A self-cleavable and multiple viral structural protein expressing PDCoV viral mRNA vaccine and a preparation method thereof

Abstract

The application provides a PDCoV virus mRNA vaccine which can be self-cleaved and expresses multiple virus structure proteins and a preparation method thereof, the vaccine comprises mRNA for expressing PDCoV virus S, M and N proteins, and LNP for encapsulating the mRNA, denoted as SMN-mRNA-LNP. The application firstly proposes a PDCoV mRNA vaccine strategy based on S, M and N combination, connects three structure proteins of PDCoV S, M and N by using a self-cleaving peptide P2A, mutates the S protein with double proline, enables a single mRNA to express multiple PDCoV antigens, and utilizes the neutralizing antibody induction ability of the S protein, the immune regulation function of the M protein and the cell immune activation characteristics of the N protein to construct a multi-level defense system. Through evaluation of immunogenicity, antibody level and challenge protection effect of the vaccine in mice, suckling piglets and pregnant sows, a new idea for developing a broad-spectrum and high-efficiency PDCoV vaccine is provided, and practical basis for research and development of a coronavirus multi-antigen mRNA vaccine is provided.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to a PDCoV virus mRNA vaccine that can self-cleave and express multiple viral structural proteins. Background Technology

[0002] Coronaviruses are an important class of pathogens that can cause severe, fatal, and highly prevalent diseases in humans and other animals. Studies have shown that many animal coronaviruses have the potential to cross species and spread to humans; therefore, controlling the spread of animal coronaviruses is crucial for human health. Porcine deltacoronavirus (PDCoV) is a newly emerging porcine enteric pathogen that can infect pigs of all ages, causing watery diarrhea, vomiting, and intestinal pathological damage, with particularly high mortality rates in newborn piglets. Since its initial discovery and reporting in 2012, it has gradually spread globally, posing a serious threat to the global pig farming industry in recent years. Other studies have shown that PDCoV can also infect chickens, turkeys, calves, and children, possessing the potential for cross-species transmission and posing a serious threat to public health security. Vaccination is the most effective method for preventing and controlling this disease, but currently there is no commercially available vaccine against PDCoV. Therefore, developing an effective and safe vaccine is an urgent need for the prevention and control of this disease.

[0003] The development strategies for coronavirus vaccines mainly include traditional inactivated and attenuated vaccines, as well as novel genetically engineered vaccines. Genetically engineered vaccines primarily include subunit vaccines, nucleic acid vaccines, and live viral vector vaccines. In recent years, mRNA vaccine technology has demonstrated revolutionary potential in the field of infectious disease control due to its short development cycle, ease of industrialization, simple production process, flexibility in responding to new variants, and advantages in combining humoral and cellular immune activation.

[0004] PDCoV belongs to the genus *delta* of the order *Nidovirales* in the family *Coronairidae*. It is a single-stranded, positive-sense RNA virus. Mature viral particles contain four structural proteins: S (spike glycoprotein), M (membrane protein), N (nucleoprotein protein), and E (envelope protein). The coronavirus S protein is a type I membrane glycoprotein, distributed in spikes on the surface of the viral particle. It is mainly responsible for recognizing and binding to host receptors and fusing with host cells, mediating viral entry into host cells. The M protein is the most abundant transmembrane protein on the surface of the coronavirus envelope, exhibiting high conservation among different coronaviruses. It is responsible for the assembly and release of viral particles. During viral entry, the M protein plays a crucial role in the interaction between the viral envelope and the host cell membrane. Studies have shown that the M protein contains T-cell epitopes and neutralizing antibody epitopes, which can induce a strong T-cell immune response, making it a potential target for coronavirus vaccine research. The N protein is the most abundant and functionally diverse protein in coronaviruses, playing an important role in viral replication. Although vaccine development has primarily focused on the S protein, research indicates that the N protein is also a major target antigen for antibody responses and T-cell epitopes. Vaccines targeting the N protein can induce strong humoral and cellular immune responses and provide protection against coronavirus infection. Therefore, the coronavirus N protein can also serve as a candidate vaccine antigen. Current coronavirus mRNA vaccine research is largely limited to the single S protein antigen, while the viral M and N proteins also participate in key immune responses in natural infection. The M and N proteins possess abundant and conserved T-cell epitopes, which can enhance cross-protective capabilities. Therefore, the design of mRNA vaccines integrating multiple antigenic components holds promise for overcoming the limitations of single-target vaccines. Summary of the Invention

[0005] Based on the above, the purpose of this invention is to provide a PDCoV virus mRNA vaccine that can self-cleave and express multiple viral structural proteins, thereby enhancing the breadth and durability of immune protection against PDCoV virus through the synergistic effect of multiple components.

[0006] To achieve its purpose, the present invention adopts the following technical solution:

[0007] The present invention provides a PDCoV virus mRNA vaccine that can self-cleave and express multiple viral structural proteins, comprising mRNA that simultaneously expresses PDCoV virus S, M, and N proteins, and LNPs encapsulating the mRNA, denoted as SMN-mRNA-LNP.

[0008] The method for preparing the above-mentioned PDCoV virus mRNA vaccine that can self-cleave and express multiple viral structural proteins includes the following steps:

[0009] (1) Plasmid DNA construction

[0010] Using the PDCoV virus sequence as a reference, and codon optimization of the S, M, and N gene sequences, a double proline mutation (E855P, V856P) was introduced into the S gene to design and synthesize the mRNA in vitro transcription plasmid pSMN; the nucleotide sequence of the plasmid pSMN is shown in SEQ ID NO.1.

