PDCoV virus mRNA (messenger Ribonucleic Acid) vaccine capable of self-cutting and expressing multiple virus structural proteins and preparation method of PDCoV virus mRNA vaccine

Abstract

The invention provides a PDCoV virus mRNA (messenger Ribonucleic Acid) vaccine capable of self-cleaving and expressing a plurality of virus structural proteins and a preparation method of the PDCoV virus mRNA vaccine, and the vaccine comprises mRNA for expressing S, M and N proteins of a PDCoV virus and LNP for encapsulating the mRNA, and the LNP is marked as SMN-mRNA-LNP. The invention provides a PDCoV mRNA vaccine strategy based on combination of S, M and N for the first time, the S, M and N structural proteins of the PDCoV are connected by using a self-cleavage peptide P2A, the S protein is subjected to double proline mutation, so that a single mRNA can express multiple PDCoV antigens, and a multi-level defense system is constructed by using the neutralizing antibody induction capability of the S protein, the immune regulation function of the M protein and the cellular immune activation characteristic of the N protein. Through evaluation of immunogenicity, antibody level and challenge protection effect of the vaccine in mice, suckling piglets and pregnant sows, a new idea is provided for development of broad-spectrum and efficient PDCoV vaccines, and a practical basis is provided for research and development of coronavirus multi-antigen mRNA vaccines.

Description

Technical Field

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

[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 to humans, making controlling the spread of animal coronaviruses crucial for human health. Porcine deltacoronavirus (PDCoV) is an emerging enteric pathogen in pigs of all ages, causing watery diarrhea, vomiting, and intestinal pathology, with a particularly high mortality rate in newborn piglets. Since its initial discovery and report in 2012, it has gradually spread globally, posing a serious threat to the global swine industry in recent years. Other studies have shown that PDCoV can also infect chickens, turkeys, calves, and children, demonstrating its potential for cross-species transmission and posing a serious threat to public health. Vaccination is the most effective method for preventing and controlling the disease, but there is currently no commercially available vaccine against PDCoV. Therefore, the development of an effective and safe vaccine is urgently needed to prevent and control the disease.

[0003] Coronavirus vaccine development strategies primarily 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 infectious disease prevention and control due to its short development cycle, ease of industrialization, simplified production process, flexibility in responding to new variants, and combined humoral and cellular immune activation.

[0004] PDCoV, a member of the genus deltacoronavirus in the order Nidovirales, family Coronairidae, is a single-stranded, positive-sense RNA virus. Mature virions 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 as spikes on the surface of the virion. It is primarily responsible for recognizing and binding to host receptors and undergoing membrane fusion with host cells, mediating viral entry. The M protein is the most abundant transmembrane protein on the surface of the coronavirus envelope and is highly conserved among different coronaviruses. It is responsible for the assembly and release of viral particles. During viral entry, the M protein plays a key 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, can induce a strong T cell immune response, and can serve as a target for coronavirus vaccine research. The N protein is the most abundant and functional protein in coronaviruses and plays a vital role in viral replication. Although vaccine development has primarily focused on the S protein, studies have shown 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 be used as a candidate vaccine antigen. Existing coronavirus mRNA vaccine research is mostly limited to the single S protein antigen, while the virus's M and N proteins also participate in key immune responses during natural infection. The M and N proteins have rich and conserved T cell epitopes that can enhance cross-protection. Therefore, the design of mRNA vaccines that integrate multiple antigen components is expected to break through the limitations of a single target. Summary of the Invention

[0005] Based on the above, the purpose of the present 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 solutions:

[0007] The present invention provides a PDCoV virus mRNA vaccine that can self-cleave and express multiple viral structural proteins, including mRNA that simultaneously expresses PDCoV virus S, M, and N proteins, and LNP that encapsulates 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 comprises the following steps:

[0009] (1) Plasmid DNA construction

[0010] Using the PDCoV virus sequence as a reference, the S, M, and N gene sequences were codon-optimized, and double proline mutations (E855P, V856P) were 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 the pSMN plasmid as a template, the P2A self-cleaving peptide was introduced between S and M, and between M and N, respectively. After in vitro transcription, single-stranded mRNA with a 5′-m7G Cap1, a poly(A) structure, and an N1-methyl-piperazine modification was obtained. The enzyme, DNA template, and free nucleotides were removed by magnetic beads to obtain a 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, which was then mixed with a 20 mM citric acid buffer solution containing mRNA to encapsulate the mRNA to obtain mRNA-LNP. Finally, the mRNA-LNP was dialyzed with a phosphate buffer solution, centrifuged, and concentrated to obtain SMN-mRNA-LNP. As a further preferred embodiment of the technical solution 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 content of mRNA in the citric acid buffer was 106.84 μg / mL.

