PDCoV-S1 recombinant protein antigen as well as preparation method and vaccine thereof

Abstract

The invention discloses a PDCoV-S1 recombinant protein antigen as well as a preparation method and a vaccine thereof, and belongs to the technical field of recombinant protein vaccines. The amino acid sequence of the PDCoV-S1 recombinant protein antigen disclosed by the invention is as shown in SEQ ID NO: 1; the nucleotide sequence of the PDCoV-S1 recombinant protein antigen is as shown in SEQ ID NO: 2. The vaccine provided by the invention only contains recombinant PDCoV-S1 protein and does not contain genetic materials of viruses, so that all risks of virus replication, virulence reversion or gene recombination are fundamentally avoided. Live viruses do not need to be operated in the production process, so that the biological safety risk and the requirement on the production environment are greatly reduced. The constructed stable CHO engineering cell strain is combined with a serum-free suspension culture technology, so that high-density, large-scale and standardized production of the PDCoV-S1 protein can be realized in a bioreactor. The production process is stable and reliable, the batch-to-batch consistency is good, and the uncertainty and volatility caused by the virus culture link of the traditional vaccine are overcome.

Description

Technical Field

[0001] This invention belongs to the field of recombinant protein vaccine technology, specifically relating to the PDCoV-S1 recombinant protein antigen and its preparation method, and a vaccine. Background Technology

[0002] Porcine Delta coronavirus (PDCoV) is a newly emerging enteric pathogenic coronavirus in pigs that can cause acute diarrhea, vomiting, and dehydration in pigs of all ages, with particularly severe damage to newborn piglets, resulting in a mortality rate of 30%-50% and causing significant economic losses to the global pig industry. PDCoV belongs to the genus Delta coronavirus in the family Coronaviridae and is an enveloped, single-stranded, positive-sense RNA virus. Its spike protein (S protein) on the surface of the viral particle is the virus's main antigenic protein, playing a crucial role in viral adsorption, host cell receptor recognition, and membrane fusion. The S protein can be further hydrolyzed into two functional subunits, S1 and S2. The S1 subunit contains a receptor-binding domain (RBD) and a major antigenic determinant, which can effectively induce the host to produce neutralizing antibodies, making it an ideal target for developing subunit vaccines.

[0003] Currently, commercial vaccines against PDCoV are still under development, and there is a lack of safe and effective prevention and control products on the market. Traditional vaccine strategies, such as inactivated vaccines and live attenuated vaccines, have shown some effectiveness in the prevention and control of other coronaviruses, but they still have significant limitations: inactivated vaccines have weak immunogenicity, often requiring multiple immunizations and are difficult to induce mucosal immunity; while live attenuated vaccines can induce a full immune response, there are risks of virulence reversion and genetic recombination with wild-type strains, and the production process involves handling live viruses, posing biosafety risks.

[0004] With the development of molecular biology techniques, genetically engineered subunit vaccines offer a new direction for solving the aforementioned problems. These vaccines contain only the pathogen-specific antigenic components, without viral genetic material, offering a significant safety advantage while avoiding the biosafety risks of live virus culture. Furthermore, they are easy to standardize and scale up for production. However, the success of these vaccines highly depends on the correct folding, post-translational modification, and immunogenicity of the antigen protein.

[0005] Despite significant progress in the development of swine Delta coronavirus (PDCoV) vaccines, existing technological approaches still face a series of serious challenges that limit the effectiveness, safety, and widespread application of these vaccines.

[0006] First, regarding immunization efficacy, the core issues are insufficient immunogenicity and limited scope of protection. Existing vaccines, especially those based on inactivated vaccines and traditional subunit vaccines, generally suffer from weak immunogenicity. They often fail to efficiently elicit a sufficiently strong and durable protective immune response, particularly in terms of insufficient antibody levels to effectively neutralize the virus. More importantly, PDCoV, as an RNA virus, exhibits high variability, potentially leading to antigenic differences between different strains. This makes vaccines designed based on a single strain or a single antigenic epitope difficult to provide broad cross-protection, and their protective effect against constantly emerging genetically variant strains may be significantly reduced.

