S protein ancestral sequence of porcine acute diarrhea syndrome coronavirus and application of S protein ancestral sequence

Abstract

The invention belongs to the technical field of biological medicines, and provides an S protein ancestor sequence AS mRNA of a porcine acute diarrhea syndrome coronavirus, the ancestor sequence of an SADS-CoV Spike protein is constructed through phylogenetic analysis, and the sequence aims at the existing virus and also aims at the evolution pedigree of the virus; the sequence design is used for converting target defense aiming at a single pathogen into active intervention on a virus evolution trajectory; and the prepared AS mRNA vaccine has important significance on feeding safety of animals and prevention and control of zoonosis diseases.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of veterinary biomedicine, and particularly relates to a S protein ancestral sequence of porcine acute diarrhea syndrome coronavirus A S mRNA and applications thereof. BACKGROUND

[0002] Porcine acute diarrhea syndrome coronavirus (SADS-CoV) belongs to the member of alpha coronavirus of the Coronaviridae family, is an enveloped single-stranded RNA virus, and has a genome size of about 27 kb, which can encode various structural proteins and non-structural proteins including spike protein (S protein). The virus mainly infects neonatal piglets within 5 days of age, and the main clinical symptoms are severe acute diarrhea, vomiting, dehydration and rapid death, etc., which has caused serious economic losses to the global pig industry. As a new porcine enteric coronavirus, SADS-CoV, together with porcine epidemic diarrhea virus (PEDV) and other pathogens, constitutes the main threat to piglet diarrhea, and its high targeting to young piglets in the pathogenic mechanism further increases the difficulty of prevention and control.

[0003] At present, there is still a lack of effective means for the prevention and control of SADS-CoV, and no commercial vaccine has been put into application. Referring to the prevention and control experience of similar porcine coronaviruses, the traditional inactivated vaccine and attenuated live vaccine have many inherent defects: the inactivated vaccine needs to be inoculated multiple times to maintain the immune efficacy, and it is difficult to induce effective intestinal mucosal immune response; the attenuated live vaccine faces the risk of virulence return and gene recombination, and in addition, its research and development cycle is as long as 6-12 months, which cannot cope with the sudden epidemic caused by virus variation. What is particularly key is that SADS-CoV is a new virus in recent years, and its epidemic strain has not formed a stable lineage, so the problem of “strains matching lag” of traditional vaccines is more prominent.

[0004] The emergence of mRNA vaccine technology provides a new path to solve the above-mentioned difficulties. This technology synthesizes mRNA molecules encoding pathogenic antigens in vitro, and then expresses antigens after being introduced into host cells by a delivery system, thereby simultaneously inducing humoral and cellular immune responses. Compared with traditional vaccines, mRNA vaccines have three core advantages: first, the development cycle is short, and sequence design and preparation can be completed in 4-8 weeks, which can quickly match the variant strain; second, the safety is high, and the production process does not involve live virus operation, and there is no risk of reverse transcription integration and virulence return; third, the immunogenicity is comprehensive, and the secreted IgA (sIgA) response of mucosal immunity can be activated by optimizing antigen design. However, the mRNA vaccine development of SADS-CoV is still in the blank stage. The core challenge is that: as the key antigen for mediating viral invasion, the stability of the pre-fusion conformation of the S protein, the immunogenicity of the receptor binding domain (RBD), and the cell expression efficiency of the mRNA sequence have not been clearly defined, and need to be optimized in combination with structural biology and nucleic acid engineering technology. Therefore, developing an mRNA vaccine targeting key antigens of SADS-CoV with safety and broad-spectrum protection is a technical problem that needs to be solved. SUMMARY

[0005] To solve the problems in the background art, the present application provides a S protein ancestral sequence of porcine acute diarrhea syndrome coronavirus A The S mRNA and its application in vaccines are of great significance to animal feeding safety and prevention and control of zoonotic diseases.

[0006] To achieve the above-mentioned purpose, in the first aspect, the present application provides a S protein ancestral sequence of porcine acute diarrhea syndrome coronavirus A The S mRNA has an amino acid sequence of SEQ ID NO. 1.