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

[0019] The beneficial effects of the present invention are:

[0020] This paper proposes for the first time a PDCoV mRNA vaccine strategy based on the combination of three antigens: S, M, and N. 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. This strategy leverages 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 to construct a multi-layered defense system. By evaluating the immunogenicity, antibody levels, and protective efficacy of this vaccine in mice, lactating piglets, and pregnant sows, the authors aim to provide new ideas for the development of a broad-spectrum, highly effective PDCoV vaccine and a practical basis for the research and development of multi-antigen mRNA vaccines against coronaviruses.

[0021] Specifically, the present invention has developed a novel mRNA vaccine, SMN-mRNA-LNP, that expresses the three major structural proteins of PDCoV, S, M, and N, through a single mRNA. This vaccine offers superior active immune protection in piglets compared to S2P-mRNA-LNP, which expresses a single S protein. Furthermore, after immunization of pregnant sows with SMN-mRNA-LNP, IgA antibody titers in colostrum reached as high as 105.4, 25 times that of the inactivated vaccine group. This allows newborn piglets to obtain higher levels of passive immunity through breastfeeding, ultimately protecting them from PDCoV. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

[0026] Figure 5 This is the protective effect of SMN-mRNA-LNP on passive immunity in piglets. DETAILED DESCRIPTION

[0027] The present invention will be described in detail below 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-distearoyl-sn-glycero-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. TM The lipid mixture was mixed with 6 mL of 20 mM citrate buffer (pH 4.0, mRNA concentration of 106.84 μg / mL) containing mRNA to achieve mRNA encapsulation. The mRNA-LNP was then dialyzed against phosphate buffered saline (PBS) (pH 7.4) and finally concentrated to the desired concentration using a 100 kd Amicon centrifugal ultrafiltration tube (Millipore, USA), resulting in two mRNA-LNPs, named S2P-mRNA-LNP and SMN-mRNA-LNP.

[0037] 1.3 Immunoblotting 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 incubated at 37°C for 24 h. After 24 h, cells were harvested and treated with cell lysate. Polyacrylamide gel electrophoresis was performed and the membranes were transferred to nitrocellulose membranes (Pall, USA) for immunoblotting analysis. Rabbit polyclonal antibodies against S, M, or N (1:500 dilution) prepared in the applicant's laboratory were used as primary antibodies, and HRP-conjugated goat anti-rabbit IgG (1:10,000 dilution, Zhongshan Jinqiao, Beijing, China) was used as secondary antibodies. Chemiluminescent substrate (ECL, ThermoScientific, USA) was used for development.

[0039] 1.4 Immunofluorescence detection

[0040] S2P-mRNA-LNP and SMN-mRNA-LNP (2 μg / well) were transfected into LLC-PK cells at a cell density of approximately 80%. Control wells were treated with lipo3000 and incubated in a 37°C constant temperature incubator for 24 hours. After 24 hours, the culture medium was discarded, the cells were washed three times with PBS, fixed with 4% paraformaldehyde at 4°C for 1 hour, permeabilized with 0.25% Triton-100 at room temperature for 10 minutes, and then blocked with 5% BSA for 1 hour. 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. 4′,6-diamidino-2-phenylindole was used for dark staining for 5 minutes, and the cells were washed three times with PBS and observed under a fluorescence microscope.

