A tetravalent vaccine immunization composition and its use for the prevention of hand-foot-and-mouth disease

Abstract

The present application belongs to the technical field of genetic engineering vaccine, and relates to a tetravalent vaccine immune composition and use thereof for preventing hand-foot-mouth disease. The immune composition comprises a coxsackievirus A10 type capsid protein mutant and / or a coding gene thereof, a coxsackievirus A16 type capsid protein and / or a coding gene thereof, a coxsackievirus A6 type capsid protein and / or a coding gene thereof, and an enterovirus 71 type capsid protein and / or a coding gene thereof, wherein the coxsackievirus A10 type capsid protein mutant, the coxsackievirus A16 type capsid protein, the coxsackievirus A6 type capsid protein and the enterovirus 71 type capsid protein are all assembled from capsid proteins VP1, VP2, VP3 and VP4, and the amino acid sequence of the capsid protein VP1 of the coxsackievirus A10 type capsid protein mutant is shown in SEQ ID NO. 1. The immune composition of the present application has a better immune protection effect on hand-foot-mouth disease or angina.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering vaccine technology, and relates to a quadrivalent vaccine immunization composition and its use in the prevention of hand-foot-mouth disease. Background Technology

[0002] Hand, foot, and mouth disease (HFMD) is a common infectious disease in children caused by human enteroviruses, primarily affecting infants and young children under 5 years old. The main clinical symptoms include fever, blisters on the hands, feet, and mouth, and pharyngitis. Severe cases can lead to myocarditis, pulmonary edema, neurological damage, and even death. Currently, the main circulating viruses causing HFMD include enterovirus 71 (EV71), Coxsackievirus A16 (CA16), Coxsackievirus A6 (CA6), and Coxsackievirus A10 (CA10).

[0003] EV71, CA16, CA6, and CA10 viruses are all single-stranded positive-sense RNA viruses, belonging to the family Picornaviridae and the genus Enterovirus. Their genes are all composed of approximately 7400–7440 nucleotides, each with only one open reading frame, encoding a polyprotein containing approximately 2190 amino acids. This polyprotein can be further hydrolyzed into three precursor proteins: P1, P2, and P3. The P1 precursor protein can degrade into capsid proteins VP1, VP2, VP3, and VP4; the P2 and P3 precursor proteins can degrade into seven non-structural proteins, which are highly conserved during evolution.

[0004] The viral particles of EV71, CA16, CA6, and CA10 are all icosahedral spherical structures with no envelope or protrusions, and have a diameter of approximately 23–30 nm. The virus consists of an outer capsid protein and an inner RNA core. The capsid protein is initially composed of VP1, VP2, VP3, and VP4 molecules forming protomers, i.e., protein subunits. Five protomers then form a pentamer structure, and finally, 60 subunits form an icosahedral spherical capsid.

[0005] In the four capsid structures of EV71, CA16, CA6, and CA10, VP1, VP2, and VP3 are exposed on the outer surface, while the VP4 protein is embedded on the inner surface and linked to the RNA core. The VP1 protein varies considerably among different viral strains and plays a crucial role in viral immunogenicity, binding to specific receptors on the host cell surface, and viral uncoating. Meanwhile, VP2 and VP3 proteins also contain certain antigenic determinants.

[0006] Recombinant hand-foot-mouth disease virus-like particles (VLPs) are similar to natural viruses in that their viral capsid protein structure and particle size are the same, both being icosahedral spherical structures with no envelope or protrusions, and with a diameter of approximately 23-30 nm. The difference is that they only have an outer capsid protein and do not contain an infectious RNA core, therefore they are immunogenic but not infectious.

[0007] Currently, the only hand-foot-and-mouth disease (HFMD) vaccine available in China is the EV71 inactivated vaccine. Because there is no cross-immunity between different enterovirus types, this vaccine only prevents infection with the EV71 virus and cannot prevent infection with the CA6, CA16, and CA10 strains that have become increasingly prevalent in recent years. With changes in the viral epidemic spectrum, CA10 has become the dominant circulating strain for HFMD and herpetic pharyngitis, and its clinical infection rate is increasing year by year. Therefore, developing a HFMD vaccine targeting the CA10 strain, and further developing a quadrivalent HFMD vaccine covering the four major circulating strains EV71, CA6, CA16, and CA10, has significant public health value.

[0008] Traditional enterovirus vaccines are mostly prepared using virus inactivation processes, which have drawbacks such as high biosafety risks in strain culture, long production cycles, easy viral mutation, difficulty in purification, and high production costs. Virus-like particle (VLP) vaccines do not contain viral nucleic acid, have no replication and infectivity capabilities, have high safety, can highly mimic the spatial conformation of natural viruses, and have strong immunogenicity, making them the mainstream research direction for next-generation hand-foot-mouth disease vaccines.

[0009] Existing wild-type CA10 capsid protein has significant defects: poor protein solubility, low efficiency in in vitro VLP assembly, insufficient exposure of key neutralizing antigenic epitopes, and weak immune response. Meanwhile, commonly used expression systems, such as the E. coli expression system, suffer from low protein expression levels, incomplete protein folding, and poor particle uniformity. Furthermore, when common wild-type CA10 antigen is mixed with EV71, CA6, and CA16 to prepare a quadrivalent vaccine, problems such as antigenic interference, type-specific immune imbalance, and decreased overall protective efficacy are likely to occur.