[0011] (2) mRNA production

[0012] Using pSMN plasmid as a template, P2A self-cleaving peptides were introduced between S and M, and between M and N, respectively. After in vitro transcription, single-stranded mRNA with 5′-m7G Cap1, poly(A) structure and N1-methyl-piperazine modification was obtained. Enzymes, DNA template and free nucleotides were removed by magnetic bead method to obtain high-purity mRNA stock solution.

[0013] (3) LNP encapsulation

[0014] LNP, DSPC, cholesterol, and DMG-PEG2000 were dissolved in ethanol to obtain an LNP-ethanol mixture. This mixture was then mixed with 20mM citrate buffer containing mRNA to encapsulate the mRNA, yielding mRNA-LNP. Finally, the mRNA-LNP was dialyzed with phosphate buffer, centrifuged, and concentrated to obtain SMN-mRNA-LNP. As a further preferred embodiment of the present invention, in step (1), the PDCoV virus is the CH / XJYN / 2016 strain.

[0015] Preferably, in step (2), the self-cleaving peptide is a P2A self-cleaving peptide.

[0016] In step (3), the concentration of LNP in the LNP ethanol mixture is 25 μM / mL, the concentration of DSPC is 5 μM / mL, the concentration of cholesterol is 19.25 μM / mL, and the concentration of DMG-PEG2000 is 0.75 μM / mL.

[0017] The citrate buffer solution contained 106.84 μg / mL of mRNA.

[0018] The volume ratio of the LNP ethanol mixture to the citrate buffer is 1:3.

[0019] The beneficial effects of this invention are as follows:

[0020] This invention proposes for the first time a PDCoV mRNA vaccine strategy based on the combined use of S, M, and N triantigens. The three structural proteins of PDCoV, S, M, and N, are linked using the self-cleaving peptide P2A, and the S protein undergoes a double-proline mutation, enabling a single mRNA to express multiple PDCoV antigens. A multi-layered defense system is constructed by utilizing the neutralizing antibody-inducing ability of the S protein, the immunomodulatory function of the M protein, and the cellular immune activation properties of the N protein. The immunogenicity, antibody levels, and protective efficacy against viral challenge of this vaccine in mice, suckling piglets, and pregnant sows are evaluated to provide new insights for developing broad-spectrum and highly effective PDCoV vaccines and to provide practical evidence for the development of multi-antigen mRNA vaccines for coronaviruses.

[0021] Specifically, this invention develops a novel mRNA vaccine, SMN-mRNA-LNP, capable of expressing the three major structural proteins S, M, and N of PDCoV via a single mRNA. This vaccine provides superior active immunization protection for piglets compared to S2P-mRNA-LNP, which expresses only the S protein. Furthermore, after immunization of pregnant sows with SMN-mRNA-LNP, the IgA antibody titer in colostrum reached 105.4, 25 times that of the inactivated vaccine group. Newborn piglets can acquire a higher level of passive immunity through breast milk intake, ultimately protecting them from PDCoV attack. Attached Figure Description

[0022] Figure 1 Design and expression validation of PDCoV mRNA vaccine;

[0023] Figure 2 Immunogenicity of S2P-mRNA-Lnp in mice;

[0024] Figure 3 The active immune protection effect of S2P-mRNA-LNP and SMN-mRNA-LNP on piglets;

[0025] Figure 4 Immunogenicity of SMN-mRNA-LNP in pregnant sows;

[0026] Figure 5 This study aimed to assess the protective effect of SMN-mRNA-LNP on passive immunity in piglets. Detailed Implementation

[0027] The present invention will now be described in detail with reference to the accompanying drawings.

[0028] 1. Method

[0029] 1.1 Construction of plasmid DNA template

[0030]

[0031] 1.2 mRNA production and LNP encapsulation

[0032] 1.2.1 mRNA production

[0033]

[0034]

[0035] 1.2.2 LNP Encapsulation

[0036] Cationic lipids, 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), cholesterol, and DMG-PEG2000 were dissolved in 2 mL of ethanol at a molar ratio (μM) of 50:10:38.5:1.5, and then analyzed using Nano Assemblr Ignite based on NxGen microfluidic technology. TM The system encapsulated the mRNA by mixing the lipid mixture with 6 mL of 20 mM citrate buffer (pH 4.0, mRNA concentration 106.84 μg / mL). The mRNA-LNPs were then dialyzed with phosphate-buffered saline (PBS) (pH 7.4) and finally concentrated to the desired concentration using a 100 kDa Amicon ultrafiltration tube (Millipore, USA) to obtain two mRNA-LNPs, named S2P-mRNA-LNP and SMN-mRNA-LNP, respectively.

[0037] 1.3 Immunoblot Analysis

[0038] S2P-mRNA-LNP and SMN-mRNA-LNP (2 μg / well) were transfected into 293T cells at a cell density of approximately 80%. Control wells were treated with lipo3000, and cells were incubated at 37°C for 24 h. After 24 h, cells were collected, treated with cell lysis buffer, and subjected to polyacrylamide gel electrophoresis. Immunoblot analysis was then performed using nitrocellulose membranes (Pall, USA). Rabbit anti-S, M, or N polyclonal antibodies (1:500 dilution) prepared in the applicant's laboratory were used as primary antibodies, and HRP-labeled goat anti-rabbit IgG (1:10,000 dilution, Zhongshan Jinqiao, Beijing, China) was used as secondary antibodies. Imaging was performed using chemiluminescent substrate (ECL, ThermoScientific, USA).

