TWI871759B - A dna vaccine of porcine epidemic diarrhea virus and application thereof - Google Patents

Abstract

The present invention provides a polynucleotide encoding the spike protein of Porcine Epidemic Diarrhea Virus (PEDV), a vector containing the polynucleotide, and a DNA vaccine composition containing the vector. The DNA vaccine is administered orally or through injection to a subject in need thereof to prevent and/or alleviate symptoms caused by PEDV.

Description

DNA vaccine of porcine epidemic diarrhea virus and its application

The present invention relates to a DNA vaccine of porcine epidemic diarrhea virus and its application, in particular to a DNA vaccine for preventing and treating diseases caused by porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV) is a virus of the genus Alphacoronavirus. It mainly infects pigs. Pigs infected with PEDV will show symptoms such as vomiting, dehydration, loss of appetite, acute diarrhea, and fever. The impact on piglets is particularly serious, with a very high mortality rate. The virus has caused several major epidemics in Asia and other regions, causing inestimable serious losses to the pork industry and national economies.

DNA vaccine is a new type of vaccine technology that prevents and treats diseases by injecting specific pathogen gene fragments into the human or animal body to trigger the immune system's response. The gene fragments are transcribed and translated into proteins by cells, thereby inducing the immune system to produce an immune response to the antigen. The preparation process of DNA vaccine is relatively simple and flexible, and the gene fragments can be adjusted as needed. It does not contain live viruses or bacteria, thus reducing the risk of infection and inducing a long-term immune response.

However, the development of DNA vaccines also faces many challenges. For example, the design of the delivery method is an important consideration, which must ensure that the gene fragment can enter the cells and trigger an appropriate immune response. In addition, the immune response intensity of DNA vaccines is often insufficient and there are safety issues. The risk of any adverse reactions needs to be carefully evaluated.

In summary, although there are vaccines for PEDV on the market, their actual protective effects in clinical practice are poor. In addition, all current PEDV vaccines are intramuscular vaccines that require professional and trained personnel to administer, making large-scale vaccinations more expensive. Therefore, how to design an effective vaccine for the prevention and treatment of PEDV with a low operating threshold and low cost is an urgent problem to be solved in this field.

Based on the above content, the present invention provides a polynucleotide encoding the spike protein of porcine epidemic diarrhea virus, wherein the polynucleotide has a nucleotide sequence as shown in SEQ ID NO: 1.

In one aspect, the present invention provides an expression vector comprising the polynucleotide described above.

In some embodiments, the expression vector is a pTCY expression vector.

In another aspect, the present invention provides a DNA vaccine comprising the expression vector as described above, and at least one pharmaceutically acceptable carrier, adjuvant, diluent or formulation.

In some embodiments, the DNA vaccine is administered orally.

In some embodiments, the DNA vaccine comprises an adjuvant, wherein the adjuvant is a peptide nanotube.

In some embodiments, the DNA vaccine is administered by injection.

In some embodiments, the DNA vaccine comprises an adjuvant, wherein the adjuvant is a CpG motif.

In another aspect, the present invention provides a use of the DNA vaccine as described above in the preparation of a pharmaceutical composition for preventing and alleviating symptoms caused by PED virus.

In some specific embodiments, it further comprises a booster, which is the DNA vaccine or single-unit vaccine of the present invention as described above.

The present invention combines the research and development of DNA vaccines to develop a DNA vaccine that is effective in preventing and treating porcine epidemic diarrhea virus. The DNA vaccine contains a polynucleotide sequence that encodes the spike protein antigen epitope of porcine epidemic diarrhea virus. Clinical animal experiments have confirmed that the DNA vaccine can be administered orally and by injection, and can indeed enter cells to produce appropriate immune responses, improving the problem that DNA vaccines in the past were difficult to produce sufficiently strong immune responses. Furthermore, the present invention can be administered orally, greatly reducing the operational threshold and cost, and can be simply and effectively administered to pigs on a large scale. In addition, the DNA vaccine described in the present invention will not have adverse effects on pigs and is safe to implement. In summary, the DNA vaccine described in the present invention has great application prospects in the prevention and treatment of diseases caused by porcine epidemic diarrhea virus.

The existing PEDV vaccines are not clinically effective, especially against PEDV strains in Taiwan. This may be due to the variability of PEDV, which causes mutations in some protein regions, significantly reducing the effectiveness of vaccines developed in the past.

