A rotavirus multi-antigen mRNA vaccine and its application

Abstract

The present invention belongs to the technical field of mRNA vaccine preparation, and relates to a rotavirus multi-antigen mRNA vaccine and its application. The mRNA vaccine comprises a delivery vector and an mRNA immunogenic composition, wherein the composition comprises: i) an mRNA encoding a G1 rotavirus antigen protein VP7 antigenic fragment, the sequence of which is shown in SEQ ID NO: 2; ii) an mRNA encoding a G3 rotavirus antigen protein VP7 antigenic fragment, the sequence of which is shown in SEQ ID NO: 4; and iii) an mRNA encoding a G9 rotavirus antigen protein VP7 antigenic fragment, the sequence of which is shown in SEQ ID NO: 6. The mRNA vaccines prepared by the present invention have been shown to stimulate neutralizing antibody titers through humoral immunity experiments. The multi-antigen mRNA vaccine under compounding can produce higher antibody titers and has a cross-neutralizing effect on different types of rotavirus.

Description

Technical Field

[0001] The present invention belongs to the technical field of mRNA vaccine preparation, and specifically relates to a rotavirus multi-antigen mRNA vaccine and applications thereof. Background Art

[0002] Diarrhea is the second leading cause of death in children under five years of age, particularly in low-income and developing countries, killing approximately 500,000 children annually. Rotavirus infection is the leading cause of diarrheal morbidity, hospitalization, and mortality in children under five globally, accounting for one-quarter to one-third of diarrheal deaths in children under five and resulting in 120,000 to 220,000 deaths annually. Epidemiological surveys indicate that in 2003, approximately 1.14 billion children under five were infected with rotavirus worldwide, of whom 24 million required outpatient care and 2.3 million required hospitalization. In 2013, rotavirus infection alone caused 215,000 deaths in children under five worldwide. Between 2013 and 2017, an estimated 122,000 to 215,000 children died annually from rotavirus, a decrease of 59% to 77% since 2000. While there is currently no specific treatment for rotavirus infection, vaccination is the most cost-effective means of preventing it. Currently, all rotavirus vaccines marketed and used domestically and internationally are live attenuated vaccines. The use of vaccines has played a certain role in preventing and treating gastroenteritis caused by rotavirus infection, but the disease burden caused by rotavirus infection is still very heavy.

[0003] mRNA vaccines are the third generation of nucleic acid vaccines, following traditional vaccines (inactivated vaccines, live attenuated vaccines) and novel vaccines (subunit vaccines, viral vector vaccines). They work by introducing mRNA encoding one or more target antigens into the host cell cytoplasm, where the antigens are expressed and subsequently delivered to the host's immune system, activating the immune system to produce antibodies, ultimately conferring immune protection. mRNA vaccines are categorized by their design into non-replicating mRNA, self-amplifying mRNA (saRNA), trans-amplifying RNA (taRNA), and circular RNA (circRNA). As a novel nucleic acid vaccine, mRNA vaccines are characterized by their short development cycle, ease of industrialization, simple and controllable production processes, enhanced responsiveness to new variants, and the absence of nuclear integration, eliminating the risk of genomic integration. They are therefore more effective in inducing humoral and cellular immunity. They have attracted extensive research in the treatment of viruses, bacteria, parasites, metabolic genetic diseases, cardiovascular and cerebrovascular diseases, and tumors. The development of a new generation of non-replicating vaccines, such as rotavirus mRNA vaccines, could help address issues such as reversion to virulence in live vaccines, low protection in low- and middle-income countries, and the susceptibility to intussusception. Multivalent vaccines contain antigens from multiple serotypes / genotypes and possess a richer array of mutant epitopes than monovalent vaccines. This allows them to generate higher neutralizing antibody titers against different genotypes, thus providing broader cross-protection against different virus types. Currently, there are no reports on trivalent rotavirus mRNA vaccines. Summary of the Invention

[0004] The purpose of the present invention is to provide an mRNA immunogenic composition and an mRNA vaccine prepared using the composition, so as to provide new treatment and prevention of rotavirus infection.

[0005] The specific technical solutions provided by the present invention are as follows:

[0006] An mRNA immunogenic composition comprising at least one protein or fragment selected from the group consisting of i) and ii) below:

[0007] i) an mRNA encoding the G3 rotavirus antigen protein VP7 or an antigenic fragment thereof, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and expected to have the same or substantially the same immunogenicity, said mRNA being designated as RV3;

[0008] ii) an mRNA encoding the G9 rotavirus antigen protein VP7 or an antigenic fragment thereof, or an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical thereto and is expected to have the same or substantially the same immunogenicity, the mRNA being designated RV9.