[0007] Further, the nucleotide sequence is SEQ ID NO. 2.

[0008] In the second aspect, the present application provides a liposome nanoparticle containing the above-mentioned S protein ancestral sequence of porcine acute diarrhea syndrome coronavirus A S mRNA.

[0009] In the third aspect, the present application provides a biological material containing the above-mentioned S protein ancestral sequence of porcine acute diarrhea syndrome coronavirus A S mRNA.

[0010] Further, it includes an expression cassette, a recombinant vector, or a recombinant microorganism.

[0011] Specifically, the biological material is selected from any one of the following:

[0012] (A1) encodes the aforementioned mRNA molecule A DNA molecules encoding the S-code sequence;

[0013] (A2) An expression cassette containing the DNA molecule described in (A1);

[0014] (A3) A recombinant vector containing the DNA molecule described in (A1) or a recombinant vector containing the expression cassette described in (A2);

[0015] (A4) A recombinant microorganism containing the DNA molecule described in (A1), or a recombinant microorganism containing the expression cassette described in (A2), or a recombinant microorganism containing the recombinant vector described in (A3).

[0016] Fourthly, the present invention provides an ancestral sequence of the S protein of porcine acute diarrhea syndrome coronavirus. A The SmRNA vaccine contains the ancestral S protein sequence of the porcine acute diarrhea syndrome coronavirus mentioned above. A S mRNA.

[0017] This application has the following beneficial effects:

[0018] This invention, through phylogenetic analysis, constructed the ancestral sequence of the SADS-CoV Spike protein. This sequence targets not only existing viruses but also the viral evolutionary lineage. This approach shifts vaccine design from targeted defense against a single pathogen to proactive intervention in the viral evolutionary trajectory. After determining... A Following the characterization of S mRNA-LNP, studies were conducted in mice, piglets, and sows. A The S mRNA vaccine was evaluated for immunization. In pig and mouse models, maternal immunization provided complete protection to offspring against lethal SADS-CoV. Actively immunized piglets also acquired complete protection. More importantly, vaccine-induced antibodies prevented viral replication in human cell lines, with protective efficacy lasting at least 140 days. These results indicate that the prepared vaccine... A S mRNA vaccines are of great significance for animal husbandry safety and the prevention and control of zoonotic diseases. Attached Figure Description

[0019] Figure 1 for A Phylogenetic analysis diagram of S mRNA ancestral sequence;

[0020] Figure 2 for A Serum neutralization titer of S mRNA vaccine in immunized mice;

[0021] Figure 3 for ASurvival rate of F1 generation suckling mice after intracranial injection of SADS-CoV / HNNY / 2023 in S mRNA vaccine group and PBS group;

[0022] Figure 4 For A Histological immunofluorescence of F1 generation suckling mice after intracranial injection of SADS-CoV / HNNY / 2023 in S mRNA vaccine group and PBS group;

[0023] Figure 5 For A HE tissue section of F1 generation suckling mice after intracranial injection of SADS-CoV / HNNY / 2023 in S mRNA vaccine group and PBS group;

[0024] Figure 6 For A Neutralization titer of colostrum after immunization of sows with S mRNA vaccine;

[0025] Figure 7 For A Neutralization titer of serum after immunization of sows with S mRNA vaccine;

[0026] Figure 8 For A Tissue viral load of F1 generation piglets after oral administration of SADS-CoV / HNNY / 2023 in S mRNA vaccine group and PBS group;

[0027] Figure 9 For A Survival rate of F1 generation piglets after oral administration of SADS-CoV / HNNY / 2023 in S mRNA vaccine group and PBS group;

[0028] Figure 10 For A Histological immunofluorescence of F1 generation piglets after oral administration of SADS-CoV / HNNY / 2023 in S mRNA vaccine group and PBS group;

[0029] Figure 11 For A HE tissue section of F1 generation piglets after oral administration of SADS-CoV / HNNY / 2023 in S mRNA vaccine group and PBS group;

[0030] Figure 12 For A Neutralization titer of serum after immunization of piglets with S mRNA vaccine;