[0039] 1.4 Immunofluorescence detection

[0040] S2P-mRNA-LNP and SMN-mRNA-LNP (2 μg / well) were transfected into LLC-PK cells at approximately 80% confluency. Control wells were treated with Lipo3000, and the cells were incubated at 37°C for 24 h. After 24 h, the culture medium was discarded, the cells were washed three times with PBS, fixed with 4% paraformaldehyde at 4°C for 1 h, permeabilized with 0.25% Triton-100 at room temperature for 10 min, and then blocked with 5% BSA for 1 h. Rabbit anti-S, M, or N polyclonal antibodies prepared in the applicant's laboratory and 594-labeled goat anti-rabbit IgG were used as primary and secondary antibodies, respectively. The cells were stained with 4′,6-diamidinyl-2-phenylindole in the dark for 5 min, washed three times with PBS, and observed under a fluorescence microscope.

[0041] 1.5 Mouse Immunization Test

[0042] Eighteen 4-6 week old specific pathogen-free female BALB / c mice were randomly divided into three groups (6 mice / group): S2P-mRNA-LNP, PDCoV inactivated vaccine, and PBS control group. Mice in the S2P-mRNA-LNP group were vaccinated with 30 μg / mouse, mice in the PDCoV inactivated vaccine group were vaccinated with 100 μL / mouse, and the control group was injected with PBS. All three groups of mice were immunized once each via subcutaneous injection in the back on day 0 and day 14. Figure 2 A), and blood was collected from each mouse before immunization, on day 14 and day 28 after the initial immunization.

[0043] 1.6 Active immunization and virus challenge protection test for suckling piglets

[0044] Twenty 5-day-old suckling piglets were purchased from commercial pig farms with no history of PDCoV infection or vaccination. Indirect ELISA and RT-QPCR were used to detect PDCoV in serum and fecal swabs from sows and piglets to ensure PDCoV negativity. The 20 5-day-old suckling piglets were randomly divided into four groups (5 piglets / group): S2P-mRNA-LNP, SMN-mRNA-LNP, PDCoV inactivated vaccine, and PBS control group. Piglets in the S2P-mRNA-LNP and SMN-mRNA-LNP groups were vaccinated with 40 μg / piglet, while piglets in the PDCoV inactivated vaccine group were vaccinated with 1 mL / piglet (pre-inactivation viral load was 1.0 × 10⁻⁶). 6.5 TCID 50 Piglets in each group were vaccinated with PBS ( / ml) and the control group was injected with PBS. Piglets in each group were immunized once on day 0 and once on day 14. Figure 3 A) Serum was collected before immunization and on days 14 and 28 after the initial immunization. On day 28 post-immunization, all piglets were orally vaccinated with 8 mL × 10 ml. 4.0 TCID 50 / mLCH / XJYN / 2016(Gao et al.,2020). After inoculation with PDCoV, the clinical symptoms of piglets were observed for 7 consecutive days and the amount of viral RNA excreted in fecal swabs was detected.

[0045] 1.7 Passive immunization and virus challenge protection experiment in suckling piglets

[0046] Nine PDCoV antigen- and antibody-negative sows were randomly divided into three groups (3 sows per group). Forty-two days before farrowing, the three groups of sows were injected with SMN-mRNA-LNP at a dose of 100 μg / sow, and PDCoV inactivated vaccine at a dose of 2 mL / sow (with a pre-inactivation viral load of 1.0 × 10⁻⁶). 6.5 TCID 50 / ml) and PBS. All sows received booster immunizations 21 days after the primary immunization, and serum was collected before immunization, on day 21 after the primary immunization, and on the day of farrowing. Colostrum was collected from sows after farrowing for antibody testing. Five days after lactation, five piglets were randomly selected from each group (piglets from different sows within the same group were selected to minimize the influence of individual sow differences), and each piglet was orally challenged with 1mL × 10 4.0 TCID 50 / mL CH / XJYN / 2016. After challenge, the clinical symptoms of piglets were observed daily to monitor the amount of virus shed and the diarrhea score of each group of piglets. The clinical symptom diarrhea score was: 0 = normal; 1 = pasty; 2 = semi-fluid; and 3 = liquid.

[0047] 1.8 Specific antibody detection

[0048] Indirect ELISA method was used to determine the levels of specific antibodies against S, M and N proteins in serum and colostrum (Yu R, Zhang L, Zhou P, Zhang Z, Liu X, Wang Y, Guo H, Pan L, Liu X. 2024. Evaluation of the immunoprotective effects of porcine deltacoronavirus subunitvaccines. Virology 590:109955). Specifically, purified S, M, and N proteins were diluted to 2 μg / mL with 50 mM carbonate buffer (pH 9.6) and coated onto 96-well microplates (Corning, USA), 100 μl / well, and incubated overnight at 4°C. The coating solution was discarded, the plates were washed three times with PBST, and 200 μL / well of blocking buffer containing 5% skim milk powder was added. The plates were incubated at 37°C for 2 h. The blocking solution was discarded, the plates were washed three times with PBST, and 100 μL of serially diluted serum or colostrum sample was added to each well. The plates were incubated at 37°C for 1 h. The liquid in the wells was discarded, the plates were washed three times with PBST, and 100 μL of HRP-labeled goat anti-mouse IgG (1:10,000 dilution, Zhongshan Jinqiao, Beijing, China), HRP-labeled goat anti-pig IgG (1:10,000 dilution, Abcam, UK), or HRP-labeled goat anti-pig IgA (1:10,000 dilution) diluted with serum dilution was added to each well. Dilute 0.000 (Abcam, UK), incubate at 37℃ for 1 h; discard the liquid in the wells, wash 3 times with PBST, add 100 μL of TMB substrate solution (Solepro, Beijing, China) to each well, and react at 37℃ in the dark for 10 min; add 100 μL of 2M H2SO4 to each well to stop the reaction, and measure the absorbance at 450 nm using a microplate reader.