[0041] 1.5 Mouse Immunization Test

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

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

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

[0045] 1.7 Passive Immunization and Virus Challenge Protection Test in Suckling Piglets

[0046] Nine PDCoV antigen and antibody double-negative sows were randomly divided into three groups (3 sows / group). 42 days before farrowing, the three groups of sows were injected with SMN-mRNA-LNP at a dose of 100 μg / head and PDCoV inactivated vaccine at a dose of 2 mL / head (the virus content before inactivation was 1.0×10 6.5 TCID 50 / ml) and PBS. All sows were boosted 21 days after the initial vaccination and serum was collected before vaccination, 21 days after the initial vaccination, and on the day of farrowing. Colostrum was collected after farrowing for antibody detection. After 5 days of lactation, 5 piglets were randomly selected from each group (piglets from different sows in the same group were selected to minimize the influence of individual sow differences). Each piglet was orally challenged with 1mL×10 4.0 TCID 50 / mL CH / XJYN / 2016. After challenge, piglets were observed daily for clinical signs and to monitor the amount of toxin excreted and diarrhea scores in each group. Diarrhea scores were based on a scale of: 0 = normal; 1 = mushy; 2 = semifluid; and 3 = liquid.

[0047] 1.8 Specific Antibody Detection

[0048] The levels of specific antibodies to S, M, and N proteins in serum and colostrum were determined by indirect ELISA (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 subunit vaccines. Virology 590: 109955). Specifically, the purified S, M, and N proteins were diluted to 2 μg / mL with 50 mM carbonate buffer (pH 9.6), coated on a 96-well microtiter plate (Corning, USA) at 100 μL / well, and incubated at 4°C overnight. The coating solution was discarded, the plate was washed three times with PBST, and 200 μL / well of blocking solution containing 5% skim milk powder was added, and incubated at 37°C for 2 h. The blocking solution was discarded, the plate was washed three times with PBST, and 100 μL of serum diluent serially diluted serum or colostrum samples was added to each well, and incubated at 37°C for 1 h. The liquid in the well was discarded, the plate was washed three times with PBST, and 100 μL of serum diluent diluted HRP-conjugated goat anti-mouse IgG (1:10,000 dilution, Zhongshan Jinqiao, Beijing, China), HRP-conjugated goat anti-swine IgG (1:10,000 dilution, Abcam, UK), or HRP-conjugated goat anti-swine IgA (1:10,000 dilution, Abcam, UK) was added to each well. 000 dilution, Abcam, UK), incubated at 37°C for 1 h; the liquid in the wells was discarded, and the wells were washed three times with PBST. 100 μL of TMB substrate solution (Solarbio, Beijing, China) was added to each well and reacted at 37°C in the dark for 10 min; 100 μL of 2 M H2SO4 was added to each well to terminate the reaction, and the absorbance at 450 nm was measured with a microplate reader.

[0049] 1.9 Neutralization experiment

[0050] The titer of PDCoV neutralizing antibodies in serum was determined using the fixed virus-dilution serum method. The specific procedure was as follows: A 96-well plate filled with 70%-80% LLC-PK cells was prepared in advance; the serum was inactivated in a 56°C water bath for 30 minutes and then serially diluted 2-fold in a 96-well microplate, with four wells replicated for each dilution. An equal volume of diluted virus solution (200 TCID) was added to each well. 50 ), mixed and placed in a 37°C 5% CO2 incubator for 1 hour; virus control and normal cell control were set, where the virus control was set at 200 TCID 50 (all lesions), 20TCID 50 , 2TCID 50 , 0.2TCID 50Four different control concentrations (no disease) were used. Cells were washed three times with PBS, and 100 μL of the virus-serum mixture was added to each well. The cells were incubated at 37°C in a 5% CO2 incubator for 1 hour. After incubation, the wells were discarded, the cells were washed three times with PBS, and 200 μL of maintenance medium (containing 20 μg / mL trypsin) was added to each well. CPE was observed and recorded daily. Neutralization 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 the 28th day after immunization, the spleen of the mice was harvested and lymphocyte suspension was prepared using an animal lymphocyte separation kit (Solarbio, Beijing, China). The lymphocyte suspension was diluted to 5 × 10 6 100 μL of lymphocyte suspension was prepared at a concentration of 10 cells / mL. 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 according to the manufacturer's instructions, mixed thoroughly, and incubated at 4°C for 30 min. Unbound antibodies were then washed three times with ice-cold PBS to remove the cells. Cell phenotypes were then analyzed using a Beckman CytoFLEX LX 5L19C.

