Preparation and application of a live attenuated Coxsackievirus A6 vaccine vector

Abstract

This invention provides an attenuated vaccine against Coxsackievirus A6 (CVA6), its preparation method, and its application. By comparing a highly pathogenic clinical isolate (CVA6-HeB) with an attenuated strain (CVA6-TW141), the genetic basis of CVA6 virulence was systematically studied. This invention located the core virulence determinant to the P1 capsid region and identified a key lethal amino acid residue (VP3-238) that significantly weakens viral replication in target tissues. Based on this discovery, this invention designed and validated a candidate attenuated live vaccine with significantly reduced lethality.

Description

Technical Field

[0001] This invention relates to the field of viral vaccine technology, and in particular to the preparation and application of a live attenuated Coxsackievirus A6 vector. Background Technology

[0002] Hand, foot, and mouth disease (HFMD) is a major global public health problem, frequently causing large-scale outbreaks. While historically associated with enterovirus A71 (EV-A71) and Coxsackievirus A16 (CVA16), the epidemiological landscape has changed significantly. Coxsackievirus A6 (CVA6) has now become an increasingly prevalent major pathogen worldwide. Notably, CVA6 infection often presents with severe and atypical symptoms, such as widespread skin rashes, nail development, and complications including meningitis and pulmonary edema, highlighting its considerable pathogenic potential. Currently, although several experimental vaccine candidates are under investigation, there is no licensed vaccine or specific treatment for CVA6. This significant deficiency underscores the urgent need to elucidate its pathogenic mechanisms in order to develop effective countermeasures.

[0003] CVA6 is a positive-sense single-stranded RNA virus with a genome of approximately 7.4 kb. Its genome contains a single open reading frame (ORF), which is translated into a polyprotein and subsequently processed into three precursor polypeptides (P1, P2, and P3). The P1 polyprotein is cleaved to form four structural capsid proteins (VP1, VP2, VP3, and VP4), which assemble into icosahedral viral particles. VP1–VP3 are exposed on the viral surface and are key determinants of antigenicity, receptor binding, and host tropism. Conversely, the P2 and P3 precursors are processed into non-structural proteins that promote viral replication and modulate the host immune response. CVA6 employs a dual-receptor entry mechanism: heparan sulfate proteoglycans (HSPGs) mediate initial attachment, while the major receptor, Kringle-containing transmembrane protein 1 (KREMEN1 / KRM1), binds to the canyon region of the capsid, inducing uncoating.

[0004] There are currently no approved vaccines or drugs worldwide for hand, foot, and mouth disease caused by CVA6. Therefore, accelerating the development of a safe and effective CVA6 vaccine is of great significance for reducing the incidence of this disease and alleviating the public health burden. Summary of the Invention

[0005] This invention systematically investigated the genetic basis of CVA6 virulence by comparing a highly pathogenic clinical isolate (CVA6-HeB) with an attenuated strain (CVA6-TW141). The study located the core virulence determinant to the P1 capsid region and identified a key lethal amino acid residue (VP3-238) that significantly weakens viral replication in target tissues. Based on this finding, this invention designed and validated an attenuated live vaccine candidate strain that significantly reduces lethality.

[0006] The first aspect of the present invention provides a method for preparing a live attenuated Coxsackievirus A6 vaccine vector, wherein the nucleic acid sequence encoding the 238th amino acid of the A6 Coxsackievirus VP3 protein is subjected to site-directed mutagenesis, so that the 238th amino acid of the VP3 protein is mutated from the coding sequence of glutamic acid E to the coding sequence of alanine A.

[0007] A second aspect of the present invention provides a nucleic acid molecule comprising: (i) A nucleotide sequence as shown in SEQ ID NO:1; or, (ii) Having at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:1 and having nucleotides 712-714 encoding alanine; the sequence encoding alanine may be specifically selected from “GCA”, “GCU”, “GCG” or “GCC”. (iii) The complementary sequence of (i) or (ii) above.

[0008] In the most preferred embodiment of the present invention, in the sequence described in item (ii), nucleotides 712-714 are “GCA”.

[0009] And / or, preferably, the sequence described in item (ii) of the present invention has at least 98% sequence identity with the nucleotide sequence of SEQ ID NO.1; more preferably, it has at least 98%, 99%, 99.5% or 99.9% sequence identity with the nucleotide sequence of SEQ ID NO.1.