[0049] 1.9 Neutralization Experiment

[0050] The titer of PDCoV neutralizing antibodies in serum was determined using a fixed virus-diluted serum method. The specific procedures were as follows: Prepare a 96-well plate containing 70%-80% LLC-PK cells; inactivate the serum in a 56°C water bath for 30 min, then perform serial 2-fold dilutions in 96-well microplates, with each dilution replicated in 4 wells. Add an equal volume of diluted virus solution (200 TCID50) to each well. 50 After mixing, incubate at 37℃ with 5% CO2 for 1 hour; set up a virus control and a normal cell control, with the virus control set at 200 TCID50. 50 (All lesions), 20 TCID 50 2TCID 50 0.2TCID 50(Non-pathogenic) Four different concentrations of control cells were used. Cells were washed three times with PBS, and 100 μL of virus-serum mixture was added to each well. The cells were incubated at 37°C with 5% CO2 for 1 hour. After incubation, the liquid in the wells was discarded, and the cells were washed three times with PBS. 200 μL of maintenance medium (containing 20 μg / mL trypsin) was added to each well. Cell cytotoxicity (CPE) was observed and recorded daily. The neutralizing titer was defined as the reciprocal of the serum dilution required to neutralize 50% of the viral infection.

[0051] 1.10 Flow cytometry analysis

[0052] On day 28 post-immunization, mouse spleens were harvested, and lymphocyte suspensions were prepared using an animal lymphocyte separation kit (Solarbio, Beijing, China). The lymphocyte suspensions were diluted to 5 × 10⁶ cells / mL using RPMI 1640 medium (Gibco, USA). 6 Cells / mL were collected, and 100 μL of lymphocyte suspension was added according to the manufacturer's instructions. Appropriate amounts of FITC-conjugated anti-mouse CD3 antibody (BioLegend, Cat.:100204), PE-conjugated anti-mouse CD4 antibody (BioLegend, Cat.:100408), and APC-conjugated anti-mouse CD8a antibody (BioLegend, Cat.:100713) were added and mixed thoroughly. The mixture was then incubated at 4°C for 30 min. Cells were then washed three times with ice-cold PBS to remove unbound antibodies. Cell phenotype was subsequently analyzed using a Beckman CytoFLEX LX 5L19C spectrophotometer.

[0053] 1.11 Lymphocyte proliferation assay

[0054] The prepared lymphocyte suspension was added to 96-well cell culture plates at a concentration of 100 μL / well. S protein was added to a final concentration of 5 μg / mL to stimulate lymphocyte proliferation, and the reaction was repeated for 6 wells. Stimulated wells served as positive controls, and wells without stimulation served as cell control wells. The 96-well plates were incubated at 37°C in a 5% CO2 cell culture incubator for 72 h. The cell culture supernatant was collected for cytokine detection and replaced with fresh culture medium. 10 μL of CCK-8 solution (Beyotime, China) was added to each well, and the plates were incubated at 37°C for 2 h, followed by shaking for 1 min. The absorbance was measured at 490 nm using a microplate reader, and the stimulation index (SI) was calculated using the formula: SI = OD value of stimulation wells - OD value of blank control wells / OD value of control wells - OD value of blank control wells.

[0055] 1.12 Detection of Cytokine Expression Levels

[0056] After stimulating lymphocytes with S protein for 72 hours, the lymphocyte culture supernatant was collected, and the levels of IFN-γ, IL-2, IL-4, IL-21 and TGF-β1 in the supernatant were detected using a cytokine ELISA kit (refer to the kit's instruction manual for specific experimental procedures).

[0057] 1.13 Pathological tissue and immunohistochemical examination

[0058] Ileum tissue was collected from piglets and fixed with 10% neutral formaldehyde. The fixed tissue was dehydrated using a JT-12S automated tissue dehydrator, embedded, and sectioned. Sections were dewaxed to water, stained with hematoxylin for 10-20 min, differentiated with hydrochloric acid-alcohol for 5-10 s, washed with water for 1-3 min, and then placed in 50℃ warm water or a weakly alkaline aqueous solution for blue reversion until blue appeared. The sections were then treated with 85% alcohol for 3-5 min, stained with eosin for 3-5 min, washed with water for 3-5 s, dehydrated with a gradient of alcohols, cleared with xylene, and mounted with neutral resin before microscopic examination. Images of the sections were acquired using a Pannoramic 250 digital slide scanner to observe specific lesions. A 1:100 dilution of PEDV N protein monoclonal antibody prepared in the applicant's laboratory was used as the primary antibody, and HRP-labeled goat anti-mouse IgG was used as the secondary antibody. DAB staining was performed for 2 min. Images of the sections were acquired using a BA200 Digital trinocular microscopy system.

[0059] 2. Data Analysis

[0060] All statistical analyses were performed using GrappPad Prism software. Fisher's exact test or chi-square test was used to assess statistical significance (asterisk * indicates statistical significance: P > 0.05 no statistical difference, * P < 0.05 statistical difference, ** P < 0.01 statistically significant difference, *** P < 0.001 highly significant statistical difference).

[0061] 3. Ethical Approval

[0062] All experimental pigs were housed at the Lanzhou Veterinary Research Institute, received humane care, and were euthanized at the end of the experiment. The animal experiment protocol was approved by the Animal Ethics Committee of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0063] 4. Experimental Results

[0064] 4.1 A single mRNA can express three structural proteins of PDCoV: S2P, M, and N.

