Coxsackievirus a10 strain, construction method therefor, and application thereof

Abstract

Provided are a coxsackievirus A10 strain, a construction method therefor, and an application thereof. The coxsackievirus A10 strain rCv-A10-H comprises a VP1 capsid protein and VP2 capsid protein of the coxsackievirus A10 strain, wherein the VP1 capsid protein has an amino acid sequence as shown in SEQ ID NO: 1; and the VP2 capsid protein has an amino acid sequence as shown in SEQ ID NO: 2. Introducing specific amino acid substitutions into a viral structural protein adapts the CV-A10 strain to grow in Vero cells, improving humoral immunogenicity while proliferative capacity and stability are unaffected, and exhibiting a cross-protection effect on other strains of the same serotype, thereby providing a technical basis for selecting and developing coxsackievirus A10 and other enterovirus vaccine strains for vaccine production.

Description

Coxsackievirus A10 strain, its construction method, and its applications Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a Coxsackievirus A10 strain, its construction method, and its application. Background Technology

[0002] CV-A10 is a type of Coxsackievirus A, primarily associated with hand-foot-and-mouth disease (HFMD), particularly affecting children. CV-A10 is a single-stranded, positive-sense RNA virus with an icosahedral capsid composed of 60 capsid proteins (VP1, VP2, VP3, and VP4). Currently, there is no corresponding vaccine for CV-A10. Developing a CV-A10 vaccine requires screening candidate strains. Research results indicate that CV-A10 vaccine candidates generally exhibit poor humoral immunogenicity. Whether using multivalent inactivated vaccine technology or multivalent virus-like particle vaccine technology, the level of neutralizing antibodies induced by CV-A10 vaccine candidates is several times lower than that of other serotypes, severely hindering the development of multivalent HFMD vaccines, including those targeting CV-A10. Summary of the Invention

[0003] In view of this, the present invention provides a Coxsackievirus A10 strain, its construction method and application, which can improve the humoral immunogenicity of the CV-A10 strain and provide a technical basis for breeding Coxsackievirus A10 and other enterovirus vaccine strains that can be used for vaccine production.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] In a first aspect, the present invention provides a Coxsackievirus A10 strain rCV-A10-H, wherein the strain rCV-A10-H includes a VP1 capsid protein and a VP2 capsid protein, wherein the amino acid sequence of the VP1 capsid protein is shown in SEQ ID NO:1; and the amino acid sequence of the VP2 capsid protein is shown in SEQ ID NO:2.

[0006] Preferably, the strain rCV-A10-H further includes VP3 capsid protein and VP4 capsid protein, wherein the amino acid sequence of VP3 capsid protein is shown in SEQ ID NO:3; and the amino acid sequence of VP4 capsid protein is shown in SEQ ID NO:4.

[0007] Secondly, the present invention provides a genome of a Coxsackievirus A10 strain rCV-A10-H, the nucleotide sequence of which is shown in SEQ ID NO:5.

[0008] Thirdly, the present invention provides a virus-like particle of Coxsackievirus A10 strain rCV-A10-H, comprising VP1 capsid protein, VP2 capsid protein, VP3 capsid protein and VP4 capsid protein, wherein the amino acid sequence of VP1 capsid protein is shown in SEQ ID NO:1; the amino acid sequence of VP2 capsid protein is shown in SEQ ID NO:2; the amino acid sequence of VP3 capsid protein is shown in SEQ ID NO:3; and the amino acid sequence of VP4 capsid protein is shown in SEQ ID NO:4.

[0009] Fourthly, the present invention provides the application of the aforementioned virus-like particles in the preparation of medicines for the prevention of hand-foot-mouth disease.

[0010] Fifthly, the present invention provides a pharmaceutical composition for the prevention of hand-foot-mouth disease, the pharmaceutical composition comprising the aforementioned virus-like particles and a pharmaceutically acceptable carrier.

[0011] Preferably, the pharmaceutical composition is a vaccine composition.