[0007] Secondly, regarding the type of immune response, a key deficiency lies in the difficulty of inducing effective mucosal immunity. PDCoV infection and pathogenesis primarily occur in the intestinal mucosa. An ideal vaccine needs to establish the first line of immune defense at the infection gateway—the intestinal mucosa—that is, to elicit an effective mucosal immune response (especially secretory IgA antibodies). However, most existing vaccine technologies administered via non-mucosal routes (such as intramuscular injection) struggle to efficiently induce strong intestinal mucosal immunity. This means that even if systemic immunity (such as IgG antibodies in serum) exists in vaccinated animals, it cannot completely prevent the initial colonization and replication of the virus in the intestine, affecting the final protective efficacy of the vaccine.

[0008] Third, there are dual bottlenecks in safety and production processes, stemming from both traditional and modern methods. While traditional live attenuated vaccines offer relatively good immunization, they always carry the potential risk of virulence reversion, and the possibility of gene recombination between vaccine strains and circulating wild strains poses a significant biosafety hazard. On the other hand, some promising new vaccines, such as virus-like particle (VLP) vaccines, while offering higher safety profiles, typically involve more complex and costly production processes. Scaling them up for large-scale industrial production presents challenges in terms of stability and economic viability, limiting their widespread application.

[0009] Finally, gaps in understanding remain regarding fundamental research and immunization strategies. Currently, the scientific community's understanding of the immune protection mechanisms of PDCoV, particularly the crucial role of T-cell immune responses, is incomplete. This hinders the rational design of novel vaccines capable of evoking a comprehensive immune response. Furthermore, the stability and persistence of passive immunization (providing robust and reliable maternal antibody protection to susceptible newborn piglets under different rearing conditions through immunization of sows) remains a practical issue requiring continuous optimization and validation.

[0010] In summary, existing PDCoV vaccine technologies still face significant technical bottlenecks in improving immunogenicity, broadening the scope of protection, effectively stimulating mucosal immunity, ensuring absolute safety, and achieving low-cost large-scale production. Breakthroughs urgently need to be sought through innovations in antigen design, adjuvant development, delivery systems, and production processes. Summary of the Invention

[0011] In view of this, the purpose of this invention is to provide PDCoV-S1 recombinant protein antigen and its preparation method and vaccine. This invention utilizes the CHO cell expression system to construct an engineered cell line that efficiently expresses PDCoV-S1 protein, and prepares a safe, efficient and stable porcine Delta coronavirus genetically engineered subunit vaccine through protein purification and adjuvant emulsification processes.

[0012] To achieve the above objectives, the present invention provides the following technical solution:

[0013] In a first aspect, the present invention provides a PDCoV-S1 recombinant protein antigen, the amino acid sequence of which is shown in SEQ ID NO:1.

[0014] In a second aspect, the present invention provides a nucleotide sequence of a PDCoV-S1 recombinant protein antigen, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0015] Thirdly, the present invention provides a method for preparing the above-mentioned PDCoV-S1 recombinant protein antigen, comprising the following steps: synthesizing the nucleotide sequence shown in SEQ ID NO:2 into the pKS001 vector to construct the recombinant plasmid pKS001-PDCoV-S1; then introducing the recombinant plasmid pKS001-PDCoV-S1 into an engineered CHO-K1Q cell line for serum-free suspension culture, screening out monoclonal cell lines that highly express PDCoV-S1 protein; and then collecting and purifying the PDCoV-S1 protein.

