Novel coronavirus mRNA vaccine and its preparation method and application

Abstract

The present invention provides a novel coronavirus mRNA vaccine and a preparation method and application thereof. The present invention provides the following (a) or (b) proteins: (a) a protein consisting of the amino acid sequence shown in SEQ ID No.1; (b) a derivative protein having the same function as the protein consisting of the amino acid sequence shown in SEQ ID No.1 after substitution, deletion and / or addition of one or more amino acids in the amino acid sequence shown in SEQ ID No.1. The present invention designs the S protein sequence of the pre-fusion conformation in the novel coronavirus (SARS-CoV-2), and realizes efficient expression of human cells to increase immunogenicity. The mRNA of the present invention can achieve immunity to the novel coronavirus and has a wide applicability.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and specifically relates to a novel coronavirus (SARS-CoV-2) mRNA vaccine antigen design, a vaccine, and a preparation method and application thereof. Background Art

[0002] Coronavirus is a type of RNA virus that is widely present in nature. It is named because the virus morphology resembles a crown when observed under an electron microscope. Coronavirus is a large family with many members. It only infects vertebrates and can cause respiratory, digestive and nervous system diseases in humans and animals. The new coronavirus (SARS-CoV-2) is a new coronavirus discovered in 2019. Before the discovery of SARS-CoV-2, there were 6 known coronaviruses that infect humans, 4 of which are more common in the population and have low pathogenicity, generally only causing mild respiratory symptoms similar to the common cold; while the other two viruses, SARS coronavirus and MERS coronavirus, are more pathogenic. SARS-CoV-2 is highly contagious and can cause lung inflammation. In severe cases, it can cause respiratory failure and death.

[0003] The structural proteins of the novel coronavirus are composed of spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins. The S protein is presented on the surface of the virus in the form of a trimer, forming a "crown-like" spike structure, and mediates the binding of the virus to cell receptors and membrane fusion. The S protein has high immunogenicity, can activate T cell immune responses, and can induce the production of neutralizing antibodies. The S protein is an important target for the development of coronavirus vaccines and drugs. Many studies have shown that vaccines developed for SARS-CoV and MERS-CoV S proteins can provide good immune protection. Compared with other novel coronavirus structural proteins, the S protein has a larger molecular weight and is more difficult to secrete from cells. By combining with an efficient exocytotic signal peptide, the exocytosis efficiency of the S protein can be effectively improved. At the same time, after sequence optimization, the expression of the S protein in cells can be increased, and the expression of effective antigens can be enhanced. In addition, by inserting the Foldon trimerization domain at the C-terminus of the S protein, the S protein coding sequence whose translation product is a pre-fusion conformation is obtained. The mRNA sequence corresponding to the S protein in the pre-fusion conformation can induce high-titer neutralizing antibodies in vivo, which is safer.

[0004] mRNA vaccine technology can be used as a fast and flexible technology platform to effectively respond to the threats of various emerging viruses. Therefore, mRNA vaccines are considered to be the most promising vaccine against the new coronavirus. Traditional inactivated vaccines, attenuated vaccines, and peptide vaccines have long research and development cycles and complex production processes, making it difficult to meet the needs of public health emergencies. In addition, compared with DNA vaccines, mRNA vaccines have obvious advantages. First, mRNA vaccines are more effective than DNA vaccines. DNA vaccines need to pass through the cell membrane and nuclear membrane to express into antigens. Therefore, when there is not enough DNA to reach the nucleus, the vaccine will not be able to activate the body's immune system and induce the production of antibodies. mRNA only needs to pass through the cell membrane to efficiently express antigen proteins in the cytoplasm. Secondly, mRNA vaccines are more biosafe than DNA vaccines. There is a certain probability that exogenous DNA will be integrated into the human genome, so DNA vaccines have the risk of causing genomic mutations. However, mRNA does not have the risk of gene integration into the genome. Secondly, mRNA is easily degraded after being translated into protein. Its transient expression characteristics not only ensure the safety of mRNA drugs, but also make their dosage controllable, avoiding antigen immune tolerance (a state of no response to specific antigens) caused by long-term exposure of vaccine drugs. In addition, since animal-derived viruses are not required in the vaccine development process, the risks of using viruses and animals are avoided, and effective antigens can be obtained in a short period of time.

[0005] In general, compared with traditional vaccines, mRNA vaccines have a shorter production cycle, simpler production process, and lower cost. It only takes 6 weeks from antigen sequence design to production for mRNA vaccines, while traditional vaccines take at least 5-6 months to produce. mRNA vaccines can be freeze-dried and stored at room temperature for 6-18 months, while traditional vaccines need to be stored at 2-8°C. Therefore, mRNA vaccines have a longer shelf life, do not require a cold chain, and have lower transportation costs. Traditional vaccines are powerless against many new viruses, while mRNA vaccines have a wider applicability and sequences can be flexibly designed to deal with different pathogens. Summary of the invention

[0006] One object of the present invention is to provide antigens for mRNA vaccines against the new coronavirus (SARS-CoV-2).

[0007] Another object of the present invention is to provide a vaccine against the new coronavirus (SARS-CoV-2).

[0008] Another object of the present invention is to provide a method for preparing an mRNA vaccine against the new coronavirus (SARS-CoV-2).

[0009] Another object of the present invention is to provide mRNA for preparing a vaccine against the new coronavirus (SARS-CoV-2).

[0010] Another object of the present invention is to provide DNA for preparing the mRNA against the new coronavirus (SARS-CoV-2).

[0011] Another object of the present invention is to provide a vector for preparing the mRNA against the new coronavirus (SARS-CoV-2).

[0012] Another object of the present invention is to provide an application of an mRNA vaccine against the new coronavirus (SARS-CoV-2).

[0013] The new coronavirus (SARS-CoV-2) is spherical and ellipsoidal in shape, with a diameter of 80-120nm. Under an electron microscope, there are club-shaped protrusions on the surface of the virus particles, which are composed of trimeric spike glycoproteins (Spike, S). The viral envelope is composed of membrane glycoproteins (M), which are embedded in the viral envelope through three transmembrane domains. In addition, a small amount of small transmembrane protein-envelope (E) protein also appears in the envelope. Finally, the nucleocapsid (N) protein binds to the RNA genome in the form of beads to form a helically symmetrical nucleocapsid. The results show that the S protein is the main component of the coronavirus that causes the body's immune response. The novel coronavirus can use a tightly glycosylated, homotrimeric class I fusion spike protein (S protein) to enter host cells. The S protein can exist in a relatively stable prefusion conformation and undergo drastic structural rearrangements to promote the fusion of the viral membrane structure with the host cell membrane. This process is induced by the binding of the viral S1 subunit to the host receptor, which can destroy the stability of the prefusion trimer, causing the S1 subunit to fall off and promote the S2 subunit to transform into a highly stable postfusion conformation. In order to bind to the host receptor, the receptor binding domain of S1 undergoes chain-like conformational movements that temporarily hide or expose the determinants of receptor binding. These two states are considered to be "up" or "down" conformations, where the down conformation corresponds to a state that is inaccessible to the receptor, while the up conformation corresponds to a state that is accessible to the receptor, the latter of which is considered to be unstable.

[0014] In the present invention, in order to provide an mRNA vaccine for the new coronavirus (SARS-CoV-2), the amino acid sequence of the wild-type S protein is modified, the viral antigen fragment encoded by mRNA is designed, and then the mRNA encoding the viral antigen fragment is synthesized to achieve immunity to the new coronavirus. Specifically, the present invention uses genetic engineering technology to stabilize S in its pre-fusion conformation, prevent structural rearrangement, and expose antigenic epitopes with better antigenicity to cause a higher neutralizing antibody response. Through bioinformatics and genetic engineering methods, the present invention respectively transforms the S protein coding sequence so that its translation product is stabilized in the pre-fusion conformation. The relevant antigen sequence is cloned into a plasmid for mRNA synthesis by genetic engineering technology.

[0015] In one aspect, the present invention provides a protein of (a) or (b):

[0016] (a) a protein consisting of the amino acid sequence shown in SEQ ID No. 1;

[0017] (b) A derivative protein having the same function as the protein consisting of the amino acid sequence shown in SEQ ID No. 1, wherein one or more amino acids are substituted, deleted and / or added.

[0018] The protein composed of the amino acid sequence shown in SEQ ID No.1 is a modified and improved protein based on the wild-type S protein of the present invention, and is also referred to as the pre-fusion conformation S protein in the present invention. According to the genome sequencing results of the new coronavirus (SARS-CoV-2), the present invention designs and optimizes the S sequence so that the expressed S protein is in the pre-fusion conformation to achieve efficient expression in human cells and increase immunogenicity. Compared with the wild-type S protein amino acid sequence, the pre-fusion conformation S protein of the present invention is more likely to induce the production of neutralizing antibodies.

[0019] According to a specific embodiment of the present invention, in the present invention, the derivative protein composed of the amino acid sequence shown in SEQ ID No.1 by substitution, deletion and / or addition of one or several amino acids and having the same function as the protein composed of the amino acid sequence shown in SEQ ID No.1 at least comprises a sequence of at least 15 consecutive amino acids at the C-terminus of the amino acid sequence shown in SEQ ID No.1.

[0020] According to a specific embodiment of the present invention, the protein composed of the amino acid sequence shown in SEQ ID No.1 having the same function means that the protein composed of the amino acid sequence shown in SEQ ID No.1 has the function of "more easily inducing the production of neutralizing antibodies" and / or "increasing immunogenicity" (compared with the wild-type S protein).

[0021] On the other hand, the present invention also provides the use of the improved S protein (pre-fusion conformation S protein and / or its derivative protein) as an antigen in the preparation of a vaccine.

[0022] On the other hand, the present invention also provides a DNA encoding the improved S protein of the present invention. According to a preferred embodiment of the present invention, the nucleotide sequence of the DNA comprises the sequence shown in SEQ ID No.10, SEQ ID No.11, SEQ ID No.12 or SEQ ID No.13.

[0023] On the other hand, the present invention also provides an expression vector containing the DNA of the present invention (DNA encoding the improved S protein of the present invention).

[0024] On the other hand, the present invention also provides mRNA encoding the improved S protein of the present invention.

[0025] According to a preferred embodiment of the present invention, the mRNA is obtained by in vitro transcription of the aforementioned DNA of the present invention.

[0026] According to a preferred embodiment of the present invention, the nucleotide sequence of the mRNA of the present invention includes the sequence shown in SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 or SEQ ID No.5.

[0027] According to a preferred specific embodiment of the present invention, the mRNA of the present invention may further include an adjuvant sequence at the 5' end of its nucleotide sequence. For example, the adjuvant sequence may be a nucleotide sequence that promotes protein secretion and expression. According to a preferred embodiment of the present invention, the adjuvant sequence includes a sequence shown in SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 or SEQ ID No.9. According to a more specific embodiment of the present invention, the mRNA includes an mRNA shown in a sequence shown in SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 or SEQ ID No.9 and a sequence shown in a combination of SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 or SEQ ID No.5 (the sequence shown in any one of SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 or SEQ ID No.9 is located at the 5' end of any one of SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 or SEQ ID No.5). According to a more preferred embodiment of the present invention, the mRNA includes an mRNA shown in a sequence shown in SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 or SEQ ID No.9 and a sequence shown in a combination of SEQ ID No.4.

[0028] On the other hand, the present invention also provides a method for preparing the mRNA.

[0029] According to a specific embodiment of the present invention, the method for preparing the mRNA of the present invention comprises:

[0030] Synthesis of mRNA encoding viral antigen fragments: Synthesize a DNA fragment encoding the pre-fusion conformation S protein (can be synthesized independently or commercially), and clone it into a plasmid as a template for in vitro transcription (IVT) to obtain uncapped mRNA;

[0031] mRNA capping: under the catalysis of capping enzyme, a cap structure is added to the 5' end of the uncapped mRNA to obtain a capped mRNA molecule;

[0032] Purification of mRNA: The capped mRNA is subjected to lithium chloride / ethanol precipitation, centrifugal column, chlorine extraction / ethanol precipitation or gel purification to obtain purified mRNA.

[0033] On the other hand, the present invention also provides the application of the mRNA antigen design in the preparation of a new coronavirus mRNA vaccine.

[0034] On the other hand, the present invention also provides a vaccine comprising one or more of the mRNAs described in the present invention.

[0035] According to a specific embodiment of the present invention, the vaccine of the present invention is a preventive vaccine against the new coronavirus (SARS-CoV-2), which may also include a PBS solution for dissolving mRNA.

[0036] Beneficial effects of the present invention:

[0037] The technology of the present invention is to first design the S protein sequence of the pre-fusion conformation of the new coronavirus (SARS-CoV-2) through bioinformatics methods to achieve efficient expression of human cells to increase immunogenicity; then synthesize mRNA encoding viral antigen fragments through in vitro transcription methods to achieve immunity to the new coronavirus. The entire mRNA vaccine has a short production cycle, simple process operation, low production cost, long storage time, no need for cold chain, and easy transportation. Traditional vaccines cannot respond quickly to public health events caused by many new viruses, while mRNA vaccines have a wider applicability and the sequence can be flexibly designed to respond to different pathogens. It plays an extremely important role in the rapid development of vaccines for acute infectious diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Schematic diagram of the mRNA vaccine of the present invention.

[0039] Figure 2 The expression results of different mRNA vaccines encoding the pre-fusion S protein antigen fragments after sequence optimization in Example 1 of the present invention are shown. Wherein, M: protein marker, 1#: pre-fusion S protein encoded by SEQ ID No.2, 2#: pre-fusion S protein encoded by SEQ ID No.3, 3#: pre-fusion S protein encoded by SEQ ID No.4, 4#: pre-fusion S protein encoded by SEQ ID No.5.

[0040] Figure 3The results of cell extracellular secretion after 24 hours of transfection of 293T cells with mRNA (SEQ ID No.4) with different adjuvant sequences (SEQ ID No.6-9) in Example 2 of the present invention are shown. Wherein, Control: control mRNA (SEQ ID No.4), 1#: mRNA with SEQ ID No.6 adjuvant sequence (SEQ ID No.4), 2#: mRNA with SEQ ID No.7 adjuvant sequence (SEQ ID No.4), 3#: mRNA with SEQ ID No.8 adjuvant sequence (SEQ ID No.4), 4#: mRNA with SEQ ID No.9 adjuvant sequence (SEQ ID No.4).

