CN116603060A

CN116603060A - Monkey poxvirus subunit vaccine and preparation method and application thereof

Info

Publication number: CN116603060A
Application number: CN202310668344.9A
Authority: CN
Inventors: 林吉辉; 杨雪涛; 杨稀丹; 王鹏
Original assignee: Shenzhen Pengbo Biotechnology Co ltd
Current assignee: Shenzhen Pengbo Biotechnology Co ltd
Priority date: 2023-06-07
Filing date: 2023-06-07
Publication date: 2023-08-18

Abstract

The invention discloses a monkey pox virus subunit vaccine, a preparation method and application thereof, and relates to the technical field of biology. The anti-acne virus antigen comprises an antigen and an adjuvant, wherein the antigen components are monkey pox virus antigen proteins M1R, A29L, A R and B6R, his tags are added to the tail parts of the monkey pox virus antigen proteins M1R, A29L, A R or B6R, the amino acid sequence of the monkey pox virus antigen protein M1R is shown as SEQ ID NO.1, the amino acid sequence of the monkey pox virus antigen protein A29L is shown as SEQ ID NO.2, the amino acid sequence of the monkey pox virus antigen protein A35R is shown as SEQ ID NO.3, and the amino acid sequence of the monkey pox virus antigen protein B6R is shown as SEQ ID NO. 4. The mouse intramuscular injection secondary subunit vaccine can cause wide neutralizing antibody and cell immune response, induce strong cell immune and humoral immune response, can be clinically popularized and used, is used for common people to inoculate when needed, and is safer and wider in application range.

Description

Monkey poxvirus subunit vaccine and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a monkey pox virus subunit vaccine, a preparation method and application thereof.

Background

Monkey Poxvirus (MPXV) belongs to the family poxviridae, orthopoxvirus, and also to smallpox, vaccinia, and the like, and the MPXV genome is a double stranded DNA up to 196kb long comprising 190 open reading frames encoding about 200 proteins, of which about 100 are nonstructural proteins, and since basic studies on MPXV are not much, only a few proteins of MPXV currently have a known function.

Currently, there are no specific vaccines and drugs against MPXV infection. Due to the cross-protective effect of the antigen of the orthopoxvirus member, the smallpox vaccine has a certain protective activity on the monkey pox. Studies show that smallpox vaccine has 85% protection efficiency for MPXV. Epidemiological studies have shown that about 90% of MPXV diagnosed cases have not been infected with other poxviruses, most of which were born after the WHO announced the end of the smallpox eradication program in 1980 and have not been vaccinated with smallpox vaccine. Two types of vaccines were approved by the FDA for pre-exposure vaccination against orthopoxviruses including monkey pox, the second generation vaccine ACAM2000 and the third generation vaccine Jynneos, respectively. ACAM2000 has been shown to alleviate symptoms of MPXV during the first U.S. MPXV epidemic in this century. There are side effects, including myocarditis and pericarditis, however, with a higher risk for some populations (e.g. eczema patients and pregnant women), resulting in the vaccine not being offered to the public nor used in areas where MPXV is prevalent. The danish Bavarian Nordic company developed a non-replicating smallpox-monkey pox vaccine Jynneos based on its own modified vaccinia ankara virus (modified vaccinia virus Ankara-Bavarian Nordic, MVA-BN) technology platform. Compared with the early vaccine, the JYNNEOS and other third-generation attenuated vaccines have better safety. Jynneos vaccine was approved by the FDA in the united states at month 9 of 2019, and vaccinated by subcutaneous injection, can elicit body fluid and cellular immune responses that are highly effective against MPXV, and is used to prevent smallpox (smallpox) and monkey pox (monkey pox) infections in high-risk adult populations over 18 years of age. The international personal matter (ACIP) consensus voting supports Jynneos as an alternative to ACAM2000, month 11 2021. This is the only non-replicating smallpox vaccine approved by the FDA and is the only monkey pox vaccine marketed worldwide. Unlike ACAM2000, jynneos can be used for atopic dermatitis patients and immunodeficiency patients. However, the function and effect of some immunomodulatory proteins in humans is not clear, as the protective targets are not yet defined. Furthermore, JYNNEOS was vaccinated at 28 day intervals, so in the case of post-exposure vaccination, the second dose could be too late to help prevent the disease, it is not clear whether the first dose was sufficient to achieve therapeutic effect. Both ACAM2000 and Jynneos were only approved for emergency immunization of the exposed population, and not for the general population. No complete clinical study data (vaccinator vs control group) was available for ACAM2000, jynneos and subunit and DNA vaccines under investigation to prevent MPXV infection, whether these vaccines could effectively prevent MPXV epidemics in the general population was yet to be determined.