In view of the above shortcomings of the existing vaccines, the present invention provides a polynucleotide encoding the spike protein of porcine epidemic diarrhea virus, wherein the polynucleotide has a nucleotide sequence as shown in SEQ ID NO: 1. The polynucleotide is used to produce an effective antigenic epitope of the spike protein of porcine epidemic diarrhea virus for preparing a DNA vaccine of porcine epidemic diarrhea virus.

In one aspect, the present invention provides an expression vector comprising the polynucleotide described above, wherein the expression vector may be in any form, including but not limited to plasmid vectors, viral vectors, cosmid vectors, phage plasmid vectors, artificial human chromosomes, etc.

In another aspect, the present invention provides a DNA vaccine comprising the expression vector as described above, and at least one pharmaceutically acceptable carrier, adjuvant, diluent or formulation.

In some embodiments, the DNA vaccine is administered orally. Oral administration is simple to operate, low in cost, and reduces the stress of pigs.

In some embodiments, the DNA vaccine comprises an adjuvant, wherein the adjuvant is a peptide nanotube.

In some embodiments, the DNA vaccine is administered by injection.

In some embodiments, the DNA vaccine comprises an adjuvant, wherein the adjuvant is a CpG motif.

In another aspect, the present invention provides a use of the DNA vaccine described above in the preparation of a pharmaceutical composition for preventing and alleviating symptoms caused by PED virus, wherein the symptoms caused by the PED virus include but are not limited to vomiting, dehydration, anorexia, weight loss, diarrhea, and fever.

In certain embodiments, the pharmaceutical composition is administered orally to induce the production of gastrointestinal mucosal protective IgA antibodies.

In some specific embodiments, it further comprises a booster, which is the DNA vaccine or single-unit vaccine of the present invention as described above. Wherein, the booster is used in a common vaccine immunization strategy, which is a Prime-boost immunization strategy.

It should be understood that the foregoing general description and the following detailed description are exemplary and illustrative, but not intended to limit the rights claimed by the present invention. Certain details of one or more embodiments of the present invention are described in the following description. Other features or advantages of the present invention will be apparent from the following non-exhaustive list of representative embodiments and from the attached claims.

Unless otherwise defined below, all scientific and technical terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. References to the techniques used herein are intended to indicate techniques commonly understood in the art, including variants or equivalents of those techniques or alternative techniques developed later, which are obvious to those skilled in the art.

It should be noted that, as used herein, the singular terms "a", "an", and "the" include plural referents unless expressly limited to one referent. Unless the context clearly indicates otherwise, the term "or" is used interchangeably with the term "and/or".

The term "about", "approximately" or "approximately" as used herein substantially represents that the numerical value or range described is within 20%, preferably within 10%, and more preferably within 5%. The numerical quantities provided herein are approximate values, which means that they can be inferred even if the term "about", "approximately" or "approximately" is not used.

As used herein, the term "comprising" is open-ended, indicating that such embodiments may include additional elements. Conversely, the term "consisting of" is closed-ended, indicating that such embodiments do not include additional elements (except for trace impurities). The term "consisting essentially of" is partially closed-ended, indicating that such embodiments may also include elements that do not substantially alter the basic characteristics of such embodiments.

Unless otherwise defined herein, scientific and technical terms used in conjunction with this document shall have the meanings commonly understood by persons of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular. The methods and techniques of the present invention can generally be performed according to conventional methods known in the art. In general, the nomenclature described herein to link the following techniques, as well as the techniques of biochemistry, enzymology, molecular and cellular biology, microbiology, genetics, immunology, protein and nucleic acid chemistry and hybrid reactions are all known and frequently used in the art. Unless otherwise specified, the methods and techniques of the present invention can generally be performed according to conventional methods known in the art, and are described in various general and more specific references cited and discussed in this specification.

As used herein, the term "nucleotide sequence" also includes nucleotide sequences having at least 95% identity, including nucleotide sequences having at least 95%, 96%, 97%, 98% or 99% identity.

As used herein, the term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable for use in living organisms and generally does not cause allergic reactions, such as gastrointestinal problems, fever, etc. As used herein, the term "carrier, excipient and diluent" includes but is not limited to lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinyl hydropyrrolidone, hydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, it may also contain fillers, anticoagulants, lubricants, humectants, fragrances, emulsifiers, preservatives, etc.