[0041] Figure 4 The results of S-specific antibody titer after mice were inoculated with different adjuvants in Example 3 of the present invention and the mRNA (SEQ ID No. 4) of the preferred embodiment in Example 2 are shown.

[0042] Figure 5 The results of neutralizing antibody titers after mice were inoculated with different adjuvants in Example 4 of the present invention and the mRNA (SEQ ID No. 4) of the preferred embodiment in Example 2 are shown. Each curve in the figure represents one mouse. DETAILED DESCRIPTION

[0043] The following will be combined with the embodiments of the present invention and the accompanying drawings to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] The instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, are all conventional instruments, reagents, materials, etc. in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art.

[0045] In the following examples, the DNA fragments used were synthesized on commission. The mRNA used was prepared according to the following method:

[0046] Synthesize a DNA fragment encoding the pre-fusion conformation S protein, clone it into a plasmid as a template, and perform in vitro transcription (IVT) by T7 RNA polymerase to obtain uncapped mRNA; wherein the specific sequence of the DNA fragment preferably includes the sequences shown in SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, and SEQ ID No.13, and the 5' end of the DNA fragment may also include a DNA fragment corresponding to the adjuvant sequence (preferably the sequence shown in SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 or SEQ ID No.9) as required;

[0047] mRNA capping: adding a cap structure to the 5' end of the uncapped mRNA under the catalysis of a capping enzyme to obtain a capped mRNA molecule; the capping enzyme includes but is not limited to one or a combination of dioxymethyltransferase (2'-OMethyltransferase) or RNA triphosphatase (triphosphatase), RNA glutamyltransferase (guanyltransferase) and guanine-7-methyltransferase (Guanine-7-methyltransferase);

[0048] Purification of mRNA: The capped mRNA is subjected to lithium chloride / ethanol precipitation, centrifugal column, chlorine extraction / ethanol precipitation or gel purification to obtain purified mRNA.

[0049] SEQ ID No.1: Pre-fusion conformation S protein sequence

[0050]

[0051] SEQ ID No.2: mRNA encoding the pre-fusion conformation S protein sequence (optimized solution 1)

[0052] AAG CAC AAG GAC CAC GAC GAG GGG GAC CAC UCG UCG GUC ACG CAC UUG GACUGG UGG GCC UGG GUC GAC GGG GGG CGG AUG UGG UUG UCG AAG UGG GCC CCG CAC AUGAUG GGG CUG UUC CAC AAG GCC UCG UCG CAC GAG GUG UCG UGG GUC CUG GAC AAG GACGGG AAG AAG UCG UUG CAC UGG ACC AAG GUG CGG UAG GUG CAC UCG CCG UGG UUG CCGUGG UUC GCC AAG CUG UUG GGG CAC GAC GGG AAG UUG CUG CCG CAC AUG AAG CGG UCGUGG CUC UUC UCG UUG UUG UCG UUG UUG UUG UGC UGC UGC CCG ACC UAG AAG CCG UGG UGG GAC CUG UCG UUCUGG GUC UCG GAC GAC UUG UUG UUG CGG UGG UUG CAC CAC UAG UUC CAC ACG CUCAAG GUC AAG ACG UUG CUG GGG AAG GAC CCG CAC AUG AUG GUG UUC UUG UUG UUC UCGACC UAC CUC UCG CUC AAG GCC CAC AUG UCG UCG CGG UUG UUG ACG UGG AAG CUC AUGCAC UCG GUC GGG AAG GAC UAC CUG GAC CUC CCG UUC GUC CCG UUG AAG UUC UUG GACGCC CUC AAG CAC AAG UUC UUG UAG CUG CCG AUG AAG UUC UAG AUG UCG UUC GUG UGGGGG UAG UUG GAC CAC GCC CUG GAC GGG GUC CCG AAG UCG CGG GAC CUC GGG GAC CACCUG GAC GGG UAG CCG UAG UUG UAG UGG GCC AAG GUC UGG GGG GAC CGG GAC GAG GAG GUG GCCUCG AUG GAC UGG GGG CCG CUG UCGUCG UCG CCG ACC UGG CGG CCG CGG CGG CGG AUGAUG CAC CCG AUG GAC GUC GGG GCC UGG AAG GAC GAC UUC AUG UUG CUC UUG CCG UGGUAG UGG CUG CGG CAC CUG ACG CGG GAC CUG GGG GAC UCG CUC UGG UUC ACG UGG GACUUC UCG AAG UGG CAC CUC UUC CCG UAG AUG GUC UGG UCG UUG AAG GCC CAC GUC GGGUGG CUC UCG UAG CAC GCC AAG GGG UUG UAG UGG UUG GAC ACG GGG AAG CCG CUC CACAAG UUG CGG UGG GCC AAG CGG UCG CAC AUG CGG ACC UUG GCC UUC GCC UAG UCG UUGACG CAC CGG CUG AUG UCG CAC GAC AUG UUG UCG CGG UCG AAG UCG UGG AAG UUC ACGAUG CCG CAC UCG GGG UGG UUC GAC UUG CUG GAC ACG AAG UGG UUG CAC AUG CGG CUGUCG AAG CAC UAG GCC CCG CUG CUC CAC GCC GUC UAG CGG GGG CCG GUC UGG CCG UUCUAG CGG CUG AUG UUG AUG UUC GAC GGG CUG CUG AAG UGG CCG ACG CAC UAG CGG ACCUUG UCG UUG UUG GAC CUG UCG UUC CAC CCG CCG UUG AUG UUG AUG GAC AUG GCC GACAAG GCC UUC UCG UUG GAC UUC GGG AAG CUC GCC CUG UAG UCG UGG CUC UAG AUG GUCCGG CCG UCG UGG GGG ACG UUG CCG CAC CUC CCG AAG UUG ACG AUG AAG GGG GAC GUCUCG AUG CCG AAG GUC GGG UGG UUG CCG CAC CCG AUG GUC GGGAUG GCC CAC CAC CACGAC UCG AAG CUC GAC GAG GUG CGG GGG CGG UGG CAC ACG CCG GGG UUC UUC UCG UGGUUG GAC CAC UUC UUG UUC ACG CAC UUG AAG UUG AAG UUG CCG GAC UGG CCG UGG CCGCAC GAC UGG CUC UCG UUG UUC UUC AAG GAG GGG AAG GUC GUC AAG CCG GCC CUG UAGCGG CUG UGG UGG CUG CGG CAC GCC CUG GGG GUC UGG GAC CUC UGG UAG GAC CUG UGG GGG CCG UGG UGG UCG UUG GUCCAC CGG CAC GAC AUG GUC CUG CAC UUG ACG UGG CUC CAC GGG CAC GGG CAC CGG UGG UUG GUG CGGCUG GUC GAC UGG GGG UGG ACC GCC CAC AUG UCG UGG CCG UCG UUG CAC AAG GUC UGGGCC CGG CCG ACG GAC UAG CCG CGG CUC GUG CAC UUG UUG UCG AUG CUC ACG CUG UAGGGG UAG CCG CGG CCG UAG ACG CGG UCG AUG GUC UGG GUC UGG UUG UCG GGG GCC GCCCGG GCC UCG CAC CGG UCG GUC UCG UAG UAG CGG AUG UGG UAC UCG GAC CCG CGG CUUG UCG CAC CGG AUG UCG UUG UUG UCG UAG CGG UAG GGG UGG UUG AAG UGG UAG UCGCAC UGG UGG CUC UAG GAC GGG CAC UCG UAC UGG UUC UGG UCG CAC CUG ACG UGG UACAUG UAG ACG CCG CUG UCG UGG CUC ACG UCG UUG GAC GAC GAC GUC AUG CCG UCG AREAUGG GUC GAC UUG GCC CGG GAC UGG CCG UAG CGG CAC CUC GUC CUG UUC UUG UGGGUC CAC CAC AAG CGG GUC CAC UUC GUC UAG AUG UUC UGG GGG GGG UAG UUC CUG AAGCCG CCG AAG UUG AAG UCG GUC UAG GAC GGG CUG GGG UCG UUC GGG UCG UUC GCC UCGAAG UAG CUC CUG GAC GAC AAG UUG UUC CAC UGG GAC CGG CUG CGG CCG AAG UAG UUCGUC AUG CCG CUG ACG GAC CCG CUG UAG CGG CGG GCC CUG GAC UAG ACG CGG GUC UUCAAG UUG CCG GAC UGG CAC GAC GGG GGG GAC GAC UGG CUC UAC UAG CGG GUC AUGUGG UCG CGG GAC GAC CGG CCG UGG UAG UGG UCG CCG ACC UGG AAG CCG CGG CCG CGGCGG GAC GUC UAG GGG AAG CGG UAC GUC UAC CGG AUG GCC AAG UUG CCG UAG CCG CACUGG GUC UUG CAC GAC AUG CUC UUG GUC UUC GAC UAG CGG UUG GUC AAG UUG UCG CGGUAG CCG UUC UAG GUC CUG UCG GAC UCG UGG CGG UCG CGG GAC CCG UUC GAC GUCCUG CAC CAC UUG GUC UUG CUG UUG UGG GAC CAC UUC GUC GAC UCG UCGUUG AAG CCG CGG UAG UCG UCG CAC GAC UUG CUG UUG GAC UCG GCC GAC CUG UUC CACCUC CGG CUC CAC GUC UAG CUG GCC GAC UAG UGG CCG GAC GUC UCG GAC GUC UGGAUG CAC UGG GUC GUC GAC UAGGCC CGG CGG CUC UAG GCC CGG UCG CGG UUG GAC CGGCGG UGG UUC UAC UCG CUC ACG CAC GAC CCG GUC UCG UUC GCC CAC CUG AAG ACG CCGUUC CCG AUG GUG GAC UAC UCG AAG GGG GUC UCG CGG GGG GUG CCG CAC CAC AAG GACGUG CAC UGG AUG CAC GGG CGG GUC CUC UUC UUG AAG UGG UGG CGG GGG CGG UAG ACGGUG CUG CCG UUC CGG GUG AAG GGG GCC CUC CCG CAC AAG CAC UCG UUG CCG UGG GUGACC AAG CAC UGG GUC GCC UUG AAG AUG CUC GGG GUC UAG UAG UGG UGG CUG UUG UGGAAG CAC UCG CCG UUG ACG CUG CAC CAC UAG CCG UAG CAC UUG UUG UGG CAC AUG CUGGGG GAC GUC GGG CUC GAC CUG UCG AAG UUC CUC CUC GAC CUG UUC AUG AAG UUC UUGGUG UGG UCG GGG CAC CUG GAC CCG CUG UAG UCG CCG UAG UUG CGG UCG CAC CACUUG UAG GUC UUC CUC UAG CUG GCC GAC UUG CUC CAC CGG UUC UUG GAC UUG CUC UCGGAC UAG CUG GAC GUC CUC GAC CCG UUC AUG CUC GUC AUG UAG UUC ACC GGG ACC AUGUAG ACC GAC CCG AAG UAG CGG CCG GAC CGG UAG CAC UAC CAC UGG UAC GACACG ACG UAC UGG UCG ACG ACG UCG ACG GAC UUC CCG ACG UCG CGG AUG CAC GCCUUC CUG CCG CUC ACC CAC AAG GAC ACU

[0053] SEQ ID No.3: mRNA encoding the pre-fusion conformation S protein sequence (optimized solution 2)