Therefore, there remains a need to develop an effective and safe new generation of monkey pox specific vaccines for convenient, when needed, vaccination of the general population.

Disclosure of Invention

The invention provides a monkey pox virus subunit vaccine, a preparation method and application thereof, and aims to solve the problems in the background technology. Compared with other vaccines, subunit vaccines have good safety and are also suitable for the population with low immunity. The subunit vaccine provided by the invention comprises M1R, A L antigen protein from MPXV intracellular mature virus particles (Intracellular mature virus, IMV) and A35R, B R antigen protein from extracellular envelope virus particles (Extracellular enveloped virus, EEV), and is mixed with aluminum hydroxide (alum) or CpG. Intramuscular injection of the secondary subunit vaccine into mice can cause extensive neutralizing antibodies and cellular responses, inducing strong cellular and humoral immune responses.

In order to achieve the technical purpose, the invention mainly adopts the following technical scheme:

in a first aspect, the invention discloses a monkey pox virus subunit vaccine, which comprises an antigen and an adjuvant, wherein the antigen components are monkey pox virus antigen proteins M1R, A29L, A R and B6R, his tags are added to the tail parts of the monkey pox virus antigen proteins M1R, A29L, A R or B6R, the amino acid sequence of the monkey pox virus antigen protein M1R is shown as SEQ ID NO.1, the amino acid sequence of the monkey pox virus antigen protein A29L is shown as SEQ ID NO.2, the amino acid sequence of the monkey pox virus antigen protein A35R is shown as SEQ ID NO.3, the amino acid sequence of the monkey pox virus antigen protein B6R is shown as SEQ ID NO.4,

preferably, the adjuvant is aluminum hydroxide or CpG.

More preferably, the adjuvant is CpG.

In a second aspect, the invention discloses a nucleic acid sequence capable of encoding a monkey pox virus antigen protein M1R as shown in SEQ ID NO.5, a nucleic acid sequence encoding a monkey pox virus antigen protein A29L as shown in SEQ ID NO.6, a nucleic acid sequence encoding a monkey pox virus antigen protein A35R as shown in SEQ ID NO.7, and a nucleic acid sequence encoding a monkey pox virus antigen protein M1R as shown in SEQ ID NO. 8.

In a third aspect, the invention discloses a method for preparing a monkey poxvirus subunit vaccine according to the first aspect, comprising the steps of:

(1) Constructing expression vectors containing nucleotide sequences capable of encoding the monkey poxvirus antigen protein M1R, A L, A R or B6R respectively;

(2) Transferring each expression vector into a host cell for expression culture to obtain recombinant thalli containing the monkey pox virus proteins;

(3) Culturing the recombinant bacterial liquid to express monkey pox virus antigen protein M1R, A29L, A R or B6R;

(4) The monkey pox virus subunit vaccine is prepared by separating and purifying the monkey pox virus antigen protein M1R, A29L, A R or B6R and assisting with an adjuvant.

Preferably, the expression vector is pET-9a.

Preferably, the host cell is an E.coli BL21 (DE 3) competent cell.

In a preferred embodiment of the invention, in step (4), when the adjuvant is aluminium hydroxide, the monkey poxvirus antigen protein amounts to 10 μg each of M1R, A29L, A R and B6R, 40 μg of aluminium hydroxide adjuvant; when the adjuvant is CpG, the monkey pox virus antigen protein is 10 mug of each of M1R, A29L, A R and B6R, and CpG790910 mug, and the adjuvant and each monkey pox virus antigen protein are fully and uniformly mixed.

In a fourth aspect, the invention discloses the use of a monkey poxvirus subunit vaccine according to the first aspect in monkey poxvirus immunization or for the preparation of a medicament for the prevention of diseases caused by infection by monkey poxviruses.

Furthermore, when the monkey pox virus subunit vaccine is applied to monkey pox virus immunization, 2 injection is inoculated.