[0012] In a sixth aspect, the present invention also provides a method for constructing a Coxsackievirus A10 strain rCV-A10-H, including a step of replacing key amino acid sites in the structural protein region of the Coxsackievirus A10 strain CV-A10-L, or a step of introducing nucleotide mutations in the structural protein region of the Coxsackievirus A10 strain CV-A10-L to achieve replacement of key amino acid sites.

[0013] The key amino acid sites are those that determine humoral immunogenicity.

[0014] Preferably, the determination of key sites includes the following steps:

[0015] By comparing the binding sites of the structural protein regions of the Coxsackievirus A10 strain CV-A10-FJ-01 with those of the neutralizing monoclonal antibody, and the corresponding amino acid sites of the structural protein regions of the low humoral immunogenic Coxsackievirus A10 strain CV-A10-L, sites with different amino acids were identified as potential key amino acid sites.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] (1) This invention provides a Coxsackievirus A10 strain rCV-A10-H that can improve the humoral immunogenicity of the CV-A10 strain, providing a technical basis for the selection of Coxsackievirus A10 and other enterovirus vaccine strains that can be used for vaccine production.

[0018] (2) This invention introduces an amino acid substitution into the P1 site of the structural protein coding region of the CV-A10-L strain. The modified strain's proliferation ability and stability in cells are not affected, but its humoral immunogenicity is significantly improved. Furthermore, the cross-protective effect of inactivated immunization against other CV-A10 strains within the same serotype is better than that of other vaccine candidate strains. Attached Figure Description

[0019] Figure 1 shows the alignment results of strains CV-A10-L and CV-A10-FJ-01 provided in Example 1 of the present invention in the 2G8 monoclonal antibody binding region.

[0020] Figure 2 is a schematic diagram of the infectious clones pBR322-rCV-A10-L and pBR322-rCV-A10-H provided in Embodiment 2 of the present invention;

[0021] Figure 3 shows the growth curves of the recombinant viruses rCV-A10-L and rCV-A10-H provided in Example 3 of the present invention on Vero cells;

[0022] Figure 4 shows the heat resistance curves of rCV-A10-L and rCV-A10-H provided in Example 4 of the present invention;

[0023] Figure 5 is a comparison of the levels of neutralizing antibodies induced by rCV-A10-L and rCV-A10-H provided in Example 5 of the present invention;

[0024] Figure 6 is a comparison of the cross-neutralization levels of neutralizing antibodies induced in mice by the three CV-A10 vaccine candidate strains provided in Example 6 of this invention. Detailed Implementation

[0025] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0026] Virus materials:

[0027] The Coxsackievirus A10 strain CV-A10-L can adapt to Vero cell growth, but the level of induced neutralizing antibodies is low, so it was preserved in our laboratory.

[0028] Coxsackievirus A10 strains CV-A10-C1 and CV-A10-C2 can adapt to Vero cell growth and are candidate vaccine strains preserved in this experiment.

[0029] Strain and monoclonal antibody structural information:

[0030] The sequence of Coxsackievirus A10 strain CV-A10-FJ-01 was downloaded from NCBI (GenBank accession no. KY012321).

[0031] The structural analysis information for monoclonal antibodies 2G8 and CV-A10-FJ-01 was downloaded from the PDB database (PDB accession numbers: 6AD0).

[0032] Cell culture:

[0033] Vero cells were cultured in DMEM medium supplemented with 10% newborn calf serum, with no serum added to the maintenance medium. RD cells were cultured in MEM medium supplemented with 10% newborn calf serum, with no serum added to the maintenance medium.

[0034] Main reagents:

[0035] Viral RNA extraction kit, DNA gel extraction kit, and plasmid extraction kit were purchased from QIAGEN GmbH, Germany. Reverse transcription kit... II. DNA polymerase The ClonExpress II seamless ligation kit was purchased from Nanjing Novizan Biotechnology Co., Ltd. The Lipofectamine transfection reagent was also used. TM Purchased from Thermo in the United States in 2000.