[0065] P2A self-cleaving peptides are short peptides with self-cleaving capabilities, enabling the translation of a single mRNA into multiple peptide chains, ultimately expressing multiple proteins. The applicant innovatively designed PDCoV S, M, and N proteins into a single ORF and introduced P2A self-cleaving peptides. To stabilize the pre-fusion conformation of the S protein and improve its immunogenicity, two consecutive proline mutations (E855P, V856P) were introduced at the turn between the central helix of the S2 subunit and the hepta-repetition sequence 1 (HR1), denoted as S2P protein. This ultimately resulted in the expression of S, M, and N structural proteins within a single mRNA. Two PDCoV mRNA vaccines were designed and prepared: S2P-mRNA-LNP and SMN-mRNA-LNP. Figure 1 A). The two prepared LNPs were transfected into 293T or LLC-PK cells, respectively. Western blotting and IFA analyses showed that S2P-mRNA-LNP could correctly express the S protein, and SMN-mRNA-LNP could correctly express the S, M, and N structural proteins. Figure 1 B,C).

[0066] 4.2S2P-mRNA-LNP induced a stronger immune response in mice than PDCoV inactivated vaccines.

[0067] To assess the level of immune response induced by mRNA-LNP in animals, the applicant first monitored the immune level of S2P-mRNA-LNP in mice and compared it with a PDCoV inactivated vaccine. Figure 2 A). The results showed that, compared with the PBS control group, both the S2P-mRNA-LNP and PDCoV inactivated vaccine immunization groups induced the production of PDCoV S-specific IgG and IgA antibodies after primary immunization, and antibody levels increased after booster immunization. Among them, the antibody titers in the S2P-mRNA-LNP group were significantly higher than those in the PDCoV inactivated vaccine group after both primary and booster immunization, especially the IgA antibody titer. Figure 2 B, C). In vitro neutralization assays also showed that the S2P-mRNA-LNP group induced higher neutralizing antibody titers after two immunizations, with a highly significant difference compared to the PDCoV inactivated vaccine group. Figure 2 D).

[0068] To evaluate the cellular immunity induced by S2P-mRNA-LNP and PDCoV inactivated vaccines in mice, splenic lymphocytes were isolated from mice 28 days after primary immunization. T lymphocyte subsets were identified and counted by flow cytometry. Lymphocyte proliferation was stimulated in vitro using eukaryotically expressed PDCoV S protein, and the expression levels of IFN-γ, IL-2, and IL-4 in the lymphocyte culture supernatant were detected using an ELISA kit. The results showed that the percentages of CD3+CD4+ and CD3+CD8+ T cells in the S2P-mRNA-LNP and PDCoV inactivated vaccine groups were significantly higher than those in the PBS control group, and the percentages of CD3+CD4+ and CD3+CD8+ T cells in the S2P-mRNA-LNP group were significantly higher than those in the PDCoV inactivated vaccine group. Figure 2 (E, F). The lymphocyte stimulation indices of both the S2P-mRNA-LNP and PDCoV inactivated vaccine groups were significantly higher than those of the PBS control group. The stimulation index of the S2P-mRNA-LNP group was significantly higher than that of the PDCoV inactivated vaccine group. Cytokine expression levels showed that the levels of IFN-γ, IL-2, and IL-4 cytokines in the S2P-mRNA-LNP group were significantly higher than those in the PDCoV inactivated vaccine group. These data all indicate that S2P-mRNA-LNP induced higher levels of humoral and cellular immune responses than the PDCoV inactivated vaccine.

[0069] 4.3 SMN-mRNA-LNP provides better active immune protection for piglets than S2P-mRNA-LNP.

[0070] The applicant designed an mRNA vaccine expressing three PDCoV antigens on a single mRNA: SMN-mRNA-LNP, and compared and evaluated the active immunization protective effects of SMN-mRNA-LNP, S2P-mRNA-LNP, and PDCoV inactivated vaccine in piglets. Figure 3 A). The results showed that, except for the PBS control group, the SMN-mRNA-LNP, S2P-mRNA-LNP, and PDCoV inactivated vaccine groups all induced high levels of PDCoV S protein-specific IgG and IgA antibodies after immunization. After booster immunization, the IgG and IgA antibody titers induced in the SMN-mRNA-LNP group were significantly higher than those in the S2P-mRNA-LNP and PDCoV inactivated vaccine groups. Figure 3 B, C). In vitro neutralization assay results showed that SMN-mRNA-LNP induced higher neutralizing antibody titers than S2P-mRNA-LNP and PDCoV inactivated vaccines. Figure 3 D).

[0071] To evaluate the active immunization protection effect of the mRNA vaccine in this invention on piglets, a challenge protection test was conducted on day 28 after two doses of immunization. Piglets were orally inoculated with 8 mL × 10 ml. 4.0 TCID 50 Table 1 shows the clinical symptoms and fecal viral shedding of PDCoV CH / XJYN / 2016 after inoculation. In the S2P-mRNA-LNP group, one piglet developed mild diarrhea at 4 dpi (CT = 26.81 ± 3.87; FC = 0.2 ± 0.45) and 5 dpi (CT = 28.40 ± 2.79; FC = 0.2 ± 0.45), but recovered and the diarrhea symptoms disappeared at 6 dpi. In the SMN-mRNA-LNP group, all piglets were in good condition and had normal appetite from 0 to 7 dpi, and no diarrhea symptoms were observed (FC = 0). Mild viral shedding was detected in anal swabs from 3 to 5 dpi. In the PDCoV inactivated vaccine group, one piglet developed mild diarrhea at 3 dpi (CT = 21.01 ± 6.85; FC = 0.20 ± 0.45), two piglets developed diarrhea symptoms at 4 dpi (CT = 23.64 ± 3.92; FC = 0.60 ± 0.89), and three piglets developed diarrhea symptoms at 5 dpi (CT = 23.10 ± 4.24; FC = 1.00 ± 1.00). In the PBS group, one piglet began to show clinical symptoms at 3 dpi, and by 4 dpi, all piglets in the PBS group had diarrhea symptoms (CT = 21.13 ± 2.63; FC = 1.40 ± 0.55), which persisted until 6 dpi. Through clinical observation and monitoring for 7 days after challenge, the protection rate of piglets in the S2P-mRNA-LNP group was 4 / 5, the protection rate of piglets in the SMN-mRNA-LNP group was 5 / 5, the protection rate of piglets in the PDCoV inactivated vaccine group was 2 / 5, and all piglets in the PBS group developed the disease (5 / 5).