[0054] AAG CAC AAG GAC CAC GAC GAG GGG GAC CAC UCG UCG GUC ACG CAC UUG GACUGG UGG UCC UGG GUC GAC GGG GGG CGG AUG UGG UUG UCG AAG UGG UCC CCG CAC AUGAUG GGG CUG UUC CAC AAG UCC UCG UCG CAC GAG GUG UCG UGG GUC CUG GAC AAG GACGGG AAG AAG UCG UUG CAC UGG ACC AAG GUG CGG UAG GUG CAC UCG CCG UGG UUG CCGUGG UUC UCC AAG CUG UUG GGG CAC GAC GGG AAG UUG CUG CCG CAC AUG AAG CGG UCGUGG CUC UUC UCG UUG UUG UCG UUG UUG CCG ACC UAG AAG CCG UGG UGG GAC CUG UCG UUCUGG GUC UCG GAC GAC UUG UUG CGG UGG UUG CAC CAC UAG UUC CAC ACG CUCAAG GUC AAG ACG UUG CUG GGG AAG GAC CCG CAC AUG AUG GUG UUC UUG UUG UUC UCGACC UAC CUC UCG CUC AAG UCC CAC AUG UCG UCG CGG UUG UUG ACG UGG AAG CUC AUGCAC UCG GUC GGG AAG GAC UAC CUG GAC CUC CCG UUC GUC CCG UUG AAG UUC UUG GACUCC CUC AAG CAC AAG UUC UUG UAG CUG CCG AUG AAG UUC UAG UCG UUC GUG UGGGGG UAG UUG GAC CAC UCC CUG GAC GGG GUC CCG AAG UCG CGG GAC CUC GGG GAC CACCUG GAC GGG UAG CCG UAG UUG UAG UGG UCC AAG GUC UGG UGG GAC GAC CGG GAC GAG GUG UCCUCG AUG GAC UGG GGG CCG CUG UCGUCG UCG CCG ACC UGG CGG CCG CGG CGG CGG AUGAUG CAC CCG AUG GAC GUC GGG UCC UGG AAG GAC GAC UUC AUG UUG CUC UUG CCG UGGUAG UGG CUG CGG CAC CUG ACG CGG GAC CUG GGG GAC UCG CUC UGC ACG UGG GACUUC UCG AAG UGG CAC CUC UUC CCG UAG AUG GUC UGG UCG UUG AAG UCC CAC GUC GGGUGG CUC UCG UAG CAC UCC AAG GGG UUG UUG UUG UGG UUG GAC ACG GGG AAG CCG CUC CACAAG UUG CGG UGG UGG UCC AAG CGG UCG CAC AUG CGG ACC UUG UCC UUC UCC UAG UCG UUGAG CAC CGG CUG AUG UCG CAG GAC AUG UUG UCG CGG UCG AAG UCG UGG AAG UUC ACGAUG CCG CAC UCG GGG UGG UUC GAC UUG CUG GAC ACG AAG UGG UUG CAC AUG CGG CUGUCG AAG CAC UAG UCC CCG CUG CUC CAC UCC GUC UAG CGG GGG CCG GUC UGG CCG UUCUAG CGG CUG AUG UUG AUG UUC GAC GGG CUG CUG AAG UGG CCG ACG CAC UAG CGG ACCUUG UCG UUG UUG GAC AUG UUG GAC AUG UUC GACAAG UCC UCG UUG GAG UUC GGG AAG CUC UCC CUG UAG UCG UGG CAC CUC CCG AUG GUC GGG CCG UCG UGG GGG ACG UUG CCG CAC CUC CCG AAG UUG ACG AUG AAG GGG GAC GUCUCG AUG CCG AAG GUC GGG UGG CCG CAC CCG AUG GUC GGGAUG UCC CAC CAC CACGAC UCG AAG CUC GAC GAG GUG CGG GGG CGG UGG CAC ACG CCG GGG UUC UUC UCG UGGUUG GAC CAC UUC UUG UUC ACG CAC UUG AAG UUG AAG UUG CCG GAC UGG CCG UGG CCGCAC GAC UGG CUC UCG UUG UUC UUC AAG GGG AAG GUC GUC AAG CCG UGC CUG UGG UGG CUG CGG CAC UCC CUG GGG GUC UGG GAC CUC UGG UGC UAG GAC CUG UGG GGG ACG UCG AAG CCG CCG CAC UCG CAC UAG UGG GGG CCG UGG UGG UCG UUG GUCCAC CGG CAC GAC AUG GUC CUG CAC UUG ACG UGG CUC CAC GGG CAC CGG UGG UGG UGG UGG GUCCAC CGG CAC GAG AUG GUC CUG CAC UUG ACG UGG CUC CAC GGG CAC CGG UGG UGG GUG CGGCUG GUC GAC UGG GGG UGG ACC UCC CAC AUG UCG UGG CCG UCG UUG CAC AAG GUC UGGUCC CGG CCG ACG GAC UAG CCG CGG CUC GUG CAC UUG UUG UCG AUG CUC ACG CUG UAGGGG UAG CCG CGG CCG UAG ACG CGG UCG AUG GUC UGG GUC UGG UUG UCG GGG UCC UCCCGG UCC UCG CAC CGG UCG GUC UCG UAG UAG CGG AUG UGG UAC UCG GAC CCG CGG CUUG UCG CAC CGG AUG UCG UUG UUG UCG UAG CGG UAG GGG UGG UUG AAG UGG UAG UCGCAC UGG UGG CUC UAG GAC GGG CAC UCG UAC UGG UUC UGG UCG CAC CUG ACG UGG UACAUG UAG ACG CCG CUG UCG UGG CUC ACG UCG UUG GAC GAC GAC GUC AUG CCG UCG AREAUGG GUC GAC UUG UCC CGG GAC UGG CCG UAG CGG CAC CUC GUC CUG UUC UUG UGGGUC CUC CAC AAG CGG GUC CAC UUC GUC UAG AUG UUC UGG GGG GGG UAG UUC CUG AAGCCG CCG AAG UUG AAG UCG GUC UAG GAC GGG CUG GGG UCG UUC GGG UCG UUC UCC UCGAAG UAG CUC CUG GAC GAC AAG UUG UUC CAC UGG GAC CGG CUG CGG CCG AAG UUG UUC GAC AUG CCG CUG ACG GAC CCG CUG UAG CGG CGG UCC CUG GAC UAG ACG CGG GUC UUCAAG UUG CCG GAC UGG CAC GAG GGG GGG GAC GAC UGG CUG CUC UAC UAG CGG GUC AUGUGG UCG CGG GAC GAC CGG CCG UGG UGG UCG CCG ACC UGG AAG CCG CGG CCG CGGCGG GAC GUC UAG GGG AAG CGG UAC GUC UAC CGG AUG UCC AAG UUG CCG UAG CCG CACUGG GUC UUG CAC GAC AUG CUC UUG GUC UUC GAC UAG CGG UUG GUC AAG UUG UCG CGGUAG CCG UUC UAG GUC CUG UCG GAC UCG UGG CGG UCG CGG GAC CCG UUC GAC GUCCUG CAC CAC UUG GUC UUG CUG UGG UGG GAC CAC UUC GUC GAC UCG UCGUUG AAG CCG CGG UAG UCG UCG CAC GAC UUG CUG UAG GAC UCG UCC GAC CUG UUC CACCUC CGG CUC CAC GUC UAG CUG UCC GAC UGG UGG CCG UCC GAC GUC UGGAUG CAC UGG GUC GUC GAC UAGUCC CGG CGG CUC UAG UCC CGG UCG CGG UUG GAC CGGCGG UGG UUC UAC UCG CUC ACG CAC GAC CCG GUC UCG UUC UCC CAC CUG AAG ACG CCGUUC CCG AUG GUG GAC UAC UCG AAG GGG GUC UCG CGG GGG GUG CCG CAC CAC AAG GACGUG CAC UGG AUG CAC GGG CGG GUC CUC UUC UUG AAG UGG UGG CGG GGG CGG UAG ACGGUG CUG CCG UUC CGG GUG AAG GGG UCC CUC CCG CAC AAG CAC UCG UUG CCG UGG GUGACC AAG CAC UGG GUC UCC UUG AAG AUG CUC GGG GUC UGC UAG UGG UGG CUG UUG UGGAAG CAC UCG CCG UUG ACG CUG CAC CAC UAG CCG UAG CAC UUG UUG UGG UGG CAC AUG CUGGGG GAC GUC GGG CUC GAC CUG UCG AAG UUC CUC CUC GAC CUG UUC AUG AAG UUC UUGGUG UGG UCG GGG CUG CAC CUG GAC CCG CUG UAG UCG CCG UAG UUG CGG UCG CAC CACUUG UAG GUC UUC CUC UAG CUG UCC GAC UUG CUC CAC CGG UUC UUG GAC UUG CUC UCGGAC UAG CUG GAC GUC CUC GAC CCG UUC AUG CUC GUC AUG UAG UUC ACC GGG ACC AUGUAG ACC GAC CCG AAG UAG CGG CCG GAC UAG CGG UAG CAC UAC CAC UGG UAG UAC GACACG ACG UAC UGG UCG ACG ACG UCG ACG GAC UUC CCG ACG ACG UCG CGG AUG CAC UCCUUC CUG CCG CUC ACC CAC AAG GIVE YOUR HAND

[0055] SEQ ID No.4: mRNA encoding the pre-fusion conformation S protein sequence (optimization scheme 3)

[0056] AAG CAC AAG GAC CAC GAC GGA GAC CAC UCG UCG GUC ACG CAC UUG GACUGG UGG UCC UGG GUC GAC GGA GGA CGG AUG UGG UUG UCG AAG UGG UCC CCG CAC AUGAUG GGA CUG UUC CAC AAG UCC UCG UCG CAC GAG GUG UCG UGG GUC CUG GAC AAG GACGGA AAG AAG UCG UUG CAC UGG ACC AAG GUG CGG UAG GUG CAC UCG CCG UGG UUG CCGUGG UUC UCC AAG CUG UUG GGA CAC GAC GGA AAG UUG CUG CCG CAC AUG AAG CGG UCGUGG CUC UUC UCG UUG UUG UGC CCG ACC UAG AAG CCG UGG UGG GAC CUG UCG UUCUGG GUC UCG GAC GAC UUG UUG CGG UUG CAC CAC UAG UUC CAC ACG CUCAAG GUC AAG ACG UUG CUG GGA AAG GAC CCG CAC AUG AUG GUG UUC UUG UUG UUC UCGACC UAC CUC UCG CUC AAG UCC CAC AUG UCG UCG CGG UUG UUG ACG UGG AGG CUC AUGCAC UCG GUC GGA AAG GAC UAC CUG GAC CUC CCG UUC GUC CCG UUG AAG UUC UUG GACUCC CUC AAG CAC AAG UUC UUG UAG CUG CCG AUG AAG UUC UAG UAG UCG UUC GUG UGGGGA UAG UUG GAC CAC UCC CUG GAC GGA GUC CCG AAG UCG CGG GAC CUC GGA GAC CACCUG GAC GGA UAG CCG UAG UUG UGG UCC AAG GUC UGG GGA CCG CUG UCGUCG UCG CCG ACC UGG CGG CCG CGG CGG CGG AUGAUG CAC CCG AUG GAC GUC GGA UCC UGG AAG GAC GAC UUC AUG UUG CUC UUG CCG UGGUAG UGG CUG CGG CAC CUG ACG CGG GAC CUG GGA GAC UCG CUC UGG UUC ACG UGG GACUUC UCG AAG UGG CAC CUC UUC CCG UAG AUG GUC UGG UCG UUG AAG UCC CAC GUC GGAUGG CUC UCG UAG CAC UCC AAG GGA UUG UUG UGG UGG UUG GAC ACG GGA AAG CCG CUC CACAAG UUG CGG UGG UGG UCC AAG CGG UCG CAC AUG CGG ACC UUG UCC UUC UCC UAG UUGACG CAC CGG CUG AUG UCG CAG GAC AUG UUG UCG CGG UCG AAG UCG UGG AAG UUC ACGAUG CCG CAC UCG GGA UGG UUC GAC UUG CUG GAC ACG AAG UGG UUG CAC AUG CGG CUGUCG AAG CAC UAG UCC CCG CUG CUC CAC UCC GUC UAG CGG GGA CCG GUC UGG CCG UUCUAG CGG CUG AUG UUG AUG UUC GAC GGA CUG CUG AAG UGG CCG ACG CAC UAG CGG ACCUUG UCG UUG UUG GAC CUG UCG UUC CAC CCG CCG UUG AUG UUG AUG GAC AUG UCC GACAAG UCC UCG UUG GAC UUC GGA AAG CUC UCC CUG UAG UCG UGG CUC UAG AUG GUCCGG CCG UCG UGG GGA ACG UUG CCG CAC CUC CCG AAG UUG ACG AUG AAG GGA GAC GUCUCG AUG CCG AAG GUC GGA UGG UUG CCG CAC CCG AUG GUC GGAAUG UCC CAC CAC CACGAC UCG AAG CUC GAC GUG CGG GGA CGG UGG CAC ACG CCG GGA UUC UUC UCG UGGUUG GAC CAC UUC UUG UUC ACG CAC UUG AAG UUG AAG UUG CCG GAC UGG CCG UGG CCGCAC GAC UGG CUC UCG UUG UUC UUC AAG GGA AAG GUC GUC AAG CCG UGC UGG UGG CAC UCC CUG GGA GUC UGG GAC CUC UGG UGC UAG GAC CUG UGG UGG UGG UCG UUG GUCCAC CGG CAC GAG AUG GUC CUG CAC UUG ACG UGG CUC CAC GGA CAC CGG UAG GUG CGGCUG GUC GAC UGG GGA UGG ACC UCC CAC AUG UCG UGG CCG UCG UUG CAC AAG GUC UGGUCC CGG CCG ACG GAC UAG CCG CGG CUC GUG CAC UUG UUG UCG AUG CUC ACG CUG UAGGGA UAG CCG CGG CCG UAG ACG CGG UCG AUG GUC UGG GUC UGG UUG UCG GGA UCC UCCCGG UCC UCG CAC CGG UCG GUC UCG UAG UAG CGG AUG UGG UAC UCG GAC CCG CGG CUUG UCG CAC CGG AUG UCG UUG UUG UCG UAG CGG UAG GGA UGG UUG AAG UGG UAG UCGCAC UGG UGG CUC UAG GAC GGA CAC UCG UAC UGG UUC UGG UCG CAC CUG ACG UGG UACAUG UAG ACG CCG CUG UCG UGG CUC ACG UCG UUG GAC GAC GAC GUC AUG CCG UCG AREAUGG GUC GAC UUG UCC CGG GAC UGG CCG UAG CGG CAC CUC GUC CUG UUC UUG UGGGUC CUC CAC AAG CGG GUC CAC UUC GUC UAG AUG UUC UGG GGA GGA UAG UUC CUG AAGCCG CCG AAG UUG AAG UCG GUC UAG GAC GGA CUG GGA UCG UUC GGA UCG UUC UCC UCGAAG UAG CUC CUG GAC GAC AAG UUG UUC CAC UGG GAC CGG CUG CGG CCG AAG UUG UUC GAC UUGUC AUG CCG CUG ACG GAC CCG CUG UAG CGG CGG UCC CUG GAC UAG ACG CGG GUC UUCAAG UUG CCG GAC UGG CAC GGA GGA GGA GCA GCA GCA UGG UGG CUG CUC UAC UAG CGG GUC AUGUGG UCG CGG GAC GGA GGA GCA GAC CGG CCG UGG UGG UCG CCG ACC UGG AAG CCG CGG CCG CGGCGG GAC GUC UAG GGA AAG CGG UAC GUC UAC CGG AUG UCC AAG UUG CCG UAG CCG CACUGG GUC UUG CAC GAC AUG CUC UUG GUC UUC GAC UAG CGG UUG GUC AAG UUG UCG CGGUAG CCG UUC UAG GUC CUG UCG GAC UCG UGG CGG UCG CGG GAC CCG UUC GAC GUCCUG CAC CAC UUG GUC UUG CUG UUG CUG UGG UGG GAC CAC UUC GUC GAC UCG UCGUUG AAG CCG CGG UAG UCG UCG CAC GAC UUG CUG UAG GAC UCG UCC GAC CUG UUC CACCUC CGG CUC CAC GUC UAG CUG UCC GAC UGG UGG CCG UCC GAC GUC UGGAUG CAC UGG GUC GUC GAC UAGUCC CGG CGG CUC UAG UCC CGG UCG CGG UUG GAC CGGCGG UGG UUC UAC UCG CUC ACG CAC GAC CCG GUC UCG UUC UCC CAC CUG AAG ACG CCGUUC CCG AUG GUG GAC UAC UCG AAG GGA GUC UCG CGG GGA GUG CCG CAC CAC AAG GACGUG CAC UGG AUG CAC GGA CGG GUC CUC UUC UUG AAG UGG UGG CGG GGA CGG UAG ACGGUG CUG CCG UUC CGG GUG AAG GGA UCC CUC CCG CAC AAG CAC UCG UUG CCG UGG GUGACC AAG CAC UGG GUC UCC UUG AAG AUG CUC GGA GUC UGG UGG UGG CAG UGG AUG CUC GGA GUC UGG UGG AUG CUC GGA GUC UGG UGG AUG CUGGGA GAC GUC GGA CUC GAC CUG UCG AAG UUC CUC CUC GAC CUG UUC AUG AAG UUC UUGGUG UGG UCG GGA CUC CAC CUG GAC CCG CUG UAG UCG CCG UAG UUG CGG UCG CAC CACUUG UAG GUC UUC CUC UAG CUC CAC CGG UUC UUG GAC UUG CUC UCGGAC UAG CUC GAC GU C CUC GAC CCG UUC AUG CUC GUC AUG CUC GUC AUG UAG UUC ACC GGA ACC AUGUAG ACC GAC CCG AAG UAG CGG CCG GAC UGG UAG CAC UAC CAC UGG UAC GACACG ACG UAC UGG UCG ACG ACG UCG ACG GAC UUC CCG ACG UCG CGG AUG CAC UCCUUC CUG CCG CUC ACC CAC AAG GAC ACU