Compared with the prior art, the invention has the following beneficial effects:

the monkey pox virus subunit vaccine prepared by the invention can cause wide neutralizing antibody and cell immune response after intramuscular injection of mice with the subunit vaccine for the second time, and can induce strong cell immune and humoral immune response.

The monkey pox virus subunit vaccine prepared by the invention can be used for common people to inoculate when needed, and is safer and wider in application range.

Drawings

FIG. 1 is a schematic diagram of the structure of a constructed expression vector plasmid;

FIG. 2 is a protein gel electrophoresis detection diagram of the isolated and purified monkey pox virus antigen protein M1R, A29L, A R and B6R;

FIG. 3 is a flow chart of immunization and blood collection of mice;

FIG. 4 is a graph showing the change of antibodies against each antigen in the serum of mice after immunization;

FIG. 5 is a graph showing the results of a specific T cell immunoassay for a monkey pox vaccine using a flow assay technique;

FIG. 6 is a graph showing the results of an enzyme-linked immunospot (ELISPOT) experiment;

FIG. 7 is a comparison of infection cells measured for monkey poxvirus neutralizing antibodies.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described with reference to examples. It should be understood that the embodiments described herein are for illustration only and are not intended to limit the invention.

EXAMPLE 1 expression and purification of monkey poxvirus antigen

(1) Prokaryotic expression plasmid construction

His tag is added to the tail of the monkey pox virus antigen protein M1R (aa 1-182), A29L (aa 1-110), A35R (aa 57-181) and B6R (aa 19-280), the amino acid sequence is synthesized by Kirschner Biotechnology Co., ltd, and the amino acid sequence is subjected to codon optimization, and a prokaryotic expression vector pET-9a (4.3 kb) is inserted, and the cleavage site is selected from NdeI and BamHI on a plasmid. The constructed plasmids were designated pET-9a-M1R, pET-9a-A29L, pET-9a-B6R, pET-9a-A35R, respectively, see FIG. 1.

Wherein, the amino acid sequence of the M1R ORF is shown as SEQ ID NO. 1:

MGAAASIQTTVNTLSERISSKLEQEANASAQTKCDIEIGNFYIRQNHGCNITVKNMCSADADAQLDAVLSAATETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENYVKQTCNSSAVVDNKLKIQNVIIDECYGAPGSPTNLEFINTGSSKGNCAIKALMQLTTKATTQIAPRQVAGTGGGGSHHHHHH

the amino acid sequence of the A29L ORF is shown as SEQ ID NO. 2:

MDGTLFPGDDDLAIPATEFFSTKAAKNPETKREAIVKAYGDDNEETLKQRLTNLEKKITNITTKFEQIEKCCKRNDEVLFRLENHAETLRAAMISLAKKIDVQTGRHPYEGGGGSHHHHHH

the amino acid sequence of the A35R ORF is shown as SEQ ID NO. 3:

MVRLNQCMSANKAAITDSAVAVAAASSTHRKVVSSTTQYDHKESCNGLYYQGSCYILHSDYKSFEDAKANCAAESSTLPNKSDVLTTWLIDYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCTGGGGSHHHHHH

the amino acid sequence of the B6R ORF is shown as SEQ ID NO. 4:

MSTCTVPTMNNAKLTSTETSFNDKQKVTFTCDSGYHSLDPNAVCETDKWKYENPCKKMCTVSDYVSELYDKPLYEVNSTMTLSCNGETKYFRCEEKNGNTSWNDTVTCPNAECQPLQLEHGSCQPVKEKYSFGEYMTINCDVGYEVIGVSYISCTANSWNVIPSCQQKCDIPSLSNGLISGSTFSIGGVIHLSCKSGFTLTGSPSSTCIDGKWNPILPTCVRSNEEFDPVDDGPDDETDLSKLSKDVVQYEQEIESLEATYHIGGGGSHHHHHH

the nucleic acid sequence of the M1R ORF of the antigen protein of the monkey poxvirus is shown in SEQ ID NO. 5:

ATGGGAGCAGCTGCGTCAATACAAACTACAGTCAACACCCTGAGCGAACGTATTAGCTCTAAGTTGGAGCAAGAGGCTAATGCAAGCGCCCAAACTAAGTGTGATATTGAGATTGGTAATTTCTACATCCGTCAAAACCATGGTTGTAACATCACCGTTAAGAACATGTGCAGCGCGGACGCCGACGCGCAATTGGACGCGGTCCTGAGCGCGGCTACCGAAACGTACAGCGGTCTGACCCCGGAACAGAAAGCGTATGTTCCGGCAATGTTCACCGCAGCGTTAAATATCCAGACCTCCGTTAATACCGTTGTGCGCGATTTTGAGAACTACGTGAAGCAGACGTGCAACAGCTCTGCTGTGGTGGATAATAAACTGAAAATCCAAAACGTGATCATCGACGAGTGCTATGGTGCGCCAGGTTCCCCGACCAACCTCGAATTTATCAACACCGGTTCCTCGAAAGGCAACTGCGCAATTAAAGCCCTGATGCAGCTGACTACGAAGGCGACCACACAGATTGCACCGCGTCAGGTTGCGGGCACCGGCGGCGGTGGCTCACATCACCACCACCATCAC

the nucleic acid sequence of the ORF of the monkey poxvirus antigen protein A29L is shown in SEQ ID NO. 6:

ATGGATGGAACACTATTTCCCGGGGATGACGACCTCGCGATCCCGGCGACCGAATTTTTCTCCACCAAGGCGGCTAAGAACCCGGAAACTAAGCGCGAGGCCATCGTGAAAGCGTACGGCGATGACAACGAGGAAACCCTGAAACAGCGTTTGACCAACCTGGAGAAAAAGATCACGAACATTACCACCAAGTTCGAGCAGATTGAGAAGTGCTGTAAACGTAATGATGAAGTTCTGTTTCGCTTGGAGAATCATGCGGAAACGCTGCGTGCAGCAATGATTAGCCTGGCTAAAAAAATCGACGTCCAAACCGGTCGTCATCCGTATGAAGGTGGCGGTGGCAGCCATCACCACCACCACCAC

the nucleic acid sequence of the ORF of the antigen protein A35R of the monkey poxvirus is shown in SEQ ID NO. 7:

ATGGTAAGGCTAAATCAATGTATGTCAGCTAACAAGGCGGCGATCACCGATAGCGCAGTTGCTGTGGCAGCCGCGAGCAGCACCCATCGTAAGGTCGTGAGCTCTACCACGCAGTATGATCATAAAGAGAGCTGCAACGGCCTGTATTACCAGGGTTCATGCTATATCTTGCACAGCGACTACAAATCTTTTGAGGACGCCAAAGCTAATTGTGCGGCGGAAAGCAGCACTCTGCCGAATAAGTCCGACGTACTGACCACGTGGCTCATTGATTACGTTGAAGATACCTGGGGTTCGGACGGCAACCCGATTACCAAAACCACGAGCGACTACCAAGATTCCGACGTGTCGCAAGAGGTTCGCAAGTATTTCTGCACCGGTGGCGGTGGCTCCCATCACCATCACCACCAC

the nucleic acid sequence of the monkey poxvirus antigen protein B6R ORF is shown in SEQ ID NO. 8:

ATGTCAACATGTACTGTACCCACGATGAATAACGCTAAGTTGACCTCTACGGAAACGAGCTTCAACGACAAGCAGAAGGTGACCTTCACCTGCGACAGCGGCTACCATAGTCTGGACCCGAACGCCGTTTGTGAAACCGATAAGTGGAAATACGAGAACCCGTGTAAAAAAATGTGCACGGTTTCGGATTACGTGAGCGAACTGTATGATAAACCGCTTTATGAGGTGAATAGCACCATGACCTTGTCTTGCAATGGTGAGACGAAATACTTTCGTTGTGAAGAGAAAAACGGCAACACCAGCTGGAACGACACCGTGACCTGTCCGAATGCGGAATGCCAGCCACTGCAGCTGGAGCACGGCTCCTGCCAACCGGTGAAAGAAAAGTACTCCTTTGGTGAGTATATGACCATCAACTGCGACGTGGGTTACGAGGTTATCGGCGTAAGCTACATTAGCTGCACTGCGAATTCTTGGAATGTCATCCCTTCCTGCCAACAGAAGTGCGACATCCCGAGCCTCTCCAACGGTCTGATTAGCGGCTCCACCTTCAGCATTGGCGGTGTCATCCACCTGAGCTGCAAAAGCGGTTTTACCCTGACCGGTTCGCCGTCATCTACATGCATCGACGGCAAATGGAACCCGATTCTGCCGACCTGTGTTCGCAGCAATGAAGAGTTCGATCCGGTTGATGACGGTCCGGATGATGAAACTGACCTGTCCAAGTTGTCTAAGGACGTTGTTCAGTATGAGCAAGAAATTGAGAGCCTGGAGGCAACCTATCATATCGGTGGCGGTGGCAGCCATCACCACCATCACCAC。