[0031] Figure 13 For A Tissue viral load of piglets after oral administration of SADS-CoV / HNNY / 2023 in S mRNA vaccine group and PBS group;

[0032] Figure 14 for A Immunofluorescence images of tissues from piglets in the S mRNA vaccine group and PBS group after oral administration of SADS-CoV / HNNY / 2023;

[0033] Figure 15 for A HE tissue sections of piglets in the S mRNA vaccine group and PBS group after oral administration of SADS-CoV / HNNY / 2023;

[0034] Figure 16 for A Serum neutralizing titer of S mRNA vaccine after immunization in piglets during neutralizing antibody duration experiment. Detailed Implementation

[0035] The present invention will be further described below with reference to the embodiments. Unless otherwise specified, the raw materials used in the embodiments of this application are all commercially available.

[0036] Experimental materials

[0037] Cells, viruses, and laboratory animals: Vero, CEF, and Huh7 cells were preserved in the applicant's laboratory; the porcine acute diarrhea syndrome coronavirus strain SADS-CoV / HNNY / 2023, classified as Swine acute diarrhea syndrome coronavirus strain SADS-Cov / HNNY / 2023, was deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO.V202455 on May 28, 2024; pregnant sows and 5-day-old healthy piglets (SADS-CoV nucleic acid and antibody negative) were provided by a pig farm in Nanyang City, Henan Province; 4-6 week old BALB / c mice were purchased from Zhengzhou Antu Biotechnology and raised under suitable temperature and humidity conditions.

[0038] Example 1 A S ancestral sequence and its vaccine preparation

[0039] The spike protein S of SADS-CoV was selected as the target protein. Phylogenetic analysis was used to construct the ancestral sequence of the SADS-CoV Spike protein, and it was named... A S.

[0040] A Phylogenetic analysis diagram of S ancestral sequence is shown below. Figure 1As shown. The amino acid sequences of the SADS-CoV spike protein published in GenBank (https: / / www.ncbi.nlm.nih.gov / genbank / ) were collected, and genetic evolutionary analysis and ancestral sequence reconstruction were performed, using Turkish coronavirus as the outgroup. The specific process is as follows: Sequences were aligned using MAFFT (parameters were set to global alignment algorithm and a maximum of 1000 iterations to ensure high-quality multiple sequence alignment results). Then, maximum likelihood phylogenetic analysis was performed using MEGA 11, employing the WAG+F+I evolutionary model with 500 bootstrap tests and all other parameters set to default values. Based on the obtained maximum likelihood tree and MAFFT, the ancestral sequence of the spike protein was inferred using the MEGA 11 maximum likelihood algorithm (using the WAG+F+I model). Node 1 (Clade 1) was considered the ancestral sequence of all SADS-CoV spike proteins.

[0041] To ensure effective protein expression, a cozak sequence was added to the 5' end of the sequence to significantly improve the translation initiation efficiency and accuracy of mRNA in eukaryotic host cells.

[0042] A

[0043] After optimizing the DNA sequence codons encoding the above proteins, 5′UTR, 3′UTR and polyA sequences were added. The gene was synthesized by Nanjing GenScript and cloned into the pcDNA3.1 plasmid to synthesize the mRNA vaccine template plasmid and encapsulate LNP.

[0044] coding A

[0045] Furthermore, an mRNA vaccine was designed targeting the ancestral sequence of the SADS-CoV Spike protein, namely: A S mRNA-LNP. Using this... A After pigs and mice were immunized with the S mRNA vaccine, maternal immunity provided complete protection for their offspring against the lethal attack of SADS-CoV.

[0046] A The preparation process of S mRNA vaccines is as follows: The encoding... A The DNA sequence of S, i.e., the base sequence of SEQ ID NO.2, was synthesized into the pcDNA3.1 vector with added 5'UTR, 3'UTR, and a Poly A tail, to construct pcDNA3.1- A The expression plasmid for S was constructed correctly by sequencing. The correctly sequenced plasmid was then cultured in bacterial culture and subjected to large-scale plasmid extraction. After linearization by enzyme digestion, the plasmid was transcribed in vitro using an in vitro transcription kit, followed by purification using lithium chloride precipitation. Subsequently, the mRNA was capped and packaged into mRNA-LNP using liposomes.

