mRNA encoding zika virus antigen peptide, and mRNA vaccine based on mRNA and preparation method therefor

Abstract

Provided are an mRNA encoding a Zika virus antigen peptide, an mRNA construct encoding a Zika virus antigen peptide, a recombinant expression plasmid for producing the mRNA, an mRNA vaccine against Zika virus based on the mRNA and a preparation method for the mRNA vaccine. The mRNA vaccine against Zika virus can not only avoid induced generation of FL epitope antibodies so as to significantly reduce or eliminate the ADE effect, but also generate high-level neutralizing antibodies higher than those of other vaccine vector platforms so as to effectively prevent or treat Zika virus infectious diseases, and has better clinical application values and prospects.

Description

mRNA encoding Zika virus antigen peptide, mRNA vaccine based thereon and preparation method thereof

[0001] Cross-references

[0002] This application claims priority to the Chinese patent application filed on July 14, 2023, with application number 202310871396.6, entitled “mRNA encoding Zika virus antigen peptide, mRNA vaccine based thereon and preparation method thereof”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application relates to the field of biomedicine technology, and specifically to mRNA encoding Zika virus antigen peptides, mRNA vaccines based thereon, and preparation methods thereof. Background Art

[0004] Zika fever is a mosquito-borne viral disease caused by the Zika virus (ZIKV). ZIKV was first discovered in the serum of rhesus monkeys in Uganda in 1947, and human cases of Zika virus infection were reported in Uganda and Tanzania in 1952. ZIKV infection during pregnancy can affect placental function, leading to microcephaly and miscarriage. Zika virus infection in adults can cause Guillain-Barré syndrome. From 2015 to 2016, a Zika fever pandemic occurred in Brazil, resulting in 440,000 to 1.5 million infections. The World Health Organization has declared the virus an international public health emergency.

[0005] ZIKV is an arbovirus belonging to the Flaviviridae family and the genus Flavivirus, a single-stranded, positive-sense RNA virus. The ZIKV genome is approximately 10.7 kb, encoding 3423 amino acids. Flaviviruses can induce immune cross-reactions and share similar immunogenic epitopes. Some of these cross-reactions are beneficial, resulting in cross-protection. However, some antibody cross-reactions can mediate infection enhancement (i.e., ADE) through non-neutralizing or sub-neutralizing antibodies, thereby exacerbating disease. Antibodies that cause ADE reactions are primarily produced by immune cells targeting the fusion loop (FL) of the viral envelope (E) protein during viral infection. ZIKV and dengue virus (DENV) share similar genetic composition and antigenic properties. Antibodies produced after ZIKV infection can trigger an ADE effect against DENV, posing a significant challenge to vaccine development.

[0006] Candidate ZIKV vaccines currently in phase I / II clinical trials include inactivated virus vaccines, viral vector vaccines, subunit protein vaccines, nucleic acid DNA-RNA vaccines, and vaccines based on mosquito saliva antibodies. However, to date, there have been no reports of vaccines that eliminate the ADE epitopes of ZIKV entering clinical experimental research and development.

[0007] We previously developed an adenoviral vector vaccine that eliminates the ZIKV FL epitope, effectively preventing ZIKV infection without triggering an ADE response to DENV. In the context of the COVID-19 pandemic, multi-platform vaccine approaches have rapidly developed. Data show that the Oxford University / AstraZeneca adenoviral vector vaccine ChAdOx1-S has a 70% efficacy against COVID-19, while BioNTech's mRNA vaccine (BNT162b2) and Moderna's mRNA vaccine (mRNA-1273) have an efficacy of approximately 94%. mRNA vaccines are a novel vaccine platform that requires only viral gene sequence information for development and application, making them an important new tool for combating pandemics and other infectious diseases. mRNA vaccines work by inserting viral mRNA fragments into human cells, reprogramming them to produce pathogen antigens and then stimulating an adaptive immune response against the pathogen. The mRNA molecules are typically encapsulated in drug delivery vehicles such as lipid nanoparticles to protect the fragile mRNA molecules and facilitate their uptake by human cells. Compared to traditional protein vaccines, RNA vaccines offer advantages such as rapid production, low cost, and the ability to induce both cellular and humoral immunity. However, these vaccines require cold chain distribution and low-temperature storage. Therefore, there is an urgent need to develop a new Zika virus vaccine using mRNA technology that can eliminate the ADE response to DENV.

[0008] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the application and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0009] Summary of the Invention

[0010] To overcome the problems existing in the above-mentioned prior art, the present application provides an mRNA encoding a Zika virus antigen peptide, its mRNA construct, a recombinant expression plasmid, and its use in the preparation of a vaccine, preferably an mRNA vaccine, for preventing and / or treating Zika virus infectious diseases, as well as an mRNA vaccine against Zika virus based on the aforementioned mRNA or mRNA construct and its preparation method.

[0011] Specifically, this application provides the following technical solutions:

[0012] In a first aspect, the present application provides an mRNA encoding a Zika virus antigen peptide, wherein the mRNA has an mRNA sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, or is transcribed from a DNA as shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0013] In other words, the mRNA sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 can be transcribed from the DNA shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0014] In a second aspect, the present application provides an mRNA construct encoding a Zika virus antigen peptide, wherein the mRNA construct comprises the mRNA as described in the first aspect above, and one or more elements selected from the following: a 5' UTR region, a 3' UTR region, a 5' end cap structure, a 3' end PolyA tail and a signal peptide sequence.

[0015] In a preferred embodiment, the 5' UTR region has the sequence shown in SEQ ID NO: 5;

[0016] In a preferred embodiment, the 3' UTR region has the sequence shown in SEQ ID NO: 6;

[0017] In a preferred embodiment, the 5' end cap structure is m7G(5')ppp;

[0018] In a preferred embodiment, the 3' terminal PolyA tail is 80 to 200 A;

[0019] In a preferred embodiment, the signal peptide sequence is shown as SEQ ID NO:7.

[0020] In a third aspect, the present application provides a recombinant expression plasmid for producing the mRNA as described in the first aspect above, wherein the recombinant expression plasmid comprises a plasmid backbone sequence and a DNA sequence encoding a Zika virus antigen peptide.