[0016] The Chinese hamster ovary (CHO) cell expression system is currently the most mature and widely used eukaryotic expression system for the production of recombinant protein drugs and high-end vaccine antigens. Compared with prokaryotic expression systems, CHO cells can perform complex and accurate post-translational modifications of eukaryotic proteins, such as glycosylation. These modifications are crucial for maintaining the correct spatial conformation, antigenicity, and immunogenicity of the coronavirus S protein. Furthermore, CHO cells offer advantages such as suspension culture, high expression levels, and ease of scaling up to industrial production, making them an ideal platform for producing high-quality viral antigen proteins.

[0017] Therefore, developing a genetically engineered subunit vaccine based on the PDCoV-S1 protein using the CHO cell expression system can overcome the shortcomings of traditional vaccines and provide a safe and efficient prevention and control tool. Its stable and controllable production process lays a solid foundation for the large-scale production and commercial application of the vaccine, and is of great significance for effectively controlling the spread of porcine Delta coronavirus and ensuring the healthy development of the pig farming industry.

[0018] Fourthly, the present invention provides the application of the above-mentioned PDCoV-S1 recombinant protein antigen in a porcine Delta coronavirus vaccine.

[0019] Fifthly, the present invention provides a porcine Delta coronavirus vaccine comprising the aforementioned PDCoV-S1 recombinant protein antigen.

[0020] Furthermore, it also includes water adjuvants.

[0021] Furthermore, the content of the PDCoV-S1 recombinant protein antigen is 40 μg / mL.

[0022] It contains at least the following beneficial technical effects: The vaccine of this invention contains only recombinant PDCoV-S1 protein and no viral genetic material, fundamentally eliminating all risks of viral replication, virulence reversion, or gene recombination. The production process does not require handling live virus, greatly reducing biosafety risks and requirements for the production environment.

[0023] The CHO cell eukaryotic expression system ensures that the expressed PDCoV-S1 protein undergoes correct folding and complex glycosylation modifications, maximally approximating its spatial conformation to the native protein, thus presenting a complete antigenic epitope and effectively inducing high levels of neutralizing antibodies. The S1 protein is a key antigenic protein on the viral surface; targeting it can precisely stimulate the body to generate a targeted protective immune response.

[0024] By utilizing a stable CHO engineered cell line and combining it with serum-free suspension culture technology, high-density, large-scale, and standardized production of PDCoV-S1 protein can be achieved in a bioreactor. This production process is stable and reliable, with good batch-to-batch consistency, overcoming the uncertainties and fluctuations caused by the virus culture process in traditional vaccines.

[0025] This vaccine provides a novel and powerful technological tool for controlling PDCoV, effectively filling a market gap. By immunizing sows, piglets can acquire high levels of maternal antibodies, establishing effective passive immune protection, thereby directly reducing the high mortality rate of piglets, which has significant economic and social value.

[0026] In summary, this invention, through an innovative technical approach, successfully overcomes the shortcomings of existing technologies and provides a PDCoV genetically engineered subunit vaccine that demonstrates significant advantages in terms of safety, efficacy, and production feasibility. Attached Figure Description

[0027] Figure 1 Electrophoresis image for enzyme digestion verification of recombinant expression vector pKS001-PDCoV-S1. Lane M1: DL2000 bp Marker; Lane 1: Amplified fragment using the genome of the constructed vector pKS001-PDCoV-S1 as a template; Lane M2: DL10000 bp Marker.

[0028] Figure 2 SDS-PAGE analysis of purified PDCoV-S1 protein. Lane M: Protein Marker; Lane 1: Unpurified protein sample; Lane 2: Flow-through buffer; Lane 3: Elution buffer. Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0033] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0034] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.

[0035] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0036] Example 1. Recombinant expression sequence design and vector construction Based on the porcine Delta coronavirus PDCoV-S1 protein sequence published in NCBI (accession number: ACC95468.1), a recombinant protein sequence suitable for the CHO eukaryotic expression system was designed, consisting of: signal peptide + PDCoV-S1 protein + linker + Fc tag. Using codon optimization technology, the optimized coding sequence was synthesized into the pKS001 prokaryotic vector by Sangon Biotech (Shanghai) Co., Ltd. The gene sequence contains a total of 2047 nucleotides. The recombinant plasmid pKS001-PDCoV-S1 was constructed, and the amino acid and nucleotide sequences are shown in SEQ ID NO:1; the codon-optimized nucleotide sequence is shown in SEQ ID NO:2.

