PIV3 virus f protein, mRNA vaccine and application thereof

Abstract

This invention discloses a PIV3 virus F protein, an mRNA vaccine, and their applications. The amino acid sequence of the PIV3 virus F protein is shown in SEQ ID NO:5 or 6. The mRNA vaccine provided by this invention can promote the production of anti-PIV3 virus antibodies, effectively control PIV3 virus infection and its amplification in the lungs, and can be used for the prevention and / or treatment of diseases caused by PIV3 virus infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a PIV3 virus F protein, an mRNA vaccine, and their applications. Background Technology

[0002] Intracellular delivery technologies based on biological macromolecules such as nucleic acids have received widespread attention in the field of innovative drug development in recent years. Among them, novel vaccines based on mRNA technology have seen rapid advancements in research, clinical trials, and market approval in response to the urgent need for vaccines against COVID-19, demonstrating the clinical efficacy, safety, and commercial value of the mRNA technology platform to the world.

[0003] Compared to traditional drugs that directly use proteins or peptides as active pharmaceutical ingredients, mRNA technology offers several advantages: 1) Higher bioactivity and safety: The complex protein modification processes involved in mRNA-based drugs, such as protein expression, glycosylation, terminal modification, and demethylation, are all completed naturally within the body's own cells. Whether considering the molecular structure of the raw materials, the synthetic environment, or the synthesized product, it is highly homologous to the human body. Therefore, compared to traditional protein preparations using in vitro bacterial or cell systems, endogenous protein products based on mRNA sequences have more accurate specificity, higher affinity, better safety, and more promising bioactivity. 2) Longer biological half-life: Existing experimental results confirm that proteins generated via mRNA, including antigens or antibodies, especially those using self-replicating nucleic acids or circular nucleic acids, can maintain effective blood concentrations for a longer period, thereby reducing the frequency and dosage of medication, decreasing drug side effects, and improving patient compliance. 3) Expanded Application Scenarios: The design and synthesis of mRNA itself offer high diversity and flexibility. Its components can be assembled like Lego bricks according to pharmacological needs, achieving diverse and better therapeutic effects. Furthermore, through the development of delivery-based dosage forms and diversified administration methods, mRNA formulations can reach more or more targeted clinical targets, increasing the local concentration and efficacy of the active pharmaceutical ingredient. This enriches the scope of new drug development and expands the choices of treatment options for patients in clinical practice. 4) Rapid Response: The technologies used in the product development process of mRNA vaccines are mostly basic molecular biology methods, making technology transfer relatively easy. A mature mRNA platform, from gene sequencing and design against different pathogens to production, only requires a few weeks. This allows for rapid response in time-sensitive epidemics, enabling vaccines to play a vital role in a timely manner.

[0004] Human parainfluenza viruses (hPIV), also known as human respiroviruses in recent years, belong to the Paramyxoviridae family and were first discovered in the late 1950s. Based on genetic and antigenic differences, they can be divided into four serotypes: hPIV 1-4. hPIV 2 and hPIV 4 are members of the Rubellavirus genus, while hPIV 1 and hPIV 3 are classified as members of the Respiratory Virus genus. Clinically, PIV3 is the most common (52%), followed by PIV 1 (26%), PIV 2 (12%), and PIV 4 (4%).

[0005] PIV3 is most susceptible to infants and children, causing 6.8% of hospitalizations for acute respiratory illness or fever, making it the second leading cause of respiratory hospitalizations in children under 5 years old, after respiratory syncytial virus (RSV). Infection can occur at any age; the elderly and those with chronic or weakened immune systems are particularly at risk; recurrent infections are extremely common. The virus is transmitted through coughing and sneezing, as well as through respiratory droplets produced during close contact with an infected person. Peak infection rates occur annually from March to June. In countries and regions with relatively underdeveloped healthcare, PIV3-related illnesses can be measured by mortality rates; in developing and developed countries with better economic and healthcare conditions, the economic cost of infection is estimable. It is estimated that treatment costs in the United States are around $200 million annually.

[0006] The severity of PIV3 infection varies from person to person, largely depending on age, medical history, and immune status. Clinically, it causes severe acute respiratory illness, leading to conditions such as bronchiolitis, pneumonia, and croup. Symptoms may include fever, cough, runny nose, and difficulty breathing. Infections typically begin in the upper respiratory tract and can spread downwards, causing pneumonia and secondary infections. Eustachian tube obstruction can lead to otitis media, a common sequela of PIV3 infection in children. In most healthy individuals, PIV3 results in self-limiting rhinitis.

[0007] PIV3 is an antisense, single-stranded enveloped RNA virus with a particle size of approximately 150-200 nm and a genome containing about 15,000 nucleotides. The gene sequence is arranged as follows: 3'NP-PMF-HN-L 5', encoding six viral proteins. The viral RNA is completely encapsulated by the nucleocapsid protein NP, forming an active viral polymerase complex; this is combined with the large protein L and the phosphoprotein P to form the nucleocapsid complex; the outermost layer is a lipid envelope lined with a matrix protein M, which anchors the fusion protein F and the tail of the hemagglutinin neuraminidase HN to the membrane. Although the HN protein is involved in viral entry into cells and budding and is considered a major viral surface antigen, the HN gene exhibits high antigenic and genetic variability, making it unsuitable for use as a vaccine antigen. In contrast, the fusion protein F is crucial for facilitating the fusion of the host cell membrane and the viral membrane, possesses strong antigenicity, and is relatively stable among strains, ultimately becoming a popular antigen gene.

[0008] The F protein of PIV3 belongs to class I fusion proteins. Initially, it is synthesized in an inactive form, F0, which is then cleaved through proteolysis into active forms F1 and F2, linked by disulfide bonds. The three F1-F2 monomers then non-covalently construct a metastable conformation of the F protein fusion precursor, pre-F. Upon recognition by the virus and cell receptor, the fusion precursor refolds, and this structural change draws the viral and cell membranes closer together, leading to fusion. At this point, the F protein also transforms into a more stable post-fusion conformation, post-F.

[0009] Since the discovery of PIV3, research on vaccines against this virus, while receiving the same widespread attention as that for RSV, has never ceased. Attempts have been made inactivated vaccines, live attenuated vaccines, viral vector vaccines, subunit vaccines, and virus-like particle vaccines, among others, but no vaccine or specific drug has yet been developed. In recent years, with the rise and involvement of structural molecular biology, breakthroughs have been made in understanding the working principles of vaccines. Taking RSV as an example, by utilizing point mutations in the F protein to introduce proline in regions with α-helical tendency, introducing disulfide bonds, cavity filling to replace buried polar residues, and C-terminal heterotrimerization technology, a more stable and more realistic pre-F antigen protein has been created, significantly improving the vaccine's induction effect on the human immune system. Currently, there are no reports of incorporating the same design concept into PIV3, and the results are unknown. Summary of the Invention

[0010] To address the aforementioned technical problems, this invention provides a PIV3 virus F protein, mRNA vaccine, and its application. It is an innovative work on developing a PIV3 vaccine using mRNA formulation technology, and designs, synthesizes, and validates a unique anti-PIV3 mRNA / LNP vaccine.

[0011] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0012] The first aspect of the present invention provides a PIV3 virus F protein, the amino acid sequence of which is shown in SEQ ID NO:5 or 6.

[0013] A second aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the PIV3 viral F protein as described in the first aspect of the present invention.

[0014] In some embodiments of the present invention, the nucleic acid molecule is double-stranded DNA.

[0015] In some embodiments of the present invention, the nucleotide sequence encoding the PIV3 viral F protein comprises any of the sequences shown in SEQ ID NO: 1-4.

[0016] In some embodiments of the present invention, the nucleic acid molecule further comprises one or more selected from the following:

[0017] (1) T7 promoter; the preferred nucleotide sequence of the T7 promoter is shown in SEQ ID NO:17;

[0018] (2) 5'UTR; the 5'UTR is preferably the 5'UTR of human cytochrome B-245a polypeptide, whose nucleotide sequence is shown, for example, as shown in SEQ ID NO:15;

[0019] (3) 3'UTR; the 3'UTR is preferably the 3'UTR of human AES / TLE5, whose nucleotide sequence is shown for example in SEQ ID NO:16;

[0020] (4) poly A; the nucleotide sequence of the poly A preferably has spacers, the nucleotide sequence of which is, for example, as shown in SEQ ID NO:19 (GCATATGACT); more preferably, the nucleotide sequence of the poly A is shown in SEQ ID NO:18;

[0021] (5) Kozak sequence; the Kozak sequence is preferably GCCGCCACC.

[0022] In some embodiments of the present invention, the nucleic acid molecule comprises, from the 5' end to the 3' end, a 5' UTR, a Kozak sequence, a nucleotide sequence encoding the PIV3 viral F protein, and a 3' UTR.

[0023] In some embodiments of the present invention, the 5' end of the nucleic acid molecule is connected to the T7 promoter, and the 3' end is connected to the poly A; preferably, the T7 promoter is connected to the 5'UTR via AGG or GGG.

[0024] A third aspect of the present invention provides a nucleic acid construct containing nucleic acid molecules as described in the second aspect of the present invention.

[0025] In some embodiments of the present invention, the nucleic acid molecule is constructed into the nucleic acid construct using, for example, restriction endonucleases selected from HindIII and EcoRI.

[0026] A fourth aspect of the present invention provides a recombinant expression vector containing a nucleic acid molecule as described in the second aspect of the present invention or a nucleic acid construct as described in the third aspect of the present invention.