(2) Prokaryotic expression of proteins

E.coli BL21 (DE 3) competent cells were transformed with each expression vector, and after overnight culture in an incubator at 37℃positive clones were selected the next day and inoculated in 3ml LB medium (1.0% tryptone, 1.0% sodium chloride, 0.5% yeast extract, 1.5% agar, kanamycin 50. Mu.g/ml) containing 50. Mu.g/ml kanamycin, and shake-cultured overnight at 37℃at 180 rpm; the third day is followed by 1:100 is inoculated into LB culture medium (1.0% tryptone, 0.5% yeast powder, 1% sodium chloride, 50. Mu.g/ml kanamycin) containing 50. Mu.g/ml kanamycin, the temperature is regulated to 37 ℃, the rotation speed is regulated to 240rpm, shaking culture is carried out for 3 hours until the OD value measured when the bacterial liquid is 450nm in wavelength reaches about 0.8, IPTG is added to the final concentration of 1mmol/L, and induced culture is carried out for 2 hours under a constant temperature incubator at 37 ℃, so as to obtain bacterial liquid.

(3) SDS-PAGE and Western Blot identification of prokaryotic expression proteins

200. Mu.g of the bacterial solutions were collected, centrifuged for 2min, resuspended in 80. Mu.l of PBS, and mixed with 20. Mu.l of 5×loading buffer, boiled for 5min, and sampled for 10. Mu.g. And performing conventional SDS-PAGE identification, adjusting the voltage to 80V at the beginning of electrophoresis, adjusting the voltage to 120V after bromophenol blue enters the separation gel, and continuing electrophoresis until bromophenol blue reaches the bottom of the separation gel. SDS-PAGE gels were also transferred to Nitrocellulose (NC) membranes, and shaken for 2h at room temperature with 5% skim milk (PBST with 5% skim milk powder) and antibody skim milk 1: the 10000 released His-HRP labeled mouse monoclonal antibody is incubated and shaken for 3h at room temperature, PBST is washed for 3 times, 10min each time, the water on the surface of the membrane is absorbed, and after the membrane is dried, the developing solution is added for developing. Whether the M1R, A29L, B6R, A35R protein was expressed in E.coli was examined. The results are shown in FIG. 2B, where the size of each protein matches that expected, indicating successful expression of each antigen protein.

(4) Prokaryotic protein expression condition optimization

Amplifying and culturing the bacterial solutions, centrifuging for 30min, discarding supernatant, re-suspending with 30ml PBS, crushing bacterial cells with a high-pressure homogenizer until the appearance is clear and transparent, centrifuging for 15min with a high-speed centrifuge 10000 Xg. After centrifugation, the supernatant was purified by His-Band column and protein purifier. The ultraviolet absorbance peak-to-peak value was recorded. And (3) carrying out ultrafiltration concentration on the obtained sample, purifying again by a gel filtration chromatography system, sampling the collected sample, carrying out SDS-PAG identification, and evaluating the effect of protein purification.

The 4 kinds of expressed bacteria 1 are respectively taken: 100 is inoculated into LB culture medium containing 50 mug/ml kanamycin, the temperature is regulated to 37 ℃, the rotation speed is regulated to 240rpm, the shaking culture is carried out for 3 hours until the OD value of the bacterial liquid measured at the wavelength of 450nm reaches about 0.8, and IPTG is added to the final concentration of 0.2mmol/L,0.4mmol/L,0.8mmol/L and 1mmol/L. And respectively carrying out induction culture for 2h, 4h and 6h in a 37 ℃ constant temperature incubator and a 25 ℃ constant temperature incubator, and obtaining bacterial liquid for conventional SDS-PAGE identification. The optimal conditions for expression of the three proteins at different temperatures and different IPTG concentrations were compared as shown in fig. 2 a. The results show that the expression bacteria are expressed under the induction conditions of 37 ℃ and 25 ℃, the expression quantity of A29L and M1R is high at 37 ℃ for 4 hours, and the expression quantity of B6R and A35R is high at 25 ℃ for 6 hours (as shown in figure 2A).