As used herein, the term "adjuvant" refers to any substance that increases the mucosal, humoral, or cellular immune response to an antigen, and is generally used to achieve two purposes: slowing antigen release and stimulating immune responses. Non-limiting examples of adjuvants include: aluminum (e.g., aluminum gel and/or aluminum salts, such as aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate), lipids (e.g., squalene, monophosphoryl lipid A (MPL)), AS03 (e.g., an adjuvant comprising D,L-α-tocopherol (vitamin E), squalene, and polysorbate 80), AS04 (e.g., an adjuvant comprising a combination of aluminum hydroxide and MPL), and MF59® (e.g., an adjuvant comprising squalene).

As used herein, the term "peptide nanotubes" refers to a DNA vaccine adjuvant, wherein the peptide nanotubes are long peptide nanotubes and/or short peptide nanotubes, which are used to enhance mucosal immune responses. For a detailed description of the peptide nanotubes, please refer to, for example, Republic of China Patent No. I756948, which is incorporated herein by reference in its entirety. The term "mucosal immune response" refers to the reaction produced when the mucosal immune system of an animal (including humans) is triggered when foreign substances invade the mucosal immune system, mainly through secretory immunoglobulin A (IgA), which is mainly present in saliva, milk, gastrointestinal fluid, respiratory secretions, etc., and is the first line of defense for the mucosal site against pathogenic microorganisms and harmful substances.

As used herein, the term "CpG module" refers to a DNA vaccine adjuvant, which is characterized in that the DNA vaccine adjuvant contains multiple sets of specific immunostimulatory oligonucleotides or recombinant plasmids containing the same and does not need to be phosphosulfurized. For a detailed description of the CpG module, please refer to, for example, Republic of China Patent No. I425091, which is incorporated herein by reference in its entirety.

As used herein, the term "spike protein of porcine epidemic diarrhea virus" is composed of 1,386 amino acids, and the structural domains of the protein can be divided into the S1 region (amino acids 227-633) and the S2 region (amino acids 640-1,322).

As used herein, the term "pTCY vector" is derived from the pEGFP-N1 vector (Clontech, Mount View, California, USA), wherein the CMV immediate-early promoter on the pEGFP-N1 vector is replaced with a beta-actin promoter, and the coding region of the enhanced green fluorescent protein is removed, so that other target genes can be inserted and expressed under the control of the beta-actin promoter. For detailed construction of the pTCY vector, please refer to H.-C. Wu et al. (2019) A DNA priming and protein boosting immunization scheme to augment immune responses against parvovirus in ducks. J. Appl. Microbiol. 126(1): 49-57.

As used herein, the term "Prime-boost immunization strategy" refers to a common vaccine immunization strategy that uses vaccines in two stages to stimulate the immune system, thereby achieving a more effective and lasting immune response. The original immunization (Prime) is the first dose of vaccine received by the individual in the first stage to initiate the immune response. This stage is also called the initial immunization, which is intended to induce the initial immune memory, but the immune response may be relatively weak. The boosted immunization (Boost) is a period of time after the original immunization, when the individual receives the second dose of vaccine. This stage is also called the booster immunization, which aims to stimulate the immune system, strengthen the immune response and prolong the existence of immune memory, thereby providing longer-term protection.

As used herein, the term "booster" refers to the second dose of vaccine used in the Prime-boost immunization strategy, also known as a booster.

As used herein, the term "mucosal immune response" refers to the reaction produced when the mucosal immune system of animals (including humans) is triggered when foreign substances invade the mucosal immune system, mainly through secretory immunoglobulin A (IgA), which is mainly present in saliva, milk, gastrointestinal fluid, respiratory secretions, etc., and is the first line of defense of the mucosal site against pathogenic microorganisms and harmful substances.

The present invention is further illustrated by the following embodiments, which should not be interpreted as further limiting in any way. The entire contents of all references cited in this application (including references, approved patents, published patent applications, and patent applications in the same application) are hereby expressly incorporated into this application by reference.

The data statistics in the embodiment of the present invention are statistically analyzed by the least significant difference method (LSD method) after ANOVA analysis. Among them, the average values of the figure numbers a and b are a>b in sequence; the same figure number (for example, between a and a) indicates that there is no significant difference between the groups; on the contrary, different figure numbers (for example, a and b) indicate that there is a significant difference between the groups (p<0.05); as for the figure number ab, it means that this value is not significantly different from both a and b.