[0021] Preferably, the DNA sequence encoding the Zika virus antigen peptide is shown in SEQ ID NO: 3 or SEQ ID NO: 4;

[0022] Preferably, the plasmid backbone sequence includes a T7 promoter sequence, a 5' UTR region, a signal peptide sequence, a 3' UTR region, a 5' cap structure and a 3' PolyA tail;

[0023] In a preferred embodiment, the 5' UTR region has the sequence shown in SEQ ID NO: 5;

[0024] In a preferred embodiment, the 3' UTR region has the sequence shown in SEQ ID NO: 6;

[0025] In a preferred embodiment, the 5' end cap structure is m7G(5')ppp;

[0026] In a preferred embodiment, the 3' terminal PolyA tail is 80 to 200 A;

[0027] In a preferred embodiment, the signal peptide sequence is shown as SEQ ID NO:7.

[0028] In a fourth aspect, the present application provides the use of the mRNA as described in the first aspect above, the mRNA construct as described in the second aspect above, or the recombinant expression plasmid as described in the third aspect above in the preparation of a vaccine, preferably an mRNA vaccine, for preventing and / or treating Zika virus infectious diseases.

[0029] In a fifth aspect, the present application provides an mRNA vaccine against Zika virus, which comprises the mRNA as described in the first aspect above or the mRNA construct as described in the second aspect above, and a lipid component.

[0030] In a preferred embodiment, the mass ratio of the mRNA as described in the first aspect or the mRNA construct as described in the second aspect to the lipid component is 1:(10-30), preferably 1:20;

[0031] In a preferred embodiment, the lipid component comprises or consists of cationic lipids, phosphatidylcholine, cholesterol and PEG lipids; further preferably, in the lipid component, the molar ratio of cationic lipids, phosphatidylcholine, cholesterol and PEG lipids is (40-60):(5-15):(30-45):(1-2), preferably (45-55):(8-12):(35-40):(1-2), and more preferably 50:10:38.5:1.5.

[0032] In a preferred embodiment, the mRNA vaccine is in the form of a nasal spray, oral formulation, suppository, or parenteral formulation;

[0033] Further preferably, the nasal spray is selected from aerosols, sprays and powder sprays;

[0034] Further preferably, the oral preparation is selected from tablets, powders, pills, powders, granules, fine granules, soft / hard capsules, film-coated capsules, pellets, sublingual tablets and ointments;

[0035] Further preferably, the parenteral preparation is a transdermal preparation, an ointment, a plaster, a liquid for external use, an injectable or a pushable preparation.

[0036] In a sixth aspect, the present application provides a method for preparing the mRNA vaccine as described in the fifth aspect above, comprising:

[0037] (1) dissolving the mRNA or mRNA construct in an aqueous solution as the aqueous phase;

[0038] (2) dissolving the lipid component in an organic solvent as the organic phase;

[0039] (3) mixing the aqueous phase and the organic phase to self-assemble to obtain the mRNA vaccine;

[0040] Preferably, the aqueous solution is sodium acetate buffer;

[0041] Preferably, the organic solvent is anhydrous ethanol;

[0042] Preferably, the self-assembly is performed by nanoliposome packaging.

[0043] In a seventh aspect, the present application provides a method for preventing or treating Zika virus infectious diseases, comprising: administering a preventively or therapeutically effective amount of the mRNA vaccine as described in the fifth aspect above to a subject in need.

[0044] The "preventively or therapeutically effective amount" may vary depending on the subject, target organ, symptoms, administration method, etc., and can be determined based on the doctor's judgment, taking into account the type of dosage form, administration method, patient's age and weight, patient's symptoms, etc. Beneficial effects

[0045] The inventors of the present application have developed an mRNA encoding a Zika virus antigen peptide and an mRNA vaccine against Zika virus based on the mRNA; the mRNA vaccine against Zika virus of the present application can not only avoid the induction of FL epitope antibodies, thereby reducing or eliminating the ADE effect, but also produce a high level of neutralizing antibodies higher than other vaccine vector platforms, thereby effectively preventing or treating Zika virus infectious diseases; in view of the above, the mRNA vaccine against Zika virus of the present application has good clinical application value and industrialization prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] One or more embodiments are exemplarily illustrated by the accompanying figures, and these exemplary illustrations do not limit the embodiments. The word "exemplary" is used herein to mean "serving as an example, example, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0047] Figure 1 is a schematic diagram of the structures of the Zika virus wild-type mRNA vaccine M / E-WT, the FL mutation-based mRNA vaccines M / E-MutB and M / E-MutC constructed in Example 1 of the present application; the various segments of the mRNA vaccine are marked thereon, wherein 5'UTR represents the 5' untranslated region, 3'UTR represents the 3' untranslated region, SP represents the signal peptide sequence, and Poly(A) represents the polyadenylic acid tail.

[0048] Figure 2 is a schematic diagram of the mRNA vaccine immunization of mice and sampling process described in Example 2 of the present application.

[0049] Figure 3 shows the results of humoral immunity experiments in BalB / C mice immunized with mRNA vaccines M / E-WT, M / E-MutB and M / E-MutC; among them, Figure 3A and Figure 3B respectively show the binding antibody titer levels against Zika virus wild-type E antigen in sera collected on the 21st day (i.e., after the primary immunization) and the 42nd day (i.e., after the secondary immunization) after mice were immunized with mRNA vaccines; Figure 3C and Figure 3D respectively show the neutralizing antibody titer against Zika virus wild-type E antigen in sera collected on the 21st day (i.e., after the primary immunization) and the 42nd day (i.e., after the secondary immunization) after mice were immunized with mRNA vaccines.

[0050] Figure 4 shows the results of cellular immunity experiments in BALB / C mice immunized with mRNA vaccines M / E-WT, M / E-MutB and M / E-MutC; Figure 4A shows the ELISpot test of the number of IFN-γ+ and IL-2+ cells produced by spleen cells collected on the 42nd day after mRNA vaccine immunization of mice after being stimulated by the Zika virus wild-type E polypeptide library, and Figure 4B shows the ICS test of the number of IFN-γ+, TNF-α+, IL-4+ and IL-2+ cells produced by spleen cells collected on the 42nd day after mRNA vaccine immunization of mice after being stimulated by the Zika virus wild-type E polypeptide library.