[0027] The fifth aspect of the present invention provides a transformant comprising a nucleic acid molecule as described in the second aspect of the present invention, a nucleic acid construct as described in the third aspect of the present invention, or a recombinant expression vector as described in the fourth aspect of the present invention.

[0028] In some embodiments of the present invention, the host cells used in the construction of the transformant are selected from Escherichia coli, insect cells, yeast cells, and mammalian cells, such as Escherichia coli Stable cells.

[0029] The sixth aspect of the present invention provides an mRNA comprising an mRNA encoding the PIV3 viral F protein as described in the first aspect of the present invention.

[0030] In some embodiments of the present invention, the mRNA is mRNA obtained by transcription of nucleic acid molecules as described in the second aspect of the present invention.

[0031] In some embodiments of the present invention, the sequence of the mRNA encoding the PIV3 viral F protein comprises any of the sequences shown in SEQ ID NO: 7-10.

[0032] In some embodiments of the present invention, the sequence of the mRNA is shown in any of SEQ ID NO:11-14, preferably as shown in SEQ ID NO:11 or 14.

[0033] In some embodiments of the present invention, the 5' end of the mRNA has a cap structure; the cap structure is preferably a Cap1 structure.

[0034] The seventh aspect of the present invention provides a method for preparing nucleic acid molecules or mRNA, the method comprising culturing a transformant as described in the fifth aspect of the present invention to obtain the nucleic acid molecule or mRNA.

[0035] The eighth aspect of the present invention provides a drug-loaded lipid nanoparticle comprising mRNA as described in the sixth aspect of the present invention.

[0036] In some embodiments of the present invention, the lipid nanoparticles further include cationic lipids and auxiliary lipids.

[0037] In some embodiments of the present invention, the cationic lipid is SM-102;

[0038] And / or, the auxiliary lipid is DSPC, cholesterol and / or DMG-PEG-2000.

[0039] In some embodiments of the present invention, the molar ratio of the cationic lipid, DSPC, cholesterol and DMG-PEG-2000 is (40-60):(2-15):(30-40):(0.8-1.6), for example 50:10:38.5:1.5.

[0040] The ninth aspect of the present invention provides an mRNA vaccine against PIV3 virus, the mRNA vaccine comprising mRNA as described in the sixth aspect of the present invention and / or lipid nanoparticles as described in the eighth aspect of the present invention.

[0041] In some embodiments of the present invention, the mRNA vaccine is a virus-like particle; and / or, the mRNA vaccine further includes an adjuvant.

[0042] The tenth aspect of the present invention provides a pharmaceutical composition comprising mRNA as described in the sixth aspect of the present invention, lipid nanoparticles as described in the eighth aspect of the present invention, and / or an mRNA vaccine as described in the ninth aspect of the present invention, and optionally a pharmaceutical carrier.

[0043] The eleventh aspect of the present invention provides the use of the PIV3 virus F protein as described in the first aspect of the present invention, the nucleic acid molecule as described in the second aspect of the present invention, the nucleic acid construct as described in the third aspect of the present invention, the recombinant expression vector as described in the fourth aspect of the present invention, the transformant as described in the fifth aspect of the present invention, the mRNA as described in the sixth aspect of the present invention, the lipid nanoparticle as described in the eighth aspect of the present invention, the mRNA vaccine as described in the ninth aspect of the present invention, and / or the pharmaceutical composition as described in the tenth aspect of the present invention in the preparation of medicaments for the prevention and / or treatment of diseases caused by PIV3 virus and / or the inhibition of PIV3 virus.

[0044] In some embodiments of the present invention, the diseases caused by the PIV3 virus are acute respiratory diseases, such as bronchiolitis, pneumonia, croup, or otitis media.

[0045] The twelfth aspect of the present invention provides a method for inhibiting PIV3 virus, the method comprising contacting a sample infected with PIV3 virus with mRNA as described in the sixth aspect of the present invention, lipid nanoparticles as described in the eighth aspect of the present invention, an mRNA vaccine as described in the ninth aspect of the present invention, and / or a pharmaceutical composition as described in the tenth aspect of the present invention to inhibit the PIV3 virus.

[0046] In some embodiments of the present invention, the method is for non-therapeutic purposes.

[0047] The thirteenth aspect of the present invention provides a method for preventing and / or treating diseases caused by PIV3 virus, the method comprising administering to a patient in need an effective dose of mRNA as described in the sixth aspect of the present invention, lipid nanoparticles as described in the eighth aspect of the present invention, an mRNA vaccine as described in the ninth aspect of the present invention, and / or a pharmaceutical composition as described in the tenth aspect of the present invention.

[0048] In some embodiments of the present invention, the diseases caused by the PIV3 virus are acute respiratory diseases, such as bronchiolitis, pneumonia, croup, or otitis media.

[0049] In some embodiments of the present invention, the single-dose administration of the lipid nanoparticles may be 1-200 μg, 20-60 μg, 60-110 μg, 110-160 μg, or 160-200 μg, for example 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, or doses between these values; it may also be adjusted according to the subject's weight.

[0050] In some embodiments of the present invention, the application is performed at least once, for example once, twice, three times or four times, which can be adjusted according to the actual situation.

[0051] In some embodiments of the present invention, the application frequency is at least two weeks apart, preferably four to eight weeks, such as four, five, six or seven weeks, which can be adjusted according to the actual situation.

[0052] The fourteenth aspect of the present invention provides the PIV3 virus F protein as described in the first aspect of the present invention, the nucleic acid molecule as described in the second aspect of the present invention, the nucleic acid construct as described in the third aspect of the present invention, the recombinant expression vector as described in the fourth aspect of the present invention, the transformant as described in the fifth aspect of the present invention, the mRNA as described in the sixth aspect of the present invention, the liposome nanoparticles as described in the eighth aspect of the present invention, the mRNA vaccine as described in the ninth aspect of the present invention, and / or the pharmaceutical composition as described in the tenth aspect of the present invention, for the prevention and / or treatment of diseases caused by PIV3 virus and / or the inhibition of PIV3 virus.

[0053] In some embodiments of the present invention, the diseases caused by the PIV3 virus are acute respiratory diseases, such as bronchiolitis, pneumonia, croup, or otitis media.

[0054] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0055] The reagents and raw materials used in this invention are all commercially available.

[0056] The positive and progressive effects of this invention are as follows:

[0057] The mRNA vaccine provided by this invention can promote the production of anti-PIV3 virus antibodies, effectively control PIV3 virus infection and its amplification in the lungs, and is used to prevent and / or treat diseases caused by PIV3 virus infection. Attached Figure Description

[0058] Figure 1 The secondary structures of JY-PIV3 F mRNA sequences JY-PIV3-068 and JY-PIV3-071 are shown.

[0059] Figure 2A-2B The full sequence of plasmid JY-PIV3-068 is shown; among which Figure 2A This is a plasmid map; Figure 2B The full sequence of plasmid DNA is displayed.

[0060] Figure 3 The restriction endonuclease digestion diagram of the JY-PIV3 F series vaccine plasmid is shown.

[0061] Figure 4 The gel assay demonstrates the in vitro synthesis of mRNA induced by T7 RNA polymerase in the JY-PIV3-F series vaccines.

[0062] Figure 5 The results show the intracellular protein expression of JY-PIV3-F mRNA / LNP nanoparticles after transfection into 293T cells by Western blotting.

[0063] Figures 6A-6D The results showed that the JY-PIV3-F series mRNA / LNP vaccines induced strong specific binding antibodies in mice. Among them, Figure 6A and Figure 6B The data shown is the serum antibody level 13 days after the second immunization; Figure 6C and Figure 6D The display shows the serum antibody levels 10 days after the three vaccinations.

[0064] Figure 7A and Figure 7BThe comparative effects of the JY-PIV3-F mRNA / LNP vaccine on the immunization dose-response in mice were shown. Among them, Figure 7A Compare the specific binding antibodies after different doses of the first, second, and third immunizations; Figure 7B Compare the specific binding antibodies of the same dose after each immunization.

[0065] Figure 8 The study demonstrated that the specific antibodies induced by the JY-PIV3-F mRNA / LNP vaccine in mice are persistent.

[0066] Figures 9A-9C The study demonstrated that the JY-PIV3-F mRNA / LNP vaccine induced specific antiviral antibodies in mice, and these antibodies possessed the ability to neutralize the virus; among them, Figure 9A This is a flowchart of a mouse experiment; Figure 9B The serum antibody levels for each group were displayed; Figure 9C This demonstrates that the antibody has the ability to neutralize the virus.

[0067] Figures 10A-10D The study demonstrated that the JY-PIV3-F mRNA / LNP vaccine induced specific binding antibodies in golden hamsters, and these antibodies possessed the ability to neutralize the virus. Among these, Figure 10A The pre-infection, immunization, and challenge procedures are shown; Figure 10B The levels of F protein-specific binding antibodies were displayed for all test groups / blood samples;

[0068] Figure 10C This indicates the level of antibodies specifically binding to the F protein after pre-infection or secondary immunization;

[0069] Figure 10D The study showed the level of F protein-specific binding antibodies after pre-infection plus a booster shot or three immunizations.