[0057] SEQ ID No.5: mRNA encoding the pre-fusion conformation S protein sequence (optimized solution 4)

[0058] AAG CAC AAG GAC CAC GAC GGA GAC CAC UCG UCG GUC ACA CAC UUG GACUGU UGU UCU UGU GUC GAC GGA GGA CGA AUG UGU UUG UCG AAG UGU UCU CCU CCU CAC AUGAUG GGA CUG UUC CAC AAG UCU UCG UCG CAC GUG UCG UGU GUC CUG GAC AAG GACGGA AAG AAG UCG UUG CAC UUG ACC AAG GUG CGA UAG GUG CAC UCG CCU UUG UUG CCUUGU UUC UCU AAG CUG UUG GGA CAC GGA AAG UUG CUG CCU CAC AUG AAG CGA UCGUGU CUC UUC UCG UUG UUG UUG CCU ACC UAG AAG CGA UGU UUG GAC CUG UCG UUG GUC UCG GAC GAC UAG CAC UUG UUG CGA UUG UUG CCA CAC UAG UUC CAC ACA CUCAAG GUC AAG ACA UUG CUG GGA AAG GAC CCU CAC AUG AUG GUG UUC UUG UUC UCGACC UAC CUC UCG CUC AAG UCU CAC AUG UCG UCG CGA UUG UUG ACA UGU AAG CUC AUGCAC UCG GUC GGA AAG GAC UAC CUG GAC CCU UUC GUC CCU UUG AAG UUC UUG GACUCU CUC AAG CAC AAG UUC UUG UAG CUC GGA GUC CCU AAG UUG UUG GAC CCU AUG UGC UGC UGC UGC UGC UGC UGC UGC UGC CAC UCU CUG GAC GGA GUC CCU AAG UCG CGA GAC CUC GGA GAC CACCUG GAC GGA UAG CCU UAG UUG UGU UCU AAG GUC UGU GAC GGA GAC GAG GUG UCUUCG AUG GAC UGU GGA CCU CUG UCGUCG UCG CCU ACC UGU CGA CCU CGA CGA CGA AUGAUG CAC CCU AUG GAC GUC GGA UCU UGU AAG GAC GAC UUC AUG UUG CUC UUG CCU UGUUAG UGU CUG CGA CAC CUG ACA CGA GAC CUG GGA GAC UCG CUC UGU UUC ACA UGU GACUUC UCG AAG UGU CAC CUC UUC CCU UAG AUG GUC UGU UCG UUG AAG UCU CAC GUC GGAUGU CUC UCG UAG CAC UCU AAG GGA UUG UUG GAC ACA GGA AAG CCU CUC CACAAG UUG CGA UGU UCU AAG CGA UCG CAC AUG CGA ACC UUG UCU UUC UCU UCU UUG UUG UCG AAG UUGACA CAC CGA CUG AUG UCG CAG GAC AUG UUGACA CAC CGA CUG AUG UCG CGA UCG AAG UCG UGU AAG UUC ACAAUG CCU CAC UCG GGA UGU UUC GAC UUG CUG GAC ACA AAG UGU UUG CAC AUG CGA CUGUCG AAG CAC UAG UCU CCU CUG CAC UCU GUC UAG CGA GGA CCU GUC UGU CCU UUCUAG CGA CUG AUG UUG AUG UUC GAC GGA CUG CUG AAG UGU CCU ACA CAC UAG CGA ACCUUG UCG UUG UUG GAC CUG UCG UUC CAC CCU CCU UUG AUG UUG AUG GAC AUG UCU GACAAG UCU UCG UUG GAC UUC GGA AAG CUC UCU CUG UAG UCG UGU CUC UAG AUG GUCCGA CCU UCG UGU GGA ACA UUG CCU CAC CUC CCU AAG UUG ACA AUG AAG GGA GAC GUCUCG AUG CCU AAG GUC GGA UGU UUG CCU CAC CCU AUG GUC GGAAUG UCU CAC CAC CACGAC UCG AAG CUC GAC GUG CGA GGA CGA UGU CAC ACA CCU GGA UUC UUC UCG UGUUUG GAC CAC UUC UUG UUC ACA CAC UUG AAG UUG AAG UUG CCU GAC UUG CCU GAC UUG CCU UUC UUC AAG GGA AAG GUC GUC AAG CCU UCU CUG UGCGA CUG UUG UUG CUG CGA CAC UCU CUG GGA GUC UGU GAC CUC UUG GAG UUG GGA CCU UUG UUG UUG UCG UUG GUCCAC CGA CAC GAC AUG GUC CUG CAC UUG ACA UGU CUC CAC GGA CCA CGA UUG GUG GGA CCU UUG UUG UCG UUG GUCCAC CGA CAC GAC AUG GUC CUG CAC UUG ACA UGU CUC CAC GGA CAC CGA UAG GUG CGACUG GUC GAC UGU GGA UGU ACC UCU CAC AUG UCG UGU CCU UCG UUG CAC AAG GUC UGUUCU CGA CCU ACA GAC UAG CCU CGA CUC GUG CAC UUG UUG UCG AUG CUC ACA CUG UAGGGA UAG CCU CGA CCU UAG ACA CGA UCG AUG GUC UGU GUC UGU UUG UCG GGA UCU UCUCGA UCU UCG CAC CGA UCG GUC UCG UAG UAG CGA AUG UGU UAC UCG GAC CCU CGA CUCUUG UCG CAC CGA AUG UCG UUG UUG UUG UCG UAG CGA UGA UGU UUG AAG UGU UUG UCG UAG CGA UGA UGU UUG UAG UGCAC UGU UGU CUC UAG GGA CAC UCG UAC UGU UUC UGU UCG CAC CUG ACA UGU UACAUG UAG ACA CCU CUG UCG UGU CUC ACA UCG UUG GAC GAC GAC GUC AUG CCU UCG AAGACAUGU GUC GAC UUG UCU CGA GAC UGU CCU UAG CGA CAC CUC GUC CUG UUC UUG UGUGUC CUC CAC AAG CGA GUC CAC UUC GUC UAG AUG UUC UGU GGA GGA UAG UUC CUG AAGCCU CCU AAG UUG AAG UCG GUC UAG GAC GGA CUG GGA UCG UUC GGA UCG UUC UCU UCGAAG UAG CUC CUG GAC GAC AAG UUG UUC CAC UGU GAC CGA CUG CCA AAG UUCGUC AUG CCU CUG ACA GAC CCU CUG UAG CGA CGA UCU CUG GAC UAG ACA CGA GUC UUCAAG UUG CCU GAC UGU CAC GAC GGA GGA GGA GAC GAC UGU CUG CUC UAC UAG CGA GUC AUGUG UCG CGA GAC GCA CGA CCA CCU UGU UCG CCU ACC UGU AAG CCU CGA CCU CGACGA GAC GUC UAG GGA AAG CGA UAC GUC UAC CGA AUG UCU AAG UUG CCU UAG CCU CACUGU GUC UUG CAC GAC AUG CUC UUG GUC UUC GAC UAG CGA UUG GUC AAG UUG UCG CGAUAG CCU UUC UAG GUC CUG UCG GAC UCG UCG UGU CGA UCG CCA UUC GAC GUCCUG CAC CAC UUG GUC UUG CGA GUC CGA GAC UUG UGU GAC CAC UUC GUC GAC UCG UCGUUG AAG CCU CGA UAG UCG UCG CAC GAC UUG CUG UUG GAC UCG UCU GAC CUG UUC CACCUC CGA CUC CAC GUC UAG CUG UCU GAC UUG UCU CAG UUGAUG CAC UGU GUC GUC GAC UUGUCU CGA CGA CUC UAG UCU CGA UCG CGA UUG GAC CGACGA UGU UUC UAC UCG CUC ACA CAC GAC CCU GUC UCG UUC UCU CAC CUG AAG ACA CCUUUC CCU AUG GUG GAC UAC UCG AAG GGA GUC UCG CGA GGA GUC CCU CAC CAC AAG GACGUG CAC UGU AUG CAC GGA CGA GUC CUC UUC UUG AAG UGU UGU CGA GGA CGA UAG ACAGUG CUG CCU UUC CGA GUG AAG GGA UCU CUC CAC AAG CAC UCG UUG CCU UGU GUGACC AAG CAC UGU GUC UCU UUG AAG AUG CUC GGA GUC UAG UGU UGU CUG UUG UGUAAG CAC UCG CCU UUG ACA CUG CAC CAC UAG CCU UUG UUG UGU CAG AUG CUGGGA GAC GUC GGA CUC GAC CUG UCG AAG UUC CUC CUC GAC CUG UUC AUG AAG UUC UUGGUG UGU UCG GGA CUC CAC CUG GAC CCU CUG UAG UCG CCU UAG UUG CGA UCG CAC CACUUG UAG GUC UUC CUC UAG CUG UCU GAC UUG CUC CAC CGA UUC UUG GAC UUG CUC UCGGAC UAG CUG GAC GUC CUC GAC CCU UUC AUG CUC GUC AUG UAG UUC ACC GGA ACC AUGUAG ACC GAC CCU AAG UAG CGA CCU GAC UAG CGA UAC CAC UGU UAG UAC GACACA ACA UAC UGU UCG ACA ACA UCG ACA GAC UUC CCU ACA ACA UCG CGA AUG CAC UCUUUC CUG CCU CUC ACC CAC AAG GAC ACU

[0059] SEQ ID No.6: Adjuvant sequence (1)

[0060] UAC CGC CAC UAC CGC GGC GCG UGG GAC CAC GAC GAC GAC UCG CCG CGC GACCGC GAC UGG GUC UGG ACC CGC

[0061] SEQ ID No.7: Adjuvant sequence (2)

[0062] UAC CGC UAA UAC CGC GGC GCG UGG UGG GAC CAC GAC GAC GAC UCG CCG CGC GACCGC GAC UGG

[0063] SEQ ID No.8: Adjuvant sequence (3)

[0064] UAC GCG CAC UGG CGC GGC GCG UGG CAC GAC GAC GAC GAC UCG CGC CGC GACCGC GAC UGG CUU UGG ACC CGC

[0065] SEQ ID No.9: Adjuvant sequence (4)