[0072] Table 1

[0073]

[0074] a Days of postinoculation.

[0075] b The CT mean value for each group; A cutoff point was set at 30; CTvalues ​​greater than 30 were considered negative or below the detection limit of RT-PCR.

[0076] cClinical score for fecal consistency, as follows: 0=normal; 1=pasty; 2=semiliquid; and 3=liquid.

[0077] d Number of PDCov-possible piglets.

[0078] Seven days after viral challenge, piglets were dissected and samples were taken. The necropsy revealed SMN-mRNA-LNP,

[0079] In the S2P-mRNA-LNP group, the intestinal tissue of piglets was normal, with no obvious lesions observed; in the PDCoV inactivated vaccine group, the intestines of piglets were slightly thinned, with a small amount of pale yellow fluid in the intestinal lumen; in the PBS group, the intestines of piglets were thinned and transparent, with a large amount of pale yellow fluid in the intestinal lumen. Figure 3 E). Ileal tissue was selected for histopathological and immunohistochemical examination. Histopathological examination revealed no obvious intestinal pathological damage in piglets in the SMN-mRNA-LNP and S2P-mRNA-LNP groups; in piglets in the PDCoV inactivated vaccine group, the intestinal villus epithelial cells were edematous, the number of goblet cells was small, and there was extensive edema in the lamina propria and submucosa; in piglets in the PBS group, there was more edematous intestinal villus epithelial cells, the cell bodies were swollen, the number of goblet cells was small, the connective tissue and intestinal glands were loosely arranged, and granulocyte infiltration was occasionally observed. The muscularis mucosae separated the intestinal crypts from the submucosa. Figure 3 E). Immunohistochemical analysis showed that the intestines of piglets in the SMN-mRNA-LNP and S2P-mRNA-LNP groups had almost no PDCoV antigen, while a large amount of PDCoV antigen was visible on the intestinal epithelial cells of piglets in the PDCoV inactivated vaccine group and the PBS group. Figure 3 E).

[0080] 4.4 SMN-mRNA-LNP induces high levels of immune response in sows

[0081] The results of active immunization protection studies in piglets showed that SMN-mRNA-LNP was superior to S2P-mRNA-LNP. Therefore, the applicant further evaluated the immunogenicity of SMN-mRNA-LNP in pregnant sows and compared it with a PDCoV inactivated vaccine. The results are as follows... Figure 4The results showed that after two immunizations, PDCoV S protein-specific IgA and IgG antibodies were detected in the serum and colostrum of sows in both the SMN-mRNA-LNP and PDCoV inactivated vaccine groups. Furthermore, the levels of IgA and IgG antibodies in the serum remained high on days 14 and 28 postpartum. The titers of IgA and IgG antibodies in the serum and colostrum of sows in the SMN-mRNA-LNP group were significantly higher than those in the PDCoV inactivated vaccine group. Figure 4 B, C, E, F). In vitro neutralization assays showed that the neutralizing antibody titers in the serum and colostrum of sows in the SMN-mRNA-LNP group were significantly higher than those in the PDCoV inactivated vaccine group. Figure 4 D, G). Additionally, specific antibodies against M and N proteins were detected in the serum of sows in both the SMN-mRNA-LNP and PDCoV inactivated vaccine groups, with SMN-mRNA-LNP levels significantly higher than those in the PDCoV inactivated vaccine group. Figure 4 The results (H) indicate that the applicant's designed SMN-mRNA-LNP can effectively express the three antigens of PDCoV S, M, and N in sows and induce high levels of specific antibodies. The applicant also tested two key cytokines regulating IgA (TGF-β1 and IL-21). The results showed that SMN-mRNA-LNP induced higher levels of TGF-β1 and IL-21. Figure 4 I,J). 4.5SMN-mRNA-LNP provides complete passive immune protection for piglets.

[0082] Suckling piglets acquire passive immune protection by ingesting protective antibodies from their sow's milk. After farrowing, piglets from sows immunized with SMN-mRNA-LNP or PDCoV inactivated vaccines will naturally suckle for 5 days, followed by an oral vaccination of 1 mL × 10 ml. 4.0 TCID 50 The passive immunization efficacy of PDCoV CH / XJYN / 2016 was evaluated. Results showed that piglets in the SMN-mRNA-LNP group passively acquired significantly higher levels of IgA, IgG, and neutralizing antibodies than those in the PDCoV inactivated vaccine group. Figure 5(B, C, D). After challenge, all piglets in the SMN-mRNA-LNP group were in good condition from 0 to 10 days post-infection (dpi), with normal appetite and no diarrhea symptoms observed (FC score = 0). Piglets in the PDCoV inactivated vaccine group began to show mild diarrhea, vomiting, and loss of appetite from 2 days post-infection (dpi), and PDCoV RNA shedding was detectable in their feces (CT = 28.36 ± 4.05, FC = 0.80 ± 1.10), which persisted until 7 days post-infection (dpi). By 8 days post-infection (dpi), the sick piglets in the PDCoV inactivated vaccine group had recovered without diarrhea or other clinical symptoms, but PDCoV RNA shedding was still detectable in their feces (CT = 25.12 ± 0.46, FC = 0). Three piglets in the PBS group developed diarrhea, vomiting, and loss of appetite at 2 days post-infection (dpi), and PDCoV RNA shedding was detected in their feces (CT = 24.87 ± 5.24, FC = 1.80 ± 1.64). From 3 days post-infection (dpi), all piglets developed diarrhea (CT = 22.80 ± 1.98, FC = 1.80 ± 0.45). By 9 days post-infection (dpi), the PBS group piglets recovered without diarrhea or other clinical symptoms, but PDCoV RNA shedding was still detected in their feces (CT = 25.65 ± 3.88, FC = 0). Clinical observation and monitoring for 10 days after challenge showed that the passive protection rate in the SMN-mRNA-LNP group was 5 / 5, the passive protection rate in the PDCoV inactivated vaccine group was 2 / 5, and all piglets in the PBS group developed the disease (5 / 5) (Table 2). Autopsy pathological examination and immunohistochemistry also showed… Figure 5 E) In the SMN-mRNA-LNP group, no pathological damage was observed in the intestines of piglets, and PDCoV antigen was undetectable. In the PDCoV inactivated vaccine group, slight thinning of the intestinal wall, a small amount of fluid accumulation in the intestinal lumen, and a small amount of PDCoV antigen were observed in the intestinal epithelial cells. In the PBS group, thinning of the intestinal wall, a large amount of fluid accumulation in the intestinal lumen, and a large amount of PDCoV antigen were observed in the intestinal epithelial cells.

