CN117551677A

CN117551677A - Monkey poxvirus specific fusion protein vaccine using adenovirus type 5 as vector

Info

Publication number: CN117551677A
Application number: CN202311512262.1A
Authority: CN
Inventors: 秦玉寅; 琚姝; 王爱霞; 王蒙; 程玲俐; 赵毅; 郭玉姗; 刘鑫鑫
Original assignee: Shanghai Shangyao Kangxinuo Biopharmaceutical Co ltd
Current assignee: Shanghai Shangyao Kangxinuo Biopharmaceutical Co ltd
Priority date: 2023-11-14
Filing date: 2023-11-14
Publication date: 2024-02-13

Abstract

The invention provides a monkey pox virus specific fusion protein vaccine using adenovirus type 5 as a vector. Specifically, the invention provides an A35R-M1R fusion protein expression cassette, a vector containing the expression cassette and a monkey pox virus vaccine prepared by using the vector. The preparation method of the monkey pox virus vaccine is quick, simple and convenient, has good immunogenicity, can induce organisms to generate antibodies in a short time, and is very suitable for coping with sudden epidemic situations.

Description

Monkey poxvirus specific fusion protein vaccine using adenovirus type 5 as vector

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a recombinant monkey pox virus vaccine and application thereof in preventing monkey pox virus infection.

Background

Monkey pox virus (MPXV), also known as smallpox virus, belongs to the same group of orthopoxviruses as smallpox virus and vaccinia virus, is a zoonotic virus, and originates from and varies with the tropical rain forest of africa, and is classified into two types of viruses, i.e., midafrica and western africa, depending on the virulence and hazard levels of the virus. The monkey pox virus has a size of 200-300nm, an outer membrane with a size of 30nm is arranged on the periphery, the inner core is a homogeneous core body, and the virus can be independently replicated.

Smallpox virus and monkey pox virus have been considered two of the most potential pathogens for bioterrorism. Smallpox virus has been eradicated worldwide in the 70-80 s due to the world's large scale use of safe and inexpensive vaccines. The monkey poxvirus has poor human-to-human transmission and a lower mortality rate than smallpox virus, but its host population is broad (rodents), resulting in the risk of being genetically modified to a higher strain, so that following smallpox virus, the monkey poxvirus also becomes an important poxvirus infectious disease.

Monkey pox is a self-limiting disease, the main symptoms often including fever, severe headache, muscle soreness, back pain, energy deficit, lymphadenectasis and rash or skin lesions, where the symptoms of the lymphadenopathy are a significant feature of monkey pox. Symptoms usually last for two to four weeks, and most cases disappear by themselves without treatment. But for some people these symptoms may lead to complications or even death. Neonates, children and people with immunodeficiency based diseases may be at more serious symptoms and risk of dying from monkey pox. Complications of severe monkey pox cases include skin infections, pneumonia, confusion, and ocular infections that can lead to vision loss. About 3-6% of cases currently reported in endemic countries lead to death.

There are two forms of poxviruses: mature Virions (MV) and lipid membranes (extracellular enveloped, EV), EV have an envelope from the endoplasmic reticulum membrane that breaks down upon contact with cells, exposing the outer membrane to direct fusion with the cell membrane, followed by downstream infection. And MV can be directly fused with cell membranes, at least 12 MVs participate in fusion, and can enter cells through macropolytics endocytosis, and the outer membrane and endocytosis membrane are fused in endocytosis to release an endocardial-wrapped virus core. Replication is carried out in a porous inner membrane, after which the pores are closed, and the virus particles are subjected to the next maturation to form a typical poxvirus dumbbell shape, and the mark MV is formally formed. The final completed virion can leave the cell in two ways: the most predominant one is cell lysis, MV direct release. The other is to use a secretory transport system of cells to secrete the virus out of the cells through the Golgi apparatus, which can cause the virus to additionally acquire a layer of envelope to generate EV.

A35R is an envelope component of an intracellular and extracellular envelope virion (IEV) and EEV, homologous to chordopoxvirus A33R. A33R in vaccinia virus plays a critical role in the transmission of virus particles among cells, is an important target for vaccine development, and researches show that A35R is a potential target for monkey pox virus serological detection.

M1R is homologous to vaccinia virus L1 protein, a transmembrane protein found on the surface of mature IMV particles. Encoded by the L1R ORF, is highly conserved, is involved in virion assembly, and plays an important role in viral entry and maturation. L1 is necessary to induce low pH triggered cell-cell fusion and is critical for vaccinia virus replication.

Based on the current spread situation of the monkey pox virus, there is an urgent need in the art to develop specific vaccines against the monkey pox virus to cope with the possible future epidemic situation of the monkey pox virus.

Disclosure of Invention

The invention aims to provide a monkey pox virus vaccine which selects one protein from two infectious virus particles of a monkey pox virus as an antigen target, namely M1R protein of intracellular virus particles and A35R protein of extracellular envelope virus. The invention provides a nucleotide for optimizing fusion protein genes by covalent linkage of extracellular domain for encoding A35R and complete M1R, which takes replication defective human adenovirus 5 with combined deletion of E1 and E3 as a vector, HEK293 cells integrating adenovirus gene E1 as a packaging cell line, and a novel monkey pox virus vaccine in the form of recombinant adenovirus vector is obtained through packaging.

In a first aspect of the invention there is provided an a35R-M1R fusion protein expression cassette comprising the following elements: a Kozak sequence, an optional signal peptide coding sequence, an extracellular domain coding sequence for a35R, and an M1R full-length protein coding sequence;

wherein the amino acid sequence of the extracellular domain of A35R is shown as SEQ ID NO. 1, or has at least 90% sequence identity with the amino acid sequence shown as SEQ ID NO. 1; the amino acid sequence of the M1R full-length protein is shown as SEQ ID NO. 2, or has at least 90% sequence identity with the amino acid sequence shown as SEQ ID NO. 2.

In another preferred embodiment, the expression cassette has the structure of formula I from 5 'to 3':

Z1-Z2-Z3-Z4-Z5 (formula I)

Wherein,

each "-" is independently a bond or a nucleotide linking sequence;

z1 is a Kozak sequence;

z2 is an optional signal peptide coding sequence;

z3 is the extracellular domain coding sequence of A35R;

z4 is the coding sequence of a connecting peptide (linker);

z5 is the coding sequence of the full-length protein of M1R.

In another preferred embodiment, the signal peptide is selected from the group consisting of: a tissue plasminogen activator (tPA) signal peptide or a mutant thereof.

In another preferred embodiment, the amino acid sequence of the signal peptide is shown in SEQ ID NO. 6 or 7;

Preferably, the amino acid sequence of the tPA signal peptide is shown in SEQ ID NO. 7.

In another preferred embodiment, the extracellular domain coding sequence of A35R is a codon optimized coding sequence having a nucleotide sequence as set forth in SEQ ID NO. 3 or having more than 90% (preferably more than 95%, more preferably more than 96%, more than 97%, more than 98% or more than 99%) sequence identity thereto.

In another preferred embodiment, the full-length M1R protein coding sequence is a codon-optimized coding sequence having a nucleotide sequence as set forth in SEQ ID NO. 4 that has greater than 90% (preferably greater than 95%, more preferably greater than 96%, 97%, 98% or 99%) sequence identity thereto.

In another preferred embodiment, the amino acid sequence of the linker peptide is as shown in GGGSGGGGSGG (SEQ ID NO: 5).

In another preferred embodiment, the length of each nucleotide linkage sequence is 0-30nt, preferably 1-15nt.

In another preferred embodiment, the nucleotide sequence of the expression cassette comprises: (a) A nucleotide sequence as set forth in SEQ ID NO 9 or 10, (b) a nucleotide sequence having 90% or more (preferably 95% or more, more preferably 96% or more, 97% or more, 98% or more, or 99% or more) sequence identity with the nucleotide sequence set forth in SEQ ID NO 9 or 10; and (c) a nucleotide sequence complementary to the nucleotide sequence of (a) or (b).

In another preferred embodiment, the nucleotide sequence comprises a DNA sequence, a cDNA sequence, or an mRNA sequence.

