessarily be identical to the original parent cell due to natural, accidental, or intentional mutations and / or alterations, but are still included within the scope of the host cell. Suitable host cells are those known in the art, including: plant cells such as Arabidopsis thaliana, tobacco (Nicotiana tabacum), maize (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum), etc., but not limited to these; animal cells such as mammalian cells (e.g., Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (Vero cells), young hamster kidney cells (BHK cells), mouse breast cancer cells (C127 cells), human embryonic kidney cells (HEK293 cells), human HeLa cells, fibroblasts, bone marrow cell lines, T cells or NK cells, etc.), avian cells (e.g., chicken or duck cells), amphibian cells (e.g., African clawed frog (Xenopus laevis) cells or giant salamander (Andrias davidianus) cells), fish cells (e.g., grass carp, carp, rainbow trout or catfish cells), insect cells (e.g., Sf21 cells or Sf-9 cells), etc., but not limited to these. In one or more embodiments of the present invention, the host cell is a CHO-K1Q cell.

[0072] In a fourth aspect, the present invention provides a method for preparing any of the above-mentioned Pre-F gB mutant proteins or fusion proteins, the preparation method comprising:

[0073] 1) Construct a recombinant expression vector containing a nucleic acid molecule encoding the mutant protein or fusion protein;

[0074] 2) The recombinant expression vector is introduced into host cells to obtain recombinant cells;

[0075] 3) Culture the recombinant cells and obtain the mutant protein or fusion protein by isolation and / or purification.

[0076] The aforementioned nucleic acid molecules, vectors, and host cells are as defined in the third aspect.

[0077] In one specific embodiment, the preparation method includes the following steps: introducing a nucleic acid molecule encoding the above-mentioned mutant protein or fusion protein into CHO K1Q cells to obtain recombinant cells; culturing the recombinant cells to obtain the mutant protein or fusion protein.

[0078] Furthermore, the nucleic acid molecules encoding the mutant or fusion protein are introduced into CHOK1Q cells via recombinant plasmids.

[0079] The recombinant plasmid is a plasmid obtained by inserting the nucleic acid molecule of the mutant protein or fusion protein into the vector plasmid.

[0080] In a specific embodiment of the present invention, the vector plasmid is the pKS001 vector plasmid.

[0081] 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 includes:

[0082] (1) Use in the preparation of products for the prevention and / or treatment of diseases caused by HSV virus infection;

[0083] (2) Application in the preparation of products for inducing an immune response to HSV virus antigens;

[0084] (3) Use in the prevention and / or treatment of diseases caused by HSV virus infection;

[0085] (4) Application in inducing an immune response to HSV virus antigens.

[0086] The products described in this article may be reagents or drugs, such as diagnostic reagents or vaccines.

[0087] Preferably, the preparation further includes screening, for example, using the mutant protein or fusion protein described above as a target to screen for preventive or therapeutic candidate drugs or diagnostic reagents.

[0088] Preferably, the infectious diseases caused by the HSV virus include infectious diseases caused by HSV-1 and / or HSV-2 viruses, more preferably HSV-2.

[0089] More preferably, the sites of infection for the infectious disease include the mouth, pharynx, nose, eyes and reproductive tract, skin, digestive tract, respiratory tract, central nervous system, etc.

[0090] In a sixth aspect, the present invention also provides a medicament comprising any of the above-described Pre-F gB mutant proteins, fusion proteins, or biological materials.

[0091] Preferably, the drug is a vaccine; more preferably, the vaccine further includes an adjuvant and / or a vaccine delivery system.

[0092] More preferably, the adjuvant may be a substance that can stimulate the body to produce a stronger humoral and / or cellular immune response against the antigen co-inoculated with it. The adjuvants described herein may be those known to those skilled in the art, including but not limited to: plant adjuvants (such as alkylamines, phenolic compounds, quinine, saponins, 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), cytokine and nucleic acid adjuvants (such as monocyte clone stimulating factor, leukocyte cytokines IL-1, IL-2, IL-4, IL-5, IL-6, IFN-γ, CpG motifs, nucleic acid carriers, etc.), and emulsion adjuvants (such as Freund's adjuvant). The adjuvant may be a pharmaceutically acceptable adjuvant.