Embodiment 1 Porcine epidemic diarrhea virus Vaccine Preparation

1.1 DNA Vaccine Preparation

In this embodiment, the preparation of DNA vaccine includes: synthesizing the sequence and transferring it into the expression vector; culturing the successfully transferred expression vector in large quantities, extracting the plasmid DNA, and mixing it with an adjuvant after quantification for subsequent clinical animal experiments.

In this embodiment, the polynucleotide of the present invention has a nucleotide sequence as shown in SEQ ID NO: 1. After the nucleotide sequence is synthesized, it is transferred into the pTCY expression vector to serve as the expression vector of the DNA vaccine of the present invention. The expression vector can drive the nucleotide sequence successfully transferred at the rear end to express the PEDV spike protein through the β-actin promoter.

In this embodiment, after the successful expression vectors are cultured in large quantities, plasmid DNA is extracted using a large plasmid extraction kit, and after quantification, the adjuvant is mixed at a weight ratio of 50% to complete the preparation of the DNA vaccine. In some specific embodiments, the adjuvant used in the DNA vaccine administered orally is peptide nanotubes. The exemplary configuration in the embodiment of the present invention is to take 1 mL of 260 μg/mL plasmid DNA and 1 mL of 260 μg/mL peptide nanotubes to prepare the oral DNA vaccine of the present invention, and the oral DNA vaccine administration dose in the experiment of the present invention is 2 mL per pig. In certain specific embodiments, the adjuvant used in the DNA vaccine administered by intramuscular injection is a CpG module. The exemplary configuration in the embodiments of the present invention is to mix 1 mL of 100 μg/mL plasmid DNA with 1 mL of 100 μg/mL CpG module to prepare the DNA vaccine for intramuscular injection described in the present invention, and the dosage of the DNA vaccine for intramuscular injection in the experiment of the present invention is 2 mL per pig.

1.2 Preparation of subunit vaccines

In this embodiment, the preparation of the conventional subunit vaccine is to emulsify the PEDV spike protein S1 recombinant protein (whose amino acid sequence is shown in SEQ ID NO: 2) at a weight ratio of 50% with ISA 206 adjuvant (SEPPIC, France), and after confirming sterility, administer it to pigs by intramuscular injection. The exemplary configuration in the embodiment of the present invention is to take 1 mL of 50 μg/mL PEDV spike protein S1 recombinant protein and mix it with 1 mL of ISA 206 adjuvant to prepare the subunit vaccine for intramuscular injection described in the present invention, and the dosage of the subunit vaccine administered in the experiment of the present invention is 2 mL per pig.

Embodiment 2 Porcine epidemic diarrhea virus Of DNA Vaccine trials

In this example, in order to test the efficacy and safety of the DNA vaccine of the present invention for preventing and treating porcine epidemic diarrhea virus, 3-week-old specific pathogen free (SPF) piglets were divided into 4 groups, each with 3 piglets, namely, a DNA vaccine oral group, a DNA vaccine injection group, and a blank control group (control), and a Prime-boost immunization strategy was carried out, and then the efficacy was tested by a PEDV challenge test. Among them, the blank control group was administered with 2 mL of saline. In addition, the preparation and administration of the vaccine were as described in Example 1. According to the group design, the piglets were first immunized with DNA vaccines at 3 weeks of age, and boosted with the same vaccine type and the same administration method 2 weeks after the first immunization (i.e., when the piglets were 5 weeks old). For example, the DNA vaccine oral group was orally administered with DNA vaccine at 3 weeks of age to complete the primary immunization, and was orally administered with DNA vaccine again at 5 weeks of age to complete the booster immunization. After the above groups of experimental pigs completed the Prime-boost immunization strategy, a PEDV challenge test was performed 1 week after the booster immunization (i.e., when the piglets were 6 weeks old) to verify the efficacy of the vaccine.

2.1 Safety Testing

The body temperature of piglets in each group was measured at 0 hours, 4 hours, 24 hours and 7 days after the first immunization. The results of the temperature measurement are shown in Table 1. The body temperature of each group was within the normal range of 39.4 Within C, it means that the piglets in the DNA vaccine oral group and the DNA vaccine injection group did not develop fever symptoms.