[0066] UAC CUA ACC UGG ACC UAA GAC AAA GAC CAC CGC CGC CGC UGG GCG

[0067] SEQ ID No.10: DNA encoding the pre-fusion conformation S protein sequence

[0068] AAG CAC AAG GAC CAC GAC GAC GGG GAC CAC TCG TCG GTC ACG CAC TTG GACTGG TGG GCC TGG GTC GAC GGG GGG CGG ATG TGG TTG TCG AAG TGG GCC CCG CAC ATGATG GGG CTG TTC CAC AAG GCC TCG TCG CAC GAC GTG TCG TGG GTC CTG GAC AAG GACGGG AAG AAG TCG TTG CAC TGG ACC AAG GTG CGG TAG GTG CAC TCG CCG TGG TTG CCGTGG TTC GCC AAG CTG TTG GGG CAC GAC GGG AAG TTG CTG CCG CAC ATG AAG CGG TCGTGG CTC TTC TCG TTG TAG TAG GCC CCG ACC TAG AAG CCG TGG TGG GAC CTG TCG TTCTGG GTC TCG GAC GAC TAG CAC TTG TTG CGG TGG TTG CAC CAC TAG TTC CAC ACG CTCAAG GTC AAG ACG TTG CTG GGG AAG GAC CCG CAC ATG ATG GTG TTC TTG TTG TTC TCGACC TAC CTC TCG CTC AAG GCC CAC ATG TCG TCG CGG TTG TTG ACG TGG AAG CTC ATGCAC TCG GTC GGG AAG GAC TAC CTG GAC CTC CCG TTC GTC CCG TTG AAG TTC TTG GACGCC CTC AAG CAC AAG TTC TTG TAG CTG CCG ATG AAG TTC TAG ATG TCG TTC GTG TGGGGG TAG TTG GAC CAC GCC CTG GAC GGG GTC CCG AAG TCG CGG GAC CTC GGG GAC CACCTG GAC GGG TAG CCG TAG TTG TAG TGG GCC AAG GTC TGG GAC GAC CGG GAC GTG GCCTCG ATG GAC TGG GGG CCG CTG TCGTCG TCG CCG ACC TGG CGG CCG CGG CGG CGG ATGATG CAC CCG ATG GAC GTC GGG GCC TGG AAG GAC GAC TTC ATG TTG CTC TTG CCG TGGTAG TGG CTG CGG CAC CTG ACG CGG GAC CTG GGG GAC TCG CTC TGG TTC ACG TGG GACTTC TCG AAG TGG CAC CTC TTC CCG TAG ATG GTC TGG TCG TTG AAG GCC CAC GTC GGGTGG CTC TCG TAG CAC GCC AAG GGG TTG TAG TGG TTG GAC ACG GGG AAG CCG CTC CACAAG TTG CGG TGG GCC AAG CGG TCG CAC ATG CGG ACC TTG GCC TTC GCC TAG TCG TTGACG CAC CGG CTG ATG TCG CAC GAC ATG TTG TCG CGG TCG AAG TCG TGG AAG TTC ACGATG CCG CAC TCG GGG TGG TTC GAC TTG CTG GAC ACG AAG TGG TTG CAC ATG CGG CTGTCG AAG CAC TAG GCC CCG CTG CTC CAC GCC GTC TAG CGG GGG CCG GTC TGG CCG TTCTAG CGG CTG ATG TTG ATG TTC GAC GGG CTG CTG AAG TGG CCG ACG CAC TAG CGG ACCTTG TCG TTG TTG GAC CTG TCG TTC CAC CCG CCG TTG ATG TTG ATG GAC ATG GCC GACAAG GCC TTC TCG TTG GAC TTC GGG AAG CTC GCC CTG TAG TCG TGG CTC TAG ATG GTCCGG CCG TCG TGG GGG ACG TTG CCG CAC CTC CCG AAG TTG ACG ATG AAG GGG GAC GTCTCG ATG CCG AAG GTC GGG TGG TTG CCG CAC CCG ATG GTC GGGATG GCC CAC CAC CACGAC TCG AAG CTC GAC GAC GTG CGG GGG CGG TGG CAC ACG CCG GGG TTC TTC TCG TGGTTG GAC CAC TTC TTG TTC ACG CAC TTG AAG TTG AAG TTG CCG GAC TGG CCG TGG CCGCAC GAC TGG CTC TCG TTG TTC TTC AAG GAC GGG AAG GTC GTC AAG CCG GCC CTG TAGCGG CTG TGG TGG CTG CGG CAC GCC CTG GGG GTC TGG GAC CTC TAG GAC CTG TAG TGGGGG ACG TCG AAG CCG CCG CAC TCG CAC TAG TGG GGG CCG TGG TTG TGG TCG TTG GTCCAC CGG CAC GAC ATG GTC CTG CAC TTG ACG TGG CTC CAC GGG CAC CGG TAG GTG CGGCTG GTC GAC TGG GGG TGG ACC GCC CAC ATG TCG TGG CCG TCG TTG CAC AAG GTC TGGGCC CGG CCG ACG GAC TAG CCG CGG CTC GTG CAC TTG TTG TCG ATG CTC ACG CTG TAGGGG TAG CCG CGG CCG TAG ACG CGG TCG ATG GTC TGG GTC TGG TTG TCG GGG GCC GCCCGG GCC TCG CAC CGG TCG GTC TCG TAG TAG CGG ATG TGG TAC TCG GAC CCG CGG CTCTTG TCG CAC CGG ATG TCG TTG TTG TCG TAG CGG TAG GGG TGG TTG AAG TGG TAG TCGCAC TGG TGG CTC TAG GAC GGG CAC TCG TAC TGG TTC TGG TCG CAC CTG ACG TGG TACATG TAG ACG CCG CTG TCG TGG CTC ACG TCG TTG GAC GAC GAC GTC ATG CCG TCG AAGACGTGG GTC GAC TTG GCC CGG GAC TGG CCG TAG CGG CAC CTC GTC CTG TTC TTG TGGGTC CTC CAC AAG CGG GTC CAC TTC GTC TAG ATG TTC TGG GGG GGG TAG TTC CTG AAGCCG CCG AAG TTG AAG TCG GTC TAG GAC GGG CTG GGG TCG TTC GGG TCG TTC GCC TCGAAG TAG CTC CTG GAC GAC AAG TTG TTC CAC TGG GAC CGG CTG CGG CCG AAG TAG TTCGTC ATG CCG CTG ACG GAC CCG CTG TAG CGG CGG GCC CTG GAC TAG ACG CGG GTC TTCAAG TTG CCG GAC TGG CAC GAC GGG GGG GAC GAC TGG CTG CTC TAC TAG CGG GTC ATGTGG TCG CGG GAC GAC CGG CCG TGG TAG TGG TCG CCG ACC TGG AAG CCG CGG CCG CGGCGG GAC GTC TAG GGG AAG CGG TAC GTC TAC CGG ATG GCC AAG TTG CCG TAG CCG CACTGG GTC TTG CAC GAC ATG CTC TTG GTC TTC GAC TAG CGG TTG GTC AAG TTG TCG CGGTAG CCG TTC TAG GTC CTG TCG GAC TCG TCG TGG CGG TCG CGG GAC CCG TTC GAC GTCCTG CAC CAC TTG GTC TTG CGG GTC CGG GAC TTG TGG GAC CAC TTC GTC GAC TCG TCGTTG AAG CCG CGG TAG TCG TCG CAC GAC TTG CTG TAG GAC TCG GCC GAC CTG TTC CACCTC CGG CTC CAC GTC TAG CTG GCC GAC TAG TGG CCG GCC GAC GTC TCG GAC GTC TGGATG CAC TGG GTC GTC GAC TAGGCC CGG CGG CTC TAG GCC CGG TCG CGG TTG GAC CGGCGG TGG TTC TAC TCG CTC ACG CAC GAC CCG GTC TCG TTC GCC CAC CTG AAG ACG CCGTTC CCG ATG GTG GAC TAC TCG AAG GGG GTC TCG CGG GGG GTG CCG CAC CAC AAG GACGTG CAC TGG ATG CAC GGG CGG GTC CTC TTC TTG AAG TGG TGG CGG GGG CGG TAG ACGGTG CTG CCG TTC CGG GTG AAG GGG GCC CTC CCG CAC AAG CAC TCG TTG CCG TGG GTGACC AAG CAC TGG GTC GCC TTG AAG ATG CTC GGG GTC TAG TAG TGG TGG CTG TTG TGGAAG CAC TCG CCG TTG ACG CTG CAC CAC TAG CCG TAG CAC TTG TTG TGG CAC ATG CTGGGG GAC GTC GGG CTC GAC CTG TCG AAG TTC CTC CTC GAC CTG TTC ATG AAG TTC TTGGTG TGG TCG GGG CTG CAC CTG GAC CCG CTG TAG TCG CCG TAG TTG CGG TCG CAC CACTTG TAG GTC TTC CTC TAG CTG GCC GAC TTG CTC CAC CGG TTC TTG GAC TTG CTC TCGGAC TAG CTG GAC GTC CTC GAC CCG TTC ATG CTC GTC ATG TAG TTC ACC GGG ACC ATGTAG ACC GAC CCG AAG TAG CGG CCG GAC TAG CGG TAG CAC TAC CAC TGG TAG TAC GACACG ACG TAC TGG TCG ACG ACG TCG ACG GAC TTC CCG ACG ACG TCG CGG ATG CAC GCCTTC CTG CCG CTC ACC CAC AAG GAC ACT

[0069] SEQ ID No.11: DNA encoding the pre-fusion conformation S protein sequence

[0070] AAG CAC AAG GAC CAC GAC GAC GGG GAC CAC TCG TCG GTC ACG CAC TTG GACTGG TGG TCC TGG GTC GAC GGG GGG CGG ATG TGG TTG TCG AAG TGG TCC CCG CAC ATGATG GGG CTG TTC CAC AAG TCC TCG TCG CAC GAC GTG TCG TGG GTC CTG GAC AAG GACGGG AAG AAG TCG TTG CAC TGG ACC AAG GTG CGG TAG GTG CAC TCG CCG TGG TTG CCGTGG TTC TCC AAG CTG TTG GGG CAC GAC GGG AAG TTG CTG CCG CAC ATG AAG CGG TCGTGG CTC TTC TCG TTG TAG TAG TCC CCG ACC TAG AAG CCG TGG TGG GAC CTG TCG TTCTGG GTC TCG GAC GAC TAG CAC TTG TTG CGG TGG TTG CAC CAC TAG TTC CAC ACG CTCAAG GTC AAG ACG TTG CTG GGG AAG GAC CCG CAC ATG ATG GTG TTC TTG TTG TTC TCGACC TAC CTC TCG CTC AAG TCC CAC ATG TCG TCG CGG TTG TTG ACG TGG AAG CTC ATGCAC TCG GTC GGG AAG GAC TAC CTG GAC CTC CCG TTC GTC CCG TTG AAG TTC TTG GACTCC CTC AAG CAC AAG TTC TTG TAG CTG CCG ATG AAG TTC TAG ATG TCG TTC GTG TGGGGG TAG TTG GAC CAC TCC CTG GAC GGG GTC CCG AAG TCG CGG GAC CTC GGG GAC CACCTG GAC GGG TAG CCG TAG TTG TAG TGG TCC AAG GTC TGG GAC GAC CGG GAC GTG TCCTCG ATG GAC TGG GGG CCG CTG TCGTCG TCG CCG ACC TGG CGG CCG CGG CGG CGG ATGATG CAC CCG ATG GAC GTC GGG TCC TGG AAG GAC GAC TTC ATG TTG CTC TTG CCG TGGTAG TGG CTG CGG CAC CTG ACG CGG GAC CTG GGG GAC TCG CTC TGG TTC ACG TGG GACTTC TCG AAG TGG CAC CTC TTC CCG TAG ATG GTC TGG TCG TTG AAG TCC CAC GTC GGGTGG CTC TCG TAG CAC TCC AAG GGG TTG TAG TGG TTG GAC ACG GGG AAG CCG CTC CACAAG TTG CGG TGG TCC AAG CGG TCG CAC ATG CGG ACC TTG TCC TTC TCC TAG TCG TTGACG CAC CGG CTG ATG TCG CAC GAC ATG TTG TCG CGG TCG AAG TCG TGG AAG TTC ACGATG CCG CAC TCG GGG TGG TTC GAC TTG CTG GAC ACG AAG TGG TTG CAC ATG CGG CTGTCG AAG CAC TAG TCC CCG CTG CTC CAC TCC GTC TAG CGG GGG CCG GTC TGG CCG TTCTAG CGG CTG ATG TTG ATG TTC GAC GGG CTG CTG AAG TGG CCG ACG CAC TAG CGG ACCTTG TCG TTG TTG GAC CTG TCG TTC CAC CCG CCG TTG ATG TTG ATG GAC ATG TCC GACAAG TCC TTC TCG TTG GAC TTC GGG AAG CTC TCC CTG TAG TCG TGG CTC TAG ATG GTCCGG CCG TCG TGG GGG ACG TTG CCG CAC CTC CCG AAG TTG ACG ATG AAG GGG GAC GTCTCG ATG CCG AAG GTC GGG TGG TTG CCG CAC CCG ATG GTC GGGATG TCC CAC CAC CACGAC TCG AAG CTC GAC GAC GTG CGG GGG CGG TGG CAC ACG CCG GGG TTC TTC TCG TGGTTG GAC CAC TTC TTG TTC ACG CAC TTG AAG TTG AAG TTG CCG GAC TGG CCG TGG CCGCAC GAC TGG CTC TCG TTG TTC TTC AAG GAC GGG AAG GTC GTC AAG CCG TCC CTG TAGCGG CTG TGG TGG CTG CGG CAC TCC CTG GGG GTC TGG GAC CTC TAG GAC CTG TAG TGGGGG ACG TCG AAG CCG CCG CAC TCG CAC TAG TGG GGG CCG TGG TTG TGG TCG TTG GTCCAC CGG CAC GAC ATG GTC CTG CAC TTG ACG TGG CTC CAC GGG CAC CGG TAG GTG CGGCTG GTC GAC TGG GGG TGG ACC TCC CAC ATG TCG TGG CCG TCG TTG CAC AAG GTC TGGTCC CGG CCG ACG GAC TAG CCG CGG CTC GTG CAC TTG TTG TCG ATG CTC ACG CTG TAGGGG TAG CCG CGG CCG TAG ACG CGG TCG ATG GTC TGG GTC TGG TTG TCG GGG TCC TCCCGG TCC TCG CAC CGG TCG GTC TCG TAG TAG CGG ATG TGG TAC TCG GAC CCG CGG CTCTTG TCG CAC CGG ATG TCG TTG TTG TCG TAG CGG TAG GGG TGG TTG AAG TGG TAG TCGCAC TGG TGG CTC TAG GAC GGG CAC TCG TAC TGG TTC TGG TCG CAC CTG ACG TGG TACATG TAG ACG CCG CTG TCG TGG CTC ACG TCG TTG GAC GAC GAC GTC ATG CCG TCG AAGACGTGG GTC GAC TTG TCC CGG GAC TGG CCG TAG CGG CAC CTC GTC CTG TTC TTG TGGGTC CTC CAC AAG CGG GTC CAC TTC GTC TAG ATG TTC TGG GGG GGG TAG TTC CTG AAGCCG CCG AAG TTG AAG TCG GTC TAG GAC GGG CTG GGG TCG TTC GGG TCG TTC TCC TCGAAG TAG CTC CTG GAC GAC AAG TTG TTC CAC TGG GAC CGG CTG CGG CCG AAG TAG TTCGTC ATG CCG CTG ACG GAC CCG CTG TAG CGG CGG TCC CTG GAC TAG ACG CGG GTC TTCAAG TTG CCG GAC TGG CAC GAC GGG GGG GAC GAC TGG CTG CTC TAC TAG CGG GTC ATGTGG TCG CGG GAC GAC CGG CCG TGG TAG TGG TCG CCG ACC TGG AAG CCG CGG CCG CGGCGG GAC GTC TAG GGG AAG CGG TAC GTC TAC CGG ATG TCC AAG TTG CCG TAG CCG CACTGG GTC TTG CAC GAC ATG CTC TTG GTC TTC GAC TAG CGG TTG GTC AAG TTG TCG CGGTAG CCG TTC TAG GTC CTG TCG GAC TCG TCG TGG CGG TCG CGG GAC CCG TTC GAC GTCCTG CAC CAC TTG GTC TTG CGG GTC CGG GAC TTG TGG GAC CAC TTC GTC GAC TCG TCGTTG AAG CCG CGG TAG TCG TCG CAC GAC TTG CTG TAG GAC TCG TCC GAC CTG TTC CACCTC CGG CTC CAC GTC TAG CTG TCC GAC TAG TGG CCG TCC GAC GTC TCG GAC GTC TGGATG CAC TGG GTC GTC GAC TAGTCC CGG CGG CTC TAG TCC CGG TCG CGG TTG GAC CGGCGG TGG TTC TAC TCG CTC ACG CAC GAC CCG GTC TCG TTC TCC CAC CTG AAG ACG CCGTTC CCG ATG GTG GAC TAC TCG AAG GGG GTC TCG CGG GGG GTG CCG CAC CAC AAG GACGTG CAC TGG ATG CAC GGG CGG GTC CTC TTC TTG AAG TGG TGG CGG GGG CGG TAG ACGGTG CTG CCG TTC CGG GTG AAG GGG TCC CTC CCG CAC AAG CAC TCG TTG CCG TGG GTGACC AAG CAC TGG GTC TCC TTG AAG ATG CTC GGG GTC TAG TAG TGG TGG CTG TTG TGGAAG CAC TCG CCG TTG ACG CTG CAC CAC TAG CCG TAG CAC TTG TTG TGG CAC ATG CTGGGG GAC GTC GGG CTC GAC CTG TCG AAG TTC CTC CTC GAC CTG TTC ATG AAG TTC TTGGTG TGG TCG GGG CTG CAC CTG GAC CCG CTG TAG TCG CCG TAG TTG CGG TCG CAC CACTTG TAG GTC TTC CTC TAG CTG TCC GAC TTG CTC CAC CGG TTC TTG GAC TTG CTC TCGGAC TAG CTG GAC GTC CTC GAC CCG TTC ATG CTC GTC ATG TAG TTC ACC GGG ACC ATGTAG ACC GAC CCG AAG TAG CGG CCG GAC TAG CGG TAG CAC TAC CAC TGG TAG TAC GACACG ACG TAC TGG TCG ACG ACG TCG ACG GAC TTC CCG ACG ACG TCG CGG ATG CAC TCCTTC CTG CCG CTC ACC CAC AAG GAC ACT