In another preferred embodiment, the nucleotide sequence includes a single-stranded sequence and a double-stranded sequence.

In a second aspect of the invention there is provided a vector comprising an A35R-M1R fusion protein expression cassette according to the first aspect of the invention.

In another preferred embodiment, the vector comprises one or more promoters operably linked to the expression cassette, an enhancer, a transcription termination signal, an origin of replication, a selectable marker, a nucleic acid restriction site, and/or a homologous recombination site.

In another preferred embodiment, the vector comprises a plasmid, a viral vector.

In another preferred embodiment, the vector is a plasmid; preferably, the plasmid is a viral packaging system plasmid for the production of virus-like particles.

In another preferred embodiment, the plasmid is a shuttle plasmid of the AdMax adenovirus system.

In another preferred embodiment, the vector is a viral vector.

In another preferred embodiment, the viral vector is selected from the group consisting of: an adenovirus vector, a herpes simplex virus vector, an adeno-associated virus vector (AAV), or a combination thereof; preferably, the viral vector is an adenovirus vector.

In another preferred embodiment, the adenovirus vector is selected from the group consisting of: replication-defective human adenovirus type 5 vector, chimpanzee adenovirus vector.

In a third aspect of the invention there is provided a host cell comprising a vector according to the second aspect of the invention, or a chromosome incorporating an A35R and M1R fusion protein expression cassette according to the first aspect of the invention.

In another preferred embodiment, the host cell is a mammalian cell, including human and non-human mammals.

In another preferred embodiment, the host cell is a HEK293 cell.

In another preferred embodiment, the host cell is used to produce a virus-like particle.

In a fourth aspect of the invention, there is provided a vaccine composition comprising:

(i) A vector according to the second aspect of the invention, a host cell according to the third aspect of the invention or a virus-like particle produced by expression thereof; and

(ii) A vaccine acceptable carrier.

In another preferred embodiment, the vaccine composition is for preventing infection by a monkey poxvirus.

In another preferred embodiment, said component (i) comprises 0.1 to 99.9wt%, preferably 10 to 80wt%, more preferably 30 to 60wt% of the total weight of the vaccine composition.

In another preferred embodiment, the vaccine acceptable carrier can aid in the storage of the viral particles and administration to a subject, including solvents, dispersion media, coatings, antibacterial or antifungal agents, isotonic and absorption delaying agents, and the like.

In another preferred embodiment, the vaccine acceptable carrier may comprise any suitable component, such as, but not limited to, saline.

In another preferred embodiment, the brine includes, but is not limited to: buffered saline, physiological saline, phosphate buffer, citrate buffer, acetate buffer, bicarbonate buffer, sucrose solution, saline solution, and polysorbate solution.

In another preferred embodiment, the vaccine composition further comprises an adjuvant.

In another preferred embodiment, the adjuvant comprises: an inorganic adjuvant or an organic adjuvant.

In another preferred embodiment, the inorganic adjuvant is selected from the group consisting of: aluminum adjuvants (e.g., aluminum hydroxide, aluminum phosphate), alum, or combinations thereof.

In another preferred embodiment, the organic adjuvant is selected from the group consisting of: microorganisms and their products such as mycobacteria (tubercle bacillus, bacillus calmette guerin), bacillus pumilus, pertussis, endotoxins, bacterial extracts (muramyl dipeptide), or combinations thereof.

In another preferred embodiment, the adjuvant further comprises a synthetic adjuvant.

In another preferred embodiment, the synthetic adjuvant is selected from the group consisting of: artificially synthesized double-stranded polynucleotides (double-stranded polyadenylation, uridylic acid), levamisole, isoprinosine, or combinations thereof.

In another preferred embodiment, the adjuvant further comprises an oil.

In another preferred embodiment, the oil is selected from the group consisting of: freund's adjuvant, peanut oil emulsifying adjuvant, mineral oil, vegetable oil, or combinations thereof.

In another preferred embodiment, the adjuvant is selected from the group consisting of: calcium phosphate, hydroxyapatite, aluminum hydroxide, SDA7749, SDA GEL701, SDA 15A, YT108, or a combination thereof.

In another preferred embodiment, the vaccine composition may be a bivalent vaccine or a multi-vaccine.

In another preferred embodiment, the vaccine composition comprises: injection, nasal drop or spray.

In a fifth aspect of the invention there is provided the use of a vector as described in the second aspect of the invention or a host cell as described in the third aspect of the invention in the manufacture of a vaccine composition for the prevention of infection by a monkey poxvirus.

In a sixth aspect of the invention, there is provided a method of preparing a vaccine composition comprising the steps of:

(1) Culturing a host cell according to the third aspect of the invention under conditions suitable for expression, thereby expressing the corresponding virus-like particle;

(2) The obtained virus-like particles are mixed with a vaccine-acceptable carrier, thereby obtaining the vaccine composition.

In another preferred embodiment, the step (1) includes the steps of:

the shuttle plasmid of the adenovirus packaging system containing the expression cassette of the first aspect of the invention and the backbone plasmid of the adenovirus packaging system are simultaneously introduced into a suitable host cell by transfection to produce a recombinant adenovirus vector, thereby obtaining a packaged virus-like particle.

In another preferred embodiment, the host cell comprises: HEK293 cells.

In a seventh aspect of the invention, there is provided a method of preventing infection by a monkey poxvirus comprising the steps of: administering to a subject in need thereof an effective amount of a vector according to the second aspect of the invention, a host cell according to the third aspect of the invention, or a vaccine composition according to the fourth aspect of the invention.

In another preferred embodiment, the subject is a mammal, including but not limited to a human, non-human primate, sheep, dog, cat, horse, cow, chicken, rat, mouse, etc.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows a plasmid map of pDC316-ori 35-M1R.

FIG. 2 shows a plasmid map of pDC 316-S35R-M1R.

FIG. 3 shows a plasmid map of pDC 316-P-S35-M1R.

FIG. 4 shows Western blot results of transfected cells with each plasmid detected using the A35R antibody.

FIG. 5 shows Western blot results of transfected cells with each plasmid detected using M1R antibody.

FIG. 6 shows Western blot results of transfected cells with each plasmid detected using the β -actin antibodies.

FIG. 7 shows Western blot results of individual recombinant adenovirus-infected cells detected using the A35R antibodies.

FIG. 8 shows Western blot results of individual recombinant adenovirus-infected cells detected using M1R antibodies.

FIG. 9 shows Western blot results of individual recombinant adenovirus-infected cells detected using β -actin antibodies.

Figure 10 shows IgG antibody titers against a35R protein in serum of immunized mice of different dose groups. Wherein A shows the antibody titer in serum of the Ad5-P-S35-M1R immunized mice of the different dose groups, and B shows the antibody titer in serum of the Ad5-S35-M1R immunized mice of the different dose groups.

Figure 11 shows IgG antibody titers against M1R protein in serum of immunized mice of different dose groups. Wherein A shows the antibody titer in serum of the Ad5-P-S35-M1R immunized mice of the different dose groups, and B shows the antibody titer in serum of the Ad5-S35-M1R immunized mice of the different dose groups.

Figure 12 shows IgG antibody titers against a35R protein in serum of immunized mice of different dose groups 9 days and 14 days after immunization. Wherein A shows the antibody titer in serum of the Ad5-P-S35-M1R immunized mice of the different dose groups, and B shows the antibody titer in serum of the Ad5-S35-M1R immunized mice of the different dose groups.

Figure 13 shows IgG antibody titers against M1R protein in serum of immunized mice of different dose groups 9 days and 14 days after immunization. Wherein A shows the antibody titer in serum of the Ad5-P-S35-M1R immunized mice of the different dose groups, and B shows the antibody titer in serum of the Ad5-S35-M1R immunized mice of the different dose groups.

FIG. 14 shows the comparison of IgG antibody titers against the A35R protein in serum of Ad5-P-S35-M1R immunized mice and Ad5-S35-M1R immunized mice at 9 and 14 days post-immunization. Wherein a shows the comparison of antibody titers 9 days after immunization and B shows the comparison of antibody titers 14 days after immunization.