[0010] In a preferred embodiment of the present invention, the nucleic acid comprises the coding sequence of the recombinant protein CVA6-HeB P1, as shown in SEQ ID NO:3. This sequence contains the coding sequence of the VP3 protein after the above-mentioned mutation at amino acid position 238.

[0011] A third aspect of the present invention provides a chimeric virus comprising the nucleic acid molecule described in any one of the above-described technical solutions. Studies of the present invention show that the VP3-E238A mutation does not significantly alter the assembly, morphology, antigenicity, infectivity, or cell-binding ability of viral particles in vivo. Therefore, this site can be combined with other Coxsackievirus optimization schemes known in the art, and the chimeric virus may contain other improved sites besides VP3-E238A.

[0012] A fourth aspect of the present invention provides a recombinant protein comprising the expression product of the nucleic acid molecule described in any one of the foregoing technical solutions, or the chimeric virus described in the foregoing technical solutions, comprising at least: (1) The amino acid sequence is the recombinant sequence VP3-238A as shown in SEQ ID NO:2; or, (2) A sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:2 and the 238th amino acid being A.

[0013] Preferably, the sequence described in item (2) of the present invention has at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO:2; more preferably, it has at least 98%, 99%, 99.5% or 99.9% sequence identity with the amino acid sequence of SEQ ID NO:2.

[0014] Preferably, the recombinant protein of the present invention comprises the recombinant protein CVA6-HeB P1 with the amino acid sequence shown in SEQ ID NO:3.

[0015] The fifth aspect of the present invention provides the application of the nucleic acid molecules, chimeric viruses, and recombinant proteins described in the foregoing technical solutions in the preparation of vaccines against Coxsackievirus A6.

[0016] In this invention, the vaccine can be any one of a DNA vaccine, an mRNA vaccine, a protein subunit vaccine, or a recombinant viral vector vaccine. The vaccine may also include other active ingredients, synergistic ingredients, or adjuvants. The vaccine can be in various formulations such as injectable or oral dosage forms. Compared with the prior art, the beneficial effects of the present invention

[0017] This invention confirms that glutamate at position 238 of the VP3 protein is a key genetic determinant of CVA6 virulence, crucial for efficient viral replication and pathogenicity in vivo. A precise and stable method for preparing attenuated CVA6 strains is provided targeting the E3238A mutation site in VP3. The effectiveness of the cHeB-E3238A mutant as a protective attenuated live vaccine candidate establishes a direct translational pathway from mechanism discovery to public health intervention. Attached Figure Description