[0093] As is well known to those skilled in the art, in order to enhance the immunogenicity of antigen proteins, in addition to adding compounds with immunomodulatory effects as adjuvants, gene combinations can be adjusted to express them into particulate structures; or they can be aggregated in vitro and encapsulated in liposomes or microspheres.

[0094] Preferably, the vaccine also includes a vaccine delivery system.

[0095] The vaccine delivery system described herein can be a substance capable of carrying antigens to the body's immune system, where they can be stored and exert their antigenic effects for an extended period. 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.

[0096] Furthermore, the drug or vaccine may also include one or more pharmaceutically acceptable carriers.

[0097] The pharmaceutically acceptable carrier may be a diluent, excipient, filler, binder, humectant, disintegrant, absorption enhancer, adsorbent, surfactant, or lubricant, but is not limited thereto.

[0098] The vaccine for preventing infection described in this invention may be an intramuscular liquid injection, an intravenous liquid injection, an intranasal liquid injection, an intradermal liquid injection, or a subcutaneous liquid injection.

[0099] In a seventh aspect, the present invention also provides a method for generating an immune response, the method comprising administering any of the above-described vaccines to a subject.

[0100] In the above method, administering the vaccine to the subject can induce an immune response against HSV in the subject. This immune response can be a cellular immune response, a humoral immune response, or a combination of both.

[0101] The cellular immune response may include B cell immune response and T cell immune response.

[0102] The subjects described in this article may be humans or non-human animals.

[0103] Furthermore, the non-human animal may be a non-human mammal.

[0104] The non-human mammal may be any one of the following, but is not limited to: mouse, rat, guinea pig, hamster, pig, dog, sheep, monkey, rabbit, cat, cow, horse.

[0105] The subjects mentioned in this article include, but are not limited to, healthy subjects, symptomatic infected subjects, asymptomatic infected subjects, or recovered subjects (subjects who have recovered from HSV infection).

[0106] The administration methods described herein include, but are not limited to, intramuscular injection, subcutaneous injection, intradermal injection, intravenous injection, arterial injection, intraperitoneal injection, microneedle injection, mucosal administration, oral administration, oral or nasal spray, or nebulized inhalation.

[0107] In an eighth aspect, the present invention also provides a method for preventing and / or treating infectious diseases caused by HSV, the method comprising administering the drug or vaccine to a subject.

[0108] Preferably, the infectious diseases caused by the HSV virus include infectious diseases caused by HSV-1 and / or HSV-2 viruses, more preferably HSV-2.

[0109] More preferably, the sites of infection for the infectious disease include the mouth, pharynx, nose, eyes and reproductive tract, skin, digestive tract, respiratory tract, central nervous system, etc.

[0110] In the above method, administration of the drug to the subject can induce an immune response against HSV in the subject. The immune response may be a cellular immune response, a humoral immune response, or a combination of both.

[0111] The cellular immune response may include B cell immune response and T cell immune response.

[0112] The subjects described in this article may be humans or non-human animals.

[0113] Furthermore, the non-human animal may be a non-human mammal.

[0114] The non-human mammal may be any one of the following, but is not limited to: mouse, rat, guinea pig, hamster, pig, dog, sheep, monkey, rabbit, cat, cow, horse.

[0115] The subjects mentioned in this article include, but are not limited to, healthy subjects, symptomatic infected subjects, asymptomatic infected subjects, or recovered subjects (subjects who have recovered after infection).

[0116] The administration methods described herein include, but are not limited to, intramuscular injection, subcutaneous injection, intradermal injection, intravenous injection, arterial injection, intraperitoneal injection, microneedle injection, mucosal administration, oral administration, oral or nasal spray, or nebulized inhalation.