FIG. 15 shows the comparison of IgG antibody titers against M1R protein in serum of Ad5-P-S35-M1R immunized mice and Ad5-S35-M1R immunized mice 9 and 14 days after immunization. Wherein a shows the comparison of antibody titers 9 days after immunization and B shows the comparison of antibody titers 14 days after immunization.

Detailed Description

The invention provides a vaccine composition of monkey pox virus specific fusion protein by taking a human type 5 replication defective adenovirus vector as a vector for the first time through extensive and intensive researches, wherein the vaccine composition combines a replication defective human type 5 adenovirus rAD virus strain with a monkey pox virus A35 protein and an M1 protein of a nucleic acid optimization sequence or an immunogenic derivative thereof to construct a recombinant replication defective type 5 adenovirus, and the recombinant replication defective adenovirus is used for preventing infection of the monkey pox virus. On this basis, the present invention has been completed.

Terminology

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

As used herein, the term "polynucleotide" refers to a chain compound formed by the polymerization of nucleotides.

As used herein, the term "vaccine" refers to a biologic made against infection by a variety of pathogenic microorganisms for the prevention or treatment of vaccination. Vaccines are broadly classified into attenuated live vaccines, inactivated vaccines, recombinant protein vaccines, vector vaccines, nucleic acid vaccines, and the like.

Polynucleotide design method of the present invention

The design method of the polynucleotide can be used for preparing vector vaccines or nucleic acid vaccines. The polynucleotide constructed based on the design method can realize the in-situ expression of antigen genes so as to induce the occurrence of immune response.

In a specific embodiment, the invention discloses a method for designing the polynucleotide, which comprises the steps of connecting an extracellular domain of an antigen protein A35R from a monkey poxvirus strain and a coding sequence of an M1R full-length protein together so as to express the polynucleotide in the form of an A35R-M1R fusion protein. Further, in order to express both A35R and M1R proteins at the same time, and the expression of the two proteins does not affect each other, a linker peptide (linker) was designed to link the A35R and M1R proteins together. In order to further increase the expression level of the fusion protein, the invention adds a signal peptide coding sequence on the basis of the coding sequence of the fusion protein. In addition, the coding sequence of the antigen protein is subjected to codon optimization so as to further improve the expression level of the fusion protein.

In a specific embodiment, the amino acid sequence of the extracellular domain of A35R is shown as SEQ ID NO. 1 or a sequence having more than 90% identity thereto. Based on the above amino acid sequences, a codon optimized extracellular domain coding sequence of a35R can be obtained. It will be appreciated by those skilled in the art that the extracellular domain protein of A35R should include variant forms, fragments, derivatives or analogues thereof.

In specific embodiments, the amino acid sequence of the M1R full-length protein is shown as SEQ ID NO. 2 or a sequence having more than 90% identity thereto. Based on the amino acid sequences described above, codon optimized M1R full-length protein coding sequences can be obtained. It will be appreciated by those skilled in the art that the M1R full-length protein should include variants, fragments, derivatives or analogues thereof.

The terms "variant," "fragment," "derivative" or "analog" as used herein refer to a polypeptide that substantially retains the biological function or activity of the original protein, but has a small difference in amino acid sequence from the original protein. The variant, fragment, derivative or analogue of the extracellular domain/M1R full-length protein of SA35R according to the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence. Such variants, fragments, derivatives or analogs are within the purview of one skilled in the art, as defined herein.

In specific embodiments, the polynucleotides (alternatively referred to as A35R-M1R fusion protein expression cassettes) designed based on the above methods include a Kozak sequence, a signal peptide coding sequence, a codon-optimized A35R ectodomain coding sequence (having the nucleotide sequence set forth in SEQ ID NO: 3), a linker peptide (linker), a codon-optimized M1R full-length protein coding sequence (having the nucleotide sequence set forth in SEQ ID NO: 4).

As used herein, the term "signal peptide" refers to a short peptide chain that directs the transfer of a newly synthesized protein to the secretory pathway. In a specific embodiment, the coding sequence of the signal peptide is located 5' to the coding sequence of the antigen of interest. It will be appreciated by those skilled in the art that the use of any signal peptide that effects secretory transfer of a protein falls within the methods of designing polynucleotides described herein and are intended to fall within the scope of the invention.

In a preferred embodiment of the invention, the signal peptide is a tissue plasminogen activator (tPA) signal peptide, preferably a mutant tPA signal peptide, obtained by mutating proline (P) at position 22 to alanine (A) on a wild-type signal peptide sequence as shown in SEQ ID NO. 6, having the amino acid sequence as shown in SEQ ID NO. 7.

In a specific embodiment, the Kozak sequence is directly linked to the 5' end of the signal peptide coding sequence, prior to the start codon.

In a preferred embodiment of the invention, the polynucleotide has a sequence as shown in SEQ ID NO. 9 or 10; most preferably, the polynucleotide has the sequence shown in SEQ ID NO. 10

As used herein, the term "antigen" refers to a protein molecule that is capable of inducing an immune response in an organism and obtaining immune protection. In a specific embodiment, the antigen is encoded by the polynucleotide sequence described above.

In particular embodiments, the polynucleotides are packaged and delivered using an adenovirus (e.g., replication defective human adenovirus type 5) as a vector as described previously.

Recombinant adenovirus vectors of the invention

In the present invention, there is provided a recombinant adenovirus vector whose genome comprises a polynucleotide designed based on the methods disclosed herein. The recombinant adenovirus vector is produced by utilizing the DNA plasmid constructed by the invention, and can be used for preparing vaccines. In a specific embodiment, the recombinant adenovirus vector is a replication defective human adenovirus type 5 vector.

As used herein, the term "DNA plasmid" refers to a circular DNA molecule that loads and expresses a foreign gene of interest. In a specific embodiment, the present invention utilizes molecular cloning techniques to construct DNA plasmids comprising the polynucleotides described above, wherein the polynucleotides are artificial synthesis products. It is reasonable to expect from the prior art that the DNA plasmids according to the present invention can be used directly as vaccines, i.e. DNA vaccines.

As used herein, the term "DNA vaccine" refers to a recombinant eukaryotic expression vector encoding a protein antigen that, when injected directly into an animal, can express foreign genes in vivo, producing antigens that activate the immune system of the body, thereby inducing specific humoral and cellular immune responses. It will be appreciated by those skilled in the art that any DNA plasmid comprising a polynucleotide designed based on the methods disclosed herein that can be used as an effective DNA vaccine would fall within the scope of the present invention.

In particular embodiments, the DNA plasmid may be introduced directly into a subject and express an antigen of interest, wherein known methods of introduction include, but are not limited to, intramuscular injection, intradermal gene gun, intradermal injection, intranasal administration, intramuscular or intradermal electroporation, intravenous injection, and the like. As used herein, the term "subject" includes any human or non-human mammal, such as a non-human primate, sheep, dog, cat, horse, cow, chicken, rat, mouse, and the like.

In particular embodiments, the immune effect of the DNA vaccine may be improved by the addition of an adjuvant. Such adjuvants include, but are not limited to, aluminum salt adjuvants, cytokine adjuvants, nucleic acid adjuvants, lipid-containing adjuvants, or oil-water mixed adjuvants, among others.

In a specific embodiment, the DNA plasmids of the invention are used in the production of recombinant adenovirus vector vaccines.

As used herein, the term "adenovirus" is a double stranded DNA virus whose genome is about 25-45kb in size and which has inverted terminal repeats (Inverted terminal repeat, ITR) on both sides.

As used herein, the term "recombinant adenovirus" refers to an adenovirus in which the natural genomic portion between ITRs is replaced by an artificially synthesized DNA sequence.

As used herein, the term "vector" refers to a molecular tool that transports, transduces, and expresses an exogenous gene of interest (e.g., an antigen of interest to which the present invention relates) contained in a target cell.

In a specific embodiment of the present invention, the term "recombinant adenovirus vector of the invention" refers to a recombinant adenovirus vector in which the replication defective human adenovirus type 5 ITR has been replaced by a portion of the adenovirus genome that is replaced by a polynucleotide (A35R-M1R fusion protein expression cassette) of the design of the invention.