[0051] Figure 5 shows the results of humoral immunity experiments of Ifnar1- / - mice immunized with mRNA vaccines M / E-WT, M / E-MutB and M / E-MutC; among them, Figures 5A and 5B respectively show the binding antibody titer levels against Zika virus wild-type E antigen in sera collected on the 21st day (i.e., after the primary immunization) and the 42nd day (i.e., after the secondary immunization) after mice were immunized with mRNA vaccines; Figures 5C and 5D respectively show the neutralizing antibody titers against Zika virus wild-type E antigen in sera collected on the 21st day (i.e., after the primary immunization) and the 42nd day (i.e., after the secondary immunization) after mice were immunized with mRNA vaccines.

[0052] Figure 6 is a graph showing Ifnar1 immunized with mRNA vaccines M / E-WT, M / E-MutB and M / E-MutC. - / - Weight change curve (A) and mortality results (B) of mice after ZIKV-PRVABC59 virus infection.

[0053] Figure 7 shows the expression of Ifnar1 immunized with mRNA vaccines M / E-WT, M / E-MutB and M / E-MutC. - / - Experimental results on the resistance of mice to viremia caused by ZIKV-PRVABC59 virus infection.

[0054] Figure 8 shows the Ifnar1 immunized with mRNA vaccines M / E-WT, M / E-MutB and M / E-MutC - / - Experimental results on the resistance of mice to infection of tissues and organs after ZIKV-PRVABC59 virus infection.

[0055] Figure 9 shows the Ifnar1 immunized with mRNA vaccines M / E-WT, M / E-MutB and M / E-MutC - / - The results of changes in neutralizing antibody titers in the serum of mice before and after infection with ZIKV-PRVABC59 virus.

[0056] Figure 10 shows the experimental results of the ADE effect of the serum of BALB / c mice immunized with mRNA vaccines M / E-WT, M / E-MutB and M / E-MutC on K562 cells infected with ZIKV, DENV1, DENV2, DENV3 and DENV4.

[0057] Figure 11 shows the results of the ADE effect experiment on DENV2 infection after the serum of BALB / c mice immunized with mRNA vaccines M / E-WT, M / E-MutB and GFP was passively transferred to AG6 mice. DETAILED DESCRIPTION

[0058] To make the purpose, technical solutions, and advantages of the embodiments of this application more clear, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of this application, not all of them. Based on the embodiments of this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0059] In addition, in order to better illustrate the present application, numerous specific details are provided in the following detailed description. It should be understood by those skilled in the art that the present application can be implemented without certain specific details. In some embodiments, raw materials, components, methods, means, etc. well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present application.

[0060] Unless expressly stated otherwise, throughout the specification and claims, the term "comprise" or variations such as "include" or "comprising", etc., will be understood to include the stated elements or components but not to exclude other elements or other components.

[0061] Example 1: Preparation and identification of mRNA constructs and mRNA vaccines

[0062] According to the structural schematic diagram of the mRNA construct shown in Figure 1, the inventors constructed the mutant mRNA constructs ZIKV-M / E-mutB and ZIKV-M / E-mutC of the present application (hereinafter referred to as M / E-mutB and M / E-mutC, respectively), as well as the wild-type mRNA construct ZIKV-M / E-WT (hereinafter referred to as M / E-WT) as a control.

[0063] The specific plan is as follows:

[0064] 1) Sequence design of the mRNA coding regions of the three mRNA constructs

[0065] First, based on the ZIKV FSS13025 virus strain (GenBank: JN860885.1), the wild-type M / E antigen (ie, M / E-WT) sequence was obtained, and its amino acid sequence is shown in SEQ ID NO: 8.

[0066] Then, referring to Dai et al, Nature immunology, 2021, Aug; 22(8): 958-968, the antigenic sequences of ZIKV-M / E-MutB / C were obtained; ZIKV-M / E-MutB originated from CFAV (Cell fusing agent virus, GenBank: NC_001564.2), with mutation sites of D98N, N103T, G106F, L107K and F108W; ZIKV-M / E-MutC originated from NAKV (Nakiwogo virus, GenBank: NC_030400.1), with mutation sites of D98N, N103T, G106L, L107E and F108W; in view of this, the antigenic sequences of M / E-mutB and M / E-mutC are shown in SEQ ID NOs: 9 and 10, respectively.

[0067] The antigen sequences shown in SEQ ID NOs: 8, 9, and 10 were codon optimized to obtain the DNA coding sequences of M / E-WT, M / E-mutB, and M / E-mutC antigens, as shown in SEQ ID NOs: 11, 3, and 4, respectively.

[0068] 2) Expression plasmid construction, linearization, in vitro transcription and construction

[0069] First, pcDNA3.1 was selected as a plasmid vector, and the antigen coding sequence and other DNA expression elements of the mRNA vaccine were introduced by conventional molecular biological methods, including, in order from upstream to downstream: (1) T7 promoter, (2) 5' UTR sequence upstream of the coding region (the 5' UTR sequences of several mRNA vaccines were the same, all as shown in SEQ ID NO: 5), (3) signal peptide coding sequence (i.e., SP, as shown in SEQ ID NO: 7), (4) DNA coding sequence of vaccine antigen (the DNA coding sequences of M / E-WT, M / E-mutB and M / E-mutC mRNA vaccine antigens were shown in SEQ ID NO: 11, 3 and 4, respectively), (5) 3' UTR sequence downstream (the 3' UTR sequences of several mRNA vaccines were the same, all as shown in SEQ ID NO: 6), (6) and poly-A tail (Poly-A-tail). Finally, three Zika virus mRNA vaccine expression plasmids, pcDNA3.1-ZIKV-ME-WT, pcDNA3.1-ZIKV-ME-mutB, and pcDNA3.1-ZIKV-ME-mutC, were obtained.