[0009] In one technical solution, the present invention provides an mRNA immunogenic composition comprising:

[0010] i) an mRNA encoding the G1 rotavirus antigen protein VP7 or an antigenic fragment thereof, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identity thereto and expected to have the same or substantially the same immunogenicity, said mRNA being designated as RV1;

[0011] ii) mRNA encoding the G3 rotavirus antigen protein VP7 or an antigenic fragment thereof, or an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical thereto and is expected to have the same or substantially the same immunogenicity, the mRNA being designated RV3;

[0012] iii) mRNA encoding the G9 rotavirus antigen protein VP7 or an antigenic fragment thereof, or an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical thereto and is expected to have the same or substantially the same immunogenicity, the mRNA being designated RV9;

[0013] Furthermore, the mRNA immunogenic composition, wherein

[0014] The amino acid sequence encoding the G1 rotavirus antigen protein VP7 or an antigenic fragment thereof has the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO: 1 and has the same or substantially the same immunogenicity as the sequence shown in SEQ ID NO: 1;

[0015] and / or encoding a G3 rotavirus antigen protein VP7 or an antigenic fragment thereof having the amino acid sequence as shown in SEQ ID NO: 3, or an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the sequence as shown in SEQ ID NO: 3 and has the same or substantially the same immunogenicity as that thereof;

[0016] and / or encoding the G9 rotavirus antigen protein VP7 or its antigenic fragment having the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO:5 and has the same or substantially the same immunogenicity as that thereof.

[0017] Furthermore, the mRNA immunogenic composition further comprises a 5' cap, a 5' untranslatable region (UTR), a 3' untranslatable region (UTR), a polyA tail, a codon optimization element, or a nucleotide modification element to optimize the mRNA sequence for improved stability, translation efficiency, and immunogenicity. In some embodiments, the addition of a 5' cap to the mRNA facilitates ribosome recognition and translation initiation, a characteristic of endogenous mRNA in eukaryotes. The polyA tail contributes to mRNA stability and translation efficiency. The 5' untranslatable region (UTR) and 3' untranslatable region (UTR) elements have important cellular functions, influencing mRNA stability and translation. Codon optimization is an effective strategy for improving mRNA translation efficiency. To maximize protein translation, mRNA sequences typically include nucleoside modifications, such as pseudouracil, N1-methyl pseudouracil, or other nucleoside analogs.

[0018] Furthermore, in the mRNA immunogenic composition, the RV1 sequence is as shown in SEQ ID NO: 2; and / or the RV3 sequence is as shown in SEQ ID NO: 4; and / or the RV9 sequence is as shown in SEQ ID NO: 6.

[0019] In one embodiment, the present invention provides an mRNA vaccine comprising a delivery vector and any of the mRNA immunogenic compositions described above; preferably, the delivery vector comprises a lipid nanoparticle (LNP), a polymer carrier, a polypeptide carrier, a dendritic cell (DC) carrier, or an extracellular vesicle carrier. Further preferably, the delivery vector is a lipid nanoparticle. In some embodiments, common mRNA polymer carriers include polyethyleneimine (PEI), polyesters, polyamino acids, and carbohydrates (such as chitosan); polypeptide carriers include protamine and cell-penetrating peptides; and extracellular vesicle carriers include exosomes, microvesicles, or apoptotic bodies.

[0020] Furthermore, in the mRNA vaccine, the mass ratio of RV1, RV3 and RV9 is 1:(1-5):(1-5), preferably, the mass ratio is 1:(1-2):(1-2).

[0021] In one embodiment, the present invention provides a pharmaceutical composition comprising any of the immunogenic compositions or the mRNA vaccines.

[0022] In one embodiment, the present invention provides a kit comprising any of the immunogenic compositions, or the mRNA vaccines, or the pharmaceutical compositions.

[0023] In some embodiments, the present invention provides the use of any of the immunogenic compositions, or the mRNA vaccines, or the pharmaceutical compositions, or the kits described above in the preparation of a drug for preventing or treating rotavirus infection. Preferably, the drug is a vaccine; further preferably, the vaccine is used for single immunization or sequential immunization with other types of rotavirus vaccines.