As used herein, the term "replication defective human adenovirus type 5" refers to a modified adenovirus whose E1 and E3 genes have been removed and which still has the ability to infect cells, while the essential genes for the production of new viral particles or virions are no longer present. Replication-defective human recombinant adenoviruses have become a model system widely used in the fields of eukaryotic gene expression analysis, vaccine research, gene therapy, and the like. The use of recombinant adenoviruses to deliver genetic material to host cells has many advantages. Recombinant adenoviruses can be used both in vivo and in vitro to transduce dividing and resting cells in a variety of mammals, particularly humans. In addition, recombinant adenoviruses can be used to transduce many types of sensitive cells.

The recombinant adenovirus vector of the invention can be prepared by the following method: the recombinant adenovirus vector is produced by transfecting an adenovirus packaging system shuttle plasmid comprising the expression cassette of the first aspect of the invention with an adenovirus packaging system backbone plasmid and simultaneously introducing the plasmid into a suitable host cell. In a specific embodiment of the invention, the recombinant adenovirus vectors of the invention are prepared using an AdMax adenovirus packaging system.

The AdMax adenovirus packaging system includes shuttle plasmid pDC316 for carrying exogenous genes, and backbone plasmids pbhglox_e1,3Cre. The AdMax adenovirus packaging system utilizes the Cre/loxP recombinase system to obtain recombinant viral particles in HEK 293 cells. Specifically, the adenovirus shuttle plasmid inserted with the exogenous gene and the skeleton plasmid carrying most of adenovirus genome are transfected into 293 cells together, and recombined under the action of Cre recombinase to obtain recombinant virus particles.

Monkey poxvirus vaccine of the invention

In the present invention, a monkey poxvirus vaccine is provided, which is a recombinant adenovirus vector vaccine. The monkey poxvirus vaccine of the present invention is prepared based on polynucleotides designed by the methods of the present disclosure.

In a specific embodiment, the invention provides a monkey poxvirus vaccine composition comprising a vector according to the second aspect of the invention, a host cell according to the third aspect of the invention or virus-like particles resulting from expression thereof. In a preferred embodiment of the invention, the monkey poxvirus vaccine composition comprises a recombinant adenovirus vector of the invention, together with a vaccine acceptable carrier.

As used herein, the term "vaccine-acceptable carrier" refers to "pharmaceutically acceptable carrier" and refers to any and all pharmaceutical carriers, such as solvents, dispersion media, coatings, antibacterial or antifungal agents, isotonic and absorption delaying agents, and the like.

These vectors can aid in the storage of the viral particles and administration to a subject. The pharmaceutically acceptable carrier may include any suitable component, such as, but not limited to, saline. Illustrative examples of saline include, but are not limited to, buffered saline, physiological saline, phosphate buffer, citrate buffer, acetate buffer, bicarbonate buffer, sucrose solution, saline solution, and polysorbate solution.

In certain embodiments, the monkey poxvirus vaccine composition may further comprise additives including, but not limited to, stabilizers, preservatives, transfection promoters to aid in cellular uptake, or adjuvants to enhance immunogenicity. Suitable stabilizers include, but are not limited to, sodium glutamate, glycine, EDTA, and albumin (e.g., human serum albumin). Suitable preservatives include, but are not limited to, 2-phenoxyethanol, sodium benzoate, potassium sorbate, methylparaben, phenol, thimerosal, and antibiotics. Suitable transfection facilitating agents include, but are not limited to, calcium ions. Suitable adjuvants include, but are not limited to, aluminum salt adjuvants, cytokine adjuvants, nucleic acid adjuvants, lipid-containing adjuvants or oil-water mixed adjuvants, and the like.

In certain embodiments, the monkey poxvirus vaccine of the invention may be prepared as an injection, nasal drops or spray.

Use and method for preventing infection of monkey poxvirus

The invention also relates to a method for vaccinating a subject against a monkey pox virus infection using the polynucleotide, DNA plasmid or recombinant adenovirus vector described above for the preparation of a monkey pox virus vaccine.

In particular embodiments, the above-described monkey poxvirus vaccine may be vaccinated by any suitable method known in the art including, but not limited to, intramuscular, intradermal, intranasal, or administration via the portal vein.

The beneficial technical effects of the invention include:

(1) The invention provides a recombinant adenovirus for simultaneously expressing the A35R and M1R proteins of the monkey pox virus as a monkey pox virus vaccine for the first time.

(2) The monkey pox virus vaccine has good immunogenicity on a mouse model, and can induce organisms to generate antibodies in a short time. The vaccine has good immunoprotection effect on the monkey pox virus.

(3) The preparation method of the monkey pox virus vaccine is quick, simple and convenient, and can realize mass production in a short period for coping with sudden epidemic situations.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

Example 1: optimization and synthesis of A35R and M1R fusion proteins

The target antigen of the recombinant monkey poxvirus vaccine is the extracellular domain of A35R and the complete M1R protein sequence of the monkey poxvirus strain (strain Zaine-96-1-16, genebank accession number: NC_ 003310.1). By optimizing the A35R and M1R protein genes, the expression level of the A35R and M1R proteins is improved, so that the immunogenicity of the vaccine is improved.

First, the uniprot site was logged on to monkey poxvirus A35R (Genebank number: NP-536572.1) to obtain the extracellular domain of A35R (Arg 58-Thr 181) having the protein sequence shown in SEQ ID NO:1. The full-length protein sequence of M1R (Genebank number: NP-536507.1) was found on the uniprot website and is shown in SEQ ID NO:2. Secondly, the extracellular domain of A35R and M1R are codon optimized by using biological software, and specific gene sequences are shown in SEQ ID NO. 3 and SEQ ID NO. 4. In order to express A35R and M1R proteins simultaneously, and the expression of the two proteins does not affect each other, the invention designs a connecting peptide (linker) for connecting the A35R and M1R proteins together, and the protein sequence of the connecting peptide is shown in SEQ ID NO. 5. In order to further improve the expression level of the fusion protein, the invention increases a tissue type plasminogen activator (tPA) signal peptide sequence based on the fusion protein, and the protein sequence is shown in SEQ ID NO. 6. The 22 nd amino acid of human tissue type plasminogen activator (tPA) signal peptide is mutated from proline (P) to alanine (tPA 22P/A), and the protein sequence is shown in SEQ ID NO. 7, so that the expression and secretion level of target protein can be further improved.

To compare whether a fusion protein increased the tPA signal peptide with a site-directed mutation to the tPA signal peptide could increase the expression level of the fusion protein, 3 fusion protein designs were performed on the fusion protein, with the fusion protein sequence without the tPA signal peptide being designated ori35-M1R (SEQ ID NO: 8), the fusion protein sequence with the tPA signal peptide being designated S35-M1R (SEQ ID NO: 9), and the fusion protein sequence with the site-directed mutation tPA signal peptide being designated P-S35-M1R (SEQ ID NO: 10). After the fusion protein design was completed, kozaq sequence was added before the translation initiation codon, and the cleavage site EcoRI was inserted upstream and the cleavage site SalI was inserted downstream of the entire sequence, and the gene sequence was synthesized, and the full-length gene synthesis was performed by Jiangsu Safeo technologies, inc., and loaded into pUC18 vector.