[0070] Then, the in vitro transcription plasmid was digested with restriction endonuclease EcoR321I to linearize it; and purified using conventional DNA purification methods to obtain an in vitro transcription template;

[0071] Next, based on the template, in vitro transcription was performed using a T7 RNA in vitro transcription kit (E131-01A, Suzhou Nearshore Protein Technology Co., Ltd.) to obtain in vitro transcribed mRNA; then, the mRNA was purified by lithium chloride precipitation using a lithium chloride recovery kit (S125, Suzhou Nearshore Protein Technology Co., Ltd.) to obtain purified in vitro transcribed mRNA.

[0072] Finally, the purified in vitro transcribed mRNA was capped at the 5' end with Cap1 using a capping enzyme kit Cap1 (M082-01B, Suzhou Jinan Protein Technology Co., Ltd.) to meet the conditions for translation in eukaryotic cells. Subsequently, the mRNA was purified again using the same lithium chloride precipitation method as above to obtain purified mRNA modified by the 5' end capping.

[0073] 3) Liposome packaging of mRNA

[0074] Cationic lipid Dlin-MC3-DMA, distearoylphosphatidylcholine DSPC, cholesterol and PEGylated lipid DMPE were mixed in a molar ratio of 50:10:38.5:1.5, and then the nano drug production system INano produced by Maianna was used. TM E, mixing it with the above-mentioned 5'-end capped mRNA and packaging it.

[0075] After packaging, the buffer solution is replaced with D-PBS by centrifugation or dialysis.

[0076] Finally, the mRNA packaging efficiency was identified using Thermo Fisher's Quan-iT Ribogreen RNAreagent kit to ensure that the packaging efficiency met the standards for mRNA vaccines.

[0077] The recombinant chimpanzee adenovirus-vectored vaccine AdC7-ME, constructed with ZIKV-M / E-WT and its mutant ZIKV-M / E-mutB / C antigens, stimulated high levels of humoral immunity, essentially achieving post-challenge clearance immunity and demonstrating excellent protective efficacy. Therefore, in the following examples, the recombinant adenovirus-vectored vaccine AdC7-ZIKV-M / E-mutB was used as a control.

[0078] Example 2: Experimental Animal Immunization and Sample Collection

[0079] In this example, 6-8 week old female BALB / c mice (purchased from Vital River) were used for animal experiments.

[0080] The experimental groups were divided into: mRNA vaccine immunization group, adenovirus vector vaccine control group and negative control group, among which the mRNA vaccine immunization group included M / E-WT mRNA vaccine immunization group, M / E-mutB mRNA vaccine immunization group and M / E-mutC mRNA vaccine immunization group, the adenovirus vector vaccine control group selected AdC7-M / E-mutB (its preparation can refer to patent application CN 202011243025.6), and the negative control group was GFP mRNA immunization group. All mice in the mRNA vaccine immunization group were immunized with the same designed mRNA vaccine (i.e., M / E-WT, M / E-mutB or M / E-mutC vaccine) on days 0 and 28, respectively. The immunization process and injection dose of the mice in the negative control group were the same as those of the vaccine group, and the adenovirus vector vaccine was only immunized with a single injection on day 0. The vaccination method was intramuscular injection. The mRNA vaccine group set two vaccination doses, 2μg mRNA and 10μg mRNA / mouse, respectively. Mouse serum samples were collected on days 21 and 42, respectively. Adenovirus vector vaccine AdC7-M / E-mutB was administered on day 0 with 1.6×10 11 vp, mouse serum samples were also collected on days 21 and 42. All collected sera were inactivated by incubation at 56°C for 30 minutes and then frozen at -80°C for testing the binding antibody titer and pseudovirus neutralizing antibody titer of the immune serum. In addition, mouse spleen samples were collected on day 42 to test the cellular immune response.

[0081] The mRNA vaccine immunization of mice and the sampling procedure are shown in Figure 2.

[0082] Example 3: Evaluation of humoral immunity levels induced by mRNA vaccines in Bal B / C mice

[0083] In this example, the level of humoral immunity induced by the mRNA vaccine in mice was detected by detecting the binding antibody level and the neutralizing antibody level in the serum of the immunized mice obtained in Example 2.

[0084] First, the binding antibody titer in the serum of immunized mice was detected by ELISA. The specific method was as follows: ZIKV-E protein (amino acid sequence shown in SEQ ID NO: 13) was diluted to 3 μg / ml with ELISA coating solution (sodium carbonate-sodium bicarbonate buffer, pH 9.6), 100 μl was added to each well of a 96-well ELISA plate, and the plate was incubated at 4°C overnight. The next day, the coating solution was discarded, and the ELISA plate was blocked with 5% skim milk in PBS and incubated at room temperature for 1 hour. Serum samples from each experimental group of mice in Example 2 were diluted fourfold starting from 1:20 in a 96-well plate. The dilutions were then transferred to the ELISA plate, and the plate was incubated at 37°C for 1.5 hours. The blocking solution was discarded, and the plate was washed four times with PBST. Then, Goat Anti-Mouse HRP secondary antibody (purchased from Jinpulai) was added at a dilution of 1:2000 in blocking solution, incubated at 37°C for 1.5 hours, and washed five times with PBST. Then, 60 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added for color development. After 10 minutes, 60 μL of 2 M hydrochloric acid was added to terminate the reaction, and the OD was measured on a microplate reader (PerkinElmer). 450 The endpoint titer is defined as the serum dilution factor at which the absorbance produced by the serum is 2.5 times greater than the background value. Antibody titers below the detection limit are defined as half of the detection limit.

[0085] The binding antibody titers against the ZIKV-E protein antigen in the sera of each immunized mouse are shown in Figures 3A (primary immune sera) and 3B (secondary immune sera).

[0086] As can be seen from Figures 3A and 3B, after the first immunization, mRNA vaccines with different doses and different immunogens (M / E-WT, M / E-mutB, M / E-mutC) all produced binding antibody titers comparable to those of the adenovirus vector vaccine AdC7-M / E-mutB. Except for the binding antibody titer at the 10μg immunization dose of M / E-mutB, which was significantly higher than that of the AdC7-M / E-mutB immunization group, there was no significant difference overall. However, after the second immunization, the binding antibody titers of mRNA vaccines with different immunogens were significantly higher than those of the AdC7-M / E-mutB immunization group, and showed dose-dependent characteristics. These results suggest that mRNA vaccines can induce higher levels of antibody titers after booster immunization, indicating that they have better immune protection efficacy.