[0018] Figure 1 The experimental results show that the virulence determinant cluster of CVA6 is located in the P1 region; among them, Figure 1 The left image in panel A shows the survival rate of mice infected with CVA6 TW141 and CVA6-HeB. Figure 1 The right image in A shows the clinical symptoms of mice infected with CVA6 TW141 and CVA6-HeB. The clinical symptom scoring criteria are as follows: 0 points, healthy; 1 point, reduced activity; 2 points, limb weakness; 3 points, limb paralysis; 4 points, death; the error bar represents SEM. Figure 1 B is a schematic diagram of the infectious clone of chimeric viruses. A series of chimeric viruses were constructed by replacing the 5'UTR, P1, P2, P3 and 3'UTR regions in the CVA6-141 backbone with the corresponding segments of CVA6-HeB. Figure 1 C shows the infection results of different chimeric viruses at low doses, with 500 TCID50 as the reference. 50 At a dose of / animal, chimeric viruses were administered to 2-day-old ICR suckling mice via intraperitoneal infection, and their survival and clinical symptoms were monitored daily. Figure 1 The left image (C) shows the survival rate after infection. Figure 1 The right image (C) shows the clinical status after infection. Figure 2 This is a graph showing the results of point mutations at each point P1 in the example; where: Figure 2 A summarizes the amino acid differences between CVA6-141 and CVA6-HeB in the P1 region; a total of 15 different amino acid residues were identified (labeled as #1-#15), and these sites were individually introduced into infectious clones of CVA6-141 to construct mutant viruses; Figure 2 B is a schematic diagram of an infectious clone of CVA6-141 driven by the T7 promoter (PT7), showing the locations of each mutation. Figure 2 C represents the in vivo virulence assessment results of fifteen CVA6-141 mutant viruses, expressed as 500 TCID⁻¹. 50 The dose was administered intraperitoneally to 2-day-old newborn ICR suckling mice. Survival and clinical symptoms were monitored daily, and the clinical scoring criteria were the same as those used in the previous year. Figure 1 Caption; Figure 2 The left image (C) shows the survival rate after infection. Figure 2 The right image (C) shows the clinical symptoms after infection. Figure 2D is a schematic diagram of the construction of an infectious clone of cHeB-P1 (hereinafter referred to as cHeB-WT) driven by the T7 promoter (PT7); the VP3-E238A (E3238A) point mutation is introduced into the infectious clone; Figure 2 E represents the virulence verification diagram of the complemented mutant virus cHeB-E3238A, with 500 TCID values. 50 The appropriate dose was administered intraperitoneally to 2-day-old newborn ICR suckling mice, and survival and clinical symptoms were monitored daily; the clinical scoring criteria were the same as those used in other vaccines. Figure 1 Caption; Figure 2 The left graph (E) shows the survival rate after infection. Figure 2 The right image (E) shows the clinical symptoms after infection. All data were analyzed using a two-tailed unpaired t-test. ns, p > 0.05, no statistically significant difference; **, p < 0.01; Figure 3 Here is a biochemical characterization diagram of the purified CVA6 cHeB-E3238A mutant virus; among which, Figure 3 A represents the protein composition of the purified virus as analyzed by SDS-PAGE and stained with Coomassie blue. The 10th gradient of the enriched mature virus particles (red arrow) was used for subsequent experiments. Figure 3 B is a negative-stain electron microscopy image of purified CVA6 virus particles, with a scale bar of 100 nm. Figure 3 C represents the antigen binding spectrum of purified CVA6 virus particles to polyclonal anti-CVA6 mouse serum; data are the mean ± SEM values ​​from three replicate wells. Figure 3 D represents the antigen binding spectrum of purified CVA6 virus particles to neutralizing monoclonal antibody 3H7 mouse serum; data are the mean ± SEM values ​​from three replicate wells. Figure 3 E represents the equivalent viral RNA copy number versus infection titer plot. Purified CVA6 virus particles were diluted to the same protein concentration (0.1 µg / mL). Viral genome copy number was determined by absolute RT-qPCR (using plasmid standards), and infection titer was determined by TCID. 50 Legal testing; Figure 3 F represents the results of the equivalent cell binding capacity assay. RD cells at 4°C were bound to an equivalent number of genome copies (1×10⁻⁶). 9CVA6 virus ( / mL) was incubated. Cell-bound viral RNA was quantified by RT-qPCR and normalized to β-actin mRNA. Data are the mean ± SD of three biological replicates. Differences were compared using the Mann-Whitney test. ns indicates no statistical significance, p > 0.05; Figure 4 The results show the viral load in newborn mice; among them, Figure 4 Figure A shows the virus-induced death and clinical symptoms. Two-day-old ICR mice (n=8 per group) were intraperitoneally injected with purified CVA6 cHeB-WT or cHeB-E3238A virus particles (5 ng per mouse) and monitored for 14 days. Figure 4 The left figure shows the survival curve and... Figure 4 Figure A on the right shows the clinical symptom score, and the error bars represent SEM. Figure 4 B represents the viral load test results in the limb muscles, spinal cord, and brain after viral infection, measured on day 4 post-infection using TCID. 50 The viral titer in tissue homogenates was quantified using an assay (n=11 per group), with each symbol representing one mouse. Data were analyzed using a two-tailed unpaired t-test. *, p < 0.05; ***, p < 0.001; Figure 5 The results of the challenge experiment on mice with the cHeB-E3238A attenuated vaccine are shown; among them, Figure 5 A is a schematic diagram of the vaccination and challenge experiment; 1-day-old ICR mice were initially immunized by intraperitoneal injection of purified CVA6 cHeB-E3238A attenuated vaccine (0.5 ng / mouse) or PBS (control group). A booster immunization was given on day 6, followed by challenge with the virulent strain CVA6-S0087b on day 10. Mice used for survival analysis (… Figure 5 Part B) was monitored for 14 days, while mice used for histological analysis (parts CD) were sacrificed and their tissues collected on day 4 after challenge. Figure 5 B represents the results of the challenge experiment after vaccination, where survival rate was monitored within 14 days after challenge with CVA6-S0087b. Figure 5 (Left image B) and clinical score ( Figure 5 (See right figure B), the error bars represent SEM; Figure 5 Figure C shows an H&E stained section of the limb muscle of a mouse on day 4 after infection; the severity of myositis is scored as follows: 0 (none), 1 (mild), 2 (moderate), 3 (severe). Figure 5D is a quantitative graph of muscle pathological damage scoring, representing the histopathological scores of each mouse in graph C. Each symbol represents one animal, and statistical significance was determined using the Mann-Whitney test. ***, p < 0.001. Detailed Implementation