[0037] 2. Construction of expression vector (pKS001-PDCoV-S1) 2.1 Primers The primers used in this experiment were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and are shown in Table 1.

[0038] Table 1 Primers

[0039] 2.2 Methods Construction and validation of recombinant proteins: Plasmids were extracted from *E. coli* pKS001-PDCoV-S1. Using plasmid pKS001-PDCoV-S1 as a template, the PDCoV-S1 gene was amplified using primers PDCoV-S1-F and PDCoV-S1-R. The amplified target fragment was verified by gel electrophoresis. The electrophoresis results are shown below. Figure 1 As shown, the recombinant plasmid pKS001-PDCoV was constructed correctly in its initial stage, consistent with the expected results. It was then sent to a sequencing company for sequencing and gene sequence comparison. The results were consistent with the expected target, proving that the recombinant plasmid pKS001-PDCoV-S1 was successfully constructed.

[0040] The correct E. coli pKS001-PDCoV-S1 was cultured and verified. The recombinant plasmid pKS001-PDCoV-S1 was extracted and verified to be correct. Electroporation was used to introduce it into the engineered CHO-K1Q cell line for serum-free suspension culture. A monoclonal cell line (CHO-PDCoV-S1) that highly expresses PDCoV-S1 protein was screened out.

[0041] 3. Expression and purification of recombinant proteins High-expression clones were selected and cultured in a bioreactor for serum-free suspension culture to achieve high-density fermentation. The cell supernatant of recombinant porcine transmissible gastroenteritis PDCoV-S1 protein expressed in the CHO-PDCoV-S1 cell line was centrifuged, collected, filtered through a 0.22 μm filter, and purified using a Protein A affinity chromatography column. The column was equilibrated with equilibration buffer at a flow rate of 2 mL / min for approximately 3-5 column volumes. Cell supernatant was then loaded into the column at a flow rate of 2 mL / min, and the flow-through was collected. After loading, the column was equilibrated with 5 column volumes of equilibration buffer at a flow rate of 2 mL / min. After baseline equilibration, the target protein was eluted with elution buffer to obtain high-purity PDCoV-S1 protein. The eluent was collected based on the UV absorption peak. The pH of the collected sample was adjusted to 7.0 using 2M Tris-Base. This protein is the antigen for the subunit vaccine. The volume of eluted protein was recorded, and the sample was stored at 4°C.

[0042] 4. Determination of protein purity and content The purity of the collected eluted proteins was determined using SDS-PAGE gel electrophoresis and thin-layer chromatography imaging system. The results are shown in [Figure number missing]. Figure 2 The purified protein showed a clear protein expression band at approximately 75 kDa, consistent with the expected result. Analysis showed that the purity of the purified protein reached 80%. The total protein content of the purified protein was determined using a BCA protein concentration assay kit, and the result was 3.5 mg / mL. The calculated content of the target protein was 2.8 mg / mL.

[0043] 5. Vaccine preparation The purified recombinant porcine Delta coronavirus PDCoV-S1 protein was sterilely filtered through a 0.22 μm filter membrane and diluted to 80 μg / mL with sterile 0.9% physiological saline. It was then mixed with sterile aqueous adjuvant at a certain ratio to prepare the vaccine. The mixture was stirred at 500 rpm for 30–60 minutes at 30°C to prepare a stable vaccine. The antigen content of the finished vaccine should meet the requirement of 40 μg / mL for the recombinant porcine Delta coronavirus PDCoV-S1 protein.