[0024] The technical effects achieved by the present invention are:

[0025] In this study, mRNA encoding the VP7 antigen protein of rotavirus G1, G3, and G9, designated RV1, RV3, and RV9, were constructed. After in vitro expression was verified by transfection into HEK293 cells, RV1, RV3, and RV9 were encapsulated with lipid nanoparticles (LNPs) to prepare rotavirus mRNA vaccines targeting G1, G3, and G9, respectively. Physical mixing was used to formulate the three rotavirus mRNA vaccines. Immunogenicity studies were conducted in Balb / c mice using the optimal immunization strategy, and the formulation of the trivalent vaccine was investigated. Experimental results demonstrated that the trivalent rotavirus mRNA vaccine (RV1-LNP + RV3-LNP + RV9-LNP) prepared in mice induced neutralizing and IgG antibody responses against rotavirus G1, G3, and G9, demonstrating cross-neutralization. Compared with the monovalent vaccine, the trivalent rotavirus mRNA vaccine can induce a higher antibody response. The results provide experimental and theoretical basis for the design of trivalent rotavirus mRNA vaccine and the formulation of immunization strategy. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Western blotting analysis of VP7 protein expression in lysates and cell supernatants of MA104 cells transfected with RV1, RV2, and RV3. (A) Cell lysate; (B) Cell supernatant.

[0027] Figure 2 Particle size determination diagram of RV1-LNP, RV3-LNP, and RV9-LNP.

[0028] Figure 3Electron microscopy observations of RV1-LNP, RV3-LNP and RV9-LNP, scale bar is 200 nm.

[0029] Figure 4 Statistical graphs of the encapsulation efficiency and PDI of RV1-LNP, RV3-LNP, and RV9-LNP. (A) Encapsulation efficiency of RV1-LNP, RV3-LNP, and RV9-LNP. (B) PDI of RV1-LNP, RV3-LNP, and RV9-LNP.

[0030] Figure 5 Neutralizing antibody titers elicited by the three monovalent vaccines, RV1-LNP, RV3-LNP, and RV9-LNP, against G1 RV, G3 RV, and G9 RV. (A) Neutralizing antibody titers elicited by RV1-LNP against G1 RV, G3 RV, and G9 RV. (B) Neutralizing antibody titers elicited by RV3-LNP against G1 RV, G3 RV, and G9 RV. (C) Neutralizing antibody titers elicited by RV9-LNP against G1 RV, G3 RV, and G9 RV. Data are presented as geometric mean and geometric SD, and significant differences were determined by one-way ANOVA (* p < 0.05, ** p < 0.01, and *** p < 0.0001; ns indicates not significant).

[0031] Figure 6 Neutralizing antibody titers elicited by four trivalent mRNA vaccine formulations against G1 RV, G3 RV, and G9 RV. Data are presented as geometric mean and geometric SD, and significant differences were determined by two-way ANOVA (* p < 0.05, ** p < 0.01, and *** p < 0.0001; ns indicates not significant).

[0032] Figure 7 Comparison of neutralizing antibody levels among trivalent mRNA vaccines at the same dose. Data are presented as geometric mean and geometric SD, and significant differences were determined by two-way ANOVA (* p < 0.05, ** p < 0.01, and *** p < 0.0001; ns. indicates not significant).

[0033] Figure 8 The trivalent vaccine elicited higher levels of neutralizing antibody titers compared to the monovalent vaccine. Data are presented as geometric mean and geometric SD, and significant differences were determined by two-way ANOVA (* p < 0.05, ** p < 0.01, and *** p < 0.0001; ns. indicates not significant).

[0034] Figure 9Detection of IFN-γ cytokine levels in mouse spleen lymphocytes in different trivalent vaccine groups (* p<0.05, ** p<0.01; ns. indicates not significant). DETAILED DESCRIPTION

[0035] The technical scheme of the present invention is described in detail below in conjunction with the drawings and Examples, but the present invention is not limited to the scope of the embodiments described. Unless otherwise specified, the experiments and methods described in the embodiments are carried out basically according to conventional methods well known in the art and described in various references, and the reagents and raw materials used in the present invention are all commercially available. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially. Those skilled in the art will appreciate that the embodiments describe the present invention by way of example and are not intended to limit the scope of the present invention. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

[0036] In the description of the present invention, mRNA is abbreviated as "RV", and the mRNA prepared from G1, G3 and G9 rotaviruses are abbreviated as "RV1, RV3, RV9" or "G1 RV, G3 RV, G9 RV", respectively.