[0053] 1.11 Lymphocyte proliferation assay

[0054] The prepared lymphocyte suspension was added to a 96-well cell culture plate at 100 μL / well. S protein was added at a final concentration of 5 μg / mL to stimulate lymphocyte proliferation, and repeated for 6 wells. The stimulated wells served as positive controls, and the wells without stimulation served as cell control wells. The 96-well plate was placed in a 37°C, 5% CO2 cell culture incubator and cultured for 72 hours. 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, incubated at 37°C for 2 hours, and shaken on a shaker for 1 minute. The absorbance was measured at 490 nm using a microplate reader, and the stimulation index (SI) was calculated using the following formula: SI = OD value of the stimulated well - OD value of the blank control well / OD value of the control well - OD value of the blank control well.

[0055] 1.12 Detection of cytokine expression levels

[0056] After 72 hours of S protein stimulation of lymphocytes, 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 (for specific experimental steps, please refer to the instructions provided with the kit).

[0057] 1.13 Pathological tissue and immunohistochemistry examination

[0058] The ileum of piglets was collected and fixed with 10% neutral formaldehyde. The fixed tissue was dehydrated using a JT-12S automatic tissue dehydrator, embedded, and sectioned. The sections were deparaffinized and hydrated, stained with hematoxylin for 10-20 minutes, differentiated with hydrochloric acid and alcohol for 5-10 seconds, rinsed with water for 1-3 minutes, and then placed in 50°C warm water or a weak alkaline solution to rebluing until a blue color appeared. The sections were treated with 85% alcohol for 3-5 minutes, stained with eosin for 3-5 minutes, rinsed with water for 3-5 seconds, dehydrated with graded alcohols, cleared with xylene, and mounted with neutral gum for microscopic examination. Sections were imaged using a Pannoramic 250 digital slide scanner to visualize specific lesions. A 1:100 dilution of a PEDV N protein monoclonal antibody prepared in the applicant's laboratory was used as the primary antibody, followed by HRP-conjugated goat anti-mouse IgG as the secondary antibody, and developed with DAB for 2 minutes. Sections were imaged using a BA200 Digital trinocular camera microscope system.

[0059] 2. Data Analysis

[0060] All statistical analyses were performed using GraphPad Prism software. Statistical significance was assessed using Fisher's exact test or chi-square test (asterisk* indicates statistical significance: P>0.05, no statistical difference; *P<0.05, statistical difference; **P<0.01, significant statistical difference; ***P<0.001, extremely significant statistical difference).

[0061] 3. Ethical approval

[0062] All experimental pigs were raised at the Lanzhou Veterinary Research Institute, received humane care, and were euthanized at the end of the experiment. The animal experimental 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 the three structural proteins of PDCoV S2P, M, and N

[0065] P2A self-cleaving peptides (2A self-cleaving peptides) are short peptides with self-cleavage function, which can enable a single mRNA to translate into multiple peptide chains and ultimately express multiple proteins. The applicant innovatively designed PDCoV S, M and N in the same ORF, and introduced P2A self-cleaving peptides. At the same time, in order to stabilize the pre-fusion conformation of the S protein and improve its immunogenicity, two consecutive proline mutations (E855P, V856P) were introduced into the turning point between the central helix of the S2 subunit and the seven-repeat sequence 1 (HR1), which was recorded as S2P protein. Ultimately, the three structural proteins S, M and N were expressed in 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 analysis showed that S2P-mRNA-LNP could correctly express S protein, and SMN-mRNA-LNP could correctly express S, M, and N structural proteins ( Figure 1 B,C).

[0066] 4.2S2P-mRNA-LNP induces stronger immune responses in mice than inactivated PDCoV vaccine

[0067] To evaluate 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 the inactivated PDCoV vaccine ( Figure 2 A). The results showed that compared with the PBS control group, 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 the antibody levels increased after booster immunization. Among them, the antibody titers of the S2P-mRNA-LNP group after primary and booster immunization were significantly higher than those of the PDCoV inactivated vaccine group, especially the IgA antibody titer ( Figure 2 B, C). In vitro neutralization tests also showed that the S2P-mRNA-LNP group induced higher neutralizing antibody titers after two immunizations, which was significantly different from the PDCoV inactivated vaccine group ( Figure 2 D).