Table 1. Average body temperature after primary vaccination ( C) Grouping Time elapsed since first immunization 0 hours 4 hours 24 hours 7 days DNA vaccine oral group 37.7 38.2 37.8 37.8 DNA vaccine injection group 38.8 38.7 38.1 38.2 Blank control group 38.1 38.5 37.9 38.1

In addition, the weight changes of piglets in each group were measured one week after the first immunization. The weight measurement results are shown in Table 2 below. There was no significant difference in weight gain between the groups that received the DNA vaccine and the blank control group one week after the first administration, indicating that the piglets in the DNA vaccine oral group and the DNA vaccine injection group did not develop symptoms such as anorexia.

Table 2. Average weight of piglets 1 week after primary vaccination (kg) Grouping Before vaccination 1 week after vaccination Weekly weight gain DNA vaccine oral group 7.48 ± 0.58 9.08 ± 1.12 1.6 ± 0.57 DNA vaccine injection group 7.16 ± 0.64 9.21 ± 0.52 2.05 ± 0.60 Blank control group 7.01 ± 0.38 8.53 ± 0.11 1.52 ± 0.27

The results of this example demonstrate that the DNA vaccine of the present invention has good safety. The test pigs had no adverse reactions after administration of the vaccine and gained weight well.

2.2 PEDV Virus attack test

One week after the Prime-boost immunization strategy was completed, that is, when the pigs were 6 weeks old, the PEDV G2 virus strain was administered by nasal injection for a virus challenge test. The virus solution concentration was 1 x 10 ⁶ TCID ₅₀ /mL, and 5 mL of virus solution was administered to each pig. After the virus challenge, the clinical symptoms of each group were analyzed and scored. The symptoms and scoring methods are shown in Table 3 below.

Table 3. Scoring of clinical symptoms after PEDV challenge Symptoms Score Description of symptoms Diarrhea 0 The color and hardness of the stool from the anal swab were normal. 1 Anal swab stool sample color, softness or hardness is abnormal 2 The color and hardness of the stool sampled from the anal swab are abnormal Dehydration and weight loss 0 Weight loss of less than 0.5 kg or weight gain compared to the previous day 1 Weight loss of 0.5 kg~1 kg compared to the previous day 2 Weight loss of more than 1 kg compared to the previous day Anorexia 0 Eat immediately 1 Although you eat, you don’t eat right away. 2 No food at all Vomiting 0 No foreign objects near the mouth 1 Vomitus residue near the mouth Depression 0 without 1 Yes (based on the interaction between pigs and their lying position) Back hair cover 0 Smooth back 1 Rough and uneven fur

The clinical symptom scores of each group were counted for 7 days. The statistical results are shown in Table 4 below. The total clinical symptom score of the blank control group after the virus challenge was as high as 52 points, indicating that the pigs developed clinical symptoms of PEDV infection after the virus challenge; however, the total clinical symptom scores of the pigs in the DNA vaccine oral group and the DNA vaccine injection group were significantly ( p < 0.05) reduced, to 7 and 8 points, respectively, indicating that the DNA vaccine of the present invention can effectively prevent and alleviate the symptoms caused by PED virus.

Table 4. Clinical symptom records and statistical scores of each group after PEDV challenge Grouping Diarrhea Anorexia Dehydration and weight loss Back hair cover Total score DNA vaccine oral group 7 0 0 0 7 ^b DNA vaccine injection group 6 0 0 2 8 ^b Blank control group 13 15 12 12 52 ^a

In addition, in this embodiment, the weight gain of pigs was integrated and statistically analyzed before and after the challenge, and the results are shown in Table 5. The average weight gain of pigs in the blank control group was 0.8 kg 7 days after the challenge, which was significantly ( p < 0.05) lower than the weight gain of pigs in the DNA vaccine oral group and the DNA vaccine injection group (3.8-3.9 kg), indicating that the DNA vaccine of the present invention has the effect of slowing down the weight loss or anorexia caused by PED virus.

Table 5. Average body weight of each group before and after challenge (kg) Grouping Before attack After attack Average total weight gain/7 days DNA vaccine oral group 25.6 ± 3.4 29.5 ± 3.1 3.9 ^a DNA vaccine injection group 25.4 ± 1.2 29.3 ± 2.9 3.8 ^a Blank control group 22.4 ± 2.9 23.2 ± 2.7 0.8 ^b

The results of this example demonstrate that the DNA vaccine of the present invention can be effectively administered orally or by intramuscular injection to prevent and alleviate the symptoms caused by PED virus.