Extracellular domain of A35R (SEQ ID NO: 1):

RQNQCMSANEAAITDSAVAVAAASSTHRKVASSTTQYDHKESCNGLYYQGSCYILHSDYKSFE DAKANCAAESSTLPNKSDVLTTWLIDYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCT

M1R full-length protein (SEQ ID NO: 2):

MGAAASIQTTVNTLSERISSKLEQEANASAQTKCDIEIGNFYIRQNHGCNITVKNMCSADADAQLDAVLSAATETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENYVKQTCNSSAVVDNKLKIQNVIIDECYGAPGSPTNLEFINTGSSKGNCAIKALMQLTTKATTQIAPRQVAGTGVQFYMIVIGVI ILAALFMYYAKRMLFTSTNDKIKLILANKENVHWTTYMDTFFRTSPMIIATTDIQN

codon optimized extracellular domain coding sequence of A35R (SEQ ID NO: 3):

AGACAGAACCAGTGCATGAGCGCCAACGAGGCCGCCATCACAGATTCTGCTGTGGCTGTGGCC GCTGCCAGCAGCACACATAGAAAGGTGGCCAGCTCCACCACACAGTACGACCACAAAGAGAGCTGCAACGGCCTGTACTACCAGGGCAGCTGCTACATCCTGCACAGCGACTACAAGAGCTTCGAGGACGCCAAGGCCAATTGTGCCGCCGAGAGTAGCACCCTGCCTAACAAGTCTGACGTGCTGACCACCTGGCTGATCGACTACGTGGAAGATACCTGGGGCAGCGACGGCAACCCTATCACCAAGACCACCAGCGATTACCAGGACAGCGACGTGTCCCAAGAAGTGCGCAAGTACTTCTGTACA

codon-optimized M1R full-length protein coding sequence (SEQ ID NO: 4):

GGCGCTGCCGCCAGCATCCAGACCACAGTGAATACCCTGAGCGAGCGGATCAGCAGCAAGCTGGAACAAGAGGCCAATGCCAGCGCTCAGACCAAGTGCGATATCGAGATCGGCAACTTCTACATCCGGCAGAACCACGGCTGCAACATCACCGTGAAGAACATGTGCAGCGCCGACGCCGATGCTCAGCTGGATGCAGTTCTGTCTGCCGCCACCGAGACATACTCTGGCCTGACACCTGAGCAGAAAGCCTACGTGCCCGCCATGTTTACCGCCGCTCTGAATATCCAGACCTCCGTGAACACCGTCGTGCGGGACTTCGAGAACTACGTGAAGCAGACCTGCAACAGCAGCGCCGTGGTGGACAACAAGCTGAAGATCCAGAACGTGATCATCGACGAGTGCTATGGCGCCCCTGGCAGCCCTACCAATCTCGAGTTTATCAACACCGGCAGCTCCAAGGGCAACTGCGCCATTAAGGCTCTGATGCAGCTGACCACTAAGGCCACCACTCAGATCGCCCCTAGACAGGTTGCCGGAACCGGCGTGCAGTTCTACATGATCGTGATCGGAGTGATCATCCTGGCCGCTCTGTTCATGTACTACGCCAAGCGGATGCTGTTCACCAGCACCAACGACAAGATCAAGCTGATCCTGGCCAACAAAGAAAACGTGCACTGGACGACCTACATGGACACATTCTTCCGGACAAGCCCCATGATCATTGCCACCACCGACATCCAGAAC

connecting peptide (SEQ ID NO: 5):

GGGSGGGGSGG

tPA signal peptide wild-type (SEQ ID NO: 6):

MDAMKRGLCCVLLLCGAVFVSP

tPA signal peptide mutant (SEQ ID NO: 7):

MDAMKRGLCCVLLLCGAVFVSA

ori35-M1R(SEQ ID NO:8)

GCCACCATGAGACAGAACCAGTGCATGAGCGCCAACGAGGCCGCCATCACAGATTCTGCTGTGGCTGTGGCCGCTGCCAGCAGCACACATAGAAAGGTGGCCAGCTCCACCACACAGTACGACCACAAAGAGAGCTGCAACGGCCTGTACTACCAGGGCAGCTGCTACATCCTGCACAGCGACTACAAGAGCTTCGAGGACGCCAAGGCCAATTGTGCCGCCGAGAGTAGCACCCTGCCTAACAAGTCTGACGTGCTGACCACCTGGCTGATCGACTACGTGGAAGATACCTGGGGCAGCGACGGCAACCCTATCACCAAGACCACCAGCGATTACCAGGACAGCGACGTGTCCCAAGAAGTGCGCAAGTACTTCTGTACAGGCGGCGGCAGCGGCGGCGGCGGCTCCGGCGGCGGCGCTGCCGCCAGCATCCAGACCACAGTGAATACCCTGAGCGAGCGGATCAGCAGCAAGCTGGAACAAGAGGCCAATGCCAGCGCTCAGACCAAGTGCGATATCGAGATCGGCAACTTCTACATCCGGCAGAACCACGGCTGCAACATCACCGTGAAGAACATGTGCAGCGCCGACGCCGATGCTCAGCTGGATGCAGTTCTGTCTGCCGCCACCGAGACATACTCTGGCCTGACACCTGAGCAGAAAGCCTACGTGCCCGCCATGTTTACCGCCGCTCTGAATATCCAGACCTCCGTGAACACCGTCGTGCGGGACTTCGAGAACTACGTGAAGCAGACCTGCAACAGCAGCGCCGTGGTGGACAACAAGCTGAAGATCCAGAACGTGATCATCGACGAGTGCTATGGCGCCCCTGGCAGCCCTACCAATCTCGAGTTTATCAACACCGGCAGCTCCAAGGGCAACTGCGCCATTAAGGCTCTGATGCAGCTGACCACTAAGGCCACCACTCAGATCGCCCCTAGACAGGTTGCCGGAACCGGCGTGCAGTTCTACATGATCGTGATCGGAGTGATCATCCTGGCCGCTCTGTTCATGTACTACGCCAAGCGGATGCTGTTCACCAGCACCAACGACAAGATCAAGCTGATCCTGGCCAACAAAGAAAACGTGCACTGGACGACCTACATGGACACATTCTTCCGGACAAGCCCCATGATCATTGCCACCACCGACATCCAGAACTGA

S35-M1R(SEQ ID NO:9)

GCCACCATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCCCAGACAGAACCAGTGCATGAGCGCCAACGAGGCCGCCATCACAGATTCTGCTGTGGCTGTGGCCGCTGCCAGCAGCACACATAGAAAGGTGGCCAGCTCCACCACACAGTACGACCACAAAGAGAGCTGCAACGGCCTGTACTACCAGGGCAGCTGCTACATCCTGCACAGCGACTACAAGAGCTTCGAGGACGCCAAGGCCAATTGTGCCGCCGAGAGTAGCACCCTGCCTAACAAGTCTGACGTGCTGACCACCTGGCTGATCGACTACGTGGAAGATACCTGGGGCAGCGACGGCAACCCTATCACCAAGACCACCAGCGATTACCAGGACAGCGACGTGTCCCAAGAAGTGCGCAAGTACTTCTGTACAGGCGGCGGCAGCGGCGGCGGCGGCTCCGGCGGCGGCGCTGCCGCCAGCATCCAGACCACAGTGAATACCCTGAGCGAGCGGATCAGCAGCAAGCTGGAACAAGAGGCCAATGCCAGCGCTCAGACCAAGTGCGATATCGAGATCGGCAACTTCTACATCCGGCAGAACCACGGCTGCAACATCACCGTGAAGAACATGTGCAGCGCCGACGCCGATGCTCAGCTGGATGCAGTTCTGTCTGCCGCCACCGAGACATACTCTGGCCTGACACCTGAGCAGAAAGCCTACGTGCCCGCCATGTTTACCGCCGCTCTGAATATCCAGACCTCCGTGAACACCGTCGTGCGGGACTTCGAGAACTACGTGAAGCAGACCTGCAACAGCAGCGCCGTGGTGGACAACAAGCTGAAGATCCAGAACGTGATCATCGACGAGTGCTATGGCGCCCCTGGCAGCCCTACCAATCTCGAGTTTATCAACACCGGCAGCTCCAAGGGCAACTGCGCCATTAAGGCTCTGATGCAGCTGACCACTAAGGCCACCACTCAGATCGCCCCTAGACAGGTTGCCGGAACCGGCGTGCAGTTCTACATGATCGTGATCGGAGTGATCATCCTGGCCGCTCTGTTCATGTACTACGCCAAGCGGATGCTGTTCACCAGCACCAACGACAAGATCAAGCTGATCCTGGCCAACAAAGAAAACGTGCACTGGACGACCTACATGGACACATTCTTCCGGACAAGCCCCATGATCATTGCCACCACCGACATCCAGAACTGA