[0044] 6. Testing of genetically engineered subunit vaccines against porcine Delta coronavirus disease 6.1 Safety testing of porcine Delta coronavirus genetically engineered subunit vaccine in piglets and pregnant sows 6.1.1 Experimental Animals 1-month-old piglets and gestation pigs with a gestation age of 20 days before farrowing 6.1.2 Safety testing of porcine Delta coronavirus genetically engineered subunit vaccine in piglets and pregnant sows The laboratory-prepared trial vaccine was used to conduct single-dose vaccination trials, single-dose repeated (14-day interval) and overdose vaccination trials on recommended-use piglets (1 month old) and pregnant pigs (20 days before farrowing).

[0045] Table 2 Grouping of experimental animals

[0046] Twenty experimental piglets and twenty experimental pregnant sows were randomly divided into four groups, and the experiment was conducted according to Table 2, with isolated rearing. Observations and records were kept continuously until day 7 post-vaccination, with daily records of the survival, mental state, diet, and local and systemic reactions of all vaccinated experimental pigs. The safety test results for single-dose vaccination of the experimental pigs are detailed in Table 3 below. The safety test results showed that all vaccinated experimental pigs survived during the observation period, with no deaths or significant local or systemic reactions caused by the vaccine. Their mental state was good, and their feed and water intake were normal. There were no visible differences between the immunized group and the control group, indicating that the three batches of vaccine are safe for piglets with the youngest age of administration (1 month old) and pregnant sows with a gestation age of 20 days before farrowing.

[0047] Table 3. Safety test results of the porcine Delta coronavirus genetically engineered subunit trial vaccine.

[0048] Note: "R" indicates a red ear tag; "Y" indicates a yellow ear tag; "B" indicates a blue ear tag; "G" indicates a green ear tag.

[0049] 6.2 Efficacy testing of porcine Delta coronavirus genetically engineered subunit vaccine in piglets and pregnant sows To investigate the immunogenicity of a porcine Delta coronavirus genetically engineered subunit vaccine, a potency test study was conducted on the laboratory-developed vaccine. The results showed that the laboratory-developed vaccine provided good immunoprotective effects against piglets and pregnant sows.

[0050] 6.2.1 Experimental grouping and immunization Experimental animals: 1-month-old piglets and pregnant sows 20 days before farrowing Ten experimental piglets were randomly divided into two groups of 5 each: an immunization group and a control group. The immunization group received 0.5 mL / piglet / administration, with a booster immunization administered 14 days post-immunization using the same method. The control group received the same volume of 0.9% physiological saline. Ten experimental pregnant sows were also randomly divided into two groups of 5 each: an immunization group and a control group. The immunization group received 1.0 mL / piglet / administration, with a booster immunization administered 14 days post-immunization using the same method. The control group received the same volume of 0.9% physiological saline. The grouping of experimental animals is shown in Table 4.

[0051] Table 4 Grouping of experimental animals

[0052] 6.2.2 Neutralization Test Each piglet in the vaccine group received a subcutaneous injection of 0.5 mL of vaccine (1 dose) in the neck, while the control group received the same dose of 0.9% saline. A booster immunization was administered 14 days after the initial vaccination using the same method. Blood samples were collected and serum separated before the second vaccination and 14 days after the second vaccination for neutralizing antibody testing.

[0053] Pregnant pigs in the vaccine group received a subcutaneous injection of 1.0 mL of vaccine (1 dose) in the neck, while the control group received the same dose of 0.9% saline. A booster immunization was administered 14 days after the initial vaccination using the same method. Blood samples were collected and serum separated before the second vaccination and 14 days after the second vaccination for neutralizing antibody testing.

[0054] Cells in both the serum toxicity control wells and the normal cell control wells should be normal. The virus control should contain 200 TCID. 50 / 0.1 mL and 20 TCID 50 / 0.1 mL of cells should all be lesions, 2 TCID 50 / 0.1 mL wells should show 0-2 wells with cytopathic effect and 0.2 TCID. 50 The cells in each 0.1 mL well should be normal. The neutralizing antibody titer in the control group should not exceed 1:4.