[0010] In addition, Hansenula polymorpha has advantages such as strong high-density fermentation capacity, high expression level of exogenous protein, perfect protein folding modification, low fermentation cost, and suitability for industrial-scale production. Summary of the Invention

[0011] The primary objective of this invention is to provide a quadrivalent vaccine immunization composition that provides better immune protection against hand-foot-mouth disease or herpetic pharyngitis.

[0012] To achieve this objective, in a basic embodiment, the present invention provides a quadrivalent vaccine immunization composition comprising a Coxsackievirus A10 capsid protein mutant and / or its encoding gene, a Coxsackievirus A16 capsid protein and / or its encoding gene, a Coxsackievirus A6 capsid protein and / or its encoding gene, and an Enterovirus 71 capsid protein and / or its encoding gene. The Coxsackievirus A10 capsid protein mutant, the Coxsackievirus A16 capsid protein, the Coxsackievirus A6 capsid protein, and the Enterovirus 71 capsid protein are all assembled from capsid proteins VP1, VP2, VP3, and VP4. The amino acid sequence of the capsid protein VP1 of the Coxsackievirus A10 capsid protein mutant is shown in SEQ ID NO. 1.

[0013] The amino acid sequence of the wild-type capsid protein VP1 of Coxsackievirus A10 is shown in SEQ ID NO.2. Therefore, the capsid protein VP1 of the Coxsackievirus A10 capsid protein mutant of the present invention has undergone the following amino acid site mutations based on the wild-type capsid protein VP1: Val at position 23 is mutated to Ile (isoleucine), Val at position 180 is mutated to Ala (alanine), and Thr at position 284 is mutated to Asn (asparagine).

[0014] In a preferred embodiment, the present invention provides a quadrivalent vaccine immunization composition, wherein the amino acid sequences of the Coxsackievirus A16 capsid protein, the Coxsackievirus A6 capsid protein, and the Enterovirus 71 capsid protein are all wild-type amino acid sequences.

[0015] In a preferred embodiment, the present invention provides a quadrivalent vaccine immunization composition, wherein the amino acid sequences of capsid proteins VP2, VP3, and VP4 of the Coxsackievirus A10 capsid protein mutant are wild-type amino acid sequences, as shown in SEQ ID NO. 3-5, respectively.

[0016] In a preferred embodiment, the present invention provides a quadrivalent vaccine immunization composition, wherein the immunization composition comprises a Coxsackievirus A10 capsid protein mutant, a Coxsackievirus A16 capsid protein, a Coxsackievirus A6 capsid protein, and an enterovirus 71 capsid protein.

[0017] In a preferred embodiment, the present invention provides a quadrivalent vaccine immunization composition, wherein the immunization composition further comprises an adjuvant.

[0018] In a preferred embodiment, the present invention provides a quadrivalent vaccine immunization composition, wherein the adjuvant is selected from one or more of complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum hydroxide adjuvant, and aluminum phosphate adjuvant.

[0019] In a preferred embodiment, the present invention provides a quadrivalent vaccine immunization composition, wherein the molar ratio of the Coxsackievirus A10 capsid protein mutant (i.e., the VLP antigen assembled from the Coxsackievirus A10 capsid protein mutant), the Coxsackievirus A16 capsid protein (i.e., the VLP antigen assembled from the Coxsackievirus A16 wild-type capsid protein), the Coxsackievirus A6 capsid protein (i.e., the VLP antigen assembled from the Coxsackievirus A6 wild-type capsid protein), and the Enterovirus 71 capsid protein (i.e., the VLP antigen assembled from the Enterovirus 71 wild-type capsid protein) is 1:0.7-1.5:0.7-1.5:0.7-1.5, preferably 1:1:1:1.

[0020] In a preferred embodiment, the present invention provides a quadrivalent vaccine immunization composition, wherein the immunization composition further comprises a diluent and / or a stabilizer.

[0021] In a preferred embodiment, the present invention provides a quadrivalent vaccine immunization composition, wherein the diluent is selected from one or more of phosphate buffer, histidine buffer, citrate buffer, and physiological saline (0.85-0.9 wt% sodium chloride solution).

[0022] In a preferred embodiment, the present invention provides a quadrivalent vaccine immunization composition, wherein the stabilizer is selected from one or more of sucrose, trehalose, mannitol, sorbitol, glycerol, glycine, L-histidine, arginine, human serum albumin, gelatin, Tween 20, Tween 80, polyethylene glycol, and dextran.

[0023] A second object of the present invention is to provide the use of the immune composition described above for the preparation of a vaccine for the prevention of hand-foot-mouth disease or herpetic pharyngitis, so as to provide better immune protection against hand-foot-mouth disease or herpetic pharyngitis.

[0024] To achieve this objective, in a basic embodiment, the present invention provides the use of the immune composition described above for the preparation of a vaccine for the prevention of hand-foot-mouth disease or herpetic pharyngitis.

[0025] Furthermore, the immune composition of the present invention as described above can be used to prepare multivalent enterovirus prophylactic vaccines.