[0070] Figure 11 The study showed that the JY-PIV3-F mRNA / LNP vaccine can reduce PIV3 infection in the lungs of golden hamsters. Detailed Implementation

[0071] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0072] Example 1: Screening, modification, and mRNA sequence optimization of viral strains

[0073] 1.1 Screening of initial virus strains

[0074] Since the discovery of hPIV3, the National Center for Biotechnology Information (NCBI) in the United States has collected nearly 3,000 hPIV3-related sequences, many of which encode the F protein. Overall, although it is an antisense single-stranded RNA virus, the F gene sequence has not changed significantly over the decades; the appearance of a few point mutations still indicates the progress of viral evolution. After analyzing several hundred representative F protein sequences, based on the viral transmission trajectory, we designed two unique and comprehensive F protein sequences, the JY-Universal PIV3-F sequences (Table 1). Version 1 of the design emphasizes universality, closely resembling recently prevalent strains, and serves as the starting point for the design of JY-062, JY-063, and JY-064. A universal immunogen design method is crucial for preventing viral pandemics. Version 2 is closer to the PIV3 strain VR-1782 (ATCC CCL-7.1), which was isolated in 2011 from a clinical sample of a PIV3-positive patient in Virginia and subsequently included in the American Type Culture Collection (ATCC) virus seed bank. It served as the design starting point for JY-068, JY-069, JY-070, and JY-071. The starting sequences for JY-065, JY-066, and JY-067 were derived from a strain clinically isolated in Australia in 2007.

[0075] Table 1 JY-PIV3-F series mRNA / LNP design numbers

[0076]

[0077]

[0078] Where: try 1 and try 2 represent different nucleic acid coding sequences corresponding to the same protein; the structural state of proteins not specified in the table above is unknown.

[0079] 1.2 Sequence Optimization:

[0080] mRNA sequence design is one of the most crucial and critical issues in the entire mRNA pharmaceutical process, and it is inextricably linked to the drug-likeness of mRNA. It affects the yield of mRNA in vitro transcription, the immunogenicity of mRNA molecules, translation efficiency, and the stability of mRNA molecules. Currently, commonly used mRNAs are composed of five elements: 5' Cap, 5' UTR (non-coding region), ORF (open reading frame for protein coding), 3' UTR, and 3' poly A tail (polyadenylated tail).

[0081] The 5' Cap structure protects mRNA from degradation by the exonuclease Xrn1 and the nuclear Xrn2 enzyme. Capped mRNA has a longer half-life and higher stability than uncapped mRNA, significantly increasing the yield of intracellular protein synthesis. The chemical nature of the cap structure is a special structure located at the 5' end of mRNA, the m7GPPPN structure, also known as the methylguanosine cap, formed during mRNA transcription modification. It is formed under the combined catalysis of RNA triphosphatase, guanylate transferase, mRNA (guanine-N7) methyltransferase, and mRNA (nucleoside-2') methyltransferase. Based on the degree of methylation modification, cap structures can be divided into three types: Cap 0, Cap 1, and Cap 2. Since uncapped mRNA or the Cap 0 structure is recognized by the innate immune receptor RIG-1, while Cap 1 and Cap 2 protect it from recognition by innate immune sensors, our design uses the commonly used Cap 1 structure.

[0082] The T7 promoter sequence can be divided into two domains: a binding domain and a transcription initiation domain. For the transcription initiation domain, any base substitution will severely affect the promoter strength. The design of this invention is TAATACGACTCACTATA (SEQ ID NO:17), followed by agg to facilitate co-transcriptional capping. Kozak is GCCGCCACC.

[0083] The regulation of eukaryotic mRNA translation initiation is mainly determined by the 5'UTR characteristics, including secondary structure, sequence elements, and 5'UTR length, all of which affect mRNA stability. Our design utilizes the UTR of the human cytochrome B-245a polypeptide (CYBA), which has relatively high protein expression (its nucleotide sequence is shown in SEQ ID NO:15). Of course, UTR expression also depends on cell type.

[0084] Recent findings in structural molecular biology have highlighted the crucial importance of stabilizing the metastable pre-fusion three-dimensional structure of membrane proteins like viral F proteins through point mutations. For the PIV3-F protein, pre-F metastable state stabilization is achieved through the following steps: 1) Removing the C-terminal transmembrane segment (TM) and intracellular segment at site 481 of the F protein, and fusing a trimerized domain fused with GCN4, thus allowing the F protein to present as a more stable trimer; 2) Mutating sites 172 and 238, I172C and N238C, introducing two non-natural disulfide bonds; 3) Filling the cavity with amino acids A463V and I474Y.

[0085] For the PIV3-F DNA sequence, selecting appropriate codons can optimize the overall translation efficiency of mRNA. One approach is to align the viral sequence with the host cell's, meaning replacing viral codons with frequently used synonymous codons from human cells. This ensures a more consistent codon usage bias and avoids an excessive number of rare codons. However, it's also important to retain a small number of viral codons to slow ribosome movement, providing sufficient time for proper protein folding and thus more accurately reconstructing the viral antigen protein structure. Another key consideration is GC content; a slightly higher GC content is believed to increase mRNA stability and improve protein expression levels in vivo. Finally, the secondary structure of the mRNA sequence is also being considered. Hairpin structures in secondary structures have been reported to reduce mRNA degradation and contribute to mRNA stability.

[0086] Similar to the 5'UTR, the 3'UTR also contains many regulatory elements that play an important role in mRNA stability, subcellular localization, and translation efficiency. The 3'UTR we used was derived from human AES / TLE5 (its nucleotide sequence is shown in SEQ ID NO:16).

[0087] The poly-A tail plays an indispensable role in protein translation. It binds to poly-A tail-binding protein (PAPB), which in turn interacts with the translation initiation factor eIF4G to form a closed-loop structure. This recruits the 40S translation initiation complex to mRNA and works synergistically with the 5' cap structure to stimulate translation initiation. During bacterial amplification, plasmids carrying long poly-A nucleotide sequences can experience unpredictable recombination events. The poly-A tail on the plasmid shortens as bacteria amplify, causing difficulties for plasmid cloning and amplification. Studies have shown that separating the poly-A sequences can significantly reduce recombination events during plasmid DNA amplification, maintain tail length, and not affect the translation efficiency and half-life of the mRNA transcribed in vitro. Following this principle, the poly-A of this invention is a 40-spacer-80 base sequence, with the nucleotide sequence AAA ...

[0088] The optimized PIV3-F series coding sequence is as follows:

[0089] 1. JY-068

[0090]

[0091]

[0092]

[0093] Protein: MPTSILLIITTMIMASHCQIDITKLQHVGVLVNSPKGMKISQNFETRYLILSLIPKIEDSNSCGDQQIKQYKRLLDRLIIPLYDGLRLQKDVIVTNQESNENTDPRTERFFGGVIGTIALGVATSAQITAAVALVEAKQARSDIEKLKEAIRDTNKAVQSVQSSIGNLIVAIKSVQDYVNKEIVPSITRLGCEAAGLQLGIALTQHYSELTNIFGDNIGSLQEKGIKLQGIASLYRTNITEIFTTSTVDKYDIYDLLFTESIKVRVIDVDLNDYSITLQVRLPLLTRLLNTQIYKVDSISYNIQNREWYIPLPSHIMTKGAFLGGADVKECIEAFSSYICPSDPGFVLNHEMESCLSGNISQCPRTTVTSDIVPRYAFVNGGVVANCITTTCTCNGIGNRINQPPDQGVKIITHKECNTIGINGMLFNTNKEGTLAFYTPDDITLNNSVALDPIDISIELNKAKSDLEESKEWIRRSNQKLDSISAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO:5)

[0094] 2. JY-069

[0095]

[0096]

[0097]

[0098] Protein: (SEQ ID NO:5)

[0099] 3. JY-070

[0100]

[0101]

[0102]

[0103] Protein: MPTSILLIITTMIMASHCQIDITKLQHVGVLVNSPKGMKISQNFETRYLILSLIPKIEDSNSCGDQQIKQYKRLLDRLIIPLYDGLRLQKDVIVTNQESNENTDPRTERFFGGVIGTIALGVATSAQITAAVALVEAKQARSDIEKLKEAIRDTNKAVQSVQSSIGNLIVACKSVQDYVNKEIVPSITRLGCEAAGLQLGIALTQHYSELTNIFGDNIGSLQEKGIKLQGIASLYRTCITEIFTTSTVDKYDIYDLLFTESIKVRVIDVDLNDYSITLQVRLPLLTRLLNTQIYKVDSISYNIQNREWYIPLPSHIMTKGAFLGGADVKECIEAFSSYICPSDPGFVLNHEMESCLSGNISQCPRTTVTSDIVPRYAFVNGGVVANCITTTCTCNGIGNRINQPPDQGVKIITHKECNTIGINGMLFNTNKEGTLAFYTPDDITLNNSVALDPIDISIELNKVKSDLEESKEWYRRSNQKLDSISAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL(SEQ ID NO:6)

[0104] 4. JY-071

[0105]

[0106]

[0107]

[0108] Protein: (SEQ ID NO:6)

[0109] Figure 1 The secondary structures of JY-PIV3-F mRNA sequences JY-PIV3-068 and JY-PIV3-071 were shown. Among all 10 designs, JY-PIV3-068 (SEQ ID NO:11) and JY-PIV3-071 (SEQ ID NO:14) stood out, exhibiting better biological activity.

[0110] Example 2: Plasmid synthesis and DNA extraction of JY-PIV3-F series mRNA vaccines

[0111] All designed sequences were cloned into the vector using HindIII and SapI. Figure 2A-2B The image shows a plasmid constructed using JY-PIV3-068 as an example, along with the complete DNA sequence (SEQ ID NO:23).

[0112] Component Name Plasmid start and end positions (bp) T7 starter 1639-1655 5'UTR 1659-1705 Kozak 1706-1714 PIV3-F 1715-3259 3'UTR 3275-3552 poly A 3557-3687

[0113] 2.1 Plasmid Synthesis

[0114] After the plasmid sequence was designed, it was synthesized by CRO.