[0083] Table 2

[0084]

[0085] a Days of postinoculation.

[0086] b The CT mean value for each group; A cutoff point was set at 30; CTvalues ​​greater than 30 were considered negative or below the detection limit of RT-PCR.

[0087] c Clinical score for fecal consistency, as follows: 0=normal; 1=pasty; 2=semiliquid; and a=liquid.

[0088] d Number of PDCoV-positive piglets.

[0089] Enterovirus-related infectious diseases in pigs cause huge economic losses to the pig farming industry. To date, vaccination remains one of the most effective means of controlling infectious diseases. Traditional inactivated vaccines and live attenuated vaccines have played an important role in the control of infectious diseases. mRNA vaccines are a new type of nucleic acid vaccine; immunization with this type of vaccine can stimulate both humoral and cellular immunity. The successful launch of COVID-19 mRNA vaccines signifies the gradual maturation of this type of vaccine in terms of its technological system. Due to its advantages such as rapid deployment and short development cycle, it has shown great potential in the prevention of infectious diseases, especially emerging infectious diseases. This invention innovatively incorporates PDCoV M and N antigens into the vaccine design, developing an mRNA vaccine SMN-mRNA-LNP that expresses three antigens (PDCoV S2P, M, and N) in a single mRNA stream. Its effectiveness in providing active and passive immunization protection to piglets was evaluated. The results show that SMN-mRNA-LNP can provide complete active and passive immunization protection to piglets, making it a very promising PDCoV vaccine. Furthermore, this mRNA vaccine design can also be applied to the development of vaccines for other coronaviruses.

[0090] The S protein is a key structural protein of coronaviruses, recognizing host cell receptors and invading host cells. It possesses strong immunogenicity, inducing high levels of protective antibodies and making it a preferred target antigen for vaccine development. In this invention, the applicant first designed an mRNA vaccine based on the full-length PDCoV S protein and mutated the S protein with a double-proline mutation to stabilize the prefusion conformation. Immunization of mice with S2P-mRNA-LNP resulted in high levels of S protein-specific IgG / IgA and neutralizing antibodies, indicating that S2P-mRNA-LNP was correctly expressed in mice and induced a strong humoral immune response. Subsequently, the applicant examined the cellular immunity levels in mice and found that the cellular immunity induced by S2P-mRNA-LNP was stronger than that induced by the PDCoV inactivated vaccine group. The study by Li et al. (Li J, Xiao L, Chen Z, Fan L, Wang W, Guo R, He Z, Hu H, Jiang J, Zhao L, Zhong T, Fan B, Zhu X, Li B. 2024. A spike-based mRNA vaccine that induces durable and broad protection against porcine deltacoronavirus inpiglets. J Virol 98:e0053524) also showed that the PDCoV S protein-based mRNA vaccine induced high levels of IgG and neutralizing antibodies in mice, and the S mRNA vaccine also showed better protective effects than the inactivated vaccine in passive immunization of suckling piglets.Similarly, immunization of mice with the COVID-19 mRNA vaccine BNT162b2 stimulated the production of potent antibodies and antigen-specific T-cell responses, and significantly enhanced the innate immune response after secondary immunization. The protection against SARS-CoV-2 infection mediated by the mRNA vaccine was primarily mediated by humoral immunity, while cellular immunity was effective in clearing the infection (Li C, Lee A, Grigorian L, Arunachalam PS, Scott MKD, Trisal M, Wimmers F, Sanyal M, Weidenbacher PA, Feng Y, Adamska JZ, Valore E, Wang Y, Verma R, Reis N, Dunham D, O'Hara R, Park H, Luo W, Gitlin AD, Kim P, Khatri P, Nadeau KC, Pulendran B. 2022. Mechanisms of innate and adaptive immunity to the Pfizer-BioNTech BNT162b2vaccine. NatImmunol). 23:543-555).