[0026] The beneficial effect of the present invention is that the immune composition of the present invention has a better immune protection effect against hand-foot-mouth disease or herpetic pharyngitis.

[0027] The Coxsackievirus A10 capsid protein mutant of the present invention has a high expression level, complete VLP assembly, and strong antigenic immunogenicity. The quadrivalent vaccine formulated with it provides balanced immunity across all types, broad protection, and good safety, and can effectively prevent hand-foot-mouth disease caused by various enteroviruses.

[0028] This invention obtains a highly assembled and highly immunogenic mutant protein by site-directed mutagenesis of the key antigenic site and assembly site of the CA10 VP1 protein. The mutant protein is then expressed efficiently using Hansenula polymorpha and a tetravalent VLP vaccine composition is constructed. This invention solves the technical problems of weak immunogenicity, poor VLP assembly, uneven tetravalent compatibility, and narrow spectrum of prevention and control in the prior art.

[0029] The beneficial effects of this invention are specifically reflected in: (1) The Coxsackievirus A10 capsid protein mutant of the present invention is based on site-directed amino acid mutation of the VP1 capsid protein of CA10. The mutation site is located in the antigen loop region and particle assembly domain on the protein surface. After mutation, the protein solubility is improved, and it can autonomously assemble a uniformly morphological VLP, and the neutralizing antigen epitope is fully exposed.

[0030] (2) The present invention optimizes the gene sequence encoding the above-mentioned Coxsackievirus A10 capsid protein mutant and adapts the codon preference to the Hansenula polymorpha expression system.

[0031] (3) The present invention constructs a recombinant expression vector of Hansenula polymorpha containing the above gene sequence, which can be transformed to obtain a stably inherited Hansenula polymorpha engineered strain.

[0032] (4) The present invention can obtain high-purity recombinant protein of Coxsackievirus A10 capsid protein mutant through high-density fermentation, induced expression, cell disruption and chromatography purification.

[0033] (5) The quadrivalent vaccine immunization composition of the present invention contains EV71 VLP antigen, CA16 VLP antigen, CA6 VLP antigen and CA10 mutant VLP antigen, and is combined with pharmaceutically acceptable adjuvant, diluent and stabilizer to prepare a vaccine formulation with better immune protection.

[0034] (6) The immune composition of the present invention has broad application prospects in the prevention of hand-foot-mouth disease or herpetic pharyngitis, the detection of viral antibodies, and the prevention and control of enteroviruses. Attached Figure Description

[0035] Figure 1 The diagram shows the structure of the recombinant expression vector pVMHU-CA10VP4231 constructed in Example 2.

[0036] Figure 2The image shows the PCR screening and identification results of Example 3. Lane 1 is the PCR negative control (water), lane 10 is the PCR positive control (recombinant expression vector pVMHU-CA10VP4231), and lanes 2-9 and 11-24 are samples of different transformed colonies.

[0037] Figure 3 The results of SDS-PAGE electrophoresis purity detection are for Example 4.

[0038] Figure 4 The images show the results of phosphotungstic acid negative staining electron microscopy observations in Example 4. The sample on the left is the CA10 capsid protein mutant VLP particle prepared in this invention, and the sample on the right is the CA10 wild-type capsid protein VLP particle.

[0039] Figure 5 This is a graph showing the GMT trend analysis of neutralizing antibodies in the immunization assays of various vaccine formulations in Example 6.

[0040] Figure 6 This is a graph showing the trend of protection rate in the maternal antibody challenge protection test in suckling mice in Example 7.

[0041] Figure 7 This is an electron micrograph of the stability characterization study of the immune composition in Example 8. Detailed Implementation

[0042] To better understand the technical solutions and advantages of the present invention, the present invention will be further described below through embodiments and accompanying drawings.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0044] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0045] rTaq enzyme and endonucleases DraI, SnaBI, EcoRI, SalI, BamHI, and BglII were all purchased from Baori Biotechnology (Beijing) Co., Ltd.

[0046] The plasmid miniprep kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.

[0047] The magnetic bead-based DNA gel extraction kit was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0048] The *Hansenula polymorpha* expression vector pVMHU was constructed and preserved by the applicant based on the pBluescriptII(+ / -) vector. This expression vector is a shuttle plasmid of *Escherichia coli* and *Hansenula polymorpha*. The prokaryotic portion includes ColE1, Amp, and the multiple cloning site MCS, all derived from the pBluescriptII(+ / -) vector itself. The yeast portion includes the promoter (methanol oxidase promoter MOXP), terminator (methanol oxidase terminator MOXT), autonomous replication sequence (HARS), and selection marker (Ura3), all derived from the genome of wild-type *Hansenula polymorpha* (ATCC34438) purchased from the American Type Culture Collection (ATCC).

[0049] Ura3- genotype Hansenula was obtained by screening wild-type Hansenula (ATCC34438) purchased from the American Type Culture Collection (ATCC) after knocking out the orotidine 5-phosphate decarboxylase (URA3) gene.