[0037] The sequence information involved in the present invention is as follows:

[0038] RV1 amino acid sequence: SEQ ID NO: 1

[0039] RV1 nucleotide sequence: SEQ ID NO: 2

[0040] RV3 amino acid sequence: SEQ ID NO: 3

[0041] RV3 nucleotide sequence: SEQ ID NO: 4

[0042] RV9 amino acid sequence: SEQ ID NO: 5

[0043] RV9 nucleotide sequence: SEQ ID NO: 6

[0044] Example 1

[0045] 1. Construction of rotavirus VP7 mRNA

[0046] Rotavirus VP7 nucleotide sequences of G1, G3, and G9 types were synthesized in vitro and designated G1-VP7. The G3-VP7 and G9-VP7 sequences contain the native rotavirus 5′UTR, a T7 promoter site, CDS region, 3′UTR (two tandem β-globins), and a poly A tail. G1-VP7, G3-VP7, and G9-VP7 were constructed into the pUC57-Kan-SapI-free vector, respectively. Plasmids were extracted, linearized, and purified using magnetic beads. The purified products were then transcribed in vitro using the T7 High Yield RNA Transcription Kit (N¹-Me-Pseudo UTP) (Nanjing Novogene Medical Technology Co., Ltd.). After transcription, RNA was purified using magnetic beads and the purified products were analyzed by capillary electrophoresis. If qualified, the transcripts were enzymatically capped using vaccinia virus capping enzyme (DD4109; Nanjing Novozymes Medical Technology Co., Ltd.) and Cap2 Oxymethyltransferase (DD4110; Nanjing Novozymes Medical Technology Co., Ltd.). After capping, the transcripts were purified using RNA magnetic beads. The purified cap-mRNAs were designated RV1, RV3, and RV9. The concentration of the purified mRNA was determined using a NanoDrop microspectrophotometer (Thermo Fisher Scientific, USA) and stored frozen at −80°C until use.

[0047] In vitro transfection and viral protein expression analysis

[0048] After capping and purification, RV1, RV3, and RV9 mRNAs were transfected into HEK293 cells using PolyPlus' jetMESSENGER transfection reagent. 48 hours after transfection, the culture medium in the wells was collected and stored. The cells were then washed twice with PBS, the PBS discarded, and the cells were centrifuged at 10,000 g for 5 minutes. Protein concentration was determined using a BCA protein assay kit and analyzed by SDS-PAGE. Lysates were run on a 10% Tris-HCl SDS-PAGE gel and transferred to a PVDF (polyvinylidene fluoride) membrane, which was blocked in PBST (Tris-buffered saline with Tween 20) containing 5% nonfat milk. The PVDF membrane was incubated with a primary antibody (rabbit anti-VP7 antibody). A secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG, Abcam) was added to the blocking buffer and incubated for 1 hour at room temperature. After washing, the PVDF membrane was incubated with a supersensitive luminescent solution (Solarbio) and imaged using an ECL system (Thermo Fisher Scientific).

[0049] Preparation of trivalent rotavirus LNP-mRNA vaccine

[0050] After confirming the expression efficacy of the three VP7 variants, the three mRNA variants were encapsulated in LNPs. The LNP formulation consisted of lipids dissolved in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5 (ionizable lipid: DSPC: cholesterol: PEG-lipid). The lipid mixture was mixed with mRNA in 50 mmol / L sodium acetate buffer (pH 5.5) at a 1:1 ratio using a microfluidic mixer (Miana). mRNA-LNPs were prepared, while empty LNPs were also encapsulated as a control. The LNP-mRNA vaccine was diluted 100-fold using This-HCl buffered saline (pH 7.5) and concentrated to the original volume using ultrafiltration tubes. The formulation was then filtered through a 0.22 μm filter and stored at 4°C until use. Particle size analysis of the LNP-mRNA vaccine was performed using a Malvern Zetasizer Nano ZS90 nanoparticle size analyzer, and mRNA encapsulation and concentration were determined using the RiboGreen assay (Thermo Fisher Scientific). The morphology and size of LNP-mRNA were observed by transmission electron microscopy (TEM).

[0051] The G1, G3 and G9 LNP-mRNA vaccines were prepared and tested respectively, and then the trivalent vaccine was prepared by physical mixing. Based on the mass of mRNA, the mixing ratio of the three vaccines was: G1:G3:G9=1:1:1; G1:G3:G9=1:2:1; G1:G3:G9=1:1:2.