[0047] Example 2 Immunological evaluation of mRNA-LNP mice

[0048] 1. Mouse experimental protocol

[0049] To evaluate the immunogenicity of the mRNA vaccine, 4-6 week old BALB / c mice were immunized at a vaccine concentration of 100 ng / μL. The mice were divided into two groups of seven each: five females and two males. A In the S immunization group, each mouse was subcutaneously injected with 0.2 mL of vaccine (containing 20 µg of vaccine) in its back, with PBS serving as the control group. A booster immunization with the same dose of vaccine was administered two weeks later. Blood samples were collected on days 0, 7, 14, 21, and 28 after the initial immunization, and serum was separated and stored at -20°C for later use.

[0050] 2. Neutralization test

[0051] The collected serum samples were heat-inactivated at 56°C for 30 min, followed by a neutralization test using Vero cells. The serum was serially diluted twofold and mixed with 200 TCID50. 50 SADS-CoV / HNNY / 2023 virus was incubated at 37°C for 1 h, then added to 96-well cell culture plates pre-coated with Vero cells and incubated for 2 days. Cytopathic effect (CPE) was demonstrated using an immunoperoxidase monolayer assay (IPMA) with SADS-CoV-N monoclonal antibody stored in the applicant's laboratory, with the highest dilution of virus neutralization at 50% of the wells as the endpoint.

[0052] The results are as follows Figure 2 As shown, no neutralizing antibodies were produced in the mice at day 7. Neutralizing antibody titers were successfully stimulated on day 14 post-immunization, and rapidly increased after booster immunization, reaching a peak on day 28.

[0053] 3. Intracranial Injection of Virus into F1 Generation Rats

[0054] Following the mouse immunization protocol, on day 28 post-immunization, two female mice and two male mice were randomly selected from each group and housed in a 1:1 ratio. Five days after birth, the pups were challenged with intracranial injection of the virus. After disinfecting the pups' brains, the target brain region was located using the anterior fontanelle as the origin. After skin preparation and disinfection, a microsyringe was used to puncture the brain to a certain depth and inject the virus solution. After challenge, the needle was removed, and pressure was applied to stop bleeding. Clinical symptoms and other indicators were monitored according to the experimental protocol.

[0055] The results are as follows Figure 3 As shown, using 4.2×10 5 TCID 50 SADS-CoV / HNNY / 2023 attacked 5-day-old F1 generation mice. A All suckling mice in the SmRNA vaccine group (n=13) survived, while all mice in the PBS group (n=11) died. Four mice died on day 4 after challenge, and seven mice died on day 5 after challenge.

[0056] 4. Histopathological observation

[0057] Once the challenged mice died or reached the experimentally set time limit, the pups were promptly dissected. Brain, heart, liver, spleen, lungs, kidneys, small intestine, and large intestine tissues were fixed with 4% paraformaldehyde to prepare pathological sections. Immunofluorescence detection of the mouse tissues was performed using SADS-CoV-N monoclonal antibody. Simultaneously, hematoxylin and eosin (H&E) staining was used to observe histopathological changes.

[0058] The results are as follows Figure 4 , Figure 5 As shown, the results were detected using tissue immunofluorescence and pathological sections. A The protective effect of the S mRNA vaccine. PBS mice showed high levels of viral nucleoprotein expression in the brain, large intestine, and heart. A The S mRNA vaccine group almost completely blocked the proliferation of SADS-CoV in all common organs, including the brain, heart, liver, spleen, lungs, kidneys, large intestine, and small intestine.

[0059] Example 3: Immunological evaluation of mRNA-LNP sow immunization and piglet challenge.

[0060] 1. Sow immunization program

[0061] The vaccine concentration used was 100 ng / μL. One healthy pregnant sow (SADS-CoV negative) was administered the vaccine via intramuscular injection in the neck. A SmRNA-LNP, 1 mL / pig (content 100 µg / pig), was administered as a booster immunization 14 days later. Piglets were challenged with the virus 5 days after birth and after nursing. Colostrum and blood were collected from the sows, and serum was separated and stored at -20°C.