[0050] Both the CA10 mouse monoclonal antibody and the CA10 rabbit polyclonal antibody were prepared by the applicant through immunization of animals with the CA10 capsid protein mutant VLP antigen prepared according to Example 4 below. The monoclonal antibody was obtained by expressing and purifying hybridoma cells derived from hybridization of mouse spleen cells and myeloma cells; the polyclonal antibody was obtained by purifying immunized rabbit serum.

[0051] Example 1: Design of mutation sites and synthesis of encoding genes for Coxsackievirus A10 capsid protein mutant The full-length amino acid sequence of the wild-type capsid protein VP1 of Coxsackievirus A10 is shown in SEQ ID NO.2. Site-directed amino acid substitution mutations were performed on the protein's flexible antigen loop, hydrophobic aggregation region, and key VLP assembly sites in this sequence to remove easily degradable sites and optimize the antigen's spatial conformation, resulting in a mutant of the Coxsackievirus A10 capsid protein VP1 with the amino acid sequence shown in SEQ ID NO.1. Specifically, SEQ ID NO.1 is based on SEQ ID NO.2, with the following amino acid sites mutated: The 23rd position Val (valine) is mutated to Ile (isoleucine), the 180th position Val (valine) is mutated to Ala (alanine), and the 284th position Thr (threonine) is mutated to Asn (asparagine).

[0052] Based on the codon preference of Hansenula polymorpha, the coding gene of the Coxsackievirus A10 capsid protein VP1 mutant encoding the amino acid sequence shown in SEQ ID NO.1 was codon optimized to obtain the coding gene of the Coxsackievirus A10 capsid protein VP1 mutant with the nucleotide sequence shown in SEQ ID NO.6, which was then artificially synthesized (DraI and EcoRI restriction sites were added to the 5' and 3' ends, respectively).

[0053] Similarly, the coding genes of Coxsackievirus A10 capsid proteins VP2, VP3, and VP4, whose amino acid sequences are shown in SEQ ID NO.3-5 respectively, were obtained and artificially synthesized (DraI and EcoRI restriction sites were added to the 5' and 3' ends respectively).

[0054] Example 2: Construction of recombinant expression vector The coding genes of the Coxsackievirus A10 capsid protein VP1 mutant, VP2, VP3, and VP4, which were artificially synthesized in Example 1, were cloned into the pUC57 vector, and then ligated into the Hansenula polymorpha expression vector pVMHU by enzyme digestion to construct the recombinant expression vectors pVMHU-CA10VP1, pVMHU-CA10VP2, pVMHU-CA10VP3, and pVMHU-CA10VP4, respectively. Then, the expression cassette elements MOXP-CA10VP2-MOXT from pVMHU-CA10VP2, MOXP-CA10VP3-MOXT from pVMHU-CA10VP3, and MOXP-CA10VP1-MOXT from pVMHU-CA10VP1 were digested with enzymes and sequentially ligated into the expression cassette element MOXP-CA10VP4-MOXT from pVMHU-CA10VP4 in tandem, thereby constructing the recombinant expression vector pVMHU-CA10VP4231 containing four gene expression cassettes: CA10VP4-VP2-VP3-VP1. Its structure is shown below. Figure 1 As shown.

[0055] The specific construction method is as follows.

[0056] The target VP1 mutant, VP2, VP3, and VP4 encoding genes were respectively cloned into the pUC57 recombinant cloning vector containing the A10 capsid protein VP1 mutant, VP2, VP3, and VP4 encoding genes after digestion with DraI and EcoRI. The pVMHU expression vector was digested with SnaBI and EcoRI, and the large fragment of the vector was recovered. The target encoding genes and the large fragment of the vector were ligated to obtain the recombinant expression vectors pVMHU-CA10VP1, pVMHU-CA10VP2, pVMHU-CA10VP3, and pVMHU-CA10VP4, respectively.

[0057] Expression cassette elements MOXP-CA10VP2-MOXT, MOXP-CA10VP3-MOXT, and MOXP-CA10VP1-MOXT were digested and recovered from pVMHU-CA10VP2, pVMHU-CA10VP3, and pVMHU-CA10VP1 using BamHI and SalI, respectively. pVMHU-CA10VP4 was digested with BglII and SalI, and the large vector fragment was recovered. The expression cassette elements MOXP-CA10VP2-MOXT, MOXP-CA10VP3-MOXT, and MOXP-CA10VP1-MOXT were sequentially tandemly ligated into the large vector fragment to obtain the CA10VP4-VP2-VP3-VP1 recombinant co-expression vector pVMHU-CA10VP4231. Sequencing verified that the target coding gene sequence on pVMHU-CA10VP4231 was completely correct, and the reading frame was error-free.

[0058] Example 3: Construction of recombinant engineered Hansenula polymorpha The recombinant expression vector pVMHU-CA10VP4231 was transformed into uracil-deficient (Ura3-) Hansenula polymorpha to achieve simultaneous co-expression of capsid proteins VP4, VP2, VP3, and VP1 mutants in Hansenula polymorpha cells, which then automatically assembled into VLP particles. The main procedure is as follows: The recombinant expression vector pVMHU-CA10VP4231 was electroporated into uracil-deficient (Ura3-) Hansenula polymorpha competent cells. The cells were plated on selection medium, and single colonies were picked for PCR identification and sequencing verification. Through subculturing, positive engineered strains stably expressing the Coxsackievirus A10 capsid protein mutant were obtained. The specific steps are as follows: (1) Linearization of recombinant expression vectors pVMHU-CA10VP4231 was amplified and extracted, and then digested with Bgl II. The target fragment was then recovered using a magnetic bead DNA gel recovery kit, eluted with 30 μL of sterile water preheated to 55°C, and the DNA was quantified by measuring OD260 nm. The linearized target fragment was diluted to 100 ng / μL and finally stored at -20°C for later use.