(5) Purification of M1R, A29L, B6R, A35R protein

Bacterial pellet of monkey pox A29L and B6R was lysed with buffer A (50 mmol/LTris,500mmol/LNaCl, pH 7.4) and the bacteria resuspended in buffer A, broken by a protein homogenizer, 800pa,20min. The cell lysate was centrifuged at 10000 Xg for 15 minutes to remove insoluble materials. The supernatant was filtered through a 0.45 μm filter, and the filtrate was purified by passing through a nickel column. The nickel column is balanced by using the solution A with the volume of 10 times, then the protein is put on the column, the solution A with the volume of 5-10 times is used for balancing the nickel column, then the solution B with the volume of 10 times (50 mmol/LTris,500mmol/LNaCl,500mmol/LpH 7.4.4) is used for gradient elution, and the eluent is taken out to obtain the protein solution. The column was then equilibrated with 10 times the volume of solution A, eluted with 5 times the volume of deionized water, and finally eluted with 5 times the volume of 20% ethanol. Concentrating the collected protein liquid, purifying again by a gel filtration chromatography system, sampling the collected sample, performing SDS-PAG identification, evaluating the protein purification effect, detecting the protein purity by SDS-PAGE electrophoresis, concentrating the protein liquid in a ultrafiltration tube, measuring the concentration by a BCA kit, and sub-packaging and storing in a refrigerator at-20 ℃.

a35R, M1R was obtained from inclusion bodies by renaturation. And (5) crushing and centrifuging thalli, taking a precipitate, and discarding supernatant to leave the precipitate. The precipitate was taken, 40 times (g/mL) of washing buffer (50 mmol/LTris,500mmol/LNaCl,2mol/L urea, 1% Triton X-100, pH 7.4) was added, and after thorough mixing, the mixture was washed with a shaking table at room temperature for 2 hours, centrifuged at 4℃at 10000 Xg for 15min, and the supernatant was discarded to obtain inclusion bodies. The pellet and supernatant were taken from each of the above steps and subjected to 12% SDS-PAGE detection. Precisely weighing inclusion bodies, adding 20 times (g/mL) of denaturation buffer (50 mmol/LTris,500mmol/LNaCl,8mol/L urea, 50mM beta-mercaptoethanol pH 7.4), fully mixing, dissolving overnight at low temperature (8-10 ℃ C., 12h above), centrifuging at 4 ℃ C., 10000 Xg for 15min, and taking supernatant to obtain inclusion body denaturation solution. The heparin column was equilibrated with 10 volumes of denaturation buffer (50 mmol/LTris,500mmol/LNaCl,8mol/L urea, 50mM beta-mercaptoethanol pH 7.4) at a flow rate of 1mL/min on a protein purification apparatus; and (3) fully balancing the denatured protein sample at the flow rate of 0.5mL/min by using the balancing solution, and then starting gradient renaturation. And (3) solution A: 50mmol/LTris,500mmol/LNaCl,8mol/L urea, 50mM beta-mercaptoethanol pH 7.4; and (2) liquid B: 50mmol/LTris,500mmol/LNaCl, pH 7.4. After complete renaturation, the protein solution was obtained by gradient elution (10 column volumes, 1 mL/min) with imidazole-containing eluent B (50 mmol/LTris,500mmol/LNaCl,500mmol/LpH 7.4.4), and collecting the peak of each stage. The remaining steps are as described previously. The M1R, A29L, B6R, A R protein with higher purity is obtained, as shown in C in FIG. 2.

(6) And (3) fully mixing and incubating 10 mug of each of the M1R, A29L, A R and B6R proteins prepared by the method with 40 mug of aluminum hydroxide adjuvant or 40 mug of CpG adjuvant CpG790910 mug respectively to prepare the monkey pox virus subunit vaccine.