[0052] The vaccine effect obtained by the present invention is:

[0053] 1. Experimental Methods

[0054] 1. Cells and viruses

[0055] The reference virus used to test for neutralizing antibodies and the source virus for the VP7 gene sequence in this study were both field-specific human rotavirus strains isolated and cultured and maintained by the Laboratory of Molecular Biology, Institute of Medical Biology, Chinese Academy of Medical Sciences. HEK293 cells were obtained from Heyuan Biotech (Shanghai) Co., Ltd. and maintained at a low passage number according to guidelines.

[0056] Mouse experiments

[0057] We divided 6-8 week old female Balb / c mice into 8 groups. The specific group information is shown in Table 1. All groups were injected intramuscularly without adjuvant. The vaccine group and the control group used the same immunization procedure. The vaccine group mice were injected with vaccine, and the control group mice were injected with empty LNP. Each group contained 5 mice. In order to explore the optimal dose mixing ratio, three ratios of G1:G3:G9=1:1:1, G1:G3:G9=1:2:1 and G1:G3:G9=1:1:2 were set. The immunization dose was 2 injections, with an interval of 14 days. Serum was collected before immunization and after 2 doses of immunization for neutralizing antibody detection, and the weight of the mice was monitored during the period.

[0058] Table 1 Grouping of mice in the trivalent mRNA vaccine immunization experiment

[0059]

[0060] 3. Enzyme-linked immunospot assay (Elispot)

[0061] Splenic lymphocytes (2 × 10 6 Cells from immunized mice were plated in 96-well plates for further analysis using an enzyme-linked immunospot (ELISPOT) assay kit (Mabtech, catalog number 3321-4AST-2 for IFN-γ detection) according to the manufacturer's protocol. VP7 peptides at a final concentration of 20 μg / mL were used to stimulate specific T cell responses, and an equal volume of PMA plus ionomycin was used as a positive control. Spots were counted using an ELISPOT reader system (AutoimmunDiagnostika GmbH, Strasberg, Germany).

[0062] Neutralizing antibody detection

[0063] After incubation with G1, G3, and G9 rotavirus indicator viruses, the serum was added to a 96-well plate filled with cells and incubated for 7 days. After 7 days, the liquid was transferred to a plate coated with antibodies. The rotavirus antigen content was determined using a double antibody sandwich method to calculate the neutralizing antibody titer. The specific steps are as follows:

[0064] 1) Pre-coat a 96-well plate with antibodies against rotavirus types G1, G3, and G9. Incubate at 4°C overnight.

[0065] 2) Add 100 μl of 3% BSA to each well and incubate at 37°C for 1 h.

[0066] 3) Wash three times with PBST, tap the wells to remove any liquid, and store at -20°C until ready for use.

[0067] 4) Serum of different dilutions was mixed with G1, G3, and G9 indicator virus working solutions, respectively, and incubated at 37°C for 2 h.

[0068] 5) The mixture was added to a 96-well plate filled with MA104 cells and incubated at 37°C for 7 days.

[0069] 6) Remove the 96-well plate, freeze-thaw twice, and then transfer the lysate to a 96-well plate coated with rotavirus antibodies of types G1, G3, and G9, and incubate at 37°C for 1 hour.

[0070] 7) Wash three times with PBST and tap the wells to remove any liquid.

[0071] 8) Add 100 μl of the corresponding rotavirus enzyme-labeled antibody to each well of a 96-well plate according to the corresponding virus typing and incubate at 37°C for 1 hour.

[0072] 9) Wash three times with PBST and tap the wells to remove any liquid.

[0073] 10) Add 100 μl of color development solution to each well, incubate at room temperature for 5 minutes, and then add 100 μl of stop solution to each well.

[0074] Place the 96-well plate into a microplate reader and read the absorbance at 450 nm and 650 nm.