P-S35-M1R(SEQ ID NO:10)

GCCACCATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGGCCAGACAGAACCAGTGCATGAGCGCCAACGAGGCCGCCATCACAGATTCTGCTGTGGCTGTGGCCGC TGCCAGCAGCACACATAGAAAGGTGGCCAGCTCCACCACACAGTACGACCACAAAGAGAGCTGCAACGGCCTGTACTACCAGGGCAGCTGCTACATCCTGCACAGCGACTACAAGAGCTTCGAGGACGCCAAGGCCAATTGTGCCGCCGAGAGTAGCACCCTGCCTAACAAGTCTGACGTGCTGACCACCTGGCTGATCGACTACGTGGAAGATACCTGGGGCAGCGACGGCAACCCTATCACCAAGACCACCAGCGATTACCAGGACAGCGACGTGTCCCAAGAAGTGCGCAAGTACTTCTGTACAGGCGGCGGCAGCGGCGGCGGCGGCTCCGGCGGCGGCGCTGCCGCCAGCATCCAGACCACAGTGAATACCCTGAGCGAGCGGATCAGCAGCAAGCTGGAACAAGAGGCCAATGCCAGCGCTCAGACCAAGTGCGATATCGAGATCGGCAACTTCTACATCCGGCAGAACCACGGCTGCAACATCACCGTGAAGAACATGTGCAGCGCCGACGCCGATGCTCAGCTGGATGCAGTTCTGTCTGCCGCCACCGAGACATACTCTGGCCTGACACCTGAGCAGAAAGCCTACGTGCCCGCCATGTTTACCGCCGCTCTGAATATCCAGACCTCCGTGAACACCGTCGTGCGGGACTTCGAGAACTACGTGAAGCAGACCTGCAACAGCAGCGCCGTGGTGGACAACAAGCTGAAGATCCAGAACGTGATCATCGACGAGTGCTATGGCGCCCCTGGCAGCCCTACCAATCTCGAGTTTATCAACACCGGCAGCTCCAAGGGCAACTGCGCCATTAAGGCTCTGATGCAGCTGACCACTAAGGCCACCACTCAGATCGCCCCTAGACAGGTTGCCGGAACCGGCGTGCAGTTCTACATGATCGTGATCGGAGTGATCATCCTGGCCGCTCTGTTCATGTACTACGCCAAGCGGATGCTGTTCACCAGCACCAACGACAAGATCAAGCTGATCCTGGCCAACAAAGAAAACGTGCACTGGACGACCTACATGGACACATTCTTCCGGACAAGCCCCATGATCATTGCCACCACCGACATCCAGAACTGA

example 2: vector construction and in vitro expression identification of fusion proteins

2.1 fusion protein vector construction

The synthetic 3 gene sequences are subjected to double digestion by EcoR and SalI, target gene fragments are recovered, meanwhile, shuttle plasmid pDC316 of an AdMax system is subjected to double digestion by EcoR and SalI, the recovered target gene fragments ori35-M1R, S35-M1R, P-S35-M1R are connected to shuttle plasmid pDC316, the target gene fragments are respectively marked as pDC316-ori35-M1R, pDC316-S35-M1R, pDC316-P-S35-M1R, stbl3 competent cells are transformed, LB plates containing ampicillin resistance are coated, colony PCR identification is carried out by picking a monoclonal, and the clone positive in PCR identification is subjected to sequencing verification. The plasmid map of pDC316-ori35-M1R is shown in FIG. 1, the plasmid map of pDC316-S35-M1R is shown in FIG. 2, and the plasmid map of pDC316-P-S35-M1R is shown in FIG. 3.

2.2 in vitro expression identification of fusion proteins

The constructed pDC316-ori35-M1R, pDC-S35-M1R and pDC316-P-S35-M1R and pDC316 vector are subjected to plasmid large extraction, transfected by using a transfection reagent PEIpro, transfected into HEK293 cells, and the cells are harvested after 48 hours of transfection for WB detection. The specific experimental method is as follows:

transfection: the day before the experiment, HEK293 cells were taken and the cell density was adjusted to 1X 10 after pancreatin digestion ⁶ Cell/well seeding 6-well plate at 37deg.C, 5% CO ₂ Cultured overnight in a cell incubator. 1h before transfection, the medium was changed to pre-warmed fresh DMEM medium containing 2% FBS, 2ml per well. During transfection, 4ug of corresponding plasmid is taken from each transfection hole, added into 500ul of OPTI-MEM culture medium, mixed uniformly, 12ul of transfection reagent is taken, added into 500ul of OPTI-MEM culture medium, mixed uniformly lightly, the mixed transfection reagent is added into the plasmid, mixed uniformly lightly, and stood for 20min at room temperature. The plasmid and transfection reagent mixture was gently added dropwise to the 6-well plate and gently mixed. The 6-well plate was placed in a 37℃5% CO2 cell incubator for culturing, and after 5 hours the medium was changed to fresh DMEM medium containing 10% FBS, and after 48 hours the cells were collected, and the sample was prepared for WB detection.

Sample preparation: after 48h of transfection, the cell culture medium was carefully aspirated, the cells were resuspended in PBS, centrifuged at 1200rpm for 3min, after centrifugation, the supernatant was discarded, and after addition of 200 ul/well RIPA lysate, the mixture was blown down and mixed well and lysed on ice for 30min, during which 10s were vortexed every 5 min. The lysed cells were centrifuged at 12000rpm at 4℃for 20min. After centrifugation, the supernatant was transferred to a new EP tube and stored in a-20deg.C refrigerator for WB detection.

Western blot detection: 30ul of the lysate supernatant was added to 6ul of 6X protein Loading Buffer, and after mixing, the mixture was dried and incubated at 100℃for 5 minutes, SDS-PAGE was performed using 10% SDS-PAGE gel with 10 wells, and the loading amount was 30ul per well. Electrophoresis conditions: and after 80V of the bromophenol blue indicator enters the separation gel, adjusting the voltage to 120V. Until just the bromophenol blue comes out of the gel. Proteins on SDS-PAGE gels were transferred to nitrocellulose membranes by a protein transfer membrane apparatus under the following conditions: 25V, 1.3A for 12min. After the transfer was completed, the nitrocellulose membrane was blocked with 5% skimmed milk powder for 1h. Then respectively taking 1: a5000 dilution was added with anti-A35R protein murine polyclonal antibody (Baiprose, cat No. 6F 11) and a 1:2500 dilution was added with humanized anti-M1R monoclonal antibody (OuKai, cat No. R417t 6) as primary antibody, and beta-actin monoclonal antibody (Yiqiao Shenzhou, cat No. 100166-MM 10) overnight at 4 ℃. The membranes were washed 3 times, 5 min/time, 3 times with TBST on a shaker. Then respectively taking 1:10000 HRP-labeled goat anti-mouse IgG antibody (sequoyins bridge, 219760802) diluted in 5% skim milk powder and 1:5000 HRP-labeled goat anti-human IgG antibody (Shanghai, D110150) diluted in 5% nonfat milk powder was used as secondary antibody and incubated for 1h at room temperature. Wash 3 times, 5 min/time, 3 times on shaker with TBST. And placing the nitrocellulose membrane on a preservative film with the right side facing upwards, adding a freshly prepared chromogenic substrate, coating the membrane with the preservative film after reaction, and attaching the preservative film in a sheet clamp. The exposure was performed in a darkroom with X-ray film, and the exposure time was adjusted according to the intensity of fluorescence.

The Western blot detection results using the a35R antibody are shown in fig. 4, where 1: western marker;2: pre-dyeing a marker;3: A35R antigen (Yiqiaoshenzhou, 40886-V07E,600 ng); 4: a35R antigen (next holy biology, 94037ES25, 600 ng) 5: transfection of pDC316-ori35-M1R cells, 6: transfecting pDC316-P-S35-M1R cells; 7: transfecting pDC316-S35-M1R cells; 8: pDC316 empty vector cells were transfected. The results show that the A35R protein of the Yinqiao Shenzhou and the next holy organism of the positive control are expressed, and the expression of the A35R protein is not detected by both transfected plasmid pDC316 empty vector cells and transfected plasmid pDC316-ori35-M1R cells; transfection of pDC316-S35-M1R cells, the expression of the A35R protein was detected; transfected pDC316-P-S35-M1R cells (mutant tPA signal peptide), the expression level of A35R protein was further increased.