[0071] SEQ ID No.12: DNA encoding the pre-fusion conformation S protein sequence

[0072] AAG CAC AAG GAC CAC GAC GAC GGA GAC CAC TCG TCG GTC ACG CAC TTG GACTGG TGG TCC TGG GTC GAC GGA GGA CGG ATG TGG TTG TCG AAG TGG TCC CCG CAC ATGATG GGA CTG TTC CAC AAG TCC TCG TCG CAC GAC GTG TCG TGG GTC CTG GAC AAG GACGGA AAG AAG TCG TTG CAC TGG ACC AAG GTG CGG TAG GTG CAC TCG CCG TGG TTG CCGTGG TTC TCC AAG CTG TTG GGA CAC GAC GGA AAG TTG CTG CCG CAC ATG AAG CGG TCGTGG CTC TTC TCG TTG TAG TAG TCC CCG ACC TAG AAG CCG TGG TGG GAC CTG TCG TTCTGG GTC TCG GAC GAC TAG CAC TTG TTG CGG TGG TTG CAC CAC TAG TTC CAC ACG CTCAAG GTC AAG ACG TTG CTG GGA AAG GAC CCG CAC ATG ATG GTG TTC TTG TTG TTC TCGACC TAC CTC TCG CTC AAG TCC CAC ATG TCG TCG CGG TTG TTG ACG TGG AAG CTC ATGCAC TCG GTC GGA AAG GAC TAC CTG GAC CTC CCG TTC GTC CCG TTG AAG TTC TTG GACTCC CTC AAG CAC AAG TTC TTG TAG CTG CCG ATG AAG TTC TAG ATG TCG TTC GTG TGGGGA TAG TTG GAC CAC TCC CTG GAC GGA GTC CCG AAG TCG CGG GAC CTC GGA GAC CACCTG GAC GGA TAG CCG TAG TTG TAG TGG TCC AAG GTC TGG GAC GAC CGG GAC GTG TCCTCG ATG GAC TGG GGA CCG CTG TCGTCG TCG CCG ACC TGG CGG CCG CGG CGG CGG ATGATG CAC CCG ATG GAC GTC GGA TCC TGG AAG GAC GAC TTC ATG TTG CTC TTG CCG TGGTAG TGG CTG CGG CAC CTG ACG CGG GAC CTG GGA GAC TCG CTC TGG TTC ACG TGG GACTTC TCG AAG TGG CAC CTC TTC CCG TAG ATG GTC TGG TCG TTG AAG TCC CAC GTC GGATGG CTC TCG TAG CAC TCC AAG GGA TTG TAG TGG TTG GAC ACG GGA AAG CCG CTC CACAAG TTG CGG TGG TCC AAG CGG TCG CAC ATG CGG ACC TTG TCC TTC TCC TAG TCG TTGACG CAC CGG CTG ATG TCG CAC GAC ATG TTG TCG CGG TCG AAG TCG TGG AAG TTC ACGATG CCG CAC TCG GGA TGG TTC GAC TTG CTG GAC ACG AAG TGG TTG CAC ATG CGG CTGTCG AAG CAC TAG TCC CCG CTG CTC CAC TCC GTC TAG CGG GGA CCG GTC TGG CCG TTCTAG CGG CTG ATG TTG ATG TTC GAC GGA CTG CTG AAG TGG CCG ACG CAC TAG CGG ACCTTG TCG TTG TTG GAC CTG TCG TTC CAC CCG CCG TTG ATG TTG ATG GAC ATG TCC GACAAG TCC TTC TCG TTG GAC TTC GGA AAG CTC TCC CTG TAG TCG TGG CTC TAG ATG GTCCGG CCG TCG TGG GGA ACG TTG CCG CAC CTC CCG AAG TTG ACG ATG AAG GGA GAC GTCTCG ATG CCG AAG GTC GGA TGG TTG CCG CAC CCG ATG GTC GGAATG TCC CAC CAC CACGAC TCG AAG CTC GAC GAC GTG CGG GGA CGG TGG CAC ACG CCG GGA TTC TTC TCG TGGTTG GAC CAC TTC TTG TTC ACG CAC TTG AAG TTG AAG TTG CCG GAC TGG CCG TGG CCGCAC GAC TGG CTC TCG TTG TTC TTC AAG GAC GGA AAG GTC GTC AAG CCG TCC CTG TAGCGG CTG TGG TGG CTG CGG CAC TCC CTG GGA GTC TGG GAC CTC TAG GAC CTG TAG TGGGGA ACG TCG AAG CCG CCG CAC TCG CAC TAG TGG GGA CCG TGG TTG TGG TCG TTG GTCCAC CGG CAC GAC ATG GTC CTG CAC TTG ACG TGG CTC CAC GGA CAC CGG TAG GTG CGGCTG GTC GAC TGG GGA TGG ACC TCC CAC ATG TCG TGG CCG TCG TTG CAC AAG GTC TGGTCC CGG CCG ACG GAC TAG CCG CGG CTC GTG CAC TTG TTG TCG ATG CTC ACG CTG TAGGGA TAG CCG CGG CCG TAG ACG CGG TCG ATG GTC TGG GTC TGG TTG TCG GGA TCC TCCCGG TCC TCG CAC CGG TCG GTC TCG TAG TAG CGG ATG TGG TAC TCG GAC CCG CGG CTCTTG TCG CAC CGG ATG TCG TTG TTG TCG TAG CGG TAG GGA TGG TTG AAG TGG TAG TCGCAC TGG TGG CTC TAG GAC GGA CAC TCG TAC TGG TTC TGG TCG CAC CTG ACG TGG TACATG TAG ACG CCG CTG TCG TGG CTC ACG TCG TTG GAC GAC GAC GTC ATG CCG TCG AAGACGTGG GTC GAC TTG TCC CGG GAC TGG CCG TAG CGG CAC CTC GTC CTG TTC TTG TGGGTC CTC CAC AAG CGG GTC CAC TTC GTC TAG ATG TTC TGG GGA GGA TAG TTC CTG AAGCCG CCG AAG TTG AAG TCG GTC TAG GAC GGA CTG GGA TCG TTC GGA TCG TTC TCC TCGAAG TAG CTC CTG GAC GAC AAG TTG TTC CAC TGG GAC CGG CTG CGG CCG AAG TAG TTCGTC ATG CCG CTG ACG GAC CCG CTG TAG CGG CGG TCC CTG GAC TAG ACG CGG GTC TTCAAG TTG CCG GAC TGG CAC GAC GGA GGA GAC GAC TGG CTG CTC TAC TAG CGG GTC ATGTGG TCG CGG GAC GAC CGG CCG TGG TAG TGG TCG CCG ACC TGG AAG CCG CGG CCG CGGCGG GAC GTC TAG GGA AAG CGG TAC GTC TAC CGG ATG TCC AAG TTG CCG TAG CCG CACTGG GTC TTG CAC GAC ATG CTC TTG GTC TTC GAC TAG CGG TTG GTC AAG TTG TCG CGGTAG CCG TTC TAG GTC CTG TCG GAC TCG TCG TGG CGG TCG CGG GAC CCG TTC GAC GTCCTG CAC CAC TTG GTC TTG CGG GTC CGG GAC TTG TGG GAC CAC TTC GTC GAC TCG TCGTTG AAG CCG CGG TAG TCG TCG CAC GAC TTG CTG TAG GAC TCG TCC GAC CTG TTC CACCTC CGG CTC CAC GTC TAG CTG TCC GAC TAG TGG CCG TCC GAC GTC TCG GAC GTC TGGATG CAC TGG GTC GTC GAC TAGTCC CGG CGG CTC TAG TCC CGG TCG CGG TTG GAC CGGCGG TGG TTC TAC TCG CTC ACG CAC GAC CCG GTC TCG TTC TCC CAC CTG AAG ACG CCGTTC CCG ATG GTG GAC TAC TCG AAG GGA GTC TCG CGG GGA GTG CCG CAC CAC AAG GACGTG CAC TGG ATG CAC GGA CGG GTC CTC TTC TTG AAG TGG TGG CGG GGA CGG TAG ACGGTG CTG CCG TTC CGG GTG AAG GGA TCC CTC CCG CAC AAG CAC TCG TTG CCG TGG GTGACC AAG CAC TGG GTC TCC TTG AAG ATG CTC GGA GTC TAG TAG TGG TGG CTG TTG TGGAAG CAC TCG CCG TTG ACG CTG CAC CAC TAG CCG TAG CAC TTG TTG TGG CAC ATG CTGGGA GAC GTC GGA CTC GAC CTG TCG AAG TTC CTC CTC GAC CTG TTC ATG AAG TTC TTGGTG TGG TCG GGA CTG CAC CTG GAC CCG CTG TAG TCG CCG TAG TTG CGG TCG CAC CACTTG TAG GTC TTC CTC TAG CTG TCC GAC TTG CTC CAC CGG TTC TTG GAC TTG CTC TCGGAC TAG CTG GAC GTC CTC GAC CCG TTC ATG CTC GTC ATG TAG TTC ACC GGA ACC ATGTAG ACC GAC CCG AAG TAG CGG CCG GAC TAG CGG TAG CAC TAC CAC TGG TAG TAC GACACG ACG TAC TGG TCG ACG ACG TCG ACG GAC TTC CCG ACG ACG TCG CGG ATG CAC TCCTTC CTG CCG CTC ACC CAC AAG GAC ACT