[0019] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Example 1 1. Materials and Methods 1.1 Cells and Viruses

[0020] Human rhabdomyosarcoma cell line (RD cell) and human embryonic kidney cell line (293T cell) were cultured in DMEM (Gibco, USA) containing 10% fetal bovine serum.

[0021] CVA6 strains: CVA6-141 (GenBank ID:KR706309) and CVA6-HeB (GenBank ID:MK106189) were both amplified in RD cells. 1.2 Antibodies

[0022] Polyclonal anti-CVA6 mouse serum was obtained by immunizing BALB / c mice with purified CVA6-HeB virus particles formulated with aluminum adjuvant.

[0023] Monoclonal antibody 3H7 is an IgG2a antibody targeting CVA6. The polyclonal antibody and monoclonal antibody 3H7 were constructed according to the method described in the literature "Molecular mechanisms of receptor recognition and antibody neutralization of coxsackievirus A6" (Ke X, Li X, Liu Z, Liu K, Liu W, Yan X, et al.Molecular mechanisms of receptor recognition and antibody neutralization of coxsackievirus A6. Nat Commun. 2025.). 1.3 Mouse infection model for virulence assessment

[0024] To assess the virulence of the virus in vivo, 2-day-old ICR suckling mice were intraperitoneally injected with wild-type CVA6-141 or CVA6-HeB (500 TCID50).50 / mouse). Mice were observed daily for 14 days, and their survival and clinical symptoms were recorded. The clinical scoring criteria were as follows: 0 points, healthy; 1 point, reduced activity; 2 points, limb weakness; 3 points, limb paralysis; 4 points, death. 1.4 Construction and Rescue of the Chimeric CVA6 Virus

[0025] To locate the virulence determinant cluster, we constructed a chimeric virus based on the CVA6-TW141 infectious clone backbone.

[0026] First, total viral RNA was extracted from the CVA6-HeB strain using Trizol reagent (Vazyme, China). Then, the RNA was reverse transcribed into cDNA using the HiScript III first-strand cDNA synthesis kit (Vazyme, China). Next, each genomic region (5′UTR, P1 capsid region, P2 region, P3 region, or 3′UTR) was amplified from the cDNA template by PCR. Each amplified fragment was assembled into the corresponding site of the linearized CVA6-TW141 backbone plasmid using 2X MultiF Seamless Assembly Mix (ABclonal, China), thereby constructing a set of chimeric infectious clones (named cHeB-5′UTR, cHeB-P1, cHeB-P2, cHeB-P3, and cHeB-3′UTR, respectively).

[0027] To rescue the virus, HEK 293T cells plated in 24-well plates were co-transfected with a chimeric plasmid and a T7 RNA polymerase expression plasmid using Lipofectamine 2000 (Gibco). Cell culture supernatant was collected 72 hours after transfection. The rescued virus was amplified in RD cells. Sanger sequencing was performed after reverse transcription PCR amplification to confirm genome integrity. The final viral stock was titered on RD cells using the TCID50 assay, and its virulence was subsequently assessed in newborn mice as described above. 1.5 Construction and Rescue of CVA6 Mutant Virus

[0028] To identify specific virulence determinants, site-directed mutagenesis was performed on the CVA6-TW141 infectious clone backbone using 2X MultiF Seamless Assembly Mix (ABclonal, China). Figure 2 As shown in Figure A, a set of clones containing 15 mutants (mutants #1–#15) was constructed to systematically test the 19 amino acid differences in the P1 region between CVA6-HeB and CVA6-TW141.

[0029] In addition, using the same assembly strategy, the attenuated mutant VP3-E238A (E3238A) was introduced into the virulent chimeric clone cHeB-P1 (called cHeB-WT) to construct the mutant cHeB-E3238A.