[0036] Example 1: Sequence determination and sequence alignment of CV-A10 strain

[0037] Genomic RNA of the CV-A10-L strain was extracted using the QIAamp viral RNA Mini Kit (Qiagen, German), and cDNA was obtained by reverse transcription using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). Viral cDNA was amplified by PCR using Phanta Master (Vazyme, Nanjing, China) and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequences were assembled using Seqman software (DNAStar, Wisconsin, USA).

[0038] Based on the structural analysis results in the database (PDB: 6AD0) and literature reports, the main binding amino acid sites for the neutralizing monoclonal antibody 2G8 on the structural proteins of the CV-A10-FJ strain were identified, primarily in the EF loop of structural protein VP2, the AB loop of VP3, and the C-terminus of VP1. Using Megalign (DNAStar, Wisconsin, USA) software, the amino acid sites binding to the monoclonal antibody 2G8 on the structural proteins of CV-A10-L and CV-A10-FJ-01 were compared, revealing a difference at position 162 of VP2 and position 283 of VP1 (Figure 1).

[0039] Example 2: Construction of Recombinant Virus

[0040] While the CV-A10-FJ-01 strain can be used to isolate the neutralizing monoclonal antibody 2G8, it is a strain cultured in RD cells and cannot be used for human vaccine production. CV-A10-L can adapt to Vero cell growth, but its humoral immunogenicity is poor. To attempt to optimize the CV-A10-L strain to improve its humoral immunogenicity, a reverse genetics system for this strain was constructed using the CV-A10-L genome as a template. The infectious cDNA plasmid was pBR322-rCV-A10-L (Figure 2), which was rescued from Vero cells. A nucleotide mutation was introduced at position 162 of the structural protein VP2, replacing histidine (H) with tyrosine (Y), and at position 283 of VP1, replacing valine (V) with isoleucine (I), to construct the infectious cDNA plasmid pBE322-rCV-A10-H (Figure 2), from which the recombinant virus rCV-A10-H was rescued. The rescued virus was passaged to P3 in Vero cells, and the genome was extracted for RT-PCR and sequencing to verify the correctness of the rescued virus sequence.

[0041] Example 3: Growth characteristics of recombinant virus on Vero cells

[0042] Virus titration was performed using 96-well cell culture plates, with each sample area divided into two partitions (4 rows × 9 columns). The viral stock solution was serially diluted 10-fold, with four replicates for each dilution. Different dilutions were added sequentially to the sample area at a rate of 100 μL / well, with 5 × 10⁶ μL added to each well. 4 After incubation at 37°C and 5% CO2 for 7 days, the cytopathic effect was observed under a microscope. Viral titers were calculated using the Reed-Muench method and recorded as CCIDs. 50 / mL (50% cell culture infectious dose permilliliter).

[0043] Vero cells were infected with the virus at a multiplicity of infection (MOI) of 10. Cell cultures were harvested at 0 h, 6 h, 12 h, 18 h, 24 h, and 48 h post-infection. The supernatant was collected by centrifugation after three freeze-thaw cycles and virus titration was performed. The virus titer at different time points was recorded, and virus growth curves were plotted (Figure 3). The results showed that rCV-A10-H and rCV-A10-L had similar proliferation abilities in Vero cells, and the substitution of two amino acid sites did not affect the proliferation of the rCV-A10-H strain in Vero cells.

[0044] Example 4: Stability of recombinant virus

[0045] dilute rCV-A10-H and rCV-A10-L to 10. 7 CCID50 / mL was aliquoted into 1.5mL centrifuge tubes at 500μL / tube and treated at 37℃, 40℃, 43℃, 46℃, 49℃, 52℃, 54℃, 56℃, and 58℃ for 30 min, respectively, before being transferred to ice. The viral titer of each tube was determined, and heat resistance curves were plotted (Figure 4). The results showed that the viral titer of rCV-A10-H and rCV-A10-L decreased to a similar degree with increasing temperature, and the substitution of two amino acid sites did not affect the stability of the rCV-A10-H strain.