[0073] SEQ ID No.13: DNA encoding the pre-fusion conformation S protein sequence

[0074] AAG CAC AAG GAC CAC GAC GAC GGA GAC CAC TCG TCG GTC ACA CAC TTG GACTGT TGT TCT TGT GTC GAC GGA GGA CGA ATG TGT TTG TCG AAG TGT TCT CCT CAC ATGATG GGA CTG TTC CAC AAG TCT TCG TCG CAC GAC GTG TCG TGT GTC CTG GAC AAG GACGGA AAG AAG TCG TTG CAC TGT ACC AAG GTG CGA TAG GTG CAC TCG CCT TGT TTG CCTTGT TTC TCT AAG CTG TTG GGA CAC GAC GGA AAG TTG CTG CCT CAC ATG AAG CGA TCGTGT CTC TTC TCG TTG TAG TAG TCT CCT ACC TAG AAG CCT TGT TGT GAC CTG TCG TTCTGT GTC TCG GAC GAC TAG CAC TTG TTG CGA TGT TTG CAC CAC TAG TTC CAC ACA CTCAAG GTC AAG ACA TTG CTG GGA AAG GAC CCT CAC ATG ATG GTG TTC TTG TTG TTC TCGACC TAC CTC TCG CTC AAG TCT CAC ATG TCG TCG CGA TTG TTG ACA TGT AAG CTC ATGCAC TCG GTC GGA AAG GAC TAC CTG GAC CTC CCT TTC GTC CCT TTG AAG TTC TTG GACTCT CTC AAG CAC AAG TTC TTG TAG CTG CCT ATG AAG TTC TAG ATG TCG TTC GTG TGTGGA TAG TTG GAC CAC TCT CTG GAC GGA GTC CCT AAG TCG CGA GAC CTC GGA GAC CACCTG GAC GGA TAG CCT TAG TTG TAG TGT TCT AAG GTC TGT GAC GAC CGA GAC GTG TCTTCG ATG GAC TGT GGA CCT CTG TCGTCG TCG CCT ACC TGT CGA CCT CGA CGA CGA ATGATG CAC CCT ATG GAC GTC GGA TCT TGT AAG GAC GAC TTC ATG TTG CTC TTG CCT TGTTAG TGT CTG CGA CAC CTG ACA CGA GAC CTG GGA GAC TCG CTC TGT TTC ACA TGT GACTTC TCG AAG TGT CAC CTC TTC CCT TAG ATG GTC TGT TCG TTG AAG TCT CAC GTC GGATGT CTC TCG TAG CAC TCT AAG GGA TTG TAG TGT TTG GAC ACA GGA AAG CCT CTC CACAAG TTG CGA TGT TCT AAG CGA TCG CAC ATG CGA ACC TTG TCT TTC TCT TAG TCG TTGACA CAC CGA CTG ATG TCG CAC GAC ATG TTG TCG CGA TCG AAG TCG TGT AAG TTC ACAATG CCT CAC TCG GGA TGT TTC GAC TTG CTG GAC ACA AAG TGT TTG CAC ATG CGA CTGTCG AAG CAC TAG TCT CCT CTG CTC CAC TCT GTC TAG CGA GGA CCT GTC TGT CCT TTCTAG CGA CTG ATG TTG ATG TTC GAC GGA CTG CTG AAG TGT CCT ACA CAC TAG CGA ACCTTG TCG TTG TTG GAC CTG TCG TTC CAC CCT CCT TTG ATG TTG ATG GAC ATG TCT GACAAG TCT TTC TCG TTG GAC TTC GGA AAG CTC TCT CTG TAG TCG TGT CTC TAG ATG GTCCGA CCT TCG TGT GGA ACA TTG CCT CAC CTC CCT AAG TTG ACA ATG AAG GGA GAC GTCTCG ATG CCT AAG GTC GGA TGT TTG CCT CAC CCT ATG GTC GGAATG TCT CAC CAC CACGAC TCG AAG CTC GAC GAC GTG CGA GGA CGA TGT CAC ACA CCT GGA TTC TTC TCG TGTTTG GAC CAC TTC TTG TTC ACA CAC TTG AAG TTG AAG TTG CCT GAC TGT CCT TGT CCTCAC GAC TGT CTC TCG TTG TTC TTC AAG GAC GGA AAG GTC GTC AAG CCT TCT CTG TAGCGA CTG TGT TGT CTG CGA CAC TCT CTG GGA GTC TGT GAC CTC TAG GAC CTG TAG TGTGGA ACA TCG AAG CCT CCT CAC TCG CAC TAG TGT GGA CCT TGT TTG TGT TCG TTG GTCCAC CGA CAC GAC ATG GTC CTG CAC TTG ACA TGT CTC CAC GGA CAC CGA TAG GTG CGACTG GTC GAC TGT GGA TGT ACC TCT CAC ATG TCG TGT CCT TCG TTG CAC AAG GTC TGTTCT CGA CCT ACA GAC TAG CCT CGA CTC GTG CAC TTG TTG TCG ATG CTC ACA CTG TAGGGA TAG CCT CGA CCT TAG ACA CGA TCG ATG GTC TGT GTC TGT TTG TCG GGA TCT TCTCGA TCT TCG CAC CGA TCG GTC TCG TAG TAG CGA ATG TGT TAC TCG GAC CCT CGA CTCTTG TCG CAC CGA ATG TCG TTG TTG TCG TAG CGA TAG GGA TGT TTG AAG TGT TAG TCGCAC TGT TGT CTC TAG GAC GGA CAC TCG TAC TGT TTC TGT TCG CAC CTG ACA TGT TACATG TAG ACA CCT CTG TCG TGT CTC ACA TCG TTG GAC GAC GAC GTC ATG CCT TCG AAGACATGT GTC GAC TTG TCT CGA GAC TGT CCT TAG CGA CAC CTC GTC CTG TTC TTG TGTGTC CTC CAC AAG CGA GTC CAC TTC GTC TAG ATG TTC TGT GGA GGA TAG TTC CTG AAGCCT CCT AAG TTG AAG TCG GTC TAG GAC GGA CTG GGA TCG TTC GGA TCG TTC TCT TCGAAG TAG CTC CTG GAC GAC AAG TTG TTC CAC TGT GAC CGA CTG CGA CCT AAG TAG TTCGTC ATG CCT CTG ACA GAC CCT CTG TAG CGA CGA TCT CTG GAC TAG ACA CGA GTC TTCAAG TTG CCT GAC TGT CAC GAC GGA GGA GAC GAC TGT CTG CTC TAC TAG CGA GTC ATGTGT TCG CGA GAC GAC CGA CCT TGT TAG TGT TCG CCT ACC TGT AAG CCT CGA CCT CGACGA GAC GTC TAG GGA AAG CGA TAC GTC TAC CGA ATG TCT AAG TTG CCT TAG CCT CACTGT GTC TTG CAC GAC ATG CTC TTG GTC TTC GAC TAG CGA TTG GTC AAG TTG TCG CGATAG CCT TTC TAG GTC CTG TCG GAC TCG TCG TGT CGA TCG CGA GAC CCT TTC GAC GTCCTG CAC CAC TTG GTC TTG CGA GTC CGA GAC TTG TGT GAC CAC TTC GTC GAC TCG TCGTTG AAG CCT CGA TAG TCG TCG CAC GAC TTG CTG TAG GAC TCG TCT GAC CTG TTC CACCTC CGA CTC CAC GTC TAG CTG TCT GAC TAG TGT CCT TCT GAC GTC TCG GAC GTC TGTATG CAC TGT GTC GTC GAC TAGTCT CGA CGA CTC TAG TCT CGA TCG CGA TTG GAC CGACGA TGT TTC TAC TCG CTC ACA CAC GAC CCT GTC TCG TTC TCT CAC CTG AAG ACA CCTTTC CCT ATG GTG GAC TAC TCG AAG GGA GTC TCG CGA GGA GTG CCT CAC CAC AAG GACGTG CAC TGT ATG CAC GGA CGA GTC CTC TTC TTG AAG TGT TGT CGA GGA CGA TAG ACAGTG CTG CCT TTC CGA GTG AAG GGA TCT CTC CCT CAC AAG CAC TCG TTG CCT TGT GTGACC AAG CAC TGT GTC TCT TTG AAG ATG CTC GGA GTC TAG TAG TGT TGT CTG TTG TGTAAG CAC TCG CCT TTG ACA CTG CAC CAC TAG CCT TAG CAC TTG TTG TGT CAC ATG CTGGGA GAC GTC GGA CTC GAC CTG TCG AAG TTC CTC CTC GAC CTG TTC ATG AAG TTC TTGGTG TGT TCG GGA CTG CAC CTG GAC CCT CTG TAG TCG CCT TAG TTG CGA TCG CAC CACTTG TAG GTC TTC CTC TAG CTG TCT GAC TTG CTC CAC CGA TTC TTG GAC TTG CTC TCGGAC TAG CTG GAC GTC CTC GAC CCT TTC ATG CTC GTC ATG TAG TTC ACC GGA ACC ATGTAG ACC GAC CCT AAG TAG CGA CCT GAC TAG CGA TAG CAC TAC CAC TGT TAG TAC GACACA ACA TAC TGT TCG ACA ACA TCG ACA GAC TTC CCT ACA ACA TCG CGA ATG CAC TCTTTC CTG CCT CTC ACC CAC AAG GAC ACT

[0075] Example 1

[0076] About 24 hours after 293T cells were passaged, the cell status in the 6-well plate was observed. When the confluence was about 90%, transfection could be performed. Take 200μl opti-MEM, add 10μg of different mRNAs encoding S protein antigen fragments after codon optimization, gently blow and mix with a gun tip, then add 60μl PEI (concentration 1mg / ml), immediately place on a vortex oscillator and oscillate 10 times, 1s each time, mix thoroughly, and let stand for 10min. The prepared transfection system was directly and evenly added to the cultured cells, and then shaken back and forth to make the transfection system evenly distributed on the cells. Change the medium 6h after transfection, suck off the old culture medium, and replace each well with 2ml of fresh culture medium (90% DMEM + 10% FBS). Harvest 30h after transfection. Aspirate the old culture medium and wash once with 1ml PBS. Aspirate the PBS, continue to blow down the cells with 1ml PBS, collect them in a 1.5ml centrifuge tube, and centrifuge at 300g for 5min. The supernatant after centrifugation was removed as cleanly as possible, the precipitated cells were lysed, and the total protein was extracted for Western blot detection. After electrophoresis transfer, antibody incubation was performed. For the primary antibody incubation of S protein, 3 μL of SARS-CoV-2 (2019-nCoV) Spike Antibody (Sino Biological, P / N 40591-MM42) was diluted into 3 ml of 5% skim milk (dilution ratio 1:1000), and incubated overnight at 4 °C in a shaker. The internal reference protein antibody was GAPDH (proteintech, P / N 60004-1-Ig). For the secondary antibody incubation, the antibodies for S protein and GAPDH protein were both from Mouse, so the same secondary antibody was used: 1 μL of HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L) (proteintech, P / N SA00001-1) was diluted into 5 ml of 5% skim milk (dilution ratio 1:5000), and incubated at room temperature for 1 h.

[0077] The experimental results are as follows Figure 2 As shown, wherein, M: protein marker, 1#: pre-fusion configuration S protein encoded by SEQ ID No.2, 2#: pre-fusion configuration S protein encoded by SEQ ID No.3, 3#: pre-fusion configuration S (prefusion) protein encoded by SEQ ID No.4, 4#: pre-fusion configuration S (prefusion) protein encoded by SEQ ID No.5.

[0078] The results showed that the expression level of the pre-fusion conformation S protein encoded by SEQ ID No.4 was significantly higher than that of the pre-fusion conformation S proteins encoded by SEQ ID No.2, SEQ ID No.3 and SEQ ID No.5.

[0079] Example 2

[0080] In this example, mRNA (SEQ ID No. 4) with different adjuvant sequences (SEQ ID No. 6-9) was transfected into 293T cells respectively, and the cell supernatant was extracted 24 hours later to see the level of S protein excretion in the supernatant. The specific steps are as follows:

[0081] 1. Coating: Dilute the mouse anti-S protein monoclonal antibody SARS-CoV-2 (2019-nCoV) Spike Antibody (Sino Biological, P / N 40591-MM42) with coating buffer to a 1000x solution, add it to the ELISA plate, add 100ul to each well, repeat 3 wells for each dilution, cover with sealing film, and coat overnight at 4°C.

[0082] 2. Washing: Pour out the coating solution from the coated 96-well plate onto absorbent paper and tap the plate vigorously until there is no residue in the wells. Prepare the elution solution, dilute 50x washing buffer with deionized water, add it to the inlet bottle of the plate washer, set the program, set the washing volume for each well to 300ul, and repeat the washing 4 times.

[0083] 3. Blocking: After washing the plate, drain the solution inside, add 250ul of Blocking buffer to each well, then seal the plate with sealing film and block at room temperature for 2 hours.

[0084] 4. Wash the plate: Wash the sealed ELISA plate according to step 3.

[0085] 5. Serum or standard incubation: Serum or standard (Sino Biological, 40591-V08H) was diluted in a gradient manner and added to the washed 96-well plate at a volume of 100ul per well. Then, the plate was sealed with a sealing film and incubated at room temperature for 1.5h.

[0086] 6. Wash the plate: Complete the plate washing according to step 3, and increase the number of plate washing times to 6 times.

[0087] 7. Primary antibody incubation: Dilute rabbit anti-S protein antibody SARS-CoV-2 (2019-nCoV) Spike Antibody (Sino Biological, P / N 40589-T62) (1:5000) with dilution buffer, add 100ul per well into the washed 96-well plate, then seal with sealing film and incubate at room temperature for 1.5h.

[0088] 8. Wash the plate: Complete the plate washing according to step 3, and increase the number of plate washing times to 6 times.

[0089] 9. Add secondary antibody: dilute HRP-labeled goat anti-rabbit IgG (abcam, ab6721) with dilution buffer at a dilution multiple of 10000x. Add 100ul of the diluted antibody to each well of the ELISA plate, seal the plate with a sealing film, and incubate at room temperature in the dark for 1 hour.

[0090] 10. Wash the plate: Follow step 8 to complete the plate washing. Be sure to wash the plate thoroughly and dry the solution.

[0091] 11. Color development: Add 100ul of TMB buffer and develop the color for 20-30 minutes in the dark. At this time, the positive sample will appear blue.

[0092] 12. Stop: Add 100ul of Stop buffer, read the result on the ELISA plate within 10 minutes, and set the absorption wavelength to 450nm.

[0093] 13. Draw a standard curve based on the light absorption value of the standard sample. The correlation coefficient of linear regression should be greater than 0.0995. Calculate the content of S protein in the sample based on the absorbance of the test sample.

[0094] The experimental results are as follows Figure 3 As shown. Wherein, Control: control group mRNA (SEQ ID No.4), 1#: mRNA with SEQ ID No.6 adjuvant sequence (SEQ ID No.4), 2#: mRNA with SEQ ID No.7 adjuvant sequence (SEQ ID No.4), 3#: mRNA with SEQ ID No.8 adjuvant sequence (SEQ ID No.4), 4#: mRNA with SEQ ID No.9 adjuvant sequence (SEQ ID No.4). The exocytosis effect of the pre-fusion conformation S protein encoded by the mRNA with SEQ ID No.7 adjuvant sequence (SEQ ID No.4) is significantly higher than that of the pre-fusion conformation S protein encoded by the mRNA with SEQ ID No.6 / SEQ ID No.8 / SEQ ID No.9 adjuvant sequence (SEQ ID No.4).

[0095] Example 3

[0096] In this example, mRNA (SEQ ID No. 4) carrying adjuvant sequences of SEQ ID No. 5-9 was injected twice into 6-week-old balb / c mice on day 1 and day 14, and mouse serum was collected on day 14 and day 35 to detect the titer of anti-S protein specific antibodies in the serum.

[0097] 1. Coating: Dilute S protein (Sino Biological, 40591-V08H) with coating buffer to a solution of 200 ng / ml, add to the ELISA plate, add 100 ul to each well, repeat 3 wells for each dilution, cover with sealing film, and coat overnight at 4°C.