[0030] All constructed mutations were confirmed by Sanger sequencing of the target regions. Subsequently, the mutant virus was rescued, amplified, titered, and its virulence was assessed in newborn mice according to the standard procedures described above. 1.6 Virus purification

[0031] To obtain high-purity virus particles for subsequent analysis, rescued cHeB-WT and cHeB-E3238A viruses were amplified on a large scale in RD cells (500 mL each), and collected after three days of culture. Infected cell cultures underwent two freeze-thaw cycles and were clarified by centrifugation. The virus in the clarified lysate was precipitated overnight at 4°C with 10% polyethylene glycol 8000 and 200 mM NaCl. The precipitate was collected by centrifugation, resuspended in 0.15 M PBS, and clarified again by high-speed centrifugation. The concentrated virus solution was then plated on a 20% sucrose pad and ultracentrifuged at 27,000 rpm for 4 hours. After resuspending the precipitate in PBS, it was further purified by ultracentrifugation at 39,000 rpm for 3 hours using a 10–50% sucrose density gradient.

[0032] Twelve components were collected from the gradient, and the components containing mature viral particles were identified by SDS-PAGE analysis for subsequent characterization. 1.7 Negative staining electron microscopy analysis

[0033] Purified CVA6 cHeB-WT and cHeB-E3238A viral particles (from fraction 10 obtained in Section 1.6) were diluted to 50 µg / mL with PBS, dropped onto glow discharge-treated carbon-copper grids, and stained with 2% uranyl acetate. Images were acquired using a Tecnai G2 Spirit transmission electron microscope (FEI, USA) operating at an accelerating voltage of 200 kV. 1.8 ELISA analysis of antigenicity

[0034] Purified cHeB-WT and cHeB-E3238A viral particles were serially diluted in PBS and coated onto 96-well plates. The plates were incubated overnight at 4°C, then blocked with 5% skim milk in PBS containing 0.05% Tween-20. The plates were incubated for 2 hours at 37°C with either of the following primary antibodies: mouse anti-CVA6 polyclonal serum diluted 1:5,000, or 50 ng / well of mouse neutralizing monoclonal antibody 3H7.

[0035] After washing with PBST, horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody (Proteintech) was added, and the mixture was incubated at 37°C for 1 hour to detect the binding antibody. Finally, after washing, TMB substrate was added for color development. After terminating the reaction, the absorbance was measured at 450 nm using a microplate reader. 1.9 Virus Adsorption Experiment

[0036] Pre-chilled RD cell monolayers (cultured in 24-well plates) were incubated at 4°C with 500 µL of purified CVA6 virus particles (0.1 µg / mL) per well for 2 hours to allow binding. After incubation, the cells were washed twice with ice-cold PBS to remove unbound virus. Total RNA was immediately extracted from the cells using Trizol reagent (Vazyme, China) and reverse transcribed into cDNA using the PrimeScript RT kit (Takara). Cell-associated viral RNA levels were detected by quantitative RT-PCR using SYBR Premix Ex Taq (Takara) on a LightCycler 480 II instrument (Roche). Viral RNA levels were normalized using the internal control gene β-actin. The primer sequences used were: CVA6: Positive, 5′-TACTTTGGGTGTCCGTGTTT-3′ (SEQ ID NO:5); Reverse, 5′-TGGCCAATCCAATAGCTATATG-3′ (SEQ ID NO:6).

[0037] β-actin: forward, 5′-GGACTTCGAGCAAGAGATGG-3′ (SEQ ID NO:7); Reverse, 5′-AGCACTGTGTTGGCGTACAG-3′ (SEQ ID NO:8).

[0038] Use 2 -ΔΔCt The method was used for relative quantitative analysis. 1.10 Quantitative analysis of viral load in tissues

[0039] To determine viral replication levels in vivo, infected mice were sacrificed on day 4 post-infection. Muscle, spinal cord, and brain tissue were aseptically collected, weighed, and homogenized in 400 μL of DMEM supplemented with 1% fetal bovine serum per sample using a tissue homogenizer. The tissue homogenates were clarified by high-speed centrifugation at 4°C. The viral titer in the clarified tissue supernatant was determined by TCID assay on RD cells. 50 Quantification is achieved through measurement. The titer is calculated and expressed as TCID per gram of tissue. 50 (TCID) 50 / g). 1.11 Histopathological Analysis

[0040] Hind limb skeletal muscle tissue was collected from mice on day 4 post-infection or challenge. The tissues were fixed in 4% paraformaldehyde, dehydrated according to standard procedures, embedded in paraffin, and sectioned. Sections were stained with hematoxylin and eosin by Sewell Biotechnology Co., Ltd. (China). The stained sections were examined under an optical microscope.