[0075] 2. Experimental Results

[0076] 1. In vitro expression of RV1, RV3, and RV9

[0077] The three plasmids G1-VP7, G3-VP7 and G9-VP7 were linearized, transcribed in vitro and capped to form RV1, RV3 and RV9, and transfected into HEK293 cells. After 48 hours, the cell lysate and supernatant were collected and tested for their respective G-type VP7. At the same time, VP7 protein was set as a positive control, and the cell lysate and supernatant of HEK293 cells were set as negative controls. The supernatant was concentrated 20 times by ultrafiltration before loading. The results are shown in Figure 2. Figure 1 As shown in Figure 2, after transfection, RV1, RV3 and RV9, VP7 protein expression was detected in both cell lysates and cell supernatants, indicating that VP7 protein could be expressed smoothly in cells ( Figure 1 A), can also be secreted outside the cell under the action of signal peptides ( Figure 1 B).

[0078] Vaccine characteristics

[0079] After confirming that the three capped products RV1, RV3, and RV9 could be successfully expressed, the capped mRNA sequences were encapsulated by LNP using a microfluidic device. The encapsulated mRNA vaccines RV1-LNP, RV3-LNP, and RV9-LNP were then tested for particle diameter, electron microscopy, encapsulation efficiency, and PDI index. The vaccine particle size was measured using a Malvern Zetasizer Nano ZS90 nanoparticle size potentiometer. The results showed that the average diameter of RV1-LNP and RV3-LNP was 100nm, while the average particle size of RV9-LNP was 110nm ( Figure 2 ).

[0080] The morphology of the prepared RV1-LNP, RV3-LNP and RV9-LNP vaccines was observed by transmission electron microscopy. The results showed that the vaccines were uniform spherical particles with a particle size of about 100 nm ( Figure 3 ); The encapsulation efficiency was detected using the Quant-iT™ RiboGreen RNA quantification kit. The encapsulation efficiencies of RV1-LNP, RV3-LNP and RV9-LNP were 90.4%, 91.1% and 90.8%, respectively ( Figure 4 A); RV1-LNP, RV3-LNP and RV9-LNPPDI are 0.144, 0.15 and 0.056, achieving the ideal encapsulation effect ( Figure 4 B).

[0081] Evaluation of the immunogenicity of trivalent rotavirus mRNA vaccine

[0082] 3.1 Humoral immunity

[0083] Tail vein blood was collected from mice on day 0, 14, and 28 of immunization, and serum was separated. 96-well plates were coated with antibodies against rotavirus G1, G3, and G9, respectively. The neutralizing antibody levels in serum were detected using a cell-neutralizing combined with a double antibody sandwich assay. Neutralizing antibody titers were expressed as Log2GMT. The results showed that after two injections, the neutralizing antibody titers of RV1-LNP against G1 RV, G3 RV, and G9 RV were 3, 3, and 3 ( Figure 5 A), the neutralizing antibody titers of RV3-LNP against G1 RV, G3 RV and G9 RV were 3.65, 3 and 3 respectively ( Figure 5 B), the neutralizing antibody titers of RV9-LNP against G1 RV, G3 RV and G9 RV were 3.95, 3 and 8.36 respectively ( Figure 5 C). There was a statistically significant difference in the neutralizing antibody levels of RV9-LNP against G1 RV and G9 RV.

[0084] Neutralizing antibody titers were measured in sera collected from mice in the four trivalent vaccine groups. Neutralizing antibody titers are presented as Log2GMT. In the following trivalent vaccine results, the RV1-LNP vaccine is referred to as G1, the RV3-LNP vaccine as G3, and the RV9-LNP vaccine as G9. The results showed that after two immunizations, the neutralizing antibody titers of the G1 3.3μg+G3 3.3μg+G9 3.3μg group against G1 RV, G3 RV and G9 RV were 6.16, 3 and 4.23 respectively; the neutralizing antibody titers of the G1 10μg+G3 10μg+G9 10μg against G1 RV, G3 RV and G9 RV were 3.73, 3.74 and 9.69 respectively; the neutralizing antibody titers of the G1 7.5μg+G3 15μg+G9 7.5μg against G1 RV, G3RV and G9 RV were 4.22, 6.4 and 9.96 respectively. The neutralizing antibody titers of G1 7.5μg+G3 7.5μg+G9 15μg against G1 RV, G3 RV and G9 RV were 3.57, 5.62 and 9.97 respectively. For G1 and G3 viruses, there was no statistical difference in the neutralizing antibody titers stimulated by the four trivalent vaccines; for G9 virus, the neutralizing antibody titers stimulated by the three high-dose combination groups were statistically different from those of the G1 3.3μg+G3 3.3μg+G9 3.3μg group ( Figure 6 ).