[0087] Then, the neutralizing antibody titer in the serum of the immune mice was detected by microneutralization test. The specific method was as follows: VERO cells were plated on 96-well plates one day in advance. The next day, when the cell confluence reached about 90%, the immune mouse serum was serially diluted in DMEM medium (Invitrogen, C11995500BT) containing 1% FBS (Gibco, 10270-106) in 96-well plates (specifically, starting from a 10-fold dilution, the serum was serially diluted in 3-fold steps to obtain 10 dilutions in total). The virus was also diluted to 6×10 3 FFU / mL; then, 85 μL of each diluted immune mouse serum and 85 μL of the virus dilution were mixed in a round-bottom 96-well plate and incubated at 37°C for 2 hours. The supernatant culture medium was removed from the VERO cell plate at approximately 90% confluency, and the serum and virus mixture prepared above was added. After 2 hours of incubation, DMEM containing 10% FBS was added. The cells were cultured in a 37°C incubator for 4 days. After 4 days, the cell culture plate was removed, all supernatant was discarded, and the cells were washed once with PBS. The cells were fixed with 150 μL of methanol and placed in a -20°C refrigerator for 18 minutes, followed by two washes with PBS. Blocking was performed using 3% skim milk in PBS and allowed to stand at room temperature for 30 minutes. The primary antibody (Z6 antibody binding to the ZIKV E protein, as described in CN 106589116B, diluted to a working concentration of 5 μg / mL in blocking solution) was then added and incubated at 37°C for 1.5 hours. Afterwards, discard the primary antibody, wash three times with PBST, and then add the secondary antibody (the secondary antibody is a goat anti-human antibody conjugated to HRP (Proteintech, SA00001-17), diluted 1500 times with blocking solution) and incubate at room temperature for 2 hours. Afterwards, discard the secondary antibody and wash four times with PBST. Add 60μl TMB colorimetric solution (Biyuntian, P0209) and incubate at room temperature for about 20 minutes. Observe the color change. When the PBS well changes from colorless to faint light blue, add 60μl 2M hydrochloric acid to terminate the reaction and read the OD450 absorbance value on the microplate reader. Use GraphPad Prism software to perform nonlinear fitting on the OD450 data to calculate the corresponding serum dilution factor when neutralizing 50% of the cell infection, which is the neutralizing antibody titer value (MN50). When the serum with the lowest dilution factor still cannot neutralize 50% of the cell infection, the MN50 of the sample is defined as half of the lowest dilution factor.

[0088] The results of microneutralization experiments with the sera of each immunized mouse are shown in Figures 3C (primary immunization sera) and 3D (secondary immunization sera). As can be seen in Figures 3C and 3D, neutralizing antibodies were almost undetectable in the immune sera of the negative control group (denoted as Sham in the figures). After the primary immunization, neutralizing antibodies were detected in both the mRNA vaccine group and the AdC7-M / E-mutB vaccine group. Statistical analysis showed no significant differences between the vaccine groups. After the booster immunization, the neutralizing antibody level in the mRNA vaccine group was significantly higher than that in the AdC7-M / E-mutB vaccine group, demonstrating a higher level of protective immunity.

[0089] Example 4: Evaluation of mRNA vaccine-induced cellular immunity

[0090] In this example, spleen samples from mice in each experimental group collected on day 42 in Example 2 were used to detect the cellular immunity level induced by the mRNA vaccine. The specific method is as follows:

[0091] 1) Mouse spleen sample processing

[0092] Mouse spleen cells were prepared into single-cell homogenates using a cell homogenizer in serum-free 1640 medium containing 1% double-antibody. The cells were filtered through a 40 μm cell strainer and lysed with erythrocyte lysis buffer (Beijing Solebold Technology Co., Ltd., R1010). The cells were then washed with PBS + 0.5% FBS, stained with AO / PI dye, and counted using a Cell Drop FL automated cell counter.

[0093] In addition, the mouse spleen cells were resuspended in 1640 medium containing 1% double antibody and 10% fetal bovine serum to prepare a single cell suspension (the concentration of the prepared cell suspension was 1×10 7 cells / mL) for subsequent experiments.

[0094] 2) ELISpot test

[0095] The day before the experiment, the PVDF membrane of the ELISpot plate was pre-wetted with 35% ethanol, and 30 μL of 35% ethanol was added to each well to treat the membrane to make it hydrophilic. Wash with deionized water, 200 μL / well, wash 5 times, and remove the ethanol; dilute the anti-mouse IFN-γ and anti-mouse IL-2 antibodies (both purchased from BD) with PBS to 15 μg / mL, and use the diluted antibodies to coat the ELISpot plate, 100 μL / well, and incubate overnight at 4°C. The next day, pour off the coating solution and wash twice with 1640 medium containing 10% FBS. Add 200 μL of 1640 medium containing 10% FBS to each well and incubate at room temperature for 1 hour. The above freshly prepared mouse spleen single cell suspension was plated at 5×105 Antibody-precoated cells were added to 1 / well of a 96-well plate and stimulated for 24 hours with a peptide library constructed from the wild-type Zika virus E protein. Key parameters for peptide library design included short peptides of 18-20 amino acids in length and overlapping amino acid fragments of approximately 10 amino acids. The designed peptide library was synthesized by Zhongke Yaguang Biotechnology Co., Ltd. Positive control wells were stimulated with phytohemagglutinin (PMA) to generate a nonspecific cellular immune response, while negative control wells were not stimulated with the peptide library. The cells were then discarded and the 96-well plate was incubated sequentially with a biotinylated IFN-γ antibody, a streptavidin-ALP antibody, and a chromogenic substrate, with five washes in between. Once spots appeared on the bottom of the plate, the samples were thoroughly rinsed with deionized water to stop color development. Finally, images were taken and the number of spots counted using Immuno Capture 6.5.0.