[0041] Histopathological changes were assessed using a semi-quantitative scoring system: 0 points, no pathological changes; 1 point, mild; 2 points, moderate; 3 points, severe damage. 1.12 Vaccination and challenge

[0042] To evaluate vaccine efficacy, 1-day-old ICR mice were randomly divided into two groups and vaccinated with either the candidate vaccine (cHeB-E3238A) or PBS (control group).

[0043] The vaccine-treated group received a primary immunization via intraperitoneal injection of 0.5 ng of purified cHeB-E3238A virus particles, followed by a booster immunization of 5 ng on day 6; the control group received PBS at the same time point. On day 10, all mice were challenged intraperitoneally with a lethal dose of the heterologous virulent strain CVA6-S0087b. This challenge strain did not exhibit significant cytopathic effects in vitro. Therefore, to standardize the challenge dose, its median lethal dose (LD50) was first determined in 10-day-old ICR mice. The challenge dose used was 93,545 LD50. 50 After the challenge, the survival and clinical symptoms of the mice were monitored daily for 14 days according to the established scoring criteria.

[0044] For histopathological evaluation, another group of mice were euthanized on day 4 after challenge with the virus. Muscle tissue from the limbs was collected using the method described above for hematoxylin-eosin staining and analysis. 1.13 Statistical Analysis

[0045] All statistical analyses were performed using GraphPad Prism version 8. 2. Results 2.1 The toxicity-determining region of CVA6 is located in region P1.

[0046] Our laboratory obtained two CVA6 viruses: CVA6-141 and CVA6-HeB. Both viruses exhibited similar replication efficiency in two cell lines, but showed significantly different virulence in animal models. Using 500 TCID50... 50 Two-day-old newborn mice were infected with varying viral loads of CVA6-HeB. Mice infected with CVA6-HeB exhibited limb weakness and paralysis, and all died within 14 days of observation, resulting in a 100% mortality rate. In stark contrast, even when the infection dose was increased to 5000 TCID... 50Mice infected with CVA6-141 did not show any obvious clinical symptoms, and the survival rate was 100%. Figure 1 A) indicates that CVA6-HeB has significantly enhanced pathogenicity compared to CVA6-141.

[0047] To determine the genomic regions controlling viral virulence, this invention uses the infectious clone of the attenuated strain CVA6-141 as a backbone, and systematically replaces the 5'UTR, P1 (structural protein region), P2 (non-structural protein region), P3 (non-structural protein region), and 3'UTR with corresponding segments from the virulent strain CVA6-HeB, constructing five chimeric viral clones. Figure 1 B). These infectious clones were transfected into HEK293T cells to rescue the virus, and then amplified by passage once in rhabdomyosarcoma (RD) cells.

[0048] 5000TCID respectively 50 The five rescued chimeric viruses were used to infect 2-day-old newborn mice, and clinical symptoms and mortality were observed daily for 14 days. Figure 1 The results showed that only the chimeric virus containing the CVA6-HeB P1 region (cHeB-P1) caused 100% mortality in newborn mice, while the other four chimeric viruses did not cause any mortality. These results indicate that the virulence-determining region of CVA6 is located in the P1 region. 2.2 The virulence-determining site of CVA6 is located at amino acid position 238 of the VP3 protein.

[0049] Sequence alignment of the P1 region of CVA6-HeB and CVA6-141 revealed 19 differential amino acid residues. Considering that the N-termini of VP4 and VP1 are located inside the viral particle and may have a relatively small impact on virulence, we used CVA6-141 as the backbone and replaced these intact fragments with the corresponding segments from CVA6-HeB. For the remaining differential residues, we introduced single-amino acid point mutations. Based on this strategy, 15 infectious clones (mutants #1-15) were successfully constructed. Figure 2 A). The specific locations of these amino acids within the P1 region are as follows: Figure 2 As shown in B.

[0050] Fifteen infectious clones were rescued by co-transfection of T7 RNA polymerase into HEK293T cells, followed by amplification after one passage in rhabdomyosarcoma (RD) cells. The TCID of the viral stock solution after amplification was measured. 50 and at 500 TCID per unit 50 Two-day-old newborn mice were infected intraperitoneally with a dose of [specific dosage], and clinical symptoms and mortality were observed daily for 14 days post-infection. Figure 2C). The results showed that only the mutant virus carrying the VP3-A238E replacement (mutant #11) caused 100% mortality in newborn mice, while the other mutants caused only partial lethality or non-lethality.