[0046] Example 5: Determination of neutralizing antibodies in mice immunized by intraperitoneal injection of recombinant virus

[0047] Twenty-three 6-week-old female Balb / c mice were randomly divided into three groups: five mice were injected with PBS as a blank control, nine mice were injected with rCV-A10-L, and nine mice were injected with rCV-A10-H. The mice were immunized with rCV-A10-H via intraperitoneal injection at weeks 0 and 2. 8 CCID 50 / mL viral solution. Blood was collected from the orbital rim during week 3. After centrifugation to separate the serum, it was inactivated at 56°C for 30 min and then aliquoted and stored at -20°C. Neutralization assay to be performed.

[0048] Serum samples were diluted 2-fold with MEM, with two replicates per sample. 100 μL of MEM was added to column A of a 96-well plate, and 50 μL of MEM was added to each well in columns B through H. The serum samples in column A were serially diluted 2-fold up to column H, with 50 μL discarded after the last dilution. CV-A10-L was used as the neutralizing stock and diluted to 100 CCID. 50 / 50μL, pipette 50μL and vertically drop it into the diluted serum sample. Incubate the 96-well plate at 37°C for 2 hours. After neutralization, add the digested RD cells at a ratio of 1×10⁻⁵. 5 Add the solution at a density of / mL to a 96-well plate. Simultaneously, determine the titer of the neutralizing agent by serial dilution (10-fold) to ensure the neutralizing agent is diluted to 30-300 CCID.50 / hole.

[0049] Balb / c mice were immunized with supernatants of rCV-A10-H and rCV-A10-L viruses of the same titer, and the level of neutralizing antibodies against CV-A10-L in mouse serum was measured (Figure 5). The results showed that rCV-A10-H induced a significant increase in the level of neutralizing antibodies in mice compared with rCV-A10-L, and the substitution of two amino acid sites could significantly improve the humoral immunogenicity of the CV-A10 strain.

[0050] Example 6: Evaluation of cross-neutralization capacity of inactivated recombinant virus within immune serotypes

[0051] The vaccine candidate strains CV-A10-C1, CV-A10-C2, and rCV-A10-H were purified and inactivated with formaldehyde. Thirty-six experimental animals were divided into six groups and immunized with 1 or 4 μg of inactivated CV-A10-C1, CV-A10-C2, and rCV-A10-H via intraperitoneal injection at weeks 0 and 2, respectively. At week 4, blood was collected from the orbital rim and centrifuged to separate serum, which was then inactivated at 56°C for 30 min and aliquoted and stored at -20°C. The neutralization test was performed on all serum samples using the same methods as in Example 5, with rCV-A10-L, CV-A10-C1, and CV-A10-C2 as the neutralizing agents.

[0052] Mice were immunized with CV-A10 vaccine candidate strains rCV-A10-H, CV-A10-C1, and CV-A10-C2 at doses of 1 μg and 4 μg respectively, and the levels of neutralizing antibodies against different strains in mouse serum were measured. Using CV-A10-L as the neutralizing agent, the level of neutralizing antibodies induced by rCV-A10-H in mice was significantly higher than that CV-A10-C1 and CV-A10-C2. Using CV-A10-C1 as the neutralizing agent, the level of neutralizing antibodies induced by rCV-A10-H in mice was similar to that CV-A10-C1, both significantly higher than CV-A10-C2. Using CV-A10-C2 as the neutralizing agent, the level of neutralizing antibodies induced by rCV-A10-H in mice was slightly higher than that CV-A10-C2, and both were significantly higher than CV-A10-C1 (Figure 6). The results showed that the recombinant virus rCV-A10-H obtained by replacing two amino acid sites had a better cross-neutralizing ability against CV-A10 serotype endotoxin strains than other vaccine candidate strains when used as an inactivated vaccine.