[0096] The results are shown in Figure 4A; Figure 4A shows that after stimulation with the Zika E protein peptide library, IFN-γ produced by spleen cells of mice immunized with 10 μg doses of M / E-WT and M / E-mutB mRNA vaccines + The number of cells was significantly higher than that of AdC7-M / E-mutB, while that of AdC7-M / E-mutB was significantly higher than that of sham group; the IL-2 produced by spleen cells of mice immunized with 2μg and 10μg doses of M / E-WT, M / E-mutB and M / E-mutC mRNA vaccines was significantly higher than that of AdC7-M / E-mutB group. + The number of cells was higher than that of AdC7-M / E-mutB, but there was no significant difference. These results indicate that M / E-WT, M / E-mutB, and M / E-mutC mRNA vaccines can effectively stimulate cellular immune responses.

[0097] 2)ICS inspection

[0098] The cell suspension obtained in step 1) of this example was plated in a round-bottom 96-well plate, with 200 μL of cell suspension in each well containing 2×10 6 Splenocytes were isolated from the culture medium. Furthermore, a Zika virus E protein peptide library (comprising 53 peptides, whose sequences are shown in SEQ ID NOs: 12-64) was diluted with 1640 medium containing 10% FBS to a working concentration of 10 μg / mL for each peptide in the library. Twenty μL of the diluted E protein peptide library solution was added to the wells containing the cells, along with 1 μL of a Golgi blocker (available from BD Biosciences, Cat. No. 51-2301kz) per well. After 12 hours, the cells were transferred to a V-bottom 96-well plate and centrifuged at 2000 rpm for 5 minutes at room temperature. The supernatant was discarded, and the cells were washed once with PBS (200 μL PBS / well) for later use.

[0099] Then, anti-mouse CD16 / CD32 antibody (Bio X Cell, Cat. No. BE0307) was diluted in PBS containing 2% FBS, and 100 μL of the diluted antibody was added to each well of the cells. The cells were incubated on ice in the dark for 5 minutes, and then centrifuged at 2000 rpm for 5 minutes. The supernatant was discarded and the cells were directly stained. 1 μL each of anti-CD3, anti-CD4, and anti-CD8 antibodies (all purchased from Neta Scientific) was added to 47 μL of PBS containing 2% FBS (a total of 50 μL), mixed, and the cells were resuspended in this mixture and incubated on ice in the dark for 20 min. After washing the cells with PBS containing 2% FBS, 100 μL of FP solution (Invitrogen eBioscience, catalog number: 88-8824-00) was added to each well to permeabilize the cells and incubated on ice in the dark for 20 min. The blank control group was not stained but permeabilized. The permeabilized cells were washed once with 1x wash buffer and then subjected to intracellular staining. Specifically, 1 μL each of anti-IL-2, anti-IL-4, anti-IFN-γ, and anti-TNF-α antibodies (all purchased from CiteAb) was added to 46 μL of 1x wash buffer. Buffer (total 50 μL), mix thoroughly, resuspend the cells in this mixture, and incubate on ice in the dark for 20 minutes. Permeabilize and stain the PMA-treated group (positive control). Resuspend each well of cells in 200 μL of stain buffer, transfer to a flow cytometer, and measure the proportion of positive cells in the sample using flow cytometry. The results are shown in Figure 4B.

[0100] The results in Figure 4B show that the CD4+ T cells in the mRNA vaccine group secreted IFN-γ, IL-2, and TNF-α cytokines. + T cells and CD8 + The proportion of T cells increased significantly compared with the negative control group, and the mRNA vaccine stimulated more cytokine secretion than the AdC7-M / E-mutB adenovirus vector vaccine; while the CD4 + T cells and CD8 + The proportion of T cells did not differ significantly between the immunization groups. Therefore, these data suggest that the mRNA vaccine induced the production of Th1-biased cytokines.

[0101] Example 5: Detection of ZIKV mRNA vaccine in Ifnar1 - / - Immunogenicity in mice

[0102] Since ZIKV infection in BALB / c mice does not cause death or obvious disease symptoms, in order to better verify the effect of the vaccine, we selected immunodeficient mice Ifnar1- / - Mice were used as a ZIKV infection model (Lazear, Helen M et al. (2016), vol. 19, 5: 720-30.) to evaluate the mRNA vaccine-induced Ifnar1 - / - Humoral immunity levels in mice.

[0103] Specifically, Ifnar1 - / - Mice were randomly divided into 4 groups. Three groups were vaccinated with M / E-WT, M / E-mutB, or M / E-mutC mRNA vaccines by intramuscular injection at a dose of 10 μg. One group of mice was immunized with PBS as a negative control (i.e., sham group). The immunization procedure was the same as that described in Example 2. That is, immunization was performed on days 0 and 28, and blood was collected on days 21 and 42 to separate serum. Ifnar1 was detected by ELISA. - / - Binding antibody titers in mouse sera, using a microneutralization assay to detect Ifnar1 - / - The titer levels of neutralizing ZIKV antibodies in mouse sera are shown in FIG5 .

[0104] Figures 5A and 5B show the binding antibody titers induced by the three mRNA vaccines after primary immunization and booster immunization, respectively. The results show that the average Log endpoint titer after primary immunization is between 2 and 3, and the average Log endpoint titer after booster immunization is above 5.

[0105] Figures 5C and 5D show the titer levels of neutralizing ZIKV antibodies induced by the three mRNA vaccines after the primary immunization and booster immunization, respectively. The results show that after the primary immunization, the three mRNA vaccines did not produce significant neutralizing antibody responses. However, after the secondary immunization, the neutralizing antibody level increased significantly, but M / E-mutC was lower than M / E-WT and M / E-mutB.

[0106] Example 6: Detection of mRNA vaccine in Ifnar1 - / - Protection against virus infection after immunization of mice

[0107] Ifnar1 in Example 5 - / - Mice were challenged with ZIKV-PRVABC59 virus 30 days after the second immunization.

[0108] Specifically, 3×10 5 / mouse FFU ZIKV-PRVABC59 virus, and then observed the survival status of the mice and monitored the changes in weight every day. The experimental results are shown in Figure 6, where Figure 6A shows the weight change curve after ZIKV-PRVABC59 virus challenge, and Figure 6B shows the mortality results after ZIKV-PRVABC59 virus challenge.