[0059] (2) Hansenula polymorpha cell treatment Select a single clone of the uracil-deficient (Ura3-) Hansenula polymorpha host cell and inoculate it into an Erlenmeyer flask containing 10 mL of YPD liquid medium. Incubate at 30°C for 16 h. Transfer 3 mL of the bacterial culture to 100 mL of YPD medium and incubate at 30°C for 4–6 h until the OD600 nm reaches approximately 1.2. Centrifuge at 6000 rpm, 4°C for 10 min and discard the supernatant. Resuspend the bacterial cells in 50 mL of TED solution (containing 25 mmol / L DTT, pH 7.0 Tris-HCl buffer) and incubate at 37°C for 20 min. Then centrifuge at 6000 rpm, 4°C for 10 min and discard the supernatant. Wash the bacterial cells with 50 mL of pre-chilled STM solution (i.e., sucrose-TrisMgCl2 solution), centrifuge at 6000 rpm, 4°C for 10 min, discard the supernatant, and repeat the washing once. Resuspend the bacterial cells in 50–200 μL of ice-cold STM solution according to the bacterial volume. μL was dispensed into each EP tube to obtain competent Hansenula polymorpha cells for electrotransformation.

[0060] (3) Recombinant expression vector yeast electroporation Take one 100 μL of Hansenula polymorpha competent cells, add 20 μL of linearized recombinant expression vector, mix well, and then add to a pre-chilled electroporation cuvette. Incubate on ice for 2 min. Place the cuvette on an electroporator, set the yeast electroporation program, and immediately place it on ice for 2 min after electroporation. Wash the cells from the cuvette into an EP tube with 1 mL of YEPD medium and incubate at 37°C for 1 h. Centrifuge at 8000 rpm for 3 min, discard the supernatant, add 150 μL of MD medium, and mix well with the cells. Plate the transformed cells onto MDL plates and incubate at 33°C for one week.

[0061] (4) Screening of transformed yeast cells The transformed colonies were transferred to 10 mL of MD liquid medium and passaged in a shaker at 33°C. During passage, PCR was used to screen for recombinant strains. A small amount of bacterial cells was used as a template, and primers MP1 (5'-cacggtggtgacatcaatctaaagt-3') and MT1 (5'-tccttccacgtctccttgctagcg-3') were used to identify recombinant strains. The PCR screening and identification results are shown below. Figure 2 The recombinant positive strains showed three distinct amplification bands (894 bp VP1, 765 bp VP2 overlapping with 720 bp VP3, and 207 bp VP4), consistent with the positive control. PCR-positive strains were passaged for 7-10 generations to stabilize the integration of the exogenous gene. After inoculation into YPD medium, the strains were cultured at 33°C in a shaker. The resulting cultures were then frozen at -70°C for immunospecific identification. Strains with high expression levels were selected as engineered strains for the fermentation and purification of Coxsackievirus A10 capsid protein.

[0062] Example 4: Induction, purification, and detection of Coxsackievirus A10 capsid protein mutant The recombinant Hansenula polymorpha constructed in Example 3 was fermented and induced to express the strain. The resulting stock solution containing the Coxsackievirus A10 capsid protein mutant was then purified, and the product was analyzed. The specific methods are as follows.

[0063] (1) Fermentation culture and induced expression After selecting a recombinant strain with relatively stable growth performance, it was inoculated at a ratio of 0.5% into 20 mL of primary medium (0.67% yeast nitrogen source, 0.5% ammonium sulfate, 1% glucose, 1% glycerol) and cultured at 34℃ in a shaker for 24 h. Then, it was inoculated at a ratio of 10% into 200 mL of secondary medium (0.67% yeast nitrogen source, 0.5% ammonium sulfate, 2% glycerol) and cultured at 34℃ in a shaker for 24 h. Finally, it was inoculated into a 3 L tank of medium (0.3% magnesium sulfate heptahydrate, 0.4% potassium chloride, 0.04% sodium chloride, 0.67% ammonium dihydrogen phosphate, 0.35% ammonium sulfate, 0.5% glycerol) for fermentation. The fermentation temperature was set at 33℃, pH 5.5, and aeration and rotation speed were controlled to ensure dissolved oxygen (DO) was not lower than 20%. After fermentation until the carbon source was depleted and DO recovered, the pH was adjusted to 6.0, the temperature was 30℃, and methanol was added to induce fermentation for 46–58 h while maintaining DO at 30%.

[0064] (2) Purification Cell harvesting: Centrifuge at 6000 g for 20 min, discard the supernatant, and harvest the cells.

[0065] Resuspending the bacterial cells: The bacterial cells were resuspended in the lysis buffer (50 mM PB, 2 mM EDTA-Na2, 0.5% Tween 80, pH 8.0) at a ratio of 1:4.