EXAMPLE 2 analysis of the immune response of mice elicited by MPXV subunits

(1) Immunization of mice

8-week-old female BALB/c mice were selected and randomly divided into five groups, five of each group, and the mice were immunized by intramuscular injection on days 0 and 14, respectively, according to the following method (note: 40. Mu.g of AMBA, which means 10. Mu.g of each of A29L, M1R, B R and A35R). The immunization program is shown in fig. 3:

(2) Determination of mouse specific antibody titers by enzyme-linked immunosorbent assay (ELISA)

M1R, A, A35, B6R antigen-specific antibody titers were detected by indirect ELISA, 96-well ELISA plates, coated overnight at 4℃with 200ng of purified monkey pox virus protein, washed 3 times with wash buffer and blocked in Blocking buffer for 2 hours. After washing the plates 3 times with Washingbuffer, the mouse serum was diluted and incubated on the plates for 2 hours at room temperature, followed by 3 washes. Plates were incubated with 1:50000 secondary anti-HRP conjugated anti-mouse IgG antibody followed by TMB substrate. Absorbance at 450nm was measured by an enzyme-labeled instrument.

As a result, as shown in fig. 4, MPXV-specific antibodies could be detected in all vaccinated mice after 14d of immunization 1, and the antibody levels in AMBA group, AMBA/alum group and AMBA/CpG group increased to higher levels after 14d of secondary immunization, while the antibody levels in alum group and CpG group were not significantly changed from those before immunization. And AMBA/CpG groups were able to generate a more intense and durable immune response compared to AMBA/alum groups, with higher levels of antibodies against each antigen in AMBA, AMBA/alum and AMBA/CpG group mouse serum after 60d of priming. The monkey pox virus subunit vaccine prepared by the invention can stimulate organisms to produce specific antibodies aiming at M1R, A, A35 and B6R antigens.

(3) Monkeypox virus specific T cell immunoassay

Antigen-specific T cell immune responses were analyzed on a polychromatic flow cytometer. After spleen cells were collected from immunized or control mice, 2X10 mice were taken from each ⁶ The individual cells were cultured in a carbon dioxide incubator and continued to be cultured for 4 hours after stimulation with 10. Mu.g/mL of the monkey poxvirus protein polypeptide. Brefeldin a was then added to spleen cells and incubated for 6 hours. The stimulated cells were washed in PBS/0.5% BSA and stained with APC/Fire 750 anti-mouse CD3 antibody, FITC anti-mouse CD4 antibody and Brilliant and then fixed using a fixation/rupture solution kit, and stained with PE anti-mouse IFN-gamma antibody, PE anti-mouse IL-2 antibody and PE anti-mouse IL-4 antibody. Finally, the flow analysis is carried out, and the results show that each group of mice CD4 ⁺ And CD8 ⁺ There was no significant difference in the percentage of T cells, but CD4 in AMBA/CpG immunized mice ⁺ IFN-gamma produced by T cells is significantly different from adjuvant CpG treated groups, demonstrating that the monkey poxvirus subunit vaccine is capable of inducing CD4 ⁺ T cells are responsible for inducing cellular immune responses as shown in figure 5.

(4) ELISA spot (ELISPOT) assay

The spleen tissue of the mice was isolated using a commercial kit (company: shenzhen Daidae bioengineering Co., ltd. # 2210005) and the cells were filtered using a40 μm cell filter. Cell count was performed after lysing erythrocytes in spleen cells with erythrocyte lysate at 2x10 ⁵ The density of individual cells/wells was added to the medium. Three wells were set up for negative and positive controls. 10 mug of protein polypeptide stimulus is added, and the mixture is cultured in a 5% CO2 incubator at 37 ℃ for 32-48 hours. The culture broth was discarded and the cells were lysed using pre-chilled deionized water at 4℃for 10min. After washing the plate five times, the labeled detection antibody IFN-gamma was added and incubated at 37℃for 1h. After the antibody incubation is finished, pouring liquid in the hole, repeatedly washing for 5 times, and adding the enzyme-linked avidin for incubation for 1h. Washing the plate for 5 times after incubation; adding the color development liquid, and standing at room temperature in dark place for 15-45min, distilled water was added to terminate the reaction. After natural air drying, counts were performed using an ELISPOT reader system. As shown in fig. 6, the results showed that the adjuvant-only alum group and CpG group had very little IFN- γ expression, whereas AMBA group, AMBA/alum group and AMBA/CpG group all had high levels of IFN- γ expression, and that the amount of IFN- γ secreting lymphocytes was significantly higher in AMBA/CpG group than AMBA/alum group, indicating that PBMCs vaccinated with AMBA/CpG group were significantly more potent in secreting IFN- γ, inducing a more intense cellular immune response.