[0068] In order to evaluate the level of cellular immunity induced by S2P-mRNA-LNP and PDCoV inactivated vaccine in mice, mouse spleen lymphocytes were isolated 28 days after primary immunization, and T lymphocyte subsets were identified and counted by flow cytometry. The eukaryotic expressed PDCoV S protein was used to stimulate lymphocyte proliferation in vitro, and the expression levels of IFN-γ, IL-2 and IL-4 in the lymphocyte culture supernatant were detected by ELISA kits. 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). Lymphocyte stimulation indices were significantly higher in both the S2P-mRNA-LNP and inactivated PDCoV vaccine groups than in the PBS control group. The stimulation index in the S2P-mRNA-LNP group was significantly higher than that in the inactivated PDCoV vaccine group. Cytokine expression results showed that IFN-γ, IL-2, and IL-4 levels in the S2P-mRNA-LNP group were significantly higher than those in the inactivated PDCoV vaccine group. These data indicate that S2P-mRNA-LNP induces higher levels of humoral and cellular immune responses than the inactivated PDCoV vaccine.

[0069] 4.3SMN-mRNA-LNP is more effective in protecting piglets from active immunity than S2P-mRNA-LNP

[0070] The applicant designed a single mRNA expressing three PDCoV antigens mRNA vaccine: SMN-mRNA-LNP, and compared and evaluated the active immune protection 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 by the SMN-mRNA-LNP group were significantly higher than those of the S2P-mRNA-LNP and PDCoV inactivated vaccine groups ( Figure 3 B, C). The results of in vitro neutralization tests showed that SMN-mRNA-LNP induced higher neutralizing antibody titers than S2P-mRNA-LNP and PDCoV inactivated vaccine ( Figure 3 D).

[0071] In order to evaluate the active immune protection effect of the mRNA vaccine of the present invention on piglets, a challenge protection test was conducted on the 28th day after the piglets were immunized with two injections. 4.0 TCID 50 The clinical symptoms and fecal shedding of the SMN-mRNA-LNP piglets after infection with the CH / XJYN / 2016 / mLPDCoV are shown in Table 1. One piglet in the S2P-mRNA-LNP group developed mild diarrhea on dpi 4 (CT = 26.81 ± 3.87; FC = 0.2 ± 0.45) and dpi 5 (CT = 28.40 ± 2.79; FC = 0.2 ± 0.45). The piglets recovered and the diarrhea disappeared on dpi 6. All piglets in the SMN-mRNA-LNP group were in good condition and had normal appetite from 0 to 7 dpi. No diarrhea was observed (FC = 0). Mild shedding was detected in anal swabs from 3 to 5 dpi. In the PDCoV inactivated vaccine group, one piglet developed mild diarrhea on dpi (CT = 21.01 ± 6.85; FC = 0.20 ± 0.45), two developed diarrhea on dpi (CT = 23.64 ± 3.92; FC = 0.60 ± 0.89), and three developed diarrhea on dpi (CT = 23.10 ± 4.24; FC = 1.00 ± 1.00). In the PBS group, one piglet began to show clinical symptoms on dpi, and all piglets in the PBS group developed diarrhea on dpi (CT = 21.13 ± 2.63; FC = 1.40 ± 0.55), which persisted until dpi 6. Through clinical observation and monitoring 7 days after infection, 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 became ill (5 / 5).

[0072] Table 1

[0073]

[0074] a Days 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] The piglets were autopsied and sampled 7 days after the challenge.