[0115] 2.2 Plasmid DNA Extraction

[0116] 2.2.1 Reagent Preparation

[0117] Plate and liquid LB medium containing peptone, sodium chloride, yeast extract and corresponding antibiotics.

[0118] 2.2.2 Plasmid Transformation

[0119] Plasmid DNA was added to Stable competent cells and incubated on an ice bath for 30 min; then heat-shocked in a 42°C water bath for 45 seconds, immediately placed on an ice bath for 2 min, and 500 μL of antibiotic-free LB medium was added. The cells were then incubated on a shaker at 37°C for 1 h, followed by plating and overnight incubation at 37°C. Clones were picked from the plates the next day.

[0120] 2.2.3 Plasmid DNA Extraction

[0121] DNA was extracted from the bacterial culture overnight using either mini-extraction or large-extraction kits.

[0122] 2.3 Plasmid DNA Restriction Validation

[0123] Verification was performed using restriction endonucleases to digest SapI, HindIII, and EcoRI. Figure 3) Qualified plasmids proceed to the next step of in vitro transcription experiments.

[0124] Example 3: In vitro transcription capping of the JY-PIV3-F series

[0125] 3.1 Plasmid DNA Linearization

[0126] This design linearizes the plasmid by adding a reversed SapI receptor at the 3' end of the polyA plasmid. After incubation at 37°C for 3 hours, SDS-PAGE gel electrophoresis was used to determine the completeness of enzyme digestion. Purification was performed using 3M sodium acetate precipitation; subsequently, the DNA concentration was determined using the Nanodrop method.

[0127] 3.2 In vitro transcription capping and purification

[0128] The design of the JY-PIV3-F series plasmids, except for JY-062, JY-063 and JY-064 which use ggg between the T7 polymerase binding site and the 5'UTR and employ a two-step method for in vitro transcription and capping, all other plasmids use agg and are suitable for co-transcription.

[0129] 3.2.1 Two-step in vitro transcription and capping

[0130] 3.2.1.1 In vitro transcription

[0131] Add the components in order according to Table 2, mix gently, and react at 37°C for 3 hours.

[0132] Table 2 Components of the in vitro transcription system

[0133] Components Volume (μL) DEPC water Complete the system volume 10× transcription buffer solution 50.0 Template DNA 25μg ATP 37.5 N1-Methyl-pseuuridine 37.5 CTP 37.5 GTP 37.5 T7 polymerase 25.0 pyrophosphatase 5.0 RNAse inhibitors 5.0 total 500.0

[0134] After the above reaction is completed, the DNA template needs to be removed enzymatically. Add 5 μL of DNase I to every 100 μL and react at 37°C for 30 min. Use an agarose gel to determine whether the DNA template has been completely digested.

[0135] 3.2.1.2 mRNA purification

[0136] Add 150 μL of lithium chloride solution (7.5 M lithium chloride, 50 mM EDTA) and 150 μL of LEPC water per 100 μL volume, maintaining the final lithium chloride concentration at 2.5-2.8 M. After mixing, incubate at -20°C for at least 30 minutes, or overnight.

[0137] Remove the sample from -20℃ and centrifuge rapidly at 12000 rpm for 15 min. Discard the supernatant and collect the precipitate. Add 1 ml of 70% water-ethanol and wash, centrifuge at 12000 rpm for 5 min, and discard the supernatant. Repeat once. Then, leave the sample in a clean bench at room temperature for 5 min with the lid open to allow the ethanol to evaporate.

[0138] Finally, after reconstitution with an appropriate amount of DEPC water, agarose gel electrophoresis was used to determine the quality of the mRNA and to measure its concentration.

[0139] 3.2.1.3 Adding a cap

[0140] After heating 250 μg of mRNA solution at 65 °C for 10 minutes to open the secondary structure, immediately place it on ice to cool for 5 minutes. Then add the other components in order according to Table 3.

[0141] Table 3 Components of the Capped System

[0142] Components Volume (μL) DEPC water Complete the system volume 10× Capped Buffer Solution 50.0 mRNA 250μg SAM (2mM) 25.0 GTP 25.0 RNAse inhibitors 5.0 2-O-methyltransferase 25.0 Smallpox Cap Enzyme 25.0 total 500.0

[0143] After gently mixing, incubate at 37°C for 1 hour. Determine mRNA quality by agarose gel electrophoresis.

[0144] 3.2.1.4 Capped mRNA Purification

[0145] Add 150 μL of lithium chloride solution (7.5 M lithium chloride, 50 mM EDTA) and 150 μL of LEPC water per 100.0 μL volume (the final concentration of lithium chloride should be maintained at 2.5-2.8 M); mix well and place at -20℃ for at least 30 min, or overnight.

[0146] Remove from -20℃, centrifuge at 12000 rpm for 15 min, discard the supernatant, and collect the precipitate. Add 1 ml of 70% water-ethanol to wash, centrifuge at 12000 rpm for 5 min, and discard the supernatant; repeat once. Then, open the lid in a clean bench and let it stand at room temperature for 5 min to allow the ethanol to evaporate.

[0147] The mRNA was reconstituted with an appropriate amount of DEPC water, and the mRNA quality was determined by agarose gel electrophoresis, and the concentration was measured.

[0148] 3.2.2 Co-transcription and Capping

[0149] 3.2.2.1 Co-transcription and Capping

[0150] Add the components in the order shown in Table 4, and react at 37°C for 3 hours.

[0151] Table 4 Components of the co-transcription and capping system

[0152] Components Volume (μL) DEPC water Complete the system volume 10× transcription buffer 50.0 Template DNA 25μg ATP 37.5 N1-Methyl-pseuuridine 37.5 CTP 37.5 GTP 37.5 Hat-like objects 37.5 T7 enzyme 25.0 pyrophosphatase 5.0 RNAse inhibitors 5.0 total 500.0

[0153] After the reaction was complete, 5 μL of DNase I was added to every 100 μL to digest the template, and the reaction was carried out at 37°C for 30 min. Agarose gel was used to determine whether the DNA template had been completely digested.

[0154] 3.2.2.2 Capped mRNA Purification

[0155] Add 150 μL of lithium chloride solution (7.5 M lithium chloride, 50 mM EDTA) and 150 μL of LEPC water per 100.0 μL volume. The final concentration of lithium chloride should be maintained at 2.5-2.8 M. After mixing, place at -20°C for at least 30 min or overnight.

[0156] Remove the sample from -20℃ and centrifuge rapidly at 12000 rpm for 15 min. Discard the supernatant and collect the precipitate. Then add 1 ml of 70% aqueous ethanol and wash, centrifuge at 12000 rpm for 5 min and discard the supernatant; repeat once. Open the lid in a clean bench and let it stand at room temperature for 5 min to allow the ethanol to evaporate.

[0157] Reconstitute the mRNA with an appropriate amount of DEPC water, and perform agarose gel electrophoresis (e.g.) Figure 4 To determine the quality and concentration of mRNA.

[0158] Example 4: Preparation of JY-PIV3-F series mRNA / LNP nanoparticles

[0159] This study uses the industry-standard method for preparing LNP nanoparticles.

[0160] LNPs were prepared by dissolving ionizable lipid (SM-102):DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine):cholesterol:DMG-PEG-2000 in ethanol at a molar ratio of 50:10:38.5:1.5. This solution was then mixed with mRNA dissolved in pH 4 acetate buffer in a high-impact nanoparticle preparation apparatus. Subsequent dilution and pH increases resulted in the formation of lipid nanoparticles (mRNA / LNP) encapsulated within the mRNA. The sample was concentrated and purified by ultrafiltration and centrifugation. The final product was analyzed for particle size, polydispersity index (PDI), zeta potential (ZP), encapsulation efficiency, and effective mRNA concentration.

[0161] 4.1 Buffer Preparation

[0162] All reagents were autoclaved at 121°C for 20 minutes and prepared using DEPC water.

[0163] 1) 50mM acetate buffer, pH=4.0;

[0164] 2) 25mM Tris-HCl buffer, pH=7.5;

[0165] 3) 0.6 g / mL sucrose solution.

[0166] 4.2 Preparation of lipid solution and mRNA solution

[0167] Table 5 Preparation of lipid solutions

[0168]

[0169] 1) Lipids need to be brought to room temperature before weighing;

[0170] 2) Weigh all the ingredients according to Table 6 and dissolve them in anhydrous ethanol. To ensure complete dissolution, use an ultrasonic instrument.

[0171] 3) Mix the prepared lipid solutions in a ratio of 1:1:1:1 to obtain a 12 mg / mL lipid mixture solution that conforms to the formula.

[0172] Table 6 Preparation of mRNA solution

[0173]

[0174]

[0175] The in vitro transcribed and capped mRNA stock solution prepared in Example 3 was diluted to the required concentration of 0.2 mg / mL according to the calculation method and buffer type shown in Table 6 above.

[0176] 4.3 Preparation of mRNA / LNP (taking a total volume of 4 mL as an example)

[0177] 4.3.1 INano LTM Rapid Nanomedicine Preparation System. Set parameters as needed.

[0178] 4.3.2 Clean the chip before use

[0179] Prepare three 5mL syringes: one with anhydrous ethanol, one with acetate buffer, and one with air. Repeat the process of adding 5mL acetate buffer followed by air three times on the left side, and 5mL ethanol followed by air three times on the right side, manually cleaning the chip. After cleaning, insert the chip into the slot.