[0085] With the same total amount of immune antigen, after two injections, the neutralizing antibody titers of RV1-LNP against G1 RV, G3 RV and G9 RV were 3, 3 and 3, respectively; the neutralizing antibody titers of RV3-LNP against G1 RV, G3 RV and G9 RV were 3.65, 3 and 3, respectively; the neutralizing antibody titers of RV9-LNP against G1 RV, G3 RV and G9 RV were 3.95, 3 and 8.36, respectively. The neutralizing antibody titers of the G1 3.3μg+G33.3μg+G9 3.3μg group against G1 RV, G3 RV and G9 RV were 6.16, 3 and 4.23, respectively ( Figure 7 ).

[0086] For G1 RV, the neutralizing antibody titers elicited by the G1 3.3μg+G3 3.3μg+G9 3.3μg group and the G1 10μg group were statistically different; for G9 RV, the neutralizing antibody titers elicited by the G1 3.3μg+G3 3.3μg+G9 3.3μg group and the RV1-LNP 10μg and RV3-LNP 10μg groups were not statistically different, while the neutralizing antibody titers elicited by the RV9-LNP 10μg group and the RV1-LNP 10μg and RV3-LNP 10μg groups were statistically different, indicating that only the RV9-LNP vaccine can elicit high levels of neutralizing antibodies against the parental strain virus ( Figure 7 ).

[0087] When the total amount of immune antigen was different, after two injections, the neutralizing antibody titers of G1 7.5μg+G3 15μg+G9 7.5μg against G1RV, G3 RV and G9 RV were 4.22, 6.4 and 9.96 respectively, the neutralizing antibody titers of RV1-LNP against G1 RV, G3 RV and G9 RV were 3, 3 and 3 respectively, the neutralizing antibody titers of RV3-LNP against G1 RV, G3 RV and G9 RV were 3.65, 3 and 3 respectively, and the neutralizing antibody titers of RV9-LNP against G1 RV, G3 RV and G9 RV were 3.95, 3 and 8.36 respectively. Compared with the monovalent mRNA vaccine groups of various types, the trivalent G1 7.5μg+G3 15μg+G9 7.5μg group can always stimulate a higher level of neutralizing antibody titer ( Figure 8 ).

[0088] Cellular immunity

[0089] 28 days after the first immunization, the spleens of mice were collected to isolate lymphocytes, counted, and added to a 96-well plate coated with IFN-γ antibody. The screened VP7-specific stimulatory peptide (DYIIYRFLLI) was added to the cells for IFN-γ detection. The results showed that compared with the control group, the lymphocytes of the mice in the trivalent vaccine group were able to secrete IFN-γ ( Figure 9 A). The number of spots increased in mice injected with a high dose of G1:G3:G9 = 1:2:1 was statistically different not only from the number of spots increased in mice injected with LNP, but also from the number of spots increased in mice injected with a low dose of G1:G3:G9 = 1:1:1 ( Figure 9 B).

Claims

1. An mRNA vaccine comprising a delivery vector and an mRNA immunogenic composition, characterized in that: The composition comprises RV1, RV3 and RV9: i) mRNA encoding the antigenic fragment of the G1 rotavirus antigen protein VP7, the mRNA being designated as RV1, the sequence of which is shown in SEQ ID NO: 2; ii) mRNA encoding the antigenic fragment of the G3 rotavirus antigen protein VP7, the mRNA being named RV3, the RV3 sequence being shown in SEQ ID NO: 4; iii) mRNA encoding the antigenic fragment of VP7 of rotavirus G9 antigen protein, the mRNA is named RV9; the sequence of RV9 is shown in SEQ ID NO:

6.

2. The mRNA vaccine according to claim 1, characterized in that The delivery carrier includes lipid nanoparticles, polymer carriers, polypeptide carriers, dendritic cell carriers or extracellular vesicle carriers.

3. The mRNA vaccine according to claim 2, characterized in that The delivery vehicle is a lipid nanoparticle.

4. The mRNA vaccine according to claim 3, characterized in that The mass ratio of RV1, RV3 and RV9 is 1:(1-2):(1-2).

5. A pharmaceutical composition, characterized in that The composition contains the mRNA vaccine according to any one of claims 1-4.

6. A kit, characterized in that The kit contains the mRNA vaccine according to any one of claims 1-4.

7. Use of the mRNA vaccine according to any one of claims 1 to 4, or the pharmaceutical composition according to claim 5, or the kit according to claim 6 in the preparation of a medicament for preventing or treating rotavirus infection.

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