[0079] The intestinal tissue of the piglets in the S2P-mRNA-LNP group was normal, with no obvious lesions. The intestinal tissue of the piglets in the PDCoV inactivated vaccine group was slightly thinner, with a small amount of light yellow fluid in the intestinal cavity. The intestinal tissue of the piglets in the PBS group was thinner and transparent, with a large amount of light yellow fluid in the intestinal cavity. Figure 3 E). Histopathological and immunohistochemical examinations were performed on ileum tissues. Histopathological findings revealed no obvious intestinal pathological damage in the SMN-mRNA-LNP and S2P-mRNA-LNP groups. In the PDCoV inactivated vaccine group, the intestinal villus epithelial cells of the piglets were edematous, the number of goblet cells was small, the lamina propria was edematous in a large area, and the submucosa was edematous in a large area. In the PBS group, the intestinal villus epithelial cells of the piglets were more edematous, 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 mucosa separated the intestinal crypts from the submucosa ( Figure 3 E). Immunohistochemical analysis showed that there was almost no PDCoV antigen in the intestines of piglets in the SMN-mRNA-LNP and S2P-mRNA-LNP groups, while a large amount of PDCoV antigen was observed on the intestinal epithelial cells of piglets in the PDCoV inactivated vaccine and PBS groups ( Figure 3 E).

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

[0081] The results of the active immune protection study on 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 the inactivated PDCoV vaccine. Figure 4The results showed that after two immunizations, PDCoV S protein-specific IgA and IgG antibodies could be detected in the serum and colostrum of sows in the SMN-mRNA-LNP and PDCoV inactivated vaccine groups. The IgA and IgG antibodies in the serum of sows remained at high levels on the 14th and 28th days after farrowing. The IgA and IgG 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 B, C, E, F). The results of the in vitro neutralization test 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). In addition, specific antibodies to M and N proteins were also detected in the sera of sows in the SMN-mRNA-LNP and PDCoV inactivated vaccine groups, and the SMN-mRNA-LNP group was significantly higher than that in the PDCoV inactivated vaccine group ( Figure 4 H), indicating that the SMN-mRNA-LNP designed by the applicant can effectively express the three antigens of PDCoV S, M and N in sows and induce the production of high levels of specific antibodies. The applicant tested two key cytokines (TGF-β1 and IL-21) that regulate IgA. The results showed that SMN-mRNA-LNP induced the production of 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 immunity by absorbing protective antibodies in sow milk. After sows immunized with SMN-mRNA-LNP or PDCoV inactivated vaccine give birth, piglets naturally suckle breast milk for 5 days and then orally inoculate 1mL×10 4.0 TCID 50 / mLPDCoV CH / XJYN / 2016, and evaluated its passive immune protection effect. The results showed that the IgA, IgG and neutralizing antibodies passively acquired by piglets in the SMN-mRNA-LNP group were significantly higher than those in the PDCoV inactivated vaccine group ( Figure 5After challenge, all piglets in the SMN-mRNA-LNP group were in good condition, had normal appetite, and no diarrhea was observed from 0 to 10 dpi (FC score = 0). Piglets in the PDCoV inactivated vaccine group began to show mild diarrhea, vomiting, and loss of appetite at 2 dpi, and PDCoV RNA shedding was detected in their feces (CT = 28.36 ± 4.05, FC = 0.80 ± 1.10), which persisted until 7 dpi. At 8 dpi, the piglets in the PDCoV inactivated vaccine group recovered and had no clinical symptoms such as diarrhea, but PDCoV RNA shedding was still detectable in their feces (CT = 25.12 ± 0.46, FC = 0). At 2 dpi, three piglets in the PBS group developed diarrhea, vomiting, and loss of appetite, and PDCoV RNA shedding was detected in their feces (CT = 24.87 ± 5.24, FC = 1.80 ± 1.64). Starting at 3 dpi, all piglets developed diarrhea symptoms (CT = 22.80 ± 1.98, FC = 1.80 ± 0.45). By 9 dpi, the piglets in the PBS group had recovered and had no clinical symptoms such as diarrhea, but PDCoV RNA shedding was still detectable in their feces (CT = 25.65 ± 3.88, FC = 0). Through clinical observation and monitoring for 10 days after the infection, the passive protection rate of the piglets in the SMN-mRNA-LNP group was 5 / 5, the passive protection rate of the piglets in the PDCoV inactivated vaccine group was 2 / 5, and all the piglets in the PBS group became ill (5 / 5) (Table 2). Necropsy pathological tissue examination and immunohistochemistry also showed ( Figure 5 E) Piglets in the SMN-mRNA-LNP group showed no intestinal pathological damage, and no PDCoV antigen was detected. In the inactivated PDCoV vaccine group, the intestinal wall was slightly thinned, with a small amount of fluid accumulated in the intestinal lumen, and a small amount of PDCoV antigen was observed in the intestinal epithelial cells. In the PBS group, the intestinal wall was thinned, with a large amount of fluid accumulated in the intestinal lumen, and a large amount of PDCoV antigen was observed in the intestinal epithelial cells.