[0180] 4.3.3 Preparation of LNPs using microfluidic equipment

[0181] Draw 3 mL of the prepared mRNA solution into a 3 mL syringe, removing any air bubbles, and insert it into the syringe slot on the left side of the machine. Draw 1 mL of the prepared lipid mixture into a 1 mL syringe, removing any air bubbles, and insert it into the syringe slot on the right side of the machine. Insert a 15 mL centrifuge tube into the waste collection port and a sample collection port for waste and sample collection, respectively. After confirming that the parameters are correct, click "Start." The INano LTM rapid nanomedicine preparation system will immediately begin preparing LNPs. After preparation, remove the centrifuge tube from the sample collection port.

[0182] 4.3.4 Processing of mRNA / LNP samples

[0183] The collected mRNA / LNP suspension was immediately diluted with 25 mM Tris-HCl pH 7.5 buffer to approximately 25 times the volume of the mRNA / LNP, followed by ultrafiltration and centrifugation at 3000 rpm, 4°C, for 1 h. This process was repeated until the solution volume was less than or equal to the original volume (3.6 mL, i.e., a total volume of 4 mL minus 0.4 mL of waste liquid). Then, the solution was sterilized by passing it through a 0.22 μm filter membrane, and 0.2 times the volume of the solution (0.6 g / mL sucrose solution) was added to obtain the final mRNA / LNP formulation product.

[0184] 4.3.5 Detection of physical properties of mRNA / LNP

[0185] The prepared mRNA / LNP should be subjected to physical property testing as soon as possible to determine the quality of the product and facilitate further bioactivity studies. The Malvern Zetasizer was used for testing, and particle size, PDI, and ZP data were obtained (Table 4).

[0186] Encapsulation efficiency analysis:

[0187] The mRNA control (100 μg / mL) was diluted from 2000 ng / mL to seven standard curve points (2000, 1600, 1200, 800, 400, 200 ng / mL) using 1×TE Buffer and 2% Triton TE Buffer, respectively. When measuring total RNA content, the LNP sample was diluted 50-fold using 2% Triton TE Buffer; when measuring free RNA content, the sample was diluted 20-fold using 1×TE Buffer. 100 μL of each of the standard curve solution and sample solution were transferred to a black ELISA plate, and then 100 μL of 0.005×Ribogreen (200-fold dilution) was added. The plates were incubated at room temperature in the dark for 2-5 min. The fluorescence response values ​​were read using the ELISA plate (excitation wavelength 480 nm, emission wavelength 520 nm), and the encapsulation efficiency was calculated using the following formula:

[0188] Encapsulation efficiency EE (%) = [(C 总RNA -C 游离RNA ) / C 总RNA *100%

[0189] The effective concentration and encapsulation efficiency of the sample mRNA obtained by the above methods are shown in Table 7.

[0190] Table 7 Physical properties of JY-PIV3-F series mRNA / LNP lipid particles

[0191] sample Particle size PDI Content (mg / mL) Encapsulation rate % JY-062 83.19 0.075 0.203 96.6 JY-063 83.8 0.068 0.202 96.7 JY-064 83.66 0.057 0.178 98 JY-065 85.95 0.07 0.187 95.5 JY-066 83.42 0.056 0.124 95.9 JY-067 82.82 0.071 0.158 96.3 JY-068 87.38 0.052 0.235 96.2 JY-069 74.94 0.074 0.103 96.9 JY-070 78.71 0.128 0.142 96 JY-071 75.93 0.121 0.149 96.8

[0192] Example 5: JY-PIV3-F series mRNA / LNP nanoparticles transfected into 293T cells for expression.

[0193] To determine whether the mRNA in lipid nanoparticles can express the correct protein, the first step is to transfect the specific mRNA / LNP into suitable cells, and then use methods such as ELISA, Western Blot, and flow cytometry to detect the correctness of protein expression.

[0194] 5.1 Cell transfection

[0195] The day before transfection, 293T (CRL-3216, ATCC) cells were loaded at a rate of 8 x 10⁻⁶. 5 The cells were seeded into 12-well plates with complete culture medium consisting of DMEM supplemented with 10% fetal bovine serum and antibiotics. The culture conditions were 37°C and 5% CO2. The next day, the cell culture medium in each well was aspirated, and the cells were gently rinsed with PBS. 1 ml of opti-MEM medium was added to each well, followed by the addition of 3 μg of RNA / well mRNA / LNP nanoparticles. The nanoparticles were gently dispersed and the plates were incubated at 37°C and 5% CO2 for 72 hours.

[0196] 5.2 Western Blot (WB) method for detecting antigen protein expression

[0197] Western blotting (WB) can be used to qualitatively detect the translation of mRNA / LNP into the designed target antigen protein after entering the cell. Since the designed antigen protein contains a signal peptide, the extracellular protein is measured in this study.

[0198] 5.2.1 Electrophoresis Sample Preparation

[0199] Collect the cell supernatant after 72 hours of transfection, centrifuge at 2500 rpm for 5 min at 4°C, and transfer the supernatant to a new 1.5 mL tube. If the expression level is too low, a suitable Millipore ultrafiltration tube can be used for preliminary concentration. Protein content determination is then performed, with each sample adjusted to the same loading volume, and 6×SDS loading buffer added to a final concentration of 1×. Before loading, heat the sample at 92°C for 5 min to denature the protein.

[0200] 5.2.2 SDS-PAGE electrophoresis

[0201] Electrophoresis was performed using a 10% separating gel and a 4% stacking gel. Samples were loaded immediately after heating. During electrophoresis, the voltage for the stacking gel section was 110V, and the voltage for the separating gel section was 150V.

[0202] 5.2.3 Transfer of film

[0203] Depending on the molecular weight, either nitrocellulose or PVDF membranes are selected. Generally, transfer is performed at 80V for 1 hour or 60V for 2 hours, with ice packs used for heat control. After transfer, the membrane is stained with 1× Ponceau S for 5 minutes to assess the transfer efficiency.

[0204] 5.2.4 Blocking and Immune Response

[0205] First, block the membrane with 5% skim milk powder and incubate overnight at 4°C using a shaker. The next day, wash three times with PBST solution, then add an appropriate amount of diluted primary antibody (mouse anti-PIV3-F) for hybridization, and incubate at room temperature with a shaker for 90 min. Then wash three times with PBST solution, 5–10 minutes each time. Use anti-mouse HRP as the secondary antibody, and incubate at room temperature with a shaker for 60 min. Finally, wash three times with PBST solution, 5–10 minutes each time.

[0206] 5.2.5 Development and Exposure

[0207] Following the ECL kit instructions, mix solution A and solution B at a 1:1 ratio and place them in the membrane. Incubate in a dark room, gently shake to mix, and then expose and detect using a chemiluminescence imaging analyzer. The accuracy of the target protein size is determined by the marker strips.

[0208] The results are as follows Figure 5 The results showed that after transfection of 293T cells with the JY-PIV3-F series, the mRNA / LNP was endocytosed into the cells, released into the cytoplasm in the acidic environment of lysosomes, and then translated and modified in the endoplasmic reticulum and Golgi apparatus before being released extracellularly guided by a signal peptide. The encoded PIV3-F antigen protein could bind to the F antibody, and the protein specificity and molecular weight met the design requirements. Notably, designs with the same or similar protein sequences showed similar F protein expression patterns. JY-062, JY-070, and JY-071 exhibited high protein expression and stability.

[0209] Example 6: Immunization of mice with JY-PIV3-F series mRNA / LNP vaccine and identification of virus-specific antibody expression.

[0210] To detect the expression and performance of the vaccine antigen in vivo, we used mice as an animal model.

[0211] 6.1 Animal Immunization

[0212] Balb / c mice, female, 6 weeks old, were allowed to acclimatize to the animal facility for one week after arrival. The vaccine was the JY-PIV3-F series mRNA / LNP vaccine prepared in Example 4. The vaccine was administered intramuscularly at a dose of 20 μg per mouse, divided into three injections 14 days apart. Blood samples were collected one day before the first immunization, 13 days after the second immunization, and 10 days after the third immunization. Serum samples were used for subsequent antibody ELISA detection.

[0213] 6.2 ELISA detection of anti-PIV3 antibodies

[0214] One of the key indicators of a vaccine is the induction of specific antiviral antibodies in animals. We used an ELISA method to detect the expression level of anti-PIV3-F antibodies.

[0215] 6.2.1 Antigen Coating

[0216] Cell supernatant containing PIV3-F protein antigen was diluted appropriately with PBS, and 100 μL / well was used to coat 96-well microplates with antigen. The mixture was incubated at 4°C for 16 h.

[0217] 6.2.2 Washing and Sealing

[0218] Remove the antigen-coated ELISA plate and wash it once with 300 μL / well PBST. Then add 100 μL of blocking buffer (5% skim milk powder) and incubate at room temperature for 30 min. Next, wash the plate three times with 300 μL / well PBST.

[0219] 6.2.3 Sample dilution and loading

[0220] Serum samples from immunized mice were serially diluted 2-fold, and 100 μL was added to each well of an ELISA plate in triplicate. The plates were incubated at room temperature for 1.5 h, and then washed three times with 300 μL / well PBST.

[0221] 6.2.4 Add detection antibodies

[0222] Depending on the number of wells to be sampled, take an appropriate amount of matching detection antibody (goat anti-mouse IgG, α-peroxidase conjugate, H+L), dilute to 0.8 ng / mL, add 80 μL to each well, incubate at room temperature for 1 h, and then wash the plate 5 times with 300 μL / well PBST.