The Western blot detection results using M1R antibody are shown in FIG. 5, where 1: western marker;2: pre-dyeing a marker;3: M1R antigen (Yiqiaoshenzhou, 40904-V07H,600 ng); 4: M1R antigen (next holy organism, 94036ES25, 600 ng) 5: transfection of pDC316-ori35-M1R cells, 6: transfecting pDC316-P-S35-M1R cells; 7: transfecting pDC316-S35-M1R cells; 8: pDC316 empty vector cells were transfected. The results show that the M1R proteins of the Yinqiao Shenzhou and the next holy organisms of the positive control are expressed, and the expression of the M1R proteins is not detected by both transfected plasmid pDC316 empty vector cells and transfected plasmid pDC316-ori35-M1R cells; transfection of pDC316-S35-M1R cells, the expression of the M1R protein was detected; transfected pDC316-P-S35-M1R cells (mutant tPA signal peptide), the expression level of M1R protein was further increased.

The Western blot detection results using the beta-actin antibody are shown in FIG. 6, wherein 1: pre-dyeing a marker;2: western marker;3: a35R antigen (next holy biology, 94037ES25, 600 ng); 4: M1R antigen (sense warrior, 40904-V07H,600 ng) 5: transfection of pDC316-ori35-M1R cells, 6: transfecting pDC316-P-S35-M1R cells; 7: transfecting pDC316-S35-M1R cells; 8: pDC316 empty vector cells were transfected. The results showed that beta-actin was expressed uniformly.

2.3 packaging, preparation and identification of recombinant adenoviruses

2.3.1 packaging of recombinant adenovirus: and respectively co-transfecting HEK293 cells with the constructed shuttle plasmids pDC316-ori35-M1R, pDC316-S35-M1R, pDC-P-S35-M1R and skeleton plasmids pBHGlox_E1 and 3Cre of an AdMax adenovirus system to package recombinant adenovirus. The specific process is as follows:

the day before the experiment, HEK293 cells are taken, and the cell density is regulated after pancreatin digestionIs 1X 10 ⁶ Cells/well were seeded with 6-well plates and incubated overnight in a cell incubator at 37℃with 5% CO 2. 1h before transfection, the medium was changed to pre-warmed fresh DMEM medium containing 2% FBS, 2ml per well. For transfection, backbone plasmids pBHGlox_E1,3Cre and shuttle plasmid were taken and transfected according to PEIpro transfection reagent. The method comprises the following specific steps: taking 2ug of skeleton plasmid and 2ug of shuttle plasmid from each transfection hole, adding into 500ul of OPTI-MEM culture medium for dilution, mixing uniformly, taking 12ul of transfection reagent, adding into 500ul of OPTI-MEM culture medium for dilution, mixing gently, adding the uniformly mixed transfection reagent into the plasmid, mixing gently, and standing for 20min at room temperature. The plasmid and transfection reagent mixture was gently added dropwise to the 6-well plate and gently mixed. The 6-well plate was placed in a 37℃and 5% CO2 cell incubator for culturing, after 5 hours, the medium was changed to fresh DMEM medium containing 10% FBS, after 24 hours of culturing, the medium was changed to DMEM medium containing 2% FBS, and the culture was continued, and the culture was periodically observed under an inverted microscope every day, during which time 0.5ml-1ml of DMEM medium containing 2% FBS was supplemented in time when the cell culture was yellow in color, and virus plaques were observed about 10-15 days. When the virus plaque of the 6-hole plate cells reaches 80%, the cells and the supernatant are collected in time into a 15ml centrifuge tube. Repeatedly freezing and thawing the collected cells and supernatant for 3 times at-80 ℃/37 ℃, centrifuging at 4000rpm for 10min at room temperature, and collecting the supernatant to obtain recombinant adenovirus seed liquid.

The virus seed packaged by adenovirus of shuttle plasmid pDC316-ori35-M1R is marked as Ad5-ori35-M1R; the virus seed packaged by adenovirus of shuttle plasmid pDC316-S35-M1R is marked as Ad5-A35-M1R; the virus species packaged by adenovirus of shuttle plasmid pDC316-P-S35-M1R is labeled as Ad5-P-A35-M1R.

2.3.2 identification of recombinant adenoviruses: the full sequence of the A35R-M1R fusion protein was amplified using the universal primers for the pDC316 vector, the primer sequences were as follows:

pDC316-F：ACGTGGGTATAAGAGGCG(SEQ ID NO:11)

pDC316-R：CGATGGTAGACGATCCAG(SEQ ID NO:12)

500ul recombinant adenovirus seed solution is taken, a virus DNA extraction kit (Tiangen, X1122) is used for extracting virus DNA, and the virus DNA is taken as a template to amplify the fusion protein A35R-M1R gene sequence.

PCR amplification conditions:

the reaction procedure:

and (3) carrying out electrophoresis detection on the PCR product, wherein the electrophoresis detection result shows that the target strip is obtained, the size of the fragment is consistent with the actual size, the target strip is subjected to glue recovery and sequencing, and the sequencing result is consistent with the actual sequence comparison sequence.

2.3.3 determination of infectious titre of recombinant adenoviruses

Using Clontech Adeno-X ^TM The recombinant adenovirus Titer was determined using the Rapid Titer Kit (cat# 632250). The specific operation is carried out according to the instruction attached to the kit.

2.3.4Western blotting method for detecting expression of recombinant monkey pox fusion protein

Infecting HEK293 cells with different constructed recombinant monkey pox fusion protein adenoviruses according to MOI of 10, collecting cells after 24h infection for Western blot detection of target antigen, performing SDS-PAGE on cell products after the infection, then performing transfer membrane, sealing for 1h after the transfer membrane is finished, respectively taking A35R monoclonal antibody diluted by 1:5000 as primary antibody, taking beta-actin monoclonal antibody diluted by 1:5000 as primary antibody, performing room temperature incubation for 1h, incubating A35R and beta-actin primary antibody for 3 times by using PBST, using goat anti-mouse IgG marked by HRP as secondary antibody (1:8000), incubating M1R primary antibody for 3 times by using PBST, using anti-human IgG marked by PBST (1:10000), and respectively detecting Western blot detection of fusion proteins of 3 virus species Ad5-ori35R-M1R, ad-S35-M1-R, ad-P35-P1R.

The Western blot detection results using the a35R antibody are shown in fig. 7, where 1: western marker;2: pre-dyeing a marker;3: pre-dyeing a marker;4: negative 5: infection of Ad5-ori35-M1R adenovirus cells, 6: infecting Ad5-S35-M1R adenovirus cells; 7: infecting Ad5-P-S35-M1R adenovirus cells; 8: A35R antigen (next holy organism, 94037ES25, 500 ng). The results showed that the positive control A35R protein was expressed, and that neither the negative control nor the cells infected with Ad5-ori35-M1R adenovirus detected the expression of the A35R protein; infection of Ad5-S35-M1R adenovirus cells, the expression of the A35R protein can be detected; infection of Ad5-P-S35-M1R adenovirus cells (mutation of tPA signal peptide) further increased the expression level of A35R protein.

The Western blot detection results using M1R antibody are shown in FIG. 8, where 1: pre-dyeing a marker;2: western marker;3: pre-dyeing a marker;4: infection of Ad5-ori35-M1R adenovirus cells, 5: infecting Ad5-S35-M1R adenovirus cells; 6: infecting Ad5-P-S35-M1R adenovirus cells; 7: M1R antigen (Yiqiaoshenzhou, 40904-V07H,500 ng); 8: negative control. The results showed that the positive control M1R protein was expressed, and that neither the negative control nor the cells infected with Ad5-ori35-M1R adenovirus detected the expression of the M1R protein; infection of Ad5-S35-M1R adenovirus cells, the expression of the M1R protein can be detected; infection of Ad5-P-S35-M1R adenovirus cells (mutation of tPA signal peptide) further increased the expression level of M1R protein.