[0083] Table 2

[0084]

[0085] a Days 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] Porcine enteroviral infectious diseases cause huge economic losses to the pig industry. To date, vaccination remains one of the most effective means of preventing and controlling infectious diseases. Traditional inactivated vaccines and live attenuated vaccines have played an important role in the prevention and control of infectious diseases. mRNA vaccine is an emerging nucleic acid vaccine that can stimulate both humoral immunity and cellular immunity. The successful launch of the new crown mRNA vaccine marks the gradual maturity of this type of vaccine in terms of technical system, and due to its advantages such as rapid deployment and short R&D cycle, it has shown great potential in the prevention of infectious diseases, especially emerging infectious diseases. The present invention innovatively incorporates PDCoV M and N antigens into the vaccine design, and develops an mRNA vaccine SMN-mRNA-LNP that expresses PDCoV S2P, M and N antigens in a single mRNA, and evaluates its effect on active and passive immune protection of piglets. The results show that SMN-mRNA-LNP can provide complete active and passive immune protection for piglets, and is a very potential PDCoV vaccine. In addition, this mRNA vaccine design can also be applied to the development of other coronavirus vaccines.

[0090] The S protein is a key structural protein for coronaviruses to recognize host cell receptors and invade host cells. It has extremely strong immunogenicity and can induce the body to produce high levels of protective antibodies. It is the preferred target antigen for vaccine development. In the present invention, the applicant first designed an mRNA vaccine based on the full-length S protein of PDCoV and performed a double proline mutation on the S protein to stabilize the prefusion conformation. After mice were immunized with S2P-mRNA-LNP, the mice produced 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. Next, the applicant tested the cellular immunity level of the mice and found that the cellular immunity induced by S2P-mRNA-LNP was stronger than that of 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 in piglets. J Virol 98: e0053524) also showed that the mRNA vaccine based on PDCoV S protein induced mice to produce high levels of IgG and neutralizing antibodies, and the S mRNA vaccine also showed better protection than the inactivated vaccine in the passive immunity protection of suckling piglets.Similarly, immunization of mice with the COVID-19 mRNA vaccine BNT162b2 stimulated potent antibody and antigen-specific T cell responses, and significantly enhanced innate immune responses after secondary immunization. mRNA vaccine-mediated protection against SARS-CoV-2 infection was primarily mediated by humoral immunity, while cellular immunity was effective in clearing the infection (Li C, Lee A, Grigoryan 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 BNT162b2 vaccine. Nat Immunol 23:543-555).

[0091] In order to achieve a better immune effect, the applicant also incorporated the PDCoV M and N antigens into the mRNA vaccine design, and designed a PDCoV mRNA vaccine SMN-mRNA-LNP in which a single mRNA simultaneously expresses three antigens. The active immune protection effects of SMN-mRNA-LNP, S2P-mRNA-LNP and PDCoV inactivated vaccine were compared and evaluated on piglets. The results showed that both SMN-mRNA-LNP and S2P-mRNA-LNP induced piglets to produce high levels of PDCoV S protein-specific IgG and IgA antibodies, indicating that both SMN-mRNA-LNP and S2P-mRNA-LNP correctly expressed the S antigen in piglets. The IgG / IgA and neutralizing antibody titers induced by SMN-mRNA-LNP were significantly higher than those induced by S2P-mRNA-LNP ( Figure 3 B, C, D). Challenge protection results demonstrated that SMN-mRNA-LNP provided superior active immune protection to piglets compared to S2P-mRNA-LNP, providing 100% protection. Postmortem histological examination and immunohistochemistry also revealed lower levels of PDCoV antigen in the intestines of piglets in the SMN-mRNA-LNP group, resulting in milder pathological lesions. Therefore, in terms of immune protection, coronavirus vaccines based on multiple structural proteins are significantly superior to those based on only a single structural protein.