[0223] 6.2.5 Color Development and Termination

[0224] Mix colorimetric solution A (containing H2O2) and colorimetric solution B (containing TMB) in a 1:1 ratio. Add 100 μL to each well and let it stand at room temperature for 10-15 minutes. Then add 30 μL of 1M HCl stop solution to each well and read the value at 450 nm using a microplate reader.

[0225] from Figures 6A-6DThe results show that, except for JY-PIV3-65, all of the JY-PIV3-F series mRNA / LNP vaccines can induce strong virus-specific binding antibodies in mice. JY-PIV3-070 and JY-PIV3-071 showed particularly strong performance, indicating that the stability and immunogenicity of the F protein resulting from the introduction of point mutations (I172C, N238C, A463V, and I474Y) are superior to the unmutated wild type. We also noted that there was a difference in efficacy between two and three doses of immunization at a dosage of 20 μg / mouse, with three doses showing better results.

[0226] Example 7: Dosage Exploration of JY-PIV3-F mRNA / LNP Vaccine

[0227] As a vaccine, dosage is a key factor to consider. We first investigated the dose-effect of the JY-PIV3-071 mRNA / LNP vaccine.

[0228] 7.1 Animal Immunization

[0229] Balb / c mice, female, 6 weeks old, were placed in the animal facility for a week to acclimatize. The vaccine was an mRNA / LNP formulation, administered intramuscularly in three doses. The first and second doses were given 21 days apart, and the second and third doses were given 14 days apart. Blood was collected for the final dose on day 7 after the third dose. The dosages were 5, 10, 20, and 30 μg / mouse.

[0230] 7.2 ELISA detection of anti-PIV3 antibodies

[0231] 7.2.1 Antigen Coating

[0232] Cell supernatant containing PIV3-F protein antigen was diluted appropriately with PBS, and 100 μL / well was used to coat 96-well microplates with antigen. The mixture was incubated at 4°C for 16 h.

[0233] 7.2.2 Washing and Sealing

[0234] Remove the antigen-coated ELISA plate and wash it once with 300 μL / well PBST. Then add 100 μL of blocking buffer (5% skim milk powder) and incubate at room temperature for 30 min. Next, wash the plate three times with 300 μL / well PBST.

[0235] 7.2.3 Sample dilution and loading

[0236] Serum samples from immunized mice were serially diluted 4-fold, and 100 μL was added to each well of an ELISA plate in triplicate. The plates were incubated at room temperature for 1.5 h, and then washed three times with 300 μL / well PBST.

[0237] 7.2.4 Add detection antibodies

[0238] Depending on the number of wells to be sampled, take an appropriate amount of matching detection antibody (goat anti-mouse IgG, peroxidase conjugated, H+L), dilute to 0.8 ng / mL, add 80 μL to each well, incubate at room temperature for 1 h, and then wash the plate 5 times with 300 μL / well PBST.

[0239] 7.2.5 Color Development and Termination

[0240] Mix colorimetric solution A (containing H2O2) and colorimetric solution B (containing TMB) in a 1:1 ratio. Add 100 μL to each well and let it stand at room temperature for 15-30 minutes. Then add 30 μL of 1M HCl stop solution to each well and read the value at 450 nm using a microplate reader.

[0241] Dosage exploration results as follows Figure 7A and Figure 7B The results show that mRNA / LNP vaccines, represented by JY-071, can induce high titers of virus-specific antibodies in animals. The differences between doses of 10μg / animal, 20μg / animal, and 30μg / animal are present but limited, especially after the second immunization. Although the low dose of 5μg / animal showed good antibody induction after the second immunization, it is still recommended to use a dose of 10μg / animal or higher.

[0242] Example 8: Exploration of the persistence of antibodies induced by JY-PIV3-F mRNA / LNP vaccine

[0243] As a vaccine, the duration of antibody retention in the body is an important consideration, as it relates to the number of immunizations required during a viral infection season. Here, we examine the JY-PIV3-068 mRNA / LNP vaccine as an example.

[0244] 8.1 Animal Immunization

[0245] Balb / c mice, female, 6 weeks old, were placed in the animal facility for a week to acclimatize. The vaccine was JY-PIV3-068mRNA / LNP, administered intramuscularly in three doses of 20 μg per mouse, 14 days apart. Blood samples were collected on day 14 and day 90 after the three doses.

[0246] 8.2 ELISA detection of anti-RSV antibodies

[0247] 8.2.1 Antigen Coating

[0248] Cell supernatant containing PIV3-F protein antigen was diluted appropriately with PBS, and 100 μL / well was used to coat 96-well microplates with antigen. The mixture was incubated at 4°C for 16 h.

[0249] 8.2.2 Washing and Sealing

[0250] Remove the antigen-coated ELISA plate and wash it once with 300 μL / well PBST. Then add 100 μL of blocking buffer (5% milk powder) and incubate at room temperature for 30 min. Finally, wash the plate three times with 300 μL / well PBST.

[0251] 8.2.3 Sample dilution and loading

[0252] Serum samples from immunized mice were serially diluted 4-fold, and 100 μL was added to each well of an ELISA plate in triplicate. The plates were incubated at room temperature for 1.5 h, and then washed three times with 300 μL / well PBST.

[0253] 8.2.4 Add detection antibodies

[0254] Depending on the number of wells to be sampled, take an appropriate amount of matching detection antibody (goat anti-mouse IgG, peroxidase conjugated, H+L), dilute to 0.8 ng / mL, add 80 μL to each well, incubate at room temperature for 1 h, and then wash the plate 5 times with 300 μL / well PBST.

[0255] 8.2.5 Color Development and Termination

[0256] Mix colorimetric solution A (containing H2O2) and colorimetric solution B (containing TMB) in a 1:1 ratio. Add 100 μL to each well and let it stand at room temperature for 15-30 minutes. Then add 30 μL of 1M HCl stop solution to each well and read the value at 450 nm using a microplate reader.

[0257] Results of viral antibody persistence exploration, such as Figure 8 The results showed that mRNA / LNP vaccines, represented by JY-068, can induce high titers of virus-specific antibodies in animals, and the antibody titers remained basically at the original level on day 90 after three immunizations, without significant decline.

[0258] Example 9: Detection of the antibody's neutralizing ability against the virus

[0259] One of the gold standard indicators for vaccine testing is determining whether the antibodies induced in animals have the ability to neutralize the virus and prevent infection. The virus neutralization test is designed for this purpose. Because PIV3 is a biosafety level 2 virus, all subsequent steps involving the virus must be completed in a biosafety level 2 laboratory according to the relevant requirements and standards.

[0260] 9.1 Animal Immunization

[0261] Balb / c mice, female, 6 weeks old, were placed in the animal facility for one week to acclimatize. The vaccine was prepared as described in Example 4. Two representative mRNA / LNP vaccines were selected: Group 1 was JY-PIV3-068, Group 2 was JY-PIV3-071, Group 3 mice were first infected with PIV3 virus (3.5 × 10^5 / mouse), and then given JY-PIV3-068 four weeks later. Group 4 was a PBS control. The dosage was 20 μg / mouse, administered in two injections 14 days apart. Figure 9A Blood samples were collected one day before the first immunization, 14 days after the first immunization, and 14 days after the second immunization. The serum was used for subsequent neutralization tests.

[0262] 9.2 Cell Preparation

[0263] LLC-MK2 cells (CCL-7, ATCC) monkey kidney cells are the most suitable cells for PIV3 growth. The day before the neutralization experiment, the cells were digested, resuspended, washed, and seeded into 96-well plates at 1×10^4 / well in DMEM complete medium containing 5% FBS, and incubated overnight at 37°C with 5% CO2.

[0264] 9.3 Antibody neutralization of virus

[0265] 9.3.1 Serum serial dilution

[0266] The serum dilution gradients and methods commonly used in our laboratory are shown in Table 8 below. For PIV3, the serum dilution medium is DMEM, without the addition of fetal bovine serum.

[0267] Table 8 Serum dilutions and dilution methods

[0268]

[0269] 9.3.2 Serum Antibodies and Virus Neutralization

[0270] The optimal dosage of PIV3 virus (VR-1782, ATCC) is 30-40 PFU / well (96-well plate). Use DMEM as the dilution medium, without fetal bovine serum. Add 60 μL of the virus dilution to each well containing diluted serum and mix gently. A virus control group (virus solution without diluted serum) is required. Incubate at 37°C for 1 hour.

[0271] 9.3.3 Cell Seeding

[0272] Add 30 μL of the above-mentioned virus-serum mixture to each of the 96-well cell culture plates in which cells have grown into monolayers, with three replicates for each dilution. An 8-well control group of normal cells and an 8-well virus control group were also included. The culture plates were incubated at 35°C in a 5% CO2 incubator for 1 hour, gently shaking every 15 minutes. After incubation, discard the mixture in each well and add 150 μL / well of pre-prepared warmed culture medium (DMEM, 3% FBS, 0.75% methylcellulose). Incubate at 35°C in a 5% CO2 incubator for 7 days.

[0273] 9.4 Results and Color Development

[0274] 9.4.1 Virus and Cell Fixation

[0275] On day 7 of incubation, discard the culture medium in the wells, wash once with PBS, and then add 100 μL of freshly prepared 1% paraformaldehyde (PFA) per well for fixation for 1 hour. After that, discard the solution in the wells and wash once with PBS.

[0276] 9.4.2 Sealing and Washing

[0277] Add 150 μL of blocking buffer (5% skim milk powder), incubate at room temperature with gentle shaking on a shaker for 30 min. Then wash the plate three times with 150 μL / well PBST.

[0278] 9.4.3 Add antibody detection

[0279] Depending on the required number of sample wells, take the matching detection antibody (anti-PIV3-F IgG, H+L), dilute it appropriately, add 100 μL to each well, incubate at room temperature for 90 minutes, and then wash the plate three times with 150 μL / well PBST.