[0117] It should be noted that any form of numbering in this invention, such as I, II, III, A, B, a, b, etc., is merely for the purpose of distinguishing each other and does not indicate a temporal or spatial order, unless otherwise stated.

[0118] In summary, compared with the prior art, the beneficial effects of the present invention are as follows:

[0119] This invention provides a method for vaccine preparation by centrally displaying a mutated pre-F (Pre-F) gB protein fragment of herpes simplex virus type 2. This invention enhances the stability and effective immunogenicity of the gB protein through mutation, and further enhances immunogenicity through display on the surface of nanoparticles.

[0120] This invention addresses the problem of poor stability of wild-type antigens, enabling them to induce the production of herpes simplex virus gB antibodies with good binding activity after entering the body, thereby providing the organism with corresponding immune protection. This invention achieves good immunogenicity at low doses by fusing the mutated HSV antigen with nanoparticles, and effectively stimulates the body's cellular immune mechanisms. Attached Figure Description

[0121] Figure 1 This is a schematic diagram of the pKS001 carrier.

[0122] Figure 2 The images show the elution products after purification of fusion proteins A, B, C, and D. The left image corresponds to the elution product after purification of fusion protein C, and the right image corresponds to the elution products after purification of fusion proteins A, B, and D.

[0123] Figure 3 Electron micrographs of fusion proteins A, B, C, D, and E.

[0124] Figure 4 The antibody titers produced after immunizing mice with fusion proteins A, B, C, D, and E are given. Detailed Implementation

[0125] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0126] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0127] Example 1: Design, preparation, and purification of a fusion protein of ferritin-HSV-2 pre-F gB mutant protein.

[0128] I. Design of a fusion protein of ferritin-HSV-2 pre-F gB mutant protein

[0129] The HSV-2 pre-F gB-related sequence was mutated and designed to obtain a pre-F gB mutant protein, which was then fused with a ferritin-related sequence to form a ferritin-pre-F gB-mutant protein (ferritin-gB) integrated subunit. Then, utilizing the self-assembly properties of ferritin, nanoparticles with good Pre-F gB antigen display capabilities were created. The specific steps are as follows:

[0130] 1. Design of pre-F gB mutant proteins

[0131] Objective: To design a stable HSV-2 pre-F gB mutant protein that can induce efficient antibody binding.

[0132] Methods: Design of pre-F gB mutant proteins

[0133] The wild-type gB was mutated. The wild-type gB can be found in positions 1-727 of GenBank Seq ID: P08666.2. The sequence of the wild-type gB is as follows:

[0134] MRGGGLICALVVGALVAAVASAAPAAPAAPRASGGVAATVAANGGPASRPPPVPSPATTKARKRKTKKPPKRPEATPPPDANATVAAGHATLRAHLREIKVENADAQFYVCPPPTGATVVQFEQPRRCPTRPEGQNYTEGIAVVFKENIAPYKFKATMYYKDVTVSQVWFGHRYSQFMGIFEDRAPVPFEEVIDKINTKGVCRSTAKYVRNNMETTAFHRDDHETDMELKPAKVATRTSRGWHTTDLKYNPSRVEAFHRYGTTVNCIVEEVDARSVYPYDEFVLATGDFVYMSPFYGYREGSHTEHTSYAADRFKQVDGFYARDLTTKARATSPTTRNLLTTPKFTVAWDWVPKRPAVCTMTKWQEVDEMLRAEYGGSFRFSSDAISTTFTTNLTEYSLSRVDLGDCIGRDAREAIDRMFARKYNATHIKVGQPQYYLATGGFLIAYQPLLSNTLAELYVREYMREQDRKPRNATPAPLREAPSANASVERIKTTSSIEFARLQFTYNHIQRHVNDMLGRIAVAWCELQNHELTLWNEARKLNPNAIASATVGRRVSARMLGDVMAVSTCVPVAPDNVIVQNSMRVSSRPGTCYSRPLVSFRYEDQGPLIEGQLGENNELRLTRDALEPCTVGHRRYFIFGGGYVYFEEYAYSHQLSRADVTTVSTFIDLNITMLEDHEFVPLEVYTRHEIKDSGLLDYTEVQRRNQLHDLRFADIDTVIRADANAA (SEQ ID No: 15);