Embodiment 3 Different types PEDV Vaccine Combinations

In order to test the efficacy of the Prime-boost immunization strategy of the DNA vaccine combined with the subunit vaccine described in the present invention, 3-week-old SPF piglets were divided into 3 groups, each with 3 piglets, and each group was a DNA vaccine oral group, a DNA vaccine injection group, and a blank control group, and the Prime-boost immunization strategy was carried out, and then the vaccine efficacy was tested by PEDV challenge test. According to the group design, the piglets were first immunized with oral DNA vaccine or injection DNA vaccine at 3 weeks of age, and the subunit vaccine was administered as a booster immunization 2 weeks after the first immunization (i.e., when the piglets were 5 weeks old). The preparation and administration of the vaccine were as described in Example 1. For example, the DNA vaccine oral group was administered orally at 3 weeks of age to complete the primary immunization, and a second unit of vaccine was administered at 5 weeks of age to complete the booster immunization. In addition, the blank control group was administered 2 mL of saline at the time of the primary immunization, and 2 mL of saline was administered again at 5 weeks of age.

3.1 In serum IgA Antibody analysis

After the first immunization, serum was collected from each group at 0, 3, and 6 weeks to analyze the IgA antibody reaction in the serum. The anti-PEDV IgA antibody titer in the serum was tested by enzyme immunoassay (ELISA). 25 µg/mL deactivated PEDV was used as the ELISA bottom antigen. The sample was a 16-fold diluted sample serum. Mouse anti-IgA secondary antibody (mouse anti-IgA-HRP, diluted 5000 times) conjugated with horseradish peroxidase (HRP) was added and the absorbance at a wavelength of 450 nm (OD 450 nm) was measured. The experimental results are shown in FIG1 . Three weeks after the first immunization, the IgA antibody of the pigs in the DNA vaccine oral group was significantly higher ( p < 0.05) than that in the blank control group, indicating that the DNA vaccine of the present invention can effectively induce the pigs to produce a mucosal immune response by oral administration to prevent and alleviate the symptoms caused by PED virus.

3.2 PEDV Virus attack test

One week after the Prime-boost immunization strategy was completed, the PEDV G2 virus strain was administered to each group of experimental pigs by nasal injection for PEDV challenge test. The concentration of the virus solution was 1 x 10 ⁶ TCID ₅₀ /mL. Each pig was given 5 mL of virus solution. The body temperature changes were measured at 0 hours, 4 hours, 1 day, 2 days, 3 days, 4 days, and 5 days after the challenge. The results are shown in Table 6 below. The body temperature of the blank control group pigs increased significantly 2 days after the challenge, and exceeded the normal body temperature range, showing symptoms of fever in the blank control group pigs, and then dropped to the normal range; the body temperature of the pigs in the DNA vaccine oral group and the DNA vaccine injection group was within the normal body temperature range after the challenge. The results indicate that after the Prime-boost immunization strategy of the present invention, pigs can effectively prevent the temperature rise caused by PEDV infection, indicating that the DNA vaccine of the present invention has the ability to prevent and alleviate the fever symptoms caused by PED virus.

Table 6. Average body temperature after PEDV challenge ( C) Grouping Time elapsed after PEDV challenge 0 hours 4 hours 1 day 2 days 3 days 4 days 5 days DNA vaccine oral group 38.7 38.7 38.5 38.8 38.9 38.7 38.5 DNA vaccine injection group 38.9 38.9 38.7 39.0 38.9 38.8 38.8 Blank control group 38.7 38.8 38.5 39.7 ^Note 38.9 38.6 38.7 Note: Body temperature is over 39.4 C is within the normal range, indicating clinical symptoms of fever.

After PEDV challenge, the clinical symptoms of pigs in each group were observed for recording and analysis. The symptoms included diarrhea, dehydration, anorexia, vomiting, depression and dandruff. The characteristics and scoring methods of each symptom are shown in Table 3 above. The clinical symptom scores of each group were counted for 7 days. The statistical results are shown in Table 7 below. The total clinical symptom score of the blank control group pigs after PEDV challenge was as high as 44 points, indicating that the pigs developed clinical symptoms of PEDV infection after the challenge. However, the total clinical symptom scores of the pigs in the DNA vaccine oral group and the DNA vaccine injection group were significantly ( p < 0.05) reduced, and the total scores dropped to 21 and 20 points, respectively, indicating that the DNA vaccine described in the present invention can effectively prevent and alleviate the symptoms caused by PED virus.