(5) Determination of monkey poxvirus neutralizing antibodies

Prior to conducting the infectious virus neutralization assay, the mouse serum is heat treated at 56℃for 30 minutes to remove complement and other potential neutralizing agents. Three-fold serial dilutions of serum from 1:10 to 1:2430 were incubated with VACV for 1 hour at 37 ℃. Mouse complement was added to the mixture at a final concentration of 2%. The mixture was then incubated with BHK 21 cells for 1 hour for adsorption. The supernatant was then replaced with fresh DMEM supplemented with 2% fbs. 72 hours after infection, cells were fixed in 4% paraformaldehyde for 15 minutes and then stained in DAPI for 5 minutes. The infected cells were assayed with the Harmony software (Perkin Elmer) by taking images with an OPeretta under a high content cell imaging system (Perkin Elmer). As shown in fig. 7, the neutralization titers of the alum group and the CpG group were zero, and the neutralization titers of the AMBA group and the AMBA/alum group were 1:270, whereas AMBA/CpG group has a higher potency of 1:810, the best protection effect.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims

1. The monkey pox virus subunit vaccine is characterized by comprising an antigen and an adjuvant, wherein the antigen components are monkey pox virus antigen proteins M1R, A29L, A R and B6R, his tags are added to the tail parts of the monkey pox virus antigen proteins M1R, A29L, A35R or B6R, the amino acid sequence of the monkey pox virus antigen protein M1R is shown as SEQ ID NO.1, the amino acid sequence of the monkey pox virus antigen protein A29L is shown as SEQ ID NO.2, the amino acid sequence of the monkey pox virus antigen protein A35R is shown as SEQ ID NO.3, and the amino acid sequence of the monkey pox virus antigen protein B6R is shown as SEQ ID NO. 4.

2. The monkey poxvirus subunit vaccine of claim 1, wherein: the adjuvant is aluminum hydroxide adjuvant or CpG adjuvant.

3. The monkey poxvirus subunit vaccine of claim 2, wherein: the adjuvant is CpG.

4. The nucleic acid sequence of encoding the monkey pox virus antigen protein M1R is shown as SEQ ID NO.5, the nucleic acid sequence of encoding the monkey pox virus antigen protein A29L is shown as SEQ ID NO.6, the nucleic acid sequence of encoding the monkey pox virus antigen protein A35R is shown as SEQ ID NO.7, and the nucleic acid sequence of encoding the monkey pox virus antigen protein M1R is shown as SEQ ID NO. 8.

5. A method of preparing a monkey poxvirus subunit vaccine according to any one of claims 1 to 3, comprising the steps of:

(2) Transferring each expression vector into a host cell for expression culture to obtain recombinant bacterial liquid containing the monkey pox virus protein;

6. The method for preparing the monkey poxvirus subunit vaccine according to claim 5, wherein the expression vector is pET-9a.

7. The method of producing a monkey poxvirus subunit vaccine according to claim 5, wherein the host cell is a competent cell of e.coli BL21 (DE 3).

8. The method of claim 5, wherein in step (4), when the adjuvant is aluminum hydroxide, the amount of the antigen protein of the monkey poxvirus is 10 μg each of M1R, A29L, A R and B6R, and the amount of the aluminum hydroxide adjuvant is 40 μg; when the adjuvant is CpG, the monkey pox virus antigen protein is 10 mug of each of M1R, A29L, A R and B6R, and CpG790910 mug, and the adjuvant and each monkey pox virus antigen protein are fully and uniformly mixed.

9. Use of a monkey poxvirus subunit vaccine according to any one of claims 1 to 3 in monkey poxvirus immunization or for the preparation of a medicament for the prevention of a disease caused by a monkey poxvirus infection.

10. The use according to claim 9, characterized in that: when the monkey pox virus subunit vaccine is applied to monkey pox virus immunization, 2 injections are inoculated.