[0066] High-pressure homogenization and disruption: Use a high-pressure homogenizer with a working pressure of 1200 bar to disrupt bacterial cells 3-5 times.

[0067] Centrifugation clarification: Centrifuge the above cell lysate at 6000 g for 40 min and collect the supernatant.

[0068] Ultrafiltration solution replacement: Use a 300 kDa membrane to ultrafilter the broken supernatant, and replace the solution with 4 times the amount of cation A solution (20 mM PB, 0.1% Tween 80, pH 7.5) each time, for a total of 3 to 6 times.

[0069] Cation chromatography: NanoGel-50SP packing material was used. After column equilibration with cation A solution, the ultrafiltrate was loaded and then eluted with cation A solution. The flow-through with UV 280 nm greater than 200 mAU was collected.

[0070] Ultrafiltration concentration: The sample after cation exchange chromatography is coated with anion A solution (20mM PB, 0.12 M Nacl, 0.1% Tween 80, pH 6.8) using a 300 kDa membrane and the solution is changed 4 times each time, for a total of 3 to 6 times.

[0071] Anion chromatography: Nuvia HP-Q packing material was used. After column equilibration with anion A solution, the ultrafiltrate was loaded onto the column and then eluted with elution buffer (20 mM PB, 0.5 M Nacl, 0.1% Tween 80, pH 6.8) to collect the UV absorption target peak at 280 nm.

[0072] Ultrafiltration concentration: The target peak sample collected after anion exchange chromatography is encapsulated in a 300 kDa membrane with molecular sieve buffer (20 mM PB, 0.9% NaCl, 0.01% Tween 80, pH 7.0) and the buffer is changed 4 times each time, for a total of 3 to 6 times.

[0073] Molecular sieve chromatography: The molecular sieve chromatography column was packed with NW Rose 6 FF packing material. After equilibrating the column with molecular sieve buffer (20 mM PB, 0.12 M Nacl, 0.01% Tween 80, pH 7.0), the ultrafiltration concentrate was loaded and the second UV 280 nm ultraviolet absorption peak was collected.

[0074] Sterilization filtration: The elution peaks collected by molecular sieve chromatography were sterilized at the end using a 0.22 μm sterilization filter to obtain the stock solution of Coxsackievirus A10 capsid protein mutant.

[0075] (3) Detection of purified products A: ELISA detection of antigen specificity of purified product The antigen-specific specific activity of the purified product was quantitatively detected using a double-antibody sandwich ELISA method, as follows: CA10 monoclonal antibody was coated overnight at 4°C using pH 9.6 carbonate buffer and PBST containing 0.5% bovine serum albumin at 37°C for 1 h. The purified product (with positive control, negative control, blank control, and standard control) was added and incubated at 37°C for 1 h, followed by washing with PBST three times for 5 minutes each time. Then, HRP-labeled anti-CA10 rabbit polyclonal antibody was added and incubated at 37°C for 1 h, followed by washing with PBST three times for 5 minutes each time. TMB was added for color development for 10-30 min, and the reaction was terminated with 2 mol / L H2SO4. The results were measured on an ELISA reader at a detection wavelength / reference wavelength of 450 nm / 630 nm.

[0076] The absorbance data of the standard and the sample were entered into an Excel spreadsheet template and analyzed using the Parallel Line Assay module 23 in StatisticalAnalysis software. The relative potency (RP) values ​​of the Coxsackievirus A10 capsid protein mutant and the wild-type capsid protein were obtained. The VLP antigen-specific specific activity of the Coxsackievirus A10 capsid protein mutant and the wild-type capsid protein were calculated to be 234.33 U / μg and 170.25 U / μg, respectively. The results showed that the VLP antigen-specific specific activity of the Coxsackievirus A10 capsid protein mutant was significantly higher than that of the wild-type capsid protein.

[0077] B: SDS-PAGE electrophoresis purity A 12% separating gel and a 6% stacking gel were prepared. The purified product was treated with SDS-PAGE buffer and loaded onto the gel, along with a standard positive control. Electrophoresis was performed at a constant current of 12 mA for 20 min, followed by 24 mA for 1.5 h. After electrophoresis, the gel was removed, stained with Coomassie Brilliant Blue, and analyzed using a gel imaging system to determine the target protein. SDS-PAGE results are shown below. Figure 3 This indicates that the VP electrophoresis bands of the recombinant mutant CA10VLP have better purity compared to the VP electrophoresis bands of the recombinant wild-type CA10VLP.

[0078] C: Identification of CA10 virus-like particles The purified CA10 capsid protein VLP stock solution and CA10 wild-type capsid protein VLP particles were observed under phosphotungstic acid negative staining electron microscopy. The results are as follows: Figure 4 As shown, the VLP particles of the CA10 capsid protein mutant are more uniform in size than the VLP particles of the CA10 wild-type capsid protein, with a size of about 30 nm, which is consistent with the theoretical value.