[0135] The gene sequence after fusion with ferritin is:

[0136]

[0137] To display and stabilize desired epitopes, or to disrupt or mask unwanted epitopes, the wild-type Pre-FgB protein of HSV-2 is mutated by at least one of the following 1)-8) to obtain the Pre-F gB mutant protein:

[0138] 1) Mutate the serine (S) at position 204 of the Pre-F gB protein amino acid sequence to phenylalanine (F).

[0139] 2) Mutate alanine (A) at position 217 of the Pre-F gB protein amino acid sequence to cysteine ​​(C).

[0140] 3) Mutate alanine (A) at position 235 of the Pre-F gB protein amino acid sequence to cysteine ​​(C).

[0141] 4) Mutate valine (V) at position 283 of the Pre-F gB protein amino acid sequence to leucine (L).

[0142] 5) Mutate histidine (H) at position 513 of the Pre-F gB protein amino acid sequence to proline (P).

[0143] 6) Mutate the leucine (L) at position 528 of the Pre-F gB protein amino acid sequence to glutamic acid (E).

[0144] 7) Mutate the glutamic acid (E) at position 604 of the Pre-F gB protein amino acid sequence to cysteine ​​(C).

[0145] 8) Mutate the glutamic acid (E) at position 611 of the Pre-F gB protein amino acid sequence to cysteine ​​(C).

[0146] In the preferred embodiment, mutations are performed at certain sites to stabilize the pre-fusion (Pre-F) gB conformation. The mutation sites of mutant proteins A, B, C, and D (corresponding to SEQ ID Nos. 1-4, respectively) are shown in Table 1.

[0147] Table 1: Mutation sites of pre-F gB mutant proteins

[0148]

[0149] Its specific structure is as follows:

[0150] SEQ ID No. 1:

[0151] MRGGGLICALVVGALVAAVASAAPAAPAAPRASGGVAATVAANGGPASRPPPVPSPATTKARKRKTKKPPKRPEATPPPDANATVAAGHATLRAHLREIKVENADAQFYVCPPPTGATVVQFEQPRRCPTRPEGQNYTEGIAVVFKENIAPYKFKATMYYKDVTVSQVWFGHRYSQFMGIFEDRAPVPFEEVIDKINTKGVCRSTAKYVRNNMETTAFHRDDHETDMELKPAKVCTRTSRGWHTTDLKYNPSRVEAFHRYGTTVNCIVEEVDARSVYPYDEFVLATGDFVYMSPFYGYREGSHTEHTSYAADRFKQVDGFYARDLTTKARATSPTTRNLLTTPKFTVAWDWVPKRPAVCTMTKWQEVDEMLRAEYGGSFRFSSDAISTTFTTNLTEYSLSRVDLGDCIGRDAREAIDRMFARKYNATHIKVGQPQYYLATGGFLIAYQPLLSNTLAELYVREYMREQDRKPRNATPAPLREAPSANASVERIKTTSSIEFARLQFTYNHIQRPVNDMLGRIAVAWCEEQNHELTLWNEARKLNPNAIASATVGRRVSARMLGDVMAVSTCVPVAPDNVIVQNSMRVSSRPGTCYSRPLVSFRYEDQGPLICGQLGENNELRLTRDALEPCTVGHRRYFIFGGGYVYFEEYAYSHQLSRADVTTVSTFIDLNITMLEDHEFVPLEVYTRHEIKDSGLLDYTEVQRRNQLHDLRFADIDTVIRADANAA。

[0152] SEQ ID No.2:

[0153] MRGGGLICALVVGALVAAVASAAPAAPAAPRASGGVAATVAANGGPASRPPPVPSPATTKARKRKTKKPPKRPEATPPPDANATVAAGHATLRAHLREIKVENADAQFYVCPPPTGATVVQFEQPRRCPTRPEGQNYTEGIAVVFKENIAPYKFKATMYYKDVTVSQVWFGHRYSQFMGIFEDRAPVPFEEVIDKINTKGVCRFTAKYVRNNMETTAFHRDDHETDMELKPAKVCTRTSRGWHTTDLKYNPSRVEAFHRYGTTVNCIVEEVDARSVYPYDEFVLATGDFVYMSPFYGYREGSHTEHTSYAADRFKQVDGFYARDLTTKARATSPTTRNLLTTPKFTVAWDWVPKRPAVCTMTKWQEVDEMLRAEYGGSFRFSSDAISTTFTTNLTEYSLSRVDLGDCIGRDAREAIDRMFARKYNATHIKVGQPQYYLATGGFLIAYQPLLSNTLAELYVREYMREQDRKPRNATPAPLREAPSANASVERIKTTSSIEFARLQFTYNHIQRPVNDMLGRIAVAWCELQNHELTLWNEARKLNPNAIASATVGRRVSARMLGDVMAVSTCVPVAPDNVIVQNSMRVSSRPGTCYSRPLVSFRYCDQGPLIEGQLGENNELRLTRDALEPCTVGHRRYFIFGGGYVYFEEYAYSHQLSRADVTTVSTFIDLNITMLEDHEFVPLEVYTRHEIKDSGLLDYTEVQRRNQLHDLRFADIDTVIRADANAA。

[0154] SEQ ID No.3:

[0155] MRGGGLICALVVGALVAAVASAAPAAPAAPRASGGVAATVAANGGPASRPPPVPSPATTKARKRKTKKPPKRPEATPPPDANATVAAGHATLRAHLREIKVENADAQFYVCPPPTGATVVQFEQPRRCPTRPEGQNYTEGIAVVFKENIAPYKFKATMYYKDVTVSQVWFGHRYSQFMGIFEDRAPVPFEEVIDKINTKGVCRFTAKYVRNNMETTCFHRDDHETDMELKPAKVATRTSRGWHTTDLKYNPSRVEAFHRYGTTVNCIVEEVDARSVYPYDEFLLATGDFVYMSPFYGYREGSHTEHTSYAADRFKQVDGFYARDLTTKARATSPTTRNLLTTPKFTVAWDWVPKRPAVCTMTKWQEVDEMLRAEYGGSFRFSSDAISTTFTTNLTEYSLSRVDLGDCIGRDAREAIDRMFARKYNATHIKVGQPQYYLATGGFLIAYQPLLSNTLAELYVREYMREQDRKPRNATPAPLREAPSANASVERIKTTSSIEFARLQFTYNHIQRHVNDMLGRIAVAWCELQNHELTLWNEARKLNPNAIASATVGRRVSARMLGDVMAVSTCVPVAPDNVIVQNSMRVSSRPGTCYSRPLVSFRYEDQGPLICGQLGENNELRLTRDALEPCTVGHRRYFIFGGGYVYFEEYAYSHQLSRADVTTVSTFIDLNITMLEDHEFVPLEVYTRHEIKDSGLLDYTEVQRRNQLHDLRFADIDTVIRADANAA。

[0156] SEQ ID No.4:

[0157] .

[0158] 2. Design of nanoparticle sequences

[0159] To improve the stability and integrity of the particles, ferritin or its mutants are linked with the aforementioned Pre-F gB mutant protein to form a fusion protein.

[0160] The definition and description of ferritin or its mutants and the preparation method of its nanoparticles in CN115850396A can be referenced. This document is incorporated herein by reference in its entirety as part of this application.

[0161] In a preferred embodiment, the ferritin sequence is:

[0162] SEQ ID No. 5:

[0163] DIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRK.