[0092] The main harm of porcine enterovirus is that it causes a large number of deaths in newborn piglets. Therefore, how to protect piglets from enterovirus is an urgent problem to be solved in veterinary clinics. Most piglets become ill within one week after birth. At this time, the piglet's own immune system is not yet mature, and the effect of vaccinating piglets at this time is poor. Therefore, the focus of prevention and control has to be placed on immunizing pregnant sows, so that piglets can obtain passive immune protection through sow colostrum. After sows were immunized with SMN-mRNA-LNP, higher levels of S antigen-specific IgG / IgA and neutralizing antibody titers were detected in the colostrum and serum of sows than inactivated vaccines, especially IgA in colostrum. The IgA antibody titer in the colostrum of sows in the SMN-mRNA-LNP group was as high as 10 5.4 , which is 25 times that of the inactivated vaccine group. Studies have shown that TGF-β1 and IL-21 may be key cytokines that regulate the production of pig IgA. The applicant tested the cytokines TGF-β1 and IL-21 produced by sow peripheral blood lymphocytes. The results showed that higher levels of TGF-β1 and IL-21 were detected in the SMN-mRNA-LNP group, which may be the reason why SMN-mRNA-LNP induces higher levels of IgA in sow colostrum. After the sows gave birth, the piglets in the SMN-mRNA-LNP group passively obtained higher levels of IgG / IgA and neutralizing antibodies through breast milk. After the virus was challenged, the piglets in the SMN-mRNA-LNP group obtained 100% (5 / 5) passive protection, while the passive protection rate of the piglets in the inactivated vaccine group was 40% (2 / 5). Autopsy pathological tissue examination and immunohistochemistry also showed that there was almost no PDCoV antigen in the intestines of the piglets in the SMN-mRNA-LNP group, and there was almost no pathological damage.

[0093] In the past, during the study of infectious gastroenteritis immunity, it was gradually discovered that the secretory IgA (sIgA) component produced by intestinal immunity is crucial for the prevention and control of TGEV infection in newborn piglets, and the concept of the Gut-mammarygland-sIgA axis was first proposed (Chattha KS, Roth JA, Saif LJ. 2015. Strategies for design and application of enteric viral vaccines. Annu Rev Anim Biosci 3: 375-95). The essence of this axis is that pathogenic microorganisms infect intestinal epithelial cells, resulting in the production of lymphoplasmablasts capable of synthesizing sIgA in the mesenteric lymph nodes. These lymphoplasmablasts can return to the intestinal lamina propria or migrate to the mammary gland to exert their effects. The lymphocytes in the basal layer of the mammary epithelium of immunized sows can continuously secrete sIgA and are not easily degraded by enzymes in milk and the intestine, thus giving piglets lasting immune protection. In the present 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 mechanisms of mRNA vaccines. Related studies have found that after vaccination with BNT162b2, exosomes containing S protein will exist in the human body, and will 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). After mice are vaccinated with BNT162b2, the antigen can be continuously detected in the 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 excellent immune protection of the SMN-mRNA-LNP and S2P-mRNA-LNP vaccines of the present invention may be due to the fact that after vaccination, the antigen is expressed in large quantities in the body and circulates through the body fluids to the mesenteric lymph nodes, thereby activating the gut-mammary gland-sIgA axis and inducing high levels of IgA antibodies to be secreted into colostrum. Therefore, if LNPs can selectively deliver mRNA to the mesenteric lymph nodes, then mRNA-LNPs may induce even better immune responses, which will be a key area of ​​future research.

[0094] In summary, the experimental results of the present invention show that the single mRNA vaccine SMN-mRNA-LNP that simultaneously expresses the three main structural proteins of PDCoV has a better immune protection effect than the mRNA vaccine S2P-mRNA-LNP based on a single S protein, and effectively avoids the complication and high cost of the production process caused by the separate delivery of multiple proteins. Furthermore, it is also shown that the PDCoV structural proteins N and M also play an important role in inducing the body to produce an immune protection response. Therefore, in the future, when developing coronavirus vaccines, in addition to the S protein, N and M proteins should be considered as important antigenic components of candidate vaccines as much as possible. The research results of the present invention provide new ideas and practical basis for the future development of new vaccines for porcine enteric coronavirus.

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