[0051] To verify the necessity of glutamic acid (E) at position 238 of VP3 for virulence, an E-to-A substitution was introduced at position 238 of VP3 in the cHeB-P1 (hereinafter referred to as cHeB-WT) backbone, constructing the cHeB-E238A mutant virus. Figure 2 D). The lethality of the rescue virus in newborn mice was then assessed. After a 14-day observation period, none of the newborn mice infected with the cHeB-E238A mutant virus died. Figure 2 E). Therefore, single-point replacement of VP3-E238A (E3238A) greatly reduces the virulence of the virus. 2.3 Preparation and characterization of purified CVA6 cHeB-WT and cHeB-E3238A viruses

[0052] CVA6 cHeB-WT and cHeB-E3238A viral particles were purified from infected RD cell lysates and supernatants by sucrose gradient ultracentrifugation. SDS-PAGE analysis of the gradient components showed that the two viruses had the same protein composition, and their structural proteins co-precipitated in the same number of sucrose gradient layers. Figure 3 A). The 10th gradient, rich in mature viral particles, was used for subsequent analysis. Negative staining electron microscopy confirmed that the purified cHeB-WT and E3238A viral preparations mainly contained intact spherical particles with a diameter of approximately 30 nm, exhibiting typical characteristics of enteroviruses. Figure 3 B).

[0053] Antigen analysis by ELISA showed that cHeB-WT and cHeB-E3238A viruses had comparable binding affinity to anti-CVA6 polyclonal serum and conformation-sensitive neutralizing monoclonal antibody 3H7(12), and this binding was dose-dependent. Figure 3 These results indicate that the E3238A mutation did not alter the overall antigenic structure.

[0054] To compare infectivity, purified cHeB-WT and cHeB-E3238A viral particles were diluted to the same protein concentration (0.1 µg / mL). Viral genome copy number was determined by absolute quantitative reverse transcription PCR, and by TCID... 50 The infection titers determined by the method were similar between the two viruses. Figure 3 E), which indicates that the ratio of the actual number of particles to the number of infectious particles has not changed.

[0055] Finally, to assess cell adhesion capacity, RD cells were subjected to a 4°C reaction with an equal number of genome copies (1×10⁻⁶). 9 Each virus was co-incubated at (copy / mL). Subsequent RT-qPCR analysis of the RNA levels of the viruses adsorbed into the cells showed no significant difference in adsorption capacity between cHeB-WT and cHeB-E3238A viruses. Figure 3 F).

[0056] In summary, these data demonstrate that the E3238A mutation does not significantly alter viral particle assembly, morphology, antigenicity, infectivity, or cell-binding ability. Therefore, the significant attenuated phenotype of this mutant in vivo is not attributable to defects in these fundamental virological characteristics. 2.4 The E3238A mutation completely prevented CVA6-induced death in mice by reducing viral load in vivo.

[0057] To determine the pathogenic impact of the E3238A mutation, newborn mice were infected with equal doses (5 ng per mouse) of purified cHeB-WT and cHeB-E3238A viral particles. All mice infected with cHeB-WT developed progressive limb weakness and paralysis, ultimately leading to 100% mortality. In contrast, all mice infected with cHeB-E3238A survived, exhibiting only mild and transient clinical symptoms. Figure 4 A).

[0058] We further examined viral load in target tissues 4 days post-infection. In all tissues tested, the cHeB-E3238A mutant titer was significantly lower than that of cHeB-WT. Figure 4 B). The viral titer in limb muscles decreased by approximately 14-fold (geometric mean titer: cHeB-WT was 1.41 × 10⁻⁶). 7 TCID 50 / g; E3238A is 1.03×10 6 TCID 50 / g). The most significant decrease was in the spinal cord, reaching 74-fold (cHeB-WT was 1.10 × 10⁻⁶ g). 6 TCID 50 / g; E3238A is 1.48×10 4 TCID 50 / g). The viral load in the brain decreased by approximately 3-fold (cHeB-WT was 2.75 × 10³ TCID). 50 / g; E3238A has a TCID of 8.69 × 10². 50 / g)( Figure 4 B). These results indicate that the E3238A mutation significantly weakens the virus's ability to replicate in target tissues, thus providing a mechanistic explanation for its complete elimination of lethality in vivo. 2.5 The protective effect of the CVA6 cHeB-E3238A attenuated vaccine against heterologous CVA6 challenge.