[0164] II. Preparation of Ferritin-gB Fusion Protein

[0165] 1. Design of Ferritin-gB gene fusion

[0166] Ferritin-gB fusion protein was prepared by fusing Pre-F gB mutant protein and ferritin through a linker (SGSGGGSG, SEQ ID No. 6). The ferritin-gB fusion protein includes Pre-F gB mutant protein, linker and ferritin from N-terminus to C-terminus.

[0167] The ferritin-Pre-F gB fusion proteins are represented by A-NP, B-NP, C-NP, and D-NP (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.

[0168] SEQ ID No. 7:

[0169]

[0170] SEQ ID No.8:

[0171]

[0172] SEQ ID No.9:

[0173]

[0174] SEQ ID No.10:

[0175]

[0176] 2) Construction of recombinant plasmids

[0177] The above four sequences were synthesized and prepared by Nanjing GenScript Biotech Co., Ltd., and sequenced for detection. They were then inserted between Hind III and Not I of the pKS001 vector plasmid (Zhongshan Kangtianshenghe Biotechnology Co., Ltd., catalog number A14101). The pKS001 vector plasmid is shown below. Figure 1 As shown.

[0178] 2. Expression of ferritin-gB fusion protein

[0179] The recombinant plasmids A, B, C, D, and E, which respectively include 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.

[0180] Using CHO K1Q cells (catalog number A14101) provided by Kang Sheng Company, and an EBXP-F1 electroporator from Yida Company, electroporation was performed according to the recommended voltage and other conditions (180V, 4 cycles, 2000μs pulse time, 611ms interval, 1X10). 7 Cell clone screening was performed using a minipool method. After electroporation and plasmid coating, positive clones were screened by ELISA. The cells were expanded from 96-well plates to 24-well plates, 6-well plates, T25 square flasks, and T25 shake flasks. Finally, the highest expression lines were selected by fed-batch culture in T25 shake flasks.

[0181] III. Purification of Ferritin-gB Fusion Protein

[0182] The supernatant of the expression cell line culture medium was purified using Capto Lentil Lectin (Cytiva, catalog number: 17548902), Q Sepharose FF (Cytiva, catalog number: 17051060), Capto Core 400 (Cytiva, catalog number: 17372402), and Superose 6 prep grade (Cytiva, catalog number: 10321079).

[0183] The specific purification steps are as follows: Centrifuge the selected cell supernatant culture medium at 8000 rpm for 20 minutes, filter using a 0.45 μm filter membrane (Jinteng, catalog number: JTSF 025013 / 014), obtaining approximately 100 mL of solution. Add equilibration buffer to a final volume of 200 mL. Equilibrate the QFF column with equilibration buffer, and load the sample using an A1 pump at a flow rate of 1.5 mL / min. After loading, rinse with equilibration buffer until the absorbance returns to and stabilizes at the pre-loading value. Elute with a gradient of elution buffer (20 mM Tris, 0.5 M NaCl, pH 8.5) at a flow rate of 2 mL / min, 0-100% B, for 50 min. Collect the elution peak. Concentrate the supernatant 5-10 times, and pass it through a Superose 6-prep grade column at a flow rate of 1 mL / min. Collect the sample with the absorption peak, and confirm the purity meets expectations using SDS-PAGE.

[0184] For example, the reduced SDS-PAGE image of the purified fusion protein product was stained with 10% protein gel.

[0185] See results Figure 2 :

[0186] In the left image, lane 1 on the left is the molecular weight marker (Solarbio, catalog number PR1910), and lane 2 on the left is the elution product of the purified fusion protein C.

[0187] In the right figure: lanes 1, 2, and 3 on the left are the purified products of fusion proteins A, B, and D, respectively, and lane 4 on the left is the molecular weight marker (Solarbio, catalog number PR1910).