[0079] Example 5: Preparation of the immunomodulatory composition The various immunizing compositions shown in Table 1 were prepared separately. The method involved diluting each capsid protein stock solution to 40 µg / mL with 0.9% sodium chloride, then slowly adding it to a 0.9% sodium chloride diluted aluminum hydroxide adjuvant solution (aluminum content 1.0 mg / mL) at a flow rate of 20–30 mL / min, while stirring at 200–600 rpm to form a mixture. After addition, stirring was continued at 200–600 rpm for 10–20 min to ensure thorough mixing and preparation of the immunizing compositions, i.e., the finished vaccine formulations. The preparation methods for CA10 wild-type capsid protein, CA6 wild-type capsid protein, CA16 wild-type capsid protein, and EV71 wild-type capsid protein were as described in Examples 1–4. The amino acid sequences of the CA6 wild-type capsid protein used are available in GenBank: AWI73800.1 (VP1: 566-870; VP2: 70-325; VP3: 326-565; VP4: 1-69), the amino acid sequences of the CA16 wild-type capsid protein are available in GenBank: UYB01042.1 (VP1: 566-862; VP2: 70-323; VP3: 324-565; VP4: 1-69), and the amino acid sequences of the EV71 wild-type capsid protein are available in GenBank: FJ606449.1 (VP1: 566-862; VP2: 70-323; VP3: 324-565; VP4: 1-69).

[0080] Table 1 Formulations of various immune compositions

[0081] Example 6: Evaluation of Immunization Effect in Mice SPF-grade BALB / c mice aged 6-8 weeks were selected and divided into 8 groups: the vaccine groups prepared according to formulations 1-7 in Example 5 and the aluminum adjuvant control group (aluminum hydroxide with an aluminum content of 0.5 mg / mL + 0.9% physiological saline), with 10 mice in each group. Each mouse received an intraperitoneal immunization dose of 0.5 ml / mouse / time, twice a day on days 0 and 14. Blood was collected from the eyeballs of each mouse after 28 days, incubated at 37°C for 1 hour, then at 4°C overnight, centrifuged at 5000 g for 10 min, and the serum was separated and stored at -20°C. The neutralizing antibody titer was determined using the microcytopathic effect method, and the neutralizing antibody GMT was calculated. The neutralizing antibody titer was the reciprocal of the serum dilution factor. Human malignant embryonic rhabdomyosarcoma (RD) cells were used for the assay, cultured at 37°C for 7 days, and cytopathic effects were observed. Results showed: 1) After immunization with vaccines formulated 1-7, mice produced high-titer specific neutralizing antibodies against the four corresponding enteroviruses, with an antibody seroconversion rate of 100%; 2) Compared with monovalent and multivalent vaccines prepared from CA10 wild-type capsid protein VLP, monovalent and multivalent vaccines prepared from CA10 capsid protein mutant VLP showed significantly increased levels of neutralizing antibodies against CA10. 3) After immunization with Formula 6 (containing CA10 capsid protein mutant VLP antigen), there was no immune interference or inhibition between different types; after immunization with Formula 7 (containing CA10 wild-type capsid protein VLP antigen), CA10 and CA6 types showed slight interference and inhibition compared with the corresponding Formula 2 and Formula 3 monovalent vaccines.

[0082] Table 2 summarizes the neutralizing antibody GMT data from the immunization assays of various vaccine formulations; trend analysis is provided below. Figure 5 .

[0083] Table 2 Summary of neutralizing antibody GMT data for each vaccine formulation

[0084] Example 7: Protection test of maternally inherited antibodies in suckling mice against viral challenge The vaccines formulated in Example 5 (Formula 6) and Formula 7 were serially diluted to prepare four doses for immunizing mother mice. Then, challenge tests were conducted on suckling mice with four types of viruses (CA10, CA6, CA16, and EV71) to evaluate the immunoprotective effect of the vaccines.

[0085] SPF-grade BALB / c mice, 6-8 weeks old, 18-22 g, 72 females and 18 males were used. Formulas 6 and 7 were used to prepare four vaccine immunization groups for each challenge serotype, and four aluminum hydroxide adjuvant control groups were also prepared, for a total of 36 groups, with 2 female mice in each group. Intraperitoneal immunization was administered at 0.5 ml / mouse / time, twice a day on days 0 and 14. Male and female mice were introduced into the same cage 7 days after the first immunization at a ratio of 4:1. After confirming pregnancy, the female mice were separated from the male mice. On the day the female gave birth, the suckling mice were injected intraperitoneally with 0.05 ml of a 50-fold LD50 solution. 50 Viral fluid (LD50 determined before experiment) 50 The mother mice were grouped and their offspring were injected with the corresponding type of virus. After the challenge, the health status of the offspring mice was observed, and the mortality of the mice was counted starting from the 3rd day.

[0086] The results showed that in the four aluminum hydroxide adjuvant control groups, neonatal mice began to show symptoms 3-4 days after challenge and all died within 7-8 days. In contrast, neonatal mice in the four dose groups of each of the three CA10, CA6, CA16, and EV71 types in Formula 6 and the first three dose groups of CA10 type in Formula 7 grew healthily after challenge and showed no signs of illness; the survival rate (protection rate) was 100%. The protection rate in the fourth dose group of CA10 type in Formula 7 (1:640, 0.015625 μg) was 70%. The protection rate statistics are shown in Table 3 below. The survival and protection rate data of mice in the 1:640 (0.015625 μg) dose groups of CA10 type in Formulas 6 and 7 and the aluminum adjuvant control group are summarized in Table 4 below. The trend of the protection rate is shown in [Table 4]. Figure 6 .