[0109] Figures 6A and 6B show that the weight of mice in the sham group began to gradually decrease on the second day after challenge and all died on the sixth day; however, the three groups of mice immunized with the M / E-WT, M / E-MutB, and M / E-MutC mRNA vaccines did not lose weight after challenge, and no mice in the three groups died during the entire experiment. These results indicate that the M / E-MutB and M / E-MutC mRNA vaccines can provide complete protection in mice challenged with the virus, just like the wild-type M / E-WT vaccine.

[0110] In order to detect whether the mRNA vaccines M / E-MutB and M / E-MutC vaccines can protect mice against viremia caused by viral infection, in this experiment, mouse blood was collected on the 3rd and 6th days after infection, the serum was separated, and then RNA was extracted using the MagaBio Plus Viral RNA Kit (Bori Technology, BSC58S1B). The viral RNA was quantified using the FastKing One-Step Reverse Transcription-Fluorescence Quantification Kit (Tiangen Biochemical Technology, FP314). The probe and primer sequences used for quantification are shown in Table 4, and the quantitative results are shown in Figure 7.

[0111] Table 4. Probe and primer sequences used for RT-PCR quantification of ZIKV-PRVABC59 nucleic acid

[0112] As shown in Figure 7, higher viral loads were detected in the sham group mice on the 3rd and 6th days after infection, while no virus was detected in the serum of mice immunized with the mRNA vaccines M / E-WT, M / E-MutB and M / E-MutC on the 3rd and 6th days after infection. This shows that the M / E-MutB and M / E-MutC mRNA vaccines can protect mice against viremia as well as the wild-type M / E-WT vaccine.

[0113] Example 7: Detection of ZIKV mRNA vaccine in Ifnar1 - / - Clearing immune effects in mice

[0114] In this example, experiments were further designed to detect whether M / E-MutB and M / E-MutC mRNA vaccines can provide clearing immunity. The specific steps are as follows.

[0115] Ifnar1 - / - Mice were randomly divided into 4 groups. Among them, 3 groups were immunized with 3 mRNA vaccines M / E-WT, M / E-MutB and M / E-MutC by intramuscular injection at a dose of 10 μg. One group of mice was immunized with GFP as a negative control (i.e., Sham group). Blood was collected and serum was separated on the 28th day after immunization. On the 30th day after immunization, 3x10 5 Mice were challenged with ZIKV-PRVABC59 at a concentration of 5FU / mouse. Four days after ZIKV-PRVABC59 challenge, mice were dissected and portions of the eyes, brain, spinal cord, kidneys, liver, spleen, testes, and ovaries were removed. The dissected tissues and organs were added to PBS and ground using a grinder (Tiangen Biotechnology, OSE-Y30). The supernatant was centrifuged and RNA was extracted using the MagaBio Plus Viral RNA Kit. RT-PCR was then performed for ZIKV RNA (primers and probes used are the same as in Table 4 above). The results are shown in Figure 8.

[0116] As shown in Figure 8, a certain amount of virus was detected in 7 organs of the Sham group mice, while no virus was detected in any tissues and organs of the three groups of mice immunized with M / E-WT, M / E-MutB and M / E-MutC mRNA vaccines.

[0117] In addition, a microneutralization experiment was performed to detect the neutralizing antibody titers in the serum 2 days before and 6 days after the infection. The results are shown in Figure 9.

[0118] As can be seen from Figure 9, no neutralizing antibodies were detected in the Sham group before challenge, but high titers of neutralizing antibodies could be detected in each mouse in the Sham group on the 6th day after challenge; while the three groups immunized with M / E-WT, M / E-MutB and M / E-MutC mRNA vaccines had no significant differences in serum neutralizing antibody titers before and after challenge, and maintained at almost the same level, which shows that the wild-type mRNA vaccine M / E-WT and the FL-engineered mRNA vaccines M / E-MutB and M / E-MutC vaccines can all achieve complete clearance immunity.

[0119] Example 8: In vitro testing of the ADE of DENV by serum from BALB / c mice immunized with ZIKV mRNA vaccine

[0120] Due to the sequence similarity between ZIKV and DENV, we further designed experiments to demonstrate whether M / E-MutB and M / E-MutC mRNA vaccines can reduce ADE to DENV. The specific steps are as follows:

[0121] The serum of BALB / c mice immunized in Example 2 was serially diluted. Specifically, starting from an 8-fold dilution, the serum was serially diluted in a 4-fold gradient to obtain a total of 8 dilutions. Then, 10 μL of serum at different dilutions was mixed with 30 μL of DENV1 (titer of 1×10 6 FFU / mL), DENV2 (titer of 1×10 5 FFU / mL), DENV3 (titer of 3×10 5 FFU / mL) and DENV4 virus solution (titer of 3×10 5 FFU / mL) were mixed and incubated, and then 3×10 4 After culturing for 4 days, K562 cells were stained with FITC-labeled Z6 antibody (Z6 antibody as described above), and the proportion of positive cells was detected by flow cytometry. The results are shown in Figure 10. Note: DENV virus cannot infect K562 cells without antibody mediation.

[0122] As can be seen from Figure 10, the serum of the Sham group does not contain antibodies that can bind to DENV, so the infection rate of the tested samples is at the background level; the mouse serum of the M / E-WT group can mediate the infection of K562 cells with four serotypes of DENV viruses at a certain concentration; and the infection rates of the samples in the M / E-MutB and M / E-MutC groups are significantly lower than that in the M / E-WT group, showing that the ADE of DENV is weakened or even eliminated; these results indicate that the M / E-MutB and M / E-MutC vaccines have obvious advantages in reducing ADE.

[0123] Example 9: In vivo testing of the ADE of DENV by serum from BALB / c mice immunized with ZIKV mRNA vaccine

[0124] To further demonstrate whether the M / E-MutB vaccine can reduce ADE of DENV under physiological conditions, we used Ifnα / βr - / - Ifnγr - / - Mice (i.e., AG6 mice, available from the Shanghai Pasteur Institute of the Chinese Academy of Sciences) were used to verify the ADE effect of the M / E-MutB mRNA vaccine against DENV.