[0188] Example 3: Nanoparticle morphology analysis of ferritin-gB fusion protein

[0189] The purified products of fusion proteins A, B, C, D, and E prepared in Example 2 were subjected to electron microscopy. The specific procedures are as follows:

[0190] Using PELCO easiGlow™, a copper grid with a carbon film was discharged at 15 mA for 90 seconds. 3 µL of sample was then added to the grid. After 40 seconds, excess liquid was aspirated with filter paper. Finally, 3 µL of 0.75% uranium formate staining solution was pipetted on and stained for 20 seconds. The stain was then aspirated dry. The grid was allowed to dry for 2 minutes.

[0191] The grid was observed using a Talos L120C electron microscope (Thermo Fisher Scientific, USA), operating at 120 kV. Images were collected using a Ceta camera.

[0192] The results are as follows Figure 3As shown in Figures A, B, C, and D, these correspond to fusion proteins A, B, C, and D, respectively. The results indicate that the purified samples of fusion proteins A, B, C, D, and E exhibit clear nanoparticle morphology and uniform protein morphology. Figure E corresponds to the nanoparticles formed by the fusion protein of wild-type pre-F gB and ferritin; however, the nanoparticle morphology and protein morphology of this fusion protein are less uniform than those of the aforementioned fusion proteins A, B, C, and D.

[0193] Example 4: Immunogenicity study of ferritin-gB fusion protein

[0194] 1. ELISA titer of antibodies in serum

[0195] Objective: To verify the effectiveness of this nanoparticle vaccine protein in animal experiments.

[0196] Methods: Balb / c mice aged 6-8 weeks (purchased from [source], catalog number [number]) were selected, with 10 mice per group. Each group was injected with 1 μg of the purified protein from the above 4 groups. The mice were injected into the thigh muscles twice on day 0 and day 21. On day 28, mouse serum was collected for ELISA analysis.

[0197] ELISA analysis used HSV 2 (strain 333) Glycoprotein B Protein (Sinochem, catalog number 40935-V08B) as the coating protein, and mouse serum as the primary antibody, serially diluted at 100-fold, 200-fold, 400-fold, 800-fold, 1600-fold, 3200-fold, 6400-fold, 12800-fold, 25600-fold, 51200-fold, 102400-fold, and 204800-fold. The secondary antibody was mouse secondary antibody (Cell Signaling Technology, catalog number: 7076S). Signal readings were performed using an ELISA reader (Shanghai Kehua, catalog number: RD-SH-012). The titer results of mouse serum after two immunizations in ELISA are shown below. Figure 4 As shown in the figure (fusion proteins A, B, C, D, and E are represented by A, B, C, D, and E respectively), E corresponds to the nanoparticles of the fusion protein formed by wild-type pre-F gB and ferritin.

[0198] The results showed that proteins C and D had the highest ELISA titers, with mean values ​​of 21,760 and 32,000, respectively, with no significant difference between them. In contrast, fusion proteins A and B had ELISA titers of 3,520 and 4,000, respectively, which were significantly different from fusion proteins C and D, and also significantly different from fusion protein E (mean value of 1,600).

[0199] 2. Mouse serum micro-fluorescence neutralization test

[0200] The viral titer of HSV-2 strain (ATCC, VR-540) cultured in EMEM medium containing 10% bovine serum using Vero cells was 1.92E+06 pfu / ml. Eight serum aliquots from each of the above groups of mice were selected and diluted with PBS. Starting with a 40-fold dilution, the cells were serially diluted 3-fold to a final volume of 29-160-fold. Each diluted aliquot was mixed with an equal volume of 400 pfu of viral solution, incubated at 37°C for 1 hour, and 200 μL was seeded into each well of a Hep-2 cell plate. Three replicates were prepared for each mouse serum sample. Cellular activity was observed at 37°C for 2 days. Specific methods can be found in the literature "Development of a microneutralization assay for HSV-2".

[0201] The results are shown in Table 2. The results show that the fusion proteins A, B, C and D in this invention all exceed the wild-type E. Among them, the neutralizing protective antibody titers of fusion proteins D and C reached 12705 and 9587, respectively, and the neutralizing protective antibody titers of fusion proteins B and A reached 2731 and 1853, respectively, which are all significantly higher than the 643 of the wild-type fusion protein E.