[0091] To achieve better immunization efficacy, the applicant incorporated both PDCoV M and N antigens into the mRNA vaccine design, creating a PDCoV mRNA vaccine, SMN-mRNA-LNP, that simultaneously expresses all three antigens in a single mRNA. The active immunization efficacy of SMN-mRNA-LNP, S2P-mRNA-LNP, and the PDCoV inactivated vaccine was compared and evaluated in piglets. Results showed that both SMN-mRNA-LNP and S2P-mRNA-LNP induced high levels of PDCoV S protein-specific IgG and IgA antibodies in piglets, indicating that both SMN-mRNA-LNP and S2P-mRNA-LNP correctly expressed the S antigen in piglets. The IgG / IgA ratio and neutralizing antibody titers induced by SMN-mRNA-LNP were significantly higher than those induced by S2P-mRNA-LNP. Figure 3 (B, C, D). The results of the challenge protection study showed that SMN-mRNA-LNP provided superior active immune protection to piglets compared to S2P-mRNA-LNP, offering 100% immune protection. Post-mortem pathological examination and immunohistochemistry also revealed lower levels of PDCoV antigen and less pathological damage in the intestines of piglets in the SMN-mRNA-LNP group. Therefore, from the perspective of immune protection efficacy, coronavirus vaccines based on multiple structural proteins are significantly superior to coronavirus vaccines based on only a single structural protein.

[0092] The main harm of porcine enteroviruses lies in causing mass mortality in newborn piglets. Therefore, how to protect piglets from enteroviruses is an urgent problem to be solved in veterinary clinical practice. Most piglets become ill within the first week of life, at which time their immune systems are not yet mature, making vaccination less effective. Therefore, the focus of prevention and control must be placed on immunizing pregnant sows, providing passive immunization to piglets through sow colostrum. After immunizing sows with SMN-mRNA-LNP, higher levels of S antigen-specific IgG / IgA and neutralizing antibody titers were detected in sow colostrum and serum than with inactivated vaccines, especially IgA in colostrum. The IgA antibody titer in the colostrum of sows in the SMN-mRNA-LNP group reached as high as 10. 5.4 The levels were 25 times higher in the SMN-mRNA-LNP group than in the inactivated vaccine group. Studies have shown that TGF-β1 and IL-21 may be key cytokines regulating porcine IgA production. The applicant detected higher levels of TGF-β1 and IL-21 in sow peripheral blood lymphocytes, which may explain the higher IgA levels induced by SMN-mRNA-LNP in sow colostrum. After farrowing, piglets in the SMN-mRNA-LNP group passively acquired higher levels of IgG / IgA and neutralizing antibodies through breast milk. After challenge, piglets in the SMN-mRNA-LNP group achieved 100% (5 / 5) passive protection, compared to 40% (2 / 5) in the inactivated vaccine group. Autopsy pathological examination and immunohistochemistry also showed that the intestines of piglets in the SMN-mRNA-LNP group had almost no PDCoV antigen and virtually no pathological damage.

[0093] Previous research on transmissible gastroenteritis (TGEV) immunization has gradually revealed that secretory IgA (sIgA) produced by intestinal immunity is crucial for the prevention and control of TGEV infection in newborn piglets. This led to the initial concept of the Gut-mammarygland-sIgA axis (Chattha KS, Roth JA, Saif LJ. 2015. Strategies for design and application of enteric viral vaccines. Annu Rev Anim Biosci 3:375-95). This axis essentially refers to the production of sIgA-synthesizing lymphoplasmacytes in the mesenteric lymph nodes due to pathogen infection of intestinal epithelial cells. These lymphoplasmacytes can return to the lamina propria of the intestine or migrate to the mammary glands to exert their effects. Lymphocytes in the basal layer of the mammary epithelium of immunized sows can continuously secrete sIgA, which is not easily degraded by enzymes in milk and the intestines, thus providing piglets with long-lasting immune protection. In this invention, both SMN-mRNA-LNP and S2P-mRNA-LNP induced higher titers of IgA antibodies than inactivated vaccines, which may be related to the unique immune properties and immune mechanisms of mRNA vaccines. Related studies have found that after vaccination with BNT162b2, exosomes containing the S protein are present in the human body and persist in the plasma for several weeks after immunization (Kent SJ, Li S, Amarasena TH, Reynaldi A, Lee WS, Leeming MG, O'Connor DH, Nguyen J, Kent HE, Caruso F, Juno JA, Wheatley AK, Davenport MP, JuY. 2024. Blood Distribution of SARS-CoV-2 Lipid Nanoparticle mRNA Vaccine in Humans. ACS Nano 18:27077-27089). In mice vaccinated with BNT162b2, the antigen can be continuously detected in draining lymph nodes and serum (Focosi D, Maggi F, Casadevall A. 2022. Mucosal Vaccines, Sterilizing Immunity, and the Future of SARS-CoV-2 Virulence. Viruses 14).Therefore, the reason why the SMN-mRNA-LNP and S2P-mRNA-LNP vaccines in this invention have such good immunoprotective effects may be that after vaccination, the antigen is expressed in large quantities in the body and reaches the mesenteric lymph nodes through the body fluid circulation, thereby activating the Gut–mammary gland–sIgA axis and inducing the production of high levels of IgA antibodies secreted into colostrum. Therefore, if LNP can selectively deliver mRNA to the mesenteric lymph nodes, then mRNA-LNP may induce even better immune effects, which will be a key direction for future research.

[0094] In summary, the experimental results of this invention demonstrate that the single mRNA vaccine SMN-mRNA-LNP, which simultaneously expresses three major PDCoV structural proteins, exhibits better immunoprotective efficacy than the mRNA vaccine S2P-mRNA-LNP based solely on the S protein, and effectively avoids the complex production process and high costs associated with delivering multiple proteins separately. Furthermore, it also shows that PDCoV structural proteins N and M play important roles in inducing a protective immune response. Therefore, in future coronavirus vaccine development, in addition to the S protein, N and M proteins should be considered as important antigenic components of candidate vaccines whenever possible. The research results of this invention provide new ideas and practical basis for the future development of novel vaccines against porcine enteric coronaviruses.

Images