[0280] 9.4.4 Add secondary antibody

[0281] Take the matching secondary antibody, dilute it at an appropriate ratio of 1:2000 to 1:5000, add 100 μL to each well, incubate at room temperature for 60 minutes, and then wash the plate 5 times with 150 μL / well PBST.

[0282] 9.4.5 Color Development and Termination

[0283] Add 100 μL of TrueBlue to each well. TM Apply peroxidase Substrate, shake gently, and virus-positive plaques will appear quickly. Then wash away the reaction solution with water. Dry in the dark. Virus plaques can be counted the next day.

[0284] from Figure 9B and Figure 9CIt can be seen that the antibodies induced by the JY-PIV3-068 and JY-PIV3-071 mRNA / LNP vaccines in the JY-PIV3-F series can effectively neutralize the PIV3 virus strain. 50 The range was between 1100 (JY-PIV3-068) and 3000 (JY-PIV3-071). Since JY-PIV3-068 is based on the wild type of JY-Universal PIV3, version 1, and JY-PIV3-071 is a relatively more stable pre-F mutant with introduced point mutations, although JY-PIV3-071 is slightly better in mice in terms of antibody binding, JY-PIV3-068 is better in terms of neutralizing antibody.

[0285] Example 10 simulates the real world by providing immune boosters to animals already infected with the PIV3 virus.

[0286] PIV3 virus respiratory infection is a very common respiratory infection. Except for newborns under one year old who are infected for the first time, almost everyone faces repeated infections from the environment. For most people with a fully developed immune system, reinfection is not a very serious problem. However, for young children whose immune systems are still developing, the elderly with weakened immune systems, and patients with compromised immune systems, PIV3 infection is often a serious threat. This study was designed to understand whether the vaccine provides enhanced protection in animals previously infected with PIV3, and to what extent that protection is provided. To more accurately understand this issue, we conducted experiments on two animal species.

[0287] 10.1 Animal immunization and sampling

[0288] 10.1.1 Mouse Experiment

[0289] This experiment and the aforementioned neutralization experiment were conducted together. Figure 9A The animals used were female Balb / c mice, 6 weeks old. After arriving at the biosafety level 2 animal facility, the animals were allowed to acclimatize there for one week. The experiment consisted of four groups: Groups 1 and 2 received two immunizations at two-week intervals, each administered intramuscularly at 20 μg / mouse, JY-PIV3-068 or JY-PIV3-071; Group 3 mice were first infected nasally with hPIV3 (HPIV3 / AUS / 9 / 2007, GeneBank: KF530225.1, ATCCVR1782) at a viral load of 3.5 × 10^5 / mouse, and blood was collected at 4 weeks for testing. A booster injection of 20 μg / mouse of JY-068 was administered the day after blood collection. Blood was collected again 14 days later for testing; the last group served as a PBS control. The challenge virus was the same as the pre-infection virus.

[0290] 10.1.2 Golden hamster experiment

[0291] The animals used were female golden hamsters, 6 weeks old. After arriving at the CRO's biosafety level 2 animal facility, the animals acclimatized there for one week. See [link to animal infection and vaccination schedule] for details. Figure 10A The experiment consisted of four groups: Groups 1 and 2 received three immunizations at two-week intervals, administered intramuscularly each time, 30 μg / rat, JY-068 or JY-071; Group 3 was initially infected nasally with hPIV3 (as above), with a viral load of 1 × 10^5 / rat. Blood samples were collected on day 31 post-infection for testing, and a booster injection of JY-068 (30 μg / rat) was administered intramuscularly the day after blood collection, with blood collected again 10 days later; Group 4 was a PBS control. The challenge virus and the pre-infection virus were from the same batch, but at a dose of 5 × 10^5 / rat.

[0292] 10.2 ELISA detection of anti-PIV antibody titer

[0293] ELISA antibody levels were measured in serum samples before viral infection, 4 weeks after infection, and after booster shots.

[0294] 10.2.1 Antigen Coating

[0295] Cell supernatant containing PIV3-F protein antigen was diluted appropriately with PBS, and 100 μL / well was used to coat 96-well microplates with antigen. The mixture was incubated at 4°C for 16 h.

[0296] 10.2.2 Washing and Sealing

[0297] Remove the antigen-coated ELISA plate and wash it once with 300 μL / well PBST. Then add 100 μL of blocking buffer (5% skim milk powder) and incubate at room temperature for 30 min. Next, wash the plate three times with 300 μL / well PBST.

[0298] 10.2.3 Sample dilution and loading

[0299] After performing appropriate serial dilutions of mouse serum samples (4-fold decrease), 100 μL of each sample was added to an ELISA plate and incubated at room temperature for 1.5 h. The plate was then washed three times with 300 μL / well PBST.

[0300] 10.2.4 Add detection antibodies

[0301] Depending on the required number of sample wells, take an appropriate amount of matching detection antibody (goat anti-mouse or anti-golden hamster IgG, peroxidase conjugated –, H+L), dilute to 0.8 ng / mL, add 60 μL to each well, incubate at room temperature for 1 h, and then wash the plate 5 times with 300 μL / well PBST.

[0302] 10.2.5 Color Development and Termination

[0303] Mix colorimetric solution A (containing H2O2) and colorimetric solution B (containing TMB) in a 1:1 ratio. Add 100 μL to each well and let it stand at room temperature for 15-30 minutes. Then add 30 μL of 1M HCl stop solution to each well and read the value at 450 nm using a microplate reader.

[0304] The results showed that in mice, the titers of serum virus-specific binding antibodies were similar for both JY-PIV3-068 and JY-PIV3-071, whether it was pre-infection with a single dose of immunization or pre-infection plus a booster dose with a second dose of immunization. A significant increase in antibody titers was observed after the second immunization or after pre-infection plus a booster dose. Figure 9B ). Figure 9C The neutralization test results further demonstrate that the specific antibodies induced by this vaccine can effectively neutralize the virus and reduce viral infection; and this protective effect is particularly pronounced when the vaccine is used as a booster shot. This fully affirms the role of the mRNA / LNP vaccine, indicating that the vaccine has a similar viral protective effect to natural PIV3 infection. In other words, in individuals already infected with PIV3, a booster immunization can greatly control PIV3 viral infection.

[0305] Next, the invention verified similar experiments on golden hamsters, and the results are shown in […]. Figures 10B-10D In general, both viral infection and immunization with JY-PIV3-068 and JY-PIV3-071 mRNA / LNP vaccines induced lower levels of virus-specific binding antibodies compared to mice. Figure 10B ), three exemptions ( Figure 10D ) is significantly higher than the second exemption ( Figure 10C The levels were also higher than in the group of animals that received a booster immunization after pre-infection with the virus. This not only reveals the potential differences between different animals, but more importantly, it suggests that in the real world, booster immunizations may require two doses, rather than one dose as is sufficient for RSV.

[0306] Example 11: JY-PIV3-F mRNA / LNP vaccine inhibits PIV3 virus infection in mouse lungs.

[0307] The ultimate test for vaccines is whether the specific immune response induced by the vaccine in the body has the ability to protect the host and reduce viral infection.

[0308] 11.1 Animal immunization, challenge and sampling

[0309] To verify the protective effect of the JY-PIV3-F vaccine against the virus in animals, we used golden hamsters in our experiment.

[0310] Golden hamster, female, 6 weeks old. The animal will acclimatize there for one week after arriving at the CRO biosafety level 2 animal facility. See [link to animal infection and vaccination schedule]. Figure 10A The hamsters were divided into four groups: Groups 1 and 2 received three immunizations at two-week intervals, administered intramuscularly each time, 30 μg / hamster, JY-PIV3-068 or JY-PIV3-071; Group 3, golden hamsters were first infected nasally with hPIV3 (same as above), with a viral load of 1×10^5 / hamster. Blood was collected on day 31 post-infection for testing, and a booster injection of JY-068, 30 μg / hamster, was administered intramuscularly the day after blood collection. Blood was collected again 10 days later for testing; the last group was a PBS control. The day after the last blood collection, the hamsters were challenged with the same viral load, 5×10^5 / hamster via nasal inhalation after isoflurane anesthesia. The experiment ended on day 4 post-challenge. After euthanasia by inhalation of the anesthetic, the animals' lungs were removed intact according to the standard operating procedure.

[0311] 11.2 Detection of viral load in the lungs

[0312] Lung viral load can be detected using plaque assays and qPCR. This study used the latter. RNA was extracted from animal lung homogenates, reverse transcribed into cDNA, and the PIV3 N gene was amplified by qPCR to quantify the virus.

[0313] 11.2.1 Lung tissue sample processing

[0314] Prepare cryovials and add 2 mL of pre-chilled 4°C virus protection solution; place fresh lung tissue into the cryovials. Homogenize the lung tissue using a high-throughput tissue homogenizer while maintaining low temperature until no obvious particles are visible. Transfer the tissue fluid into 1.5 mL EP tubes and freeze at -80°C.

[0315] 11.2.2 Extraction of total RNA from samples

[0316] Take 200 μL of lung homogenate for RNA extraction. Add GENEzoI. TM Add the reagent, then briefly vortex. Incubate at room temperature for 5 minutes. Centrifuge to remove debris, and transfer the clear supernatant to a new 15 mL centrifuge tube.

[0317] Add an equal volume of anhydrous ethanol to the supernatant at a 1:1 ratio. Vortex to mix thoroughly, then centrifuge through an RB column. Repeat this step until the sample is fully bound. Afterward, wash the RB column with 600 μL of wash buffer, repeating twice.