[0202] Table 2: Results of mouse serum micro-fluorescence neutralization experiment

[0203]

[0204] Example 5: Stability test of ferritin-gB fusion protein

[0205] To verify the stability of the ferritin-gB fusion protein prepared in this invention, the purified ferritin-gB fusion proteins were subjected to a physical stability (physical environmental challenge) test. The specific steps are as follows:

[0206] Five aliquots of the above 40 μg / μl ferritin-gB fusion protein solution and serially diluted ferritin-gB fusion protein solutions were placed in environments of pH 7.4 (25℃), pH 3.8 (25℃), pH 10 (25℃), 50℃ (pH 7.4), and 70℃ (pH 7.4) for 1 hour, respectively, and analyzed by ELISA using the same method as cell clone screening.

[0207] The results showed that the antigen-binding activity of each fusion protein remained above 75% of that of the original untreated protein after treatment with different pH and temperature conditions. This indicates that the ferritin-gB fusion proteins prepared in this invention possess sufficient physical stability.

[0208] Table 3 shows the stability results of ferritin-gB protein. It can be observed that the mutant fusion proteins A, B, C, and D are superior to the wild type (E) after pH and temperature treatment.

[0209] Table 3: Percentage of ELISA signal strength retained after physical environmental challenges

[0210]

[0211] Note: The results in the table are the average values ​​of four dilution gradients: 10, 100, 1000, and 10000 times.

[0212] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0213] 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 describe the various possible combinations separately.

[0214] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A Pre-F gB mutant protein, characterized in that, The amino acid sequence of the mutant protein is any one of SEQ ID No. 1, 2, or 4.

2. A fusion protein, characterized in that, The fusion protein comprises, from N-terminus to C-terminus, the Pre-F gB mutant protein of claim 1, a linker peptide, and ferritin, wherein the linker peptide has a linking function without affecting the function of the proteins at both ends.

3. The fusion protein according to claim 2, characterized in that, The amino acid sequence of the ferritin is shown in SEQ ID No.

5.

4. The fusion protein according to any one of claims 2-3, characterized in that, The fusion protein also includes a tag protein and / or a signal peptide.

5. A biomaterial, characterized in that, The biomaterials include: I. A nucleic acid molecule, wherein the nucleic acid molecule encodes the Pre-F gB mutant protein of claim 1 or the fusion protein of any one of claims 2-4; II. An expression cassette or vector, wherein the expression cassette or vector comprises the nucleic acid molecule described in I; or, III. Host cell, wherein the host cell contains the nucleic acid molecule described in I or the expression cassette or vector described in II.

6. The biomaterial according to claim 5, characterized in that, The nucleic acid molecules include: (I-1), the DNA molecule shown in positions 1-2181 of any sequence of SEQ ID No. 7, 8, or 10, or positions 1-2181 and 2206-2691, or the full length; Complementary or degenerate sequences of (I-2) and (I-1); (I-3) A DNA molecule that has more than 75% identity with the DNA molecule defined in (I-1) or (I-2) and encodes the mutant or fusion protein.

7. A method for preparing the Pre-F gB mutant protein of claim 1 or the fusion protein of any one of claims 2-4, characterized in that, The preparation method includes: 1) Construct a recombinant expression vector containing a nucleic acid molecule encoding the mutant protein or fusion protein; 2) The recombinant expression vector is introduced into host cells to obtain recombinant cells; 3) Culture the recombinant cells and obtain the mutant protein or fusion protein by isolation and / or purification.

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

9. A drug, characterized in that, The drug comprises the Pre-F gB mutant protein of claim 1, the fusion protein of claims 2-4, or any of the biological materials of claims 5-6.

10. The medicament according to claim 9, characterized in that, The drug in question is a vaccine.

11. The medicament according to claim 10, characterized in that, The vaccine also includes adjuvants and / or a vaccine delivery system.

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