[0053] This invention identifies key amino acid sites that potentially determine the humoral immunogenicity of CV-A10 by comparing the amino acid sequences of the structural protein regions of the CV-A10-FJ-01 strain used to isolate the neutralizing monoclonal antibody 2G8 for Coxsackievirus A10 with the low humoral immunogenicity CV-A10-L strain adapted to Vero cells. Using the low humoral immunogenicity CV-A10 strain as the parent strain, a reverse genetics system was constructed to obtain the rescue virus rCV-A10-L. Further, two amino acid substitutions (H162Y for VP2 and V283I for VP1) were introduced into the structural protein region of rCV-A10-L to obtain the recombinant virus rCV-A10-H. This strain's proliferation ability and viral stability on Vero cells remained unaffected, but its humoral immunogenicity was significantly enhanced. This invention transforms the low humoral immunogenicity rCV-A10-L strain into the enhanced humoral immunogenicity rCV-A10-H strain by introducing two amino acid substitutions in the structural protein coding region, providing a technical method for the design and optimization of CV-A10 vaccine candidate strains.

[0054] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0055] Gene sequences: all are the corresponding amino acid and nucleotide sequences of the strains mentioned in the specific embodiments above.

[0056] rCV-A10-H amino acid sequence

[0057] The amino acid sequence of the CV-A10-FJ structural protein:

[0058] The amino acid sequence of the CV-A10-L structural protein:

[0059] The nucleotide sequence of rCV-A10-L

[0060] Amino acid sequence of rCV-A10-L

[0061] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A Coxsackievirus A10 strain rCV-A10-H, characterized in that, The strain rCV-A10-H includes: VP1 capsid protein, amino acid sequence as shown in SEQ ID NO:1; and, The amino acid sequence of VP2 capsid protein is shown in SEQ ID NO:

2.

2. The rCV-A10-H strain according to claim 1, characterized in that, The rCV-A10-H strain also includes: VP3 capsid protein, amino acid sequence as shown in SEQ ID NO:3; and, The amino acid sequence of VP4 capsid protein is shown in SEQ ID NO:

4.

3. The genome of a Coxsackievirus A10 strain rCV-A10-H, characterized in that, The nucleotide sequence of the strain rCV-A10-H as described in any one of claims 1-2 is shown in SEQ ID NO:

5.

4. A virus-like particle of Coxsackievirus A10 strain rCV-A10-H, characterized in that, include: VP1 capsid protein, amino acid sequence as shown in SEQ ID NO:1; VP2 capsid protein, amino acid sequence as shown in SEQ ID NO:2; VP3 capsid protein, amino acid sequence as shown in SEQ ID NO:3; and, The amino acid sequence of VP4 capsid protein is shown in SEQ ID NO:

4.

5. The use of the virus-like particles according to claim 4 in the preparation of medicines for the prevention of hand-foot-mouth disease.

6. A pharmaceutical composition for the prevention of hand-foot-mouth disease, characterized in that, The pharmaceutical composition comprises the virus-like particles of claim 4 and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition is a vaccine composition.

8. A method for constructing a Coxsackievirus A10 strain rCV-A10-H, characterized in that, The steps include replacing key amino acid sites in the structural protein region of the Coxsackievirus A10 strain CV-A10-L, or introducing nucleotide mutations in the structural protein region of the Coxsackievirus A10 strain CV-A10-L to achieve replacement of key amino acid sites. The key amino acid sites are those that determine humoral immunogenicity.

9. The method according to claim 8, characterized in that, The identification of key sites includes the following steps: By comparing the binding sites of the structural protein regions of the Coxsackievirus A10 strain CV-A10-FJ-01 with those of the neutralizing monoclonal antibody, and the corresponding amino acid sites of the structural protein regions of the low humoral immunogenic Coxsackievirus A10 strain CV-A10-L, sites with different amino acids were identified as potential key amino acid sites.

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