[0062] 2. Neutralization test

[0063] The neutralization test was the same as in Example 2-2.

[0064] The results are as follows Figure 6 , Figure 7 As shown, both colostrum and serum of immunized sows produced high levels of neutralizing antibodies, with the level of neutralizing antibodies in colostrum being higher than that in serum.

[0065] 3. Measurement of viral load in tissues

[0066] After the piglets die, or on the 7th day after the challenge, the piglets are eviscerated and jejunum and ileum tissues are collected to determine the viral load of SADS-CoV in the intestine. The SADS-CoV genomic cDNA copy number is quantitatively detected by qRT-PCR. The main steps are as follows: (1) Take 0.3 g of jejunum / ileum tissue, add 1 mL of Trizol for homogenization and breakage, centrifuge at 12000 rpm / min for 5 min, and take the supernatant to extract tissue RNA according to the RNA extraction instructions. Complementary DNA (cDNA) was generated using a reverse transcription kit (TaKaRa, Dalian, catalog number RR036A) as a template for RT-qPCR. The content of SADS-CoV nucleocapsid protein (N) RNA was determined by absolute RT-qPCR to represent the viral RNA content of infected cells. Primers and reaction systems are shown in Tables 1 and 2, respectively. The premixed solution for the staining method was AceQ Universal SYBR qPCR master Mix (Nanjing Novizan Biotechnology Co., Ltd., catalog number Q511-03). The reaction program used was the Fast program built into the ABI quantitative PCR instrument (Applied Biosystems, USA).

[0067] The experiment was conducted independently three times, with three replicates each time. Experimental data are expressed as group means and standard deviations (SD). Statistical analysis was performed using an unpaired, two-tailed Student's t-test in GraphPad Prism 8.0 software. ****p < 0.0001 was considered statistically significant. Results are as follows... Figure 8 As shown, compared with the PBS group, A The RNA content in the jejunum and ileum tissues of the S mRNA vaccine group was significantly increased, indicating thatA S mRNA vaccines can significantly protect piglets after challenge. And, as... Figure 9 As shown, the survival rate of newborn piglets challenged with PBS after immunization of sows was 60%, with one piglet dying at 2 days of age and another at 3 days of age.

[0068] Table 1. Primer sequences for absolute real-time PCR

[0069]

[0070] Table 2. Reaction System

[0071]

[0072] 4. Histopathological observation

[0073] Once the piglets died or reached the experimentally set time limit, they were promptly dissected. The duodenum, jejunum, ileum, cecum, and colon were fixed with 4% paraformaldehyde to prepare pathological sections. Hematoxylin and eosin (H&E) staining was used to observe histopathological changes. Simultaneously, SADS-CoV-N monoclonal antibody was used to perform immunofluorescence detection on the piglet intestinal tissue.

[0074] like Figure 10 , Figure 11 As shown, A S mRNA significantly reduced viral load in the jejunum and ileum, which is consistent with Figure 8 The qPCR results were consistent. Histopathological examination showed no obvious histopathological changes in the ileum, jejunum, duodenum, cecum, or colon of piglets born to vaccinated sows. However, piglets born to control sows showed significant histopathological changes, including villus loss, blunting, atrophy, hemorrhage, inflammation, and vacuolation in the jejunum and ileum. The results indicate that newborn piglets acquired effective passive immunization, protecting them from SADS-CoV infection.

[0075] Example 4: Immunological evaluation of mRNA-LNP piglets

[0076] 1. Piglet Experimental Protocol

[0077] The vaccine concentration used was 100 ng / μL. Eight 5-day-old healthy piglets (SADS-CoV negative) were randomly divided into two groups of four each. The mRNA group received intramuscular injection in the neck. A S mRNA-LNP, 0.5 mL / head (content 50 µg / head), and the PBS group received the same dose of PBS via intramuscular injection in the neck as a control. Two weeks later, piglets were boosted with the same dose of vaccine. Two weeks after the booster immunization, each group of piglets received 5 × 10⁵ mRNA-LNP orally. 5.625 TCID50 SADS-CoV / HNNY / 2023 viral fluid was used to isolate and feed each group of piglets for 7 days. After challenge, the piglets' condition was observed daily, rectal temperature was measured, and body weight was taken.