[0125] First, 60 BALB / c mice were randomly divided into three groups, with 20 mice in each group, and immunized with GFP mRNA (as a negative control group, i.e., Sham group), M / E-WT, and M / E-MutB mRNA vaccines, respectively. The immunization dose was 10 μg / mouse. The immunization and blood collection procedures were the same as in Example 2. After blood collection, the serum was separated and heated at 56°C for 30 minutes. The serum of 20 mice in each group was mixed together, aliquoted, and frozen at -80°C for later use.

[0126] AG6 mice were randomly divided into three groups and passively immunized with the mixed serum described above. Serum from BALB / c mice immunized with M / E-WT, M / E-MutB, or GFP mRNA was diluted 1:40 with PBS, and each mouse received 200 μL of the diluted serum. Twenty-four hours later, 10,000 FFU of DENV2-NGC virus were injected subcutaneously. Each mouse was weighed and recorded before injection. The mice were observed daily for status, survival, and weight. The results are shown in Figure 11.

[0127] As shown in Figure 11, compared with the mice in the Sham group, the mice in the M / E-WT group died earlier than those in the Sham group, indicating that the serum of mice immunized with the M / E-WT vaccine enhanced DENV infection. The time and trend of death of mice in the M / E-MutB group were consistent with those in the Sham group. This suggests that the mutation of the ZIKV E protein weakened the production of antibodies that can cause ADE effects against DENV, making the performance of mice in the M / E-MutB group similar to that of the Sham group. The trend of weight changes also showed that compared with the Sham group, the M / E-WT group lost weight faster, while the weight change trend of mice in the M / E-MutB group was consistent with that of the Sham group.

[0128] These in vivo experimental results well demonstrate that the M / E-MutB vaccine can significantly reduce the ADE of DENV after immunization with the wild-type vaccine.

[0129] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the present application. Industrial Applicability

[0130] The mRNA vaccine against Zika virus provided in the present application can not only avoid the induction of FL epitope antibodies, thereby reducing or eliminating the ADE effect, but also produce high levels of neutralizing antibodies higher than other vaccine vector platforms, thereby effectively preventing or treating Zika virus infectious diseases; therefore, the mRNA vaccine against Zika virus provided in the present application has good clinical application value and industrialization prospects.

Claims

1. An mRNA encoding a Zika virus antigen peptide, characterized in that: The mRNA has an mRNA sequence as shown in SEQ ID NO:1 or SEQ ID NO:2, or is transcribed from a DNA as shown in SEQ ID NO:3 or SEQ ID NO:

4.

2. An mRNA construct encoding a Zika virus antigen peptide, characterized in that: The mRNA construct comprises the mRNA according to claim 1, and one or more elements selected from the following: a 5' UTR region, a 3' UTR region, a 5' cap structure, a 3' Poly A tail and a signal peptide coding sequence.

3. The mRNA construct according to claim 2, characterized in that The 5' UTR region has a sequence as shown in SEQ ID NO: 5; And / or, the 3' UTR region has a sequence as shown in SEQ ID NO: 6; and / or, the 5' end cap structure is m7G(5')ppp; and / or, the 3' terminal PolyA tail is 80 to 200 A; And / or, the signal peptide coding sequence is shown in SEQ ID NO:

7.

4. A recombinant expression plasmid for producing the mRNA according to claim 1, characterized in that: The recombinant expression plasmid comprises a plasmid backbone sequence and a DNA sequence encoding a Zika virus antigen peptide.

5. The recombinant expression plasmid according to claim 4, characterized in that: The DNA sequence encoding the Zika virus antigen peptide is shown in SEQ ID NO: 3 or SEQ ID NO: 4; And / or, the plasmid backbone sequence includes a T7 promoter sequence, a 5' UTR region, a signal peptide sequence, a 3' UTR region, a 5' cap structure and a 3' PolyA tail; Preferably, the 5' UTR region has a sequence as shown in SEQ ID NO: 5; Preferably, the 3' UTR region has a sequence as shown in SEQ ID NO: 6; Preferably, the 5' end cap structure is m7G(5')ppp; Preferably, the 3' terminal PolyA tail is 80 to 200 A; Preferably, the signal peptide sequence is shown as SEQ ID NO:

7.

6. Use of the mRNA according to claim 1, the mRNA construct according to claim 2 or 3, or the recombinant expression plasmid according to claim 4 or 5 in the preparation of a vaccine, preferably an mRNA vaccine, for preventing and / or treating Zika virus infectious diseases.

7. An mRNA vaccine against Zika virus, comprising the mRNA as claimed in claim 1 or the mRNA construct as claimed in claim 2 or 3, and a lipid component.

8. The mRNA vaccine according to claim 7, characterized in that The mass ratio of the mRNA according to claim 1 or the mRNA construct according to claim 2 or 3 to the lipid component is 1:(10-30), preferably 1:20; Preferably, the lipid component comprises cationic lipids, phosphatidylcholine, cholesterol and PEG lipids, or consists of cationic lipids, phosphatidylcholine, cholesterol and PEG lipids. composition; Further preferably, in the lipid component, the molar ratio of cationic lipid, phosphatidylcholine, cholesterol and PEG lipid is (40-60):(5-15):(30-45):(1-2), preferably (45-55):(8-12):(35-40):(1-2), and more preferably 50:10:38.5:1.

5.

9. The mRNA vaccine according to claim 7 or 8, characterized in that The mRNA vaccine is in the form of a nasal spray, an oral formulation, a suppository, or a parenteral formulation; Preferably, the nasal spray is selected from aerosols, sprays and powder sprays; Preferably, the oral preparation is selected from tablets, powders, pills, powders, granules, fine granules, soft / hard capsules, film-coated capsules, pellets, sublingual tablets and ointments; Preferably, the parenteral preparation is a transdermal preparation, an ointment, a plaster, an external liquid, an injectable or a pushable preparation.

10. The method for preparing an mRNA vaccine according to any one of claims 7 to 9, characterized in that: The preparation method comprises: (1) dissolving the mRNA or mRNA construct in an aqueous solution as the aqueous phase; (2) dissolving the lipid component in an organic solvent as an organic phase; (3) mixing the aqueous phase and the organic phase to self-assemble to obtain the mRNA vaccine; Preferably, the aqueous solution is a sodium acetate buffer; Preferably, the organic solvent is anhydrous ethanol; Preferably, the self-assembly is performed by nanoliposome packaging.

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