[0059] Based on the significant attenuation properties exhibited by the CVA6 cHeB-E3238A mutant ( Figure 4 We evaluated its vaccine potential. One-day-old ICR mice were initially immunized (0.5 ng of purified cHeB-E3238A virus particles or PBS), boosted on day 6 (5 ng), and challenged on day 10 with a heterologous virulent strain CVA6-S0087b at a dose of 93,545 LD50. 50 (LD) 50 (referring to the median lethal dose) Figure 5 A). It should be noted that CVA6-S0087b does not have a cytopathic effect in cell culture, but it is highly pathogenic to newborn mice.

[0060] Following immunization and lethal doses of heterologous challenge, all mice inoculated with cHeB-E3238A survived the 14-day observation period without developing serious disease. In contrast, all mice in the PBS control group died from infection. Figure 5 B). This complete protective effect confirms that the cHeB-E3238A mutant is an effective candidate strain for a live attenuated CVA6 vaccine.

[0061] Histopathological analysis of limb muscles on day 4 post-challenge showed that pathological damage in vaccinated mice was significantly reduced. Figure 5 C). Mice in the PBS control group showed moderate to severe muscle inflammation, with three animals exhibiting moderate and three exhibiting severe muscle damage. In contrast, mice vaccinated with cHeB-E3238A showed no or very mild pathological damage: seven out of eight animals had normal muscle structure, and the remaining one showed only very mild myositis. The mean pathological score of the vaccinated group was significantly lower ( Figure 5 D).

[0062] In summary, these results confirm that the cHeB-E3238A mutant can serve as a highly effective live attenuated vaccine candidate, providing strong protection against high-intensity heterologous CVA6 challenge by completely preventing death and significantly reducing histopathological damage. 3. Summary

[0063] In summary, this invention confirms that glutamate at position 238 of the VP3 protein is a key genetic determinant of CVA6 virulence, crucial for efficient viral replication and pathogenicity in vivo. From an application perspective, the E3238A mutation provides a precise and stable method for preparing attenuated CVA6 strains. The effectiveness of the cHeB-E3238A mutant as a candidate strain for a protective attenuated live vaccine establishes a direct translational pathway from mechanism discovery to public health intervention.

[0064] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a live attenuated Coxsackievirus A6 vaccine vector, characterized in that, Site-directed mutagenesis was performed on the nucleic acid sequence encoding the VP3 protein of type A6 Coxsackievirus, changing the coding sequence of glutamic acid E to alanine A.

2. A nucleic acid molecule, characterized in that, include: (i) The nucleotide sequence shown in SEQ ID NO:1; or, (ii) It has at least 90% sequence identity with the nucleotide sequence shown in SEQ ID NO:1 and the nucleotides at positions 712-714 are sequences encoding alanine A; (iii) The complementary sequence of (i) or (ii) above.

3. The nucleic acid molecule according to claim 2, characterized in that, In the sequence described in (ii), nucleotides 712-714 are GCA; and / or, the sequence described in (ii) has at least 98% sequence identity with the nucleotide sequence of SEQ ID NO.

1.

4. The nucleic acid molecule according to claim 2 or 3, characterized in that, This includes the nucleotide sequence encoding the recombinant protein CVA6-HeB P1, as shown in SEQ ID NO:

3.

5. A chimeric virus comprising the nucleic acid molecule according to any one of claims 2-4.

6. A recombinant protein comprising the expression product of the nucleic acid molecule of any one of claims 2-4, or the chimeric virus of claim 5, comprising at least: (1) The amino acid sequence is the recombinant sequence VP3-238A as shown in SEQ ID NO:2; or, (2) A sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:2 and with the amino acid at position 238 mutated to A.

7. The recombinant protein according to claim 6, characterized in that, The sequence described in item (2) has at least 98% sequence identity with the amino acid sequence shown in SEQ ID NO:

2.

8. The recombinant protein according to claim 7 or 8, characterized in that, This includes the recombinant protein CVA6-HeB P1, whose amino acid sequence is shown in SEQ ID NO:

3.

9. The use of the nucleic acid molecule according to any one of claims 2-4, the chimeric virus according to claim 5, and the recombinant protein according to claims 6-8 in the preparation of a vaccine against Coxsackievirus A6.

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