The Western blot detection results using the beta-actin antibody are shown in FIG. 9, wherein 1: pre-dyeing a marker;2: western marker;3: A35R antigen (Yiqiaoshenzhou, 40886-V07E,500 ng); 4: infecting Ad5-ori35-M1R adenovirus cells; 5: infection of Ad5-S35-M1R adenovirus cells, 6: infecting Ad5-P-S35-M1R adenovirus cells; 7: negative control cells; 8: M1R antigen (Yiqiaoshenzhou, 40904-V07H,500 ng). The results showed that beta-actin was expressed uniformly.

Example 3: preparation and identification of recombinant adenoviruses

3.1 recombinant adenovirus culture

HEK293 SF 3F6 cells at 37℃and 5% CO ₂ Suspension culture at 125rpm was carried out. Inoculation of Ad5-S35-M1R and Ad5-P-S35-M1R virus species with a cell density of 1X 10 ⁶ cells/ml, the cell viability is greater than 95% and the cell volume is 300ml. Inoculation of virus according to MOI of 3, 37 ℃,5% CO ₂ Suspension culture is carried out at 125rpm under the condition, and virus liquid is harvested after 48 hours of culture. The harvested virus liquid is placed in a refrigerator at the temperature of minus 80 ℃ and a water bath kettle at the temperature of 37 ℃ in sequence for repeated freezing and thawing for three times. Then, the virus harvest was centrifuged at 15000g for 30min at 4℃and after centrifugation, the virus-containing supernatant was collected and the pellet was discarded.

3.2 recombinant adenovirus purification

ViraTrap was used for the supernatant of the harvested virus solution ^TM Adenovirus Purification Miniprep Kit (cat# BW-V1160) kit to purify adenovirus, the specific operation steps are according to the kit operation instruction, and purified Ad5-S35-M1R and Ad5-P-S35-M1R are obtained.

3.3 detection of titres of Ad5-S35-M1R and Ad5-P-S35-M1R

3.4 viral particle count assay

The virus lysate was prepared by mixing 20mmol/L Tris-HCl, 2mmol/L EDTA (pH 7.5) solution and 2.0% SDS solution in equal volumes. And (3) taking 950ul of virus solution, adding 50ul of virus lysate, repeating 4 parts in parallel, repeatedly blowing and beating for 10 times by using a pipettor, uniformly mixing, swirling for 1min, and operating by the corresponding background solution in the same method. And (3) digesting the cracked sample and the corresponding substrate solution in a constant-temperature water bath kettle at 56 ℃ for 10min, and cracking viruses. After digestion, the mixture was centrifuged at 12000rpm/min for 5min, and the supernatant was collected. Four samples were combined into a cuvette for reading, OD values at 260nm and 280nm were measured, and the number of adenovirus particles was counted.

The number of virus particles shows that the purified recombinant adenovirus reaches 1.0X10 after concentration ¹¹ VP/ml or more.

Example 4: immunological evaluation of different constructed recombinant adenoviruses on mouse models

4.1 vaccine humoral immune response detection

35 SPF-class female BALB/c mice were randomly divided into 7 groups of 5 mice each, and the mice were immunized with Ad5-S35-M1R and Ad5-P-S35-M1R (prepared in example 3) according to the grouping conditions shown in Table 1. The intramuscular injection mode is that the injection is 50ul in the rear thigh. The grouping situation is shown in table 1.

TABLE 1 detection of mouse grouping by vaccine humoral immune response

Group of	Candidate vaccine	Dosage of	Immunization mode	Number of mice
					High dose	Ad5-S35-M1R	1×10 ⁹ VP	Intramuscular injection	5
Medium dosage	Ad5-S35-M1R	5×10 ⁸ VP	Intramuscular injection	5
					Low dose	Ad5-S35-M1R	5×10 ⁷ VP	Intramuscular injection	5
High dose	Ad5-P-S35-M1R	1×10 ⁹ VP	Intramuscular injection	5
					Medium dosage	Ad5-P-S35-M1R	5×10 ⁸ VP	Intramuscular injection	5
Low dose	Ad5-P-S35-M1R	5×10 ⁷ VP	Intramuscular injection	5
					Control	PBS	50ul	Intramuscular injection	5

Mice were bled on day 9 and day 14 after immunization, serum was isolated, and ELISA was used to detect IgG antibody titers against the A35R and M1R proteins in serum of the Ad5-S35-M1R, ad5-P-S35-M1R immunized mice, respectively. The detection results are shown in fig. 10 to 14 (ns, P. Gtoreq.0.05;, P < 0.05;, P < 0.01).

Mice intramuscular injection of Ad5-S35-M1R, ad5-P-S35-M1R produced higher serum IgG antibodies against the A35R and M1R proteins on day 9, with low dose group antibody dropletsThe average value of the degree reaches 10 ⁵ The above; serum IgG antibody levels against the a35R and M1R proteins induced by intramuscular injection of Ad5-S35-M1R, ad-P-S35-M1R showed a clear dose-dependent relationship, with higher doses and higher serum IgG antibodies against the a35R and M1R proteins (fig. 10-11). From day 9 post-immunization to day 14 post-immunization, igG antibody levels in serum against a35R and M1R proteins were further increased, with high dose group serum IgG antibody titers significantly higher at day 14 post-immunization than at day 9 (fig. 12-15).

Analysis of the serum levels of IgG antibodies against the A35R and M1R proteins induced by intramuscular injection of two recombinant adenoviruses Ad5-S35-M1R, ad5-P-S35-M1R showed that serum IgG antibodies produced by Ad5-P-S35-M1R were higher than that produced by Ad5-S35-M1R on day 9 post immunization between the two recombinant adenoviruses (FIGS. 12-15). On day 14 post immunization, the serum levels of IgG antibodies raised against the a35R and M1R proteins (fig. 12-15). The results demonstrate that the serum IgG antibody induced by Ad5-P-S35-M1R has the best titer and the best immunogenicity.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims

1. An a35R-M1R fusion protein expression cassette comprising the following elements: a Kozak sequence, an optional signal peptide coding sequence, an extracellular domain coding sequence for a35R, and an M1R full-length protein coding sequence;

2. The expression cassette of claim 1, wherein the expression cassette has a structure from 5 'to 3' of formula I:

Z1-Z2-Z3-Z4-Z5 (formula I)

Wherein,

each "-" is independently a bond or a nucleotide linking sequence;

z1 is a Kozak sequence;

z2 is an optional signal peptide coding sequence;

z3 is the extracellular domain coding sequence of A35R;

z4 is the coding sequence of a connecting peptide (linker);

z5 is the coding sequence of the full-length protein of M1R.

3. The expression cassette of claim 1, wherein the signal peptide is selected from the group consisting of: a tissue plasminogen activator (tPA) signal peptide or a mutant thereof.

4. A vector comprising the a35R-M1R fusion protein expression cassette of any one of claims 1-3.

5. The vector of claim 4, wherein the vector is a plasmid; preferably, the plasmid is a viral packaging system plasmid for the production of virus-like particles.

6. The vector of claim 4, wherein the vector is a viral vector,

the viral vector is selected from the group consisting of: an adenovirus vector, a herpes simplex virus vector, an adeno-associated virus vector (AAV), or a combination thereof; preferably, the viral vector is an adenovirus vector.

7. A host cell comprising the vector of claim 4, or having integrated into the chromosome the a35R and M1R fusion protein expression cassette of any one of claims 1-3.

8. A vaccine composition, characterized in that the vaccine composition comprises:

(i) The vector of claim 4, the host cell of claim 7, or a virus-like particle produced by expression thereof; and

(ii) A vaccine acceptable carrier.

9. Use of the vector of claim 4 or the host cell of claim 7 for the preparation of a vaccine composition for the prevention of infection by a monkey poxvirus.

10. A method of preparing a vaccine composition comprising the steps of:

(1) Culturing the host cell of claim 7 under conditions suitable for expression, thereby expressing the corresponding virus-like particle;