[0078] 2. Neutralization test

[0079] The neutralization test was the same as in Example 2-2.

[0080] The results are as follows Figure 12 As shown, high titers of neutralizing antibodies were produced 14 days after booster immunization, i.e., day 28, while they were not detected on day 14 after immunization.

[0081] 3. Measurement of viral load in tissues

[0082] The method for determining viral load in tissues is the same as in Examples 3-3.

[0083] The experiment was conducted independently three times, with three replicates each time. Experimental data are expressed as group means and standard deviations (SD). Statistical analysis was performed using an unpaired, two-tailed Student's t-test in GraphPad Prism 8.0 software. ****p < 0.0001 was considered statistically significant. Results are as follows... Figure 13 As shown, compared with the PBS group, A The RNA content in the jejunum and ileum tissues of the S mRNA vaccine group was significantly reduced, indicating that A S mRNA vaccines can significantly protect piglets after challenge.

[0084] 4. Histopathological observation

[0085] The methods for histopathological observation are the same as those in Examples 3-4.

[0086] like Figure 14 , Figure 15 As shown, A The S mRNA vaccine significantly reduced viral load in the jejunum and ileum, which is consistent with... Figure 13 The qPCR results were consistent. Histopathological examination showed that... A The S mRNA vaccine significantly reduced the pathological damage caused by the virus in the jejunum, ileum, duodenum, colon, and cecum. The results indicate that... A S mRNA vaccines can reduce clinical symptoms and pathological damage after challenge with SADS-CoV in actively immunized piglets, and reduce viral load.

[0087] Example 5: Duration of mRNA-LNP Neutralizing Antibody in Piglets

[0088] 1. Piglet Experimental Protocol

[0089] The vaccine concentration used was 100 ng / μL. Six 5-day-old healthy piglets (SADS-CoV negative) were randomly divided into two groups of three each. The mRNA group received intramuscular injection in the neck. A S mRNA-LNP, 0.5 mL / head (content 50 µg / head), and the PBS group was injected intramuscularly in the neck with the same dose of PBS as the control group. Piglets were booster immunized with the same dose of vaccine at 2-week intervals. Blood was collected from piglets via the anterior vena cava on days 14, 28, 50, 80, 120, and 140 post-immunization, and serum was separated and stored at -20°C.

[0090] 2. Neutralization test

[0091] The neutralization test was the same as in Example 2-2.

[0092] To detect the dynamic curve of antibodies after immunization, 5-day-old piglets were immunized twice. A After S mRNA is released, serum is collected at specified time points for neutralization assays. For example... Figure 16 As shown, the results indicated that high levels of neutralizing antibodies were detected in the serum of piglets on day 14 after booster immunization, which then slowly decreased and persisted until day 140 after immunization.

Claims

1. Ancestor sequence of the S protein of porcine acute diarrhea syndrome coronavirus. A S mRNA, characterized by, Its amino acid sequence is SEQ ID NO.

1.

2. The ancestral S protein sequence of porcine acute diarrhea syndrome coronavirus according to claim 1. A S mRNA, characterized by, Its nucleotide sequence is SEQ ID NO.

2.

3. A liposome nanoparticle, characterized in that, Contains the ancestral S protein sequence of porcine acute diarrhea syndrome coronavirus as described in claim 1 or 2. A S mRNA.

4. A biomaterial, characterized in that, Contains the ancestral S protein sequence of porcine acute diarrhea syndrome coronavirus as described in claim 1 or 2. A S mRNA.

5. The biomaterial according to claim 4, characterized in that, This includes expression cassettes, recombinant vectors, or recombinant microorganisms.

6. Ancestor sequence of the S protein of porcine acute diarrhea syndrome coronavirus A S mRNA vaccine, characterized by, Contains the ancestral S protein sequence of porcine acute diarrhea syndrome coronavirus as described in claim 1 or 2. A S mRNA.

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