[0087] Table 3 Summary of maternal antibody protection rate data in challenge-protective experiments in suckling mice (14 days)

[0088] Table 4. Summary of survival and protection rates of mice in the maternal antibody challenge protection test of CA10 type 1:640 (0.015625 μg) dose group and aluminum adjuvant control group.

[0089] The results showed that the quadrivalent vaccines of formulations 6 and 7 had good protective effects against four types of viruses: EV71, CA16, CA6 and CA10. Moreover, the quadrivalent vaccine prepared from the CA10 capsid protein mutant VLP had better immune protection against CA10 infection than the quadrivalent vaccine prepared from the CA10 wild-type capsid protein VLP.

[0090] Example 8: Stability Study of the Immunoassay Composition The immunogenic composition of Formula 6 prepared in Example 5 was subjected to forced high-temperature storage at 37°C for 45 days and accelerated storage at 25°C for 6 months, respectively. 2.0 ml of each composition was added to EP tubes and centrifuged for 5 min. 1.8 ml of the supernatant was collected, and 1.8 ml of desorption buffer (0.112 g disodium EDTA, 8.82 g disodium hydrogen phosphate dodecahydrate, 10 µl Tween 20, diluted to 100 ml with water, freshly prepared before use) was added to the EP tubes. The mixture was thoroughly mixed and placed in a 37°C water bath for 16–20 h. After thorough mixing, the mixture was centrifuged at 6500 g for 5 min, and the desorption supernatant was collected. The supernatant was concentrated 10-fold using a 30 kD ultrafiltration concentrator and observed under a phosphotungstic acid negative staining electron microscope. The results showed that the VLP size was 37–42 nm, consistent with the theoretical value. The VLP particles maintained their structural integrity after adsorption and desorption by aluminum adjuvant, exhibiting stable quality characterization properties, thus ensuring good and stable immunogenicity of the finished vaccine. See electron microscope images at 37℃ for 45 days. Figure 7 Left image, electron microscope image at 25℃ for 6 months (see below) Figure 7 The image on the right.

[0091] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims and their equivalents, this invention is also intended to include these modifications and variations. The above embodiments or implementations are merely illustrative examples of this invention, and it can also be implemented in other specific ways or forms without departing from its gist or essential characteristics. Therefore, the described embodiments should be considered illustrative rather than limiting in any respect. The scope of this invention should be defined by the appended claims, and any changes equivalent to the intent and scope of the claims should also be included within the scope of this invention.

Claims

1. A quadrivalent vaccine immunization composition, characterized in that: The immune composition comprises a Coxsackievirus A10 capsid protein mutant and / or its encoding gene, a Coxsackievirus A16 capsid protein and / or its encoding gene, a Coxsackievirus A6 capsid protein and / or its encoding gene, and an Enterovirus 71 capsid protein and / or its encoding gene. The Coxsackievirus A10 capsid protein mutant, the Coxsackievirus A16 capsid protein, the Coxsackievirus A6 capsid protein, and the Enterovirus 71 capsid protein are all assembled from capsid proteins VP1, VP2, VP3, and VP4. The amino acid sequence of the capsid protein VP1 of the Coxsackievirus A10 capsid protein mutant is shown in SEQ ID NO.

1.

2. The immune composition according to claim 1, characterized in that: The amino acid sequences of the Coxsackievirus A16 capsid protein, the Coxsackievirus A6 capsid protein, and the Enterovirus 71 capsid protein are all wild-type amino acid sequences.

3. The immune composition according to claim 1, characterized in that: The amino acid sequences of capsid proteins VP2, VP3, and VP4 of the Coxsackievirus A10 capsid protein mutant are wild-type amino acid sequences.

4. The immune composition according to claim 1, characterized in that: The immune composition comprises a Coxsackievirus A10 capsid protein mutant, a Coxsackievirus A16 capsid protein, a Coxsackievirus A6 capsid protein, and an Enterovirus 71 capsid protein.

5. The immune composition according to any one of claims 1-4, characterized in that: The immune composition further comprises an adjuvant.

6. The immune composition according to claim 5, characterized in that: The adjuvant is selected from one or more of complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum hydroxide adjuvant, and aluminum phosphate adjuvant.

7. The immune composition according to any one of claims 1-4, characterized in that: The immune composition further comprises a diluent and / or a stabilizer.

8. The immune composition according to claim 7, characterized in that: The diluent is selected from one or more of phosphate buffer, histidine buffer, citrate buffer, and physiological saline.

9. The immune composition according to claim 7, characterized in that: The stabilizer is selected from one or more of the following: sucrose, trehalose, mannitol, sorbitol, glycerol, glycine, L-histidine, arginine, human serum albumin, gelatin, Tween 20, Tween 80, polyethylene glycol, and dextran.

10. Use of the immune composition according to any one of claims 1-9 for the preparation of a vaccine for the prevention of hand-foot-mouth disease or herpetic pharyngitis.

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