Table 7. Clinical symptom records and statistical scores of each group after PEDV challenge Grouping Diarrhea Dehydration and weight loss Anorexia Vomiting Depression Back hair cover Total score DNA vaccine oral group 14 6 0 1 0 0 21 ^b DNA vaccine injection group 14 6 0 0 0 0 20 ^b Blank control group 29 13 0 0 0 2 44 ^a

In addition, in this example, the weight gain of each group of pigs was integrated and statistically analyzed before and after the challenge, and the results are shown in Table 8. The pigs in the blank control group lost an average of 0.93 kg in weight 7 days after the challenge. In contrast, the pigs in the DNA vaccine oral group and the DNA vaccine injection group gained 0.87 and 0.02 kg, respectively, indicating that the DNA vaccine of the present invention has a significant ( p < 0.05) effect of slowing down the weight loss symptoms caused by PED virus.

Table 8. Average body weight of each group before and after challenge (kg) Grouping Before attack After attack Average total weight gain/7 days DNA vaccine oral group 20.58 ± 4.24 21.45 ± 5.59 0.87 ^a DNA vaccine injection group 18.95 ± 1.48 18.97 ± 1.77 0.02 ^a Blank control group 20.15 ± 1.81 19.22 ± 2.53 -0.93 ^b

In summary, clinical animal experiments have confirmed that the DNA vaccine described in the present invention has good safety, does not cause adverse reactions to pigs after administration, and can effectively prevent and alleviate the symptoms caused by PED virus. In addition, the DNA vaccine described in the present invention can be administered to pigs orally and by injection. Compared with traditional subunit vaccines, the DNA vaccine described in the present invention administered orally can significantly induce pigs to produce IgA antibodies with gastrointestinal mucosal protection, and has the advantages of simple operation and low cost, and has great application prospects in combating porcine epidemic diarrhea virus.

The present invention has been specifically demonstrated and described with reference to its preferred specific embodiments, which are only some of the preferred embodiments of the present invention, but are not used to limit the present invention. Any person familiar with this technical field with common knowledge, after understanding the aforementioned technical features and embodiments of the present invention, and making equivalent changes or modifications without departing from the spirit and scope of the present invention, still falls within the scope of the present invention, and the scope of patent protection of the present invention shall be defined by the claim items attached to this specification.

In summary, the technical features disclosed in this case have fully met the statutory invention patent requirements of novelty and advancement. Therefore, an application has been filed in accordance with the law. We sincerely request that your office approve this invention patent application to encourage inventions. I would like to express my gratitude.

Figure 1 is an analysis of IgA antibody responses in pigs at 0, 3, and 6 weeks after the first administration of DNA vaccines. The average values of graphs a and b are a>b, respectively; the same graph numbers (e.g., between a and a) indicate no significant difference between the groups; on the contrary, different graph numbers (e.g., between a and b) indicate significant difference between the groups (p<0.05); and graph numbers ab indicate that the value is not significantly different from both a and b.

A0101_OR_PSEQ.xml

Claims (8)

A DNA vaccine comprises an expression vector and at least one pharmaceutically acceptable carrier, adjuvant, diluent or excipient, wherein the expression vector comprises a single polynucleotide encoding the spike protein of porcine epidemic diarrhea virus (PEDV), and the single polynucleotide has a nucleotide sequence as shown in SEQ ID NO: 1. A DNA vaccine as described in claim 1, wherein the expression vector is a pTCY expression vector. The DNA vaccine as described in claim 1 is administered orally. A DNA vaccine as described in claim 3, wherein the adjuvant is a peptide nanotube. The DNA vaccine as described in claim 1 is administered by injection. A DNA vaccine as described in claim 5, wherein the adjuvant is a CpG module. A use of the DNA vaccine as described in claim 1 in the preparation of a pharmaceutical composition for preventing and alleviating symptoms caused by PED virus. The use as described in claim 7 further comprises a booster, which is a DNA vaccine or a single-unit vaccine as described in claim 1, wherein the single-unit vaccine is prepared by mixing a PEDV spike protein S1 recombinant protein and an ISA 206 adjuvant, wherein the PEDV spike protein S1 recombinant protein has an amino acid sequence as shown in SEQ ID NO: 2.