[0318] Next, the DNA on the RB column was digested with DNase I solution at 25°C for 15 minutes.

[0319] The RB column was then washed with 400 μL of pre-wash buffer, followed by centrifugation and discarding the flow-through buffer. This process was repeated three times. Finally, the RB column was centrifuged at 14000 g for 3 minutes and the column matrix was dried.

[0320] RNA elution: Place a dry RB column in a clean 1.5 mL centrifuge tube. Add 25–50 μL of ribonuclease-free water to the center of the column matrix. Let stand for at least 3 minutes, then centrifuge at 14000 g for 1 minute to obtain RNA.

[0321] 11.2.3 Reverse Transcription

[0322] Prepare the cDNA synthesis reaction mixture according to the reverse transcription kit instructions. Each reaction contains 100-500 ng total RNA, 5 μL of 4×RT Mix, and water to bring the total volume to 20 μL. After mixing, incubate at 45°C for 30 minutes. Then inactivate at 85°C for 5 minutes to obtain cDNA.

[0323] 11.2.4 qPCR

[0324] qPCR was used to quantify PIV3 virus, using primer sequences derived from the N gene of PIV3. This allows for effective differentiation between vaccine and challenge viruses. The primer sequences are as follows:

[0325] 5'-CTGTATCCTCAGAGATCCTATACATGGT-3'(SEQ ID NO:20)

[0326] 5'-CCCCCATTGCATAACTCCATA-3'(SEQ ID NO:21)

[0327] 5'-TCGCACCAGGCAACTATCCTGCT-3'(SEQ ID NO:22)

[0328] According to the kit recommendations, the qPCR reaction system is as follows:

[0329]

[0330] qPCR reaction procedure:

[0331]

[0332] 11.2.5 Standard Curve

[0333] Using a plasmid containing the PIV3 N gene, quantitative qPCR was performed to obtain the algebraic relationship between the N gene copy number and Ct.

[0334] 11.2.6 Data Analysis

[0335] The process from lung homogenate to qPCR involves several steps, and the viral load of each lung is calculated as follows:

[0336] 2 mL of MEM was added to mouse lung tissue for homogenization, and 200 μL of the sample was used for RNA extraction, which was diluted 10 times.

[0337] The total RNA volume was 30 μL, with 2 μL used for reverse transcription, diluted 15-fold.

[0338] 20 μL of cDNA was produced by reverse transcription, and 2 μL was used for qPCR, which was diluted 10-fold.

[0339] Therefore, the total dilution factor is 1500;

[0340] The viral load of each lung was calculated using the standard curve external standard method.

[0341] The results are as follows Figure 11 The results showed that when golden hamsters received three doses of 30 μg mRNA / LNP vaccine, the induced antibodies effectively controlled the amplification of PIV3 virus in the lungs, reducing the viral load in the lungs by more than 100-fold. Given the characteristics of the qPCR virus detection method, it cannot distinguish between infectious live virus and neutralized non-infectious virus; the actual reduction may be even greater than what qPCR detects.

Claims

1. A PIV3 virus F protein, characterized in that, The amino acid sequence of the PIV3 virus F protein is shown in SEQ ID NO:5 or 6.

2. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule contains a nucleotide sequence encoding the PIV3 virus F protein as described in claim 1; preferably, the nucleic acid molecule is double-stranded DNA.

3. The nucleic acid molecule of claim 2, wherein, The nucleotide sequence encoding the PIV3 viral F protein contains any of the sequences shown in SEQ ID NO:1-4; Preferably, the nucleic acid molecule further comprises one or more selected from the following: (1) T7 promoter; the preferred nucleotide sequence of the T7 promoter is shown in SEQ ID NO:17; (2) 5'UTR; the 5'UTR is preferably the 5'UTR of human cytochrome B-245a polypeptide, whose nucleotide sequence is shown for example as SEQ ID NO:15; (3) 3'UTR; the 3'UTR is preferably the 3'UTR of human AES / TLE5, whose nucleotide sequence is shown for example in SEQ ID NO:16; (4) poly A; the nucleotide sequence of the poly A preferably has spacers, the nucleotide sequence of which is, for example, as shown in SEQ ID NO:19; more preferably, the nucleotide sequence of the poly A is shown in SEQ ID NO:18; (5) Kozak sequence; the Kozak sequence is preferably GCCGCCACC; More preferably, the nucleic acid molecule comprises, from the 5' end to the 3' end, a 5' UTR, a Kozak sequence, a nucleotide sequence encoding the PIV3 viral F protein, and a 3' UTR, in sequence. More preferably, the 5' end of the nucleic acid molecule is connected to the T7 promoter, and the 3' end is connected to the poly A; preferably, the T7 promoter and the 5'UTR are connected via AGG or GGG.

4. A nucleic acid construct, characterized in that, The nucleic acid construct contains the nucleic acid molecule as described in claim 2 or 3; Preferably, the nucleic acid molecule is constructed into the nucleic acid construct using a restriction endonuclease selected from, for example, HindIII and EcoRI.

5. A recombinant expression vector, characterized in that, The recombinant expression vector contains the nucleic acid molecule as described in claim 2 or 3 or the nucleic acid construct as described in claim 4.

6. A transformant, characterized in that, The transformant contains the nucleic acid molecule as described in claim 2 or 3, the nucleic acid construct as described in claim 4, or the recombinant expression vector as described in claim 5; Preferably, the host cells used in the construction of the transformant are selected from Escherichia coli, insect cells, yeast cells, and mammalian cells, such as Escherichia coli Stable cells.

7. An mRNA, characterized in that, The mRNA comprises an mRNA encoding the PIV3 viral F protein as described in claim 1.

8. The mRNA as described in claim 7, characterized in that, The mRNA is the mRNA obtained by transcription of the nucleic acid molecule as described in claim 2 or 3; Preferably, the sequence of the mRNA encoding the PIV3 viral F protein comprises any of the sequences shown in SEQ ID NO:7-10; More preferably, the sequence of the mRNA is shown in any of SEQ ID NO:11-14, and more preferably in SEQ ID NO:11 or 14; More preferably, the 5' end of the mRNA has a cap structure; the cap structure is preferably a Cap1 structure.

9. A method for preparing nucleic acid molecules or mRNA, characterized in that, The method includes culturing the transformant as described in claim 6 to obtain the nucleic acid molecule or mRNA.

10. A drug-loaded lipid nanoparticle, characterized in that, It contains the mRNA as described in claim 7 or 8; Preferably, the lipid nanoparticles further include cationic lipids and auxiliary lipids; More preferably, the cationic lipid is SM-102; And / or, the auxiliary lipid is DSPC, cholesterol and / or DMG-PEG-2000; More preferably, the molar ratio of the cationic lipid, DSPC, cholesterol and DMG-PEG-2000 is (40-60):(2-15):(30-40):(0.8-1.6), for example 50:10:38.5:1.

5.

11. An mRNA vaccine against PIV3 virus, characterized in that, The mRNA vaccine comprises the mRNA as described in claim 7 or 8 and / or the lipid nanoparticles as described in claim 10; Preferably, the mRNA vaccine is a virus-like particle; and / or, the mRNA vaccine further includes an adjuvant.

12. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the mRNA as described in claim 7 or 8, the lipid nanoparticles as described in claim 10, and / or the mRNA vaccine as described in claim 11, and optionally a pharmaceutical carrier.

13. The use of the PIV3 virus F protein as described in claim 1, the nucleic acid molecule as described in claim 2 or 3, the nucleic acid construct as described in claim 4, the recombinant expression vector as described in claim 5, the transformant as described in claim 6, the mRNA as described in claim 7 or 8, the lipid nanoparticles as described in claim 10, the mRNA vaccine as described in claim 11, and / or the pharmaceutical composition as described in claim 12 in the preparation of medicaments for the prevention and / or treatment of diseases caused by PIV3 virus and / or the inhibition of PIV3 virus; Preferably, the disease caused by the PIV3 virus is an acute respiratory illness, such as bronchiolitis, pneumonia, croup, or otitis media.

14. A method for inhibiting PIV3 virus, characterized in that, The method includes contacting a sample infected with PIV3 virus with the mRNA as described in claim 7 or 8, the lipid nanoparticles as described in claim 10, the mRNA vaccine as described in claim 11, and / or the pharmaceutical composition as described in claim 12 to inhibit the PIV3 virus; Preferably, the method is for non-therapeutic purposes.

15. A method for preventing or treating diseases caused by PIV3 virus, characterized in that, The method includes administering an effective dose of the mRNA as described in claim 7 or 8, the lipid nanoparticles as described in claim 10, the mRNA vaccine as described in claim 11, and / or the pharmaceutical composition as described in claim 12 to a patient in need; Preferably, the disease caused by the PIV3 virus is an acute respiratory illness, such as bronchiolitis, pneumonia, croup, or otitis media.

16. The PIV3 virus F protein as described in claim 1, the nucleic acid molecule as described in claim 2 or 3, the nucleic acid construct as described in claim 4, the recombinant expression vector as described in claim 5, the transformant as described in claim 6, the mRNA as described in claim 7 or 8, the liposome nanoparticles as described in claim 10, the mRNA vaccine as described in claim 11, and / or the pharmaceutical composition as described in claim 12, for the prevention and / or treatment of diseases caused by PIV3 virus and / or the inhibition of PIV3 virus; Preferably, the disease caused by the PIV3 virus is an acute respiratory illness, such as bronchiolitis, pneumonia, croup, or otitis media.

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