[0055] 6.2.3 Immune challenge After blood collection from piglets, each piglet, along with the control group, was orally infected with 2 mL of swine Delta coronavirus (CHN-HN-2014 strain) for 7 consecutive days. The control group should have at least 4 piglets that developed the disease, and the immunized group should have at least 4 piglets that were protected.

[0056] After blood collection from pregnant sows, each sow, along with the control group, was orally infected with 4 mL of 2 mL porcine Delta coronavirus (CHN-HN-2014 strain) for 7 consecutive days. At least 4 sows in the control group should develop the disease, and at least 4 sows in the immunized group should be protected.

[0057] 6.2.4 Detection of PDCoV neutralizing antibodies and results of protection against viral challenge Based on the results of PDCoV neutralizing antibody detection and challenge protection, the results of neutralizing antibody detection in each group of experimental piglets and pregnant sows are detailed in Table 5 below. Fourteen days after the second immunization, blood was collected from five piglets to measure PDCoV neutralizing antibodies, and they were challenged. The results showed that the neutralizing antibody titers of all five piglets were high, and 5 / 5 of the immunized piglets were protected after challenge. In contrast, the neutralizing antibody titers of the control group piglets were all below 1:4, and all five piglets developed the disease after challenge. Similarly, fourteen days after the second immunization, blood was collected from five pregnant sows to measure PDCoV neutralizing antibodies, and they were challenged. The results showed that the neutralizing antibody titers of all five pregnant sows were high, and 5 / 5 of the immunized pregnant sows were protected after challenge. In contrast, the neutralizing antibody titers of the control group piglets were all below 1:4, and all five pregnant sows developed the disease after challenge. The neutralizing antibody titer results showed that the neutralizing antibody titers measured in both piglets and pregnant sows were within acceptable limits; all were positive in the immunized group, and all were negative in the control group. This study demonstrates that a neutralizing antibody titer of 1:32 or higher against PDCoV can completely protect immunized piglets and pregnant sows from attack by virulent PDCoV. Therefore, the porcine Delta coronavirus genetically engineered subunit vaccine prepared in this invention has passed efficacy testing, and immunization with this vaccine can induce antibody production in experimental piglets and pregnant sows.

[0058] Table 5. Detection results of PDCoV neutralizing antibodies and protection against viral challenge.

[0059] Note: "O" indicates no abnormalities; "E" indicates decreased appetite; "N" indicates lethargy; "F" indicates diarrhea; "V" indicates vomiting.

[0060] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications 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 PDCoV-S1 recombinant protein antigen, characterized in that, The amino acid sequence of the PDCoV-S1 recombinant protein antigen is shown in SEQ ID NO:

1.

2. A nucleotide of a PDCoV-S1 recombinant protein antigen, characterized in that, The nucleotide sequence of the PDCoV-S1 recombinant protein antigen is shown in SEQ ID NO:

2.

3. The method for preparing the PDCoV-S1 recombinant protein antigen according to claim 1, characterized in that, The procedure includes the following steps: synthesizing the nucleotide sequence shown in SEQ ID NO:2 into the pKS001 vector to construct the recombinant plasmid pKS001-PDCoV-S1; then introducing the recombinant plasmid pKS001-PDCoV-S1 into an engineered CHO-K1Q cell line for serum-free suspension culture, screening out monoclonal cell lines that highly express PDCoV-S1 protein; and then collecting and purifying the PDCoV-S1 protein.

4. The use of the PDCoV-S1 recombinant protein antigen of claim 1 in a porcine Delta coronavirus vaccine.

5. A porcine Delta coronavirus vaccine, characterized in that, Includes the PDCoV-S1 recombinant protein antigen as described in claim 1.

6. The porcine Delta coronavirus vaccine according to claim 1, characterized in that, It also includes water-based adjuvants.

7. The porcine Delta coronavirus vaccine according to claim 6, characterized in that, The content of the PDCoV-S1 recombinant protein antigen is 40 μg / mL.

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