[0098] 2. Washing: Pour out the coating solution from the coated 96-well plate onto absorbent paper and tap the plate vigorously until there is no residue in the wells. Prepare the elution solution, dilute 50x washing buffer with deionized water, add it to the inlet bottle of the plate washer, set the program, set the washing volume for each well to 300ul, and repeat the washing 4 times.

[0099] 3. Blocking: After washing the plate, drain the solution inside, add 250ul of Blocking buffer to each well, then seal the plate with sealing film and block at room temperature for 2 hours.

[0100] 4. Wash the plate: Wash the sealed ELISA plate according to step 3.

[0101] 5. Serum incubation: dilute mouse serum to 40x, 400x, 4000x, 40000x, 400000x, 4000000x, 40000000x with dilution buffer, add 100ul per well into the washed 96-well plate, then seal with sealing film and incubate at room temperature for 1.5h.

[0102] 6. Wash the plate: Complete the plate washing according to step 3, and increase the number of plate washing times to 6 times.

[0103] 7. Add secondary antibody: dilute HRP-labeled goat anti-mouse IgG with dilution buffer at a dilution multiple of 10000x. Add the diluted antibody to the ELISA plate at a volume of 100ul per well, seal the plate with sealing film, and incubate at room temperature in the dark for 1 hour.

[0104] 8. Wash the plate: Follow step 8 to complete the plate washing. Be sure to wash the plate thoroughly and dry the solution.

[0105] 9. Color development: Add 100ul of TMB buffer and develop the color for 20-30 minutes in the dark. At this time, the positive sample will appear blue.

[0106] 10. Stop: Add 100ul of Stop buffer, read the result on the ELISA plate within 10 minutes, and set the absorption wavelength to 450nm.

[0107] Draw a standard curve based on the light absorption value of the standard sample, and the correlation coefficient of linear regression should be greater than 0.0995. Calculate the residual content of S protein-specific antibodies in the sample based on the absorbance of the test sample.

[0108] The experimental results are as follows Figure 4 As shown, Control: control group mRNA (SEQ ID No.4), 1#: mRNA with SEQ ID No.6 adjuvant sequence (SEQ ID No.4), 2#: mRNA with SEQ ID No.7 adjuvant sequence (SEQ ID No.4), 3#: mRNA with SEQ ID No.8 adjuvant sequence (SEQ ID No.4), 4#: mRNA with SEQ ID No.9 adjuvant sequence (SEQ ID No.4).

[0109] The results showed that the immune effect of the candidate vaccine mRNA (SEQ ID No. 4) with the adjuvant sequence of SEQ ID No. 9 was better than that of the candidate vaccine mRNA (SEQ ID No. 4) with the adjuvant sequence of SEQ ID No. 6 / SEQ ID No. 7 / SEQ ID No. 8.

[0110] Example 4

[0111] In this embodiment, mRNA (SEQ ID No.4) with SEQ ID No.9 adjuvant sequence was injected twice on the 1st and 14th days at low doses (1ug / mouse) and high doses (30ug / mouse) into 6-week-old balb / c mice, and mouse serum was taken on the 35th day, and the neutralizing antibody titer was detected by focus reduction neutralization tests (FRNT) experiment. The standard dose of the new coronavirus 2019-nCoV was mixed with two-fold serial dilutions of 56°C inactivated serum and incubated at 37°C for 1 hour. The virus-serum mixture (100μl) was then inoculated into the cultured 293T ACE2 overexpressing cells. After 3 days of incubation, the plate was formalin fixed, permeabilized, blocked and stained by continuous incubation with biotin-coupled S protein monoclonal antibody, streptavidin-HRP and TrueBlue peroxidase substrate (KPL). The number of foci in the positive control group without serum was the highest, and the neutralization cutoff value was set when the foci count of the experimental group was ≤50%, and the geometric mean was calculated as technical replicates.

[0112] The results are as follows Figure 5The results show that, among them, Picture A: low-dose group (1ug / mouse) of mRNA (SEQ ID No.4) with SEQ ID No.9 adjuvant sequence, Picture B: high-dose group (30ug / mouse) of mRNA (SEQ ID No.4) with SEQ ID No.9 adjuvant sequence. The neutralizing antibody titer is directly proportional to the candidate vaccine vaccination dose, and the neutralizing antibody protection effect ID50 = 260.4 in the high-dose (30ug / mouse) group is significantly better than the low-dose (1ug / mouse) group, which is 41.03. Sequence Listing <110> Shenzhen Institute of Advanced Technology <120> Novel coronavirus mRNA vaccine and its preparation method and application <130> GAI20CN2857 <160> 13 <170> PatentIn version 3.5 <210> 1 <211> 1261 <212> PRT <213> Artificial Sequence <220> <223> Antigens against the novel coronavirus (SARS-CoV-2) <400> 1 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1 5 10 15 Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 130 135 140 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 145 150 155 160 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 165 170 175 Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190 Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200 205 Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu 210 215 220 Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr 225 230 235 240 Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 245 250 255 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 260 265 270 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 275 280 285 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300 Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val 305 310 315 320 Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 325 330 335 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 340 345 350 Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu 355 360 365 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 370 375 380 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 385 390 395 400 Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 405 410 415 Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430 Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440 445 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 450 455 460 Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys 465 470 475 480 Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 485 490 495 Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val 500 505 510 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525 Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540 Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu 545 550 555 560 Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val 565 570 575 Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 580 585 590 Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 595 600 605 Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile 610 615 620 His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser 625 630 635 640 Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val 645 650 655 Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala 660 665 670 Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala 675 680 685 Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser 690 695 700 Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile 705 710 715 720 Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val 725 730 735 Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu 740 745 750 Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr 755 760 765 Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln 770 775 780 Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe 785 790 795 800 Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser 805 810 815 Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly 820 825 830 Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp 835 840 845 Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu 850 855 860 Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly 865 870 875 880 Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile 885 890 895 Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr 900 905 910 Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn 915 920 925 Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala 930 935 940 Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn 945 950 955 960 Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val 965 970 975 Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln 980 985 990 Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val 995 1000 1005 Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn 1010 1015 1020 Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys 1025 1030 1035 Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro 1040 1045 1050 Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val 1055 1060 1065 Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His 1070 1075 1080 Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn 1085 1090 1095 Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln 1100 1105 1110 Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val 1115 1120 1125 Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro 1130 1135 1140 Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn 1145 1150 1155 His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn 1160 1165 1170 Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu 1175 1180 1185 Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu 1190 1195 1200 Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu 1205 1210 1215 Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met 1220 1225 1230 Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys 1235 1240 1245 Ser Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Phe Leu 1250 1255 1260 <210> 2 <211> 3783 <212> RNA <213> Artificial Sequence <220> <223> mRNA encoding the pre-fusion conformation S protein sequence <400> 2 aagcacaagg accacgacga cggggaccac ucgucgguca cgcacuugga cuggugggcc 60 ugggucgacg gggggcggau gugguugucg aaguggggcc cgcacaugau ggggcuguuc 120 180. cacaaggccu cgucgcacga cgugucgugg guccuggaca aggacggga gaagucguug cacuggacca aggugcggua ggugcacucg ccgugguugc cgugguucgc caagcuguug gggcacgacg ggaaguugcu gccgcacaug aagcggucgu ggcucuucuc guuguaguag 360. gccccgaccu agaagccgug gugggaccug ucguucuggg ucucggacga cuagcacuug uugcgguggu ugcaccacua guuccacacg cucaagguca agacguugcu ggggaaggac ccgcacauga covered covered accused cgcucaaggc ccacaugucg 540. ucgcgguugu ugacgugga gcucaugcac ucggucggga aggacuaccu ggaccucccg 600. uucgucccgu ugaaguucuu ggacgcccuc aagcacaagu ucuuguagcu gccgaugaag 660. uucuagaugu cguucgugug gggguaguug gaccacgccc uggacggggu cccgaagucg cgggaccucg gggaccaccu ggacggguag ccguaguugu agggggccaa ggucugggac gaccgggacg uggccucgau ggacugggggg ccgcugucgu cgucgccgac cuggcggccg 780 840 uugccguggu aguggcugcg gcaccugacg cgggaccugg gggacucgcu cugguucacg 900 ugggacucu cgaaguggca ccucuucccg uagauggucu gguguugaa ggcccacgug 960 ggguggcucu cguagcacgc caagggguug uagugguugg acacggggaa gccgcuccac 1020 aaguugcggu gggccaagcg gucgcacaug cggaccuugg ccuucgccua gucguugacg 1080 caccggcuga ugucgcaacga cauguugucg cgguggaagu cguggaaguu cacgaugccg 1140 cacucgggggu gguucgacuu gcuggacacg aagugguugc acaugcggcu gucgaagcac 1200 uaggccccgc ugcuccacgc cgucuagcgg gggccggucu ggccguucua gcggcugaug 1260 uugauguucg acgggcugcu gaaguggccg acgcacuagc ggaccuuguc guuguuggac 1320 cugucguucc acccgccguu gauguugaug gacauggccg acaaggccuu cucguuggac 1380 uucgggaagc ucgcccugua gucgggcuc uagaugggucc ggccgucgug ggggacguug 1440 ccgcaccucc cgaaguugac gaugaagggg gacgucucga ugccgaaggu cgggugguug 1500 ccgcaccga uggcgggau ggcccaccac cacgacucga agcucggacga cgugcgggg 1560 cgguggcaca cgccgggguu cuucucgugg uuggaccacu ucuuguucac gcacuugaag 1620 uugaaguugc cggacuggcc guggccgcac gacuggcucu cguuguucuu caaggacggg 1680 aaggucguca agccggcccu guagcggcug ugguggcugc ggcacgcccu gggggucugg 1740 gaccucuagg accuguagug ggggacgucg aagccgccgc acucgcacua guggggccg 1800 ugguuuguggu cguuggucca ccggcacgac augguccugc acuugacgug gcuccacggg 1860 caccgguagg ugcggcuggu cgacuggggg uggaccgccc acaugucgug gccgucguug 1920 1980 acgcuguagg gguagccgcg gccguagacg cggucgaugg ucugggucug guugucgggg 2040 gccgccccgg cccgcaccg gucgguccg uaguagcgga uguggguacuc ggacccgcgg 2100 cucuugucgc accggauguc guuguugucg uagcgguagg ggugguugaa gugguagucg 2160 2220 2280 gucgacuugg cccgggacug gccguagcgg caccucgucc ugucuugugg gguccuccac 2340 aagcgggucc acuucgucua gauguucugg gggggguagu uccugaagcc gccgaaguug 2400 2460 gacaaguugu uccacuggga ccggcugcgg ccgaaguagu ucgucaugcc gcugacggac 2520 ccgcuguagc ggcgggcccu ggacuagacg cgggucuuca aguugccgga cuggcacgac 2580 ggggggggacg acuggcugcu cuacuagcgg gucauguggu cgcgggacga ccggccgugg 2640 uaguggucgc cgaccuggaa gccgcggccg cggcgggacg ucuaggggaa gcgguacguc 2700 uaccggaugg ccaaguugcc guagccgcac ugggucuugc acgacaugcu cuuggucuuc 2760 gacuagcggu uggucaaguu gucgcgguag ccguucuagg uccugucgga cucgucgugg 2820 cggucgcggg acccguucga cguccugcac cauuggucu ugcggguccg gcauugugg 2880 gaccacuucg ucgacucguc guugaagccg cgguagucgu cgcacgacuu gcuguaggac 2940 ucggccgacc uguucccaccu ccggcuccac gucuagcugg ccgacuagug gccggccgac 3000 gugucggacg ucuggucca cugggucguc gacuaggccc ggcggcucua ggcccggucg 3060 cgguuggacc ggcggugguu cuacucgcuc acgcacgacc cggucucguu cgcccaccug 3120 aagacgccgu ucccgauggu ggacuacucg aagggggcucu cgcgggggu gccgcaccac 3180 aaggaacgugc acuggaugca cgggcgguc cucuucuuga agugguggcg ggggcgguag 3240 acggugcugc cguuccgggu gaagggggcc cucccgcaca agcacucguu gccgugggug 3300 accaagcacu gggucgccuu gaaugcuc gggucuagu agugguggcu guuguggaag 3360 3420 gucggggcucg accugucgaa guuccuccuc gaccuguuca ugaaguucuu gguguggucg 3480 gggcugcacc uggacccgcu guagucgccg uaguugcggu cgcaccacuu guaggucuuc 3540 3600 cucgaccgu ucaugcucgu cauguaguuc accgggacca ugaagaccga cccgaaguag 3660 cggccggacu agcgguagca cuaccacugg uaguacgaca cgacguacug gucgacgacg 3720 ucgacggacu ucccgacgac gucgcggaug cacgccuucc ugccgcucac ccacaaggac 3780 acu 3783 <210> 3 <211> 3783 <212> RNA <213> Artificial Sequence <220> <223> mRNA encoding the pre-fusion conformation S protein sequence <400> 3 aagcacaagg accacgacga cggggaccac ucgucgguca cgcacuugga cugguggucc 60 ugggucgacg gggggcggau gugguugucg aagugguccc cgcacaugau ggggcuguuc 120 cacaaguccu cgucgcacga cgugucgugg guccuggaca aggacgggaa gaagucguug 180 cacuggacca aggugcggua ggugcacucg ccgugguugc cgugguucuc caagcuguug 240 gggcacgacg ggaaguugcu gccgcacaug aagcggucgu ggcucuucuc guuguaguag 300 uccccgaccu agaagccgug gugggaccug ucguucuggg ucucggacga cuagcacuug 360 uugcgguggu ugcaccacua guuccacacg cucaagguca agacguugcu ggggaaggac 420 ccgcacauga ugguguucuu guguucucg accuaccucu cgcucaaguc ccacaugucg 480 ucgcgguugu ugacguggaa gcucaugcac ucggucggga aggacuaccu ggaccucccg 540 uucgucccgu ugaaguucuu ggacucccuc aagcacaagu ucuuguagcu gccgaugaag 600 ucuagaugu cguucgugug ggguaguug gaccacuccc uggacggggu cccgaagucg 660 cgggaccucg gggaccaccu ggacggguag ccguaguugu agugguccaa ggucugggac 720 gaccgggacg uguccucgau ggacugggggg ccgcugucgu cgucgccgac cuggcggccg 780 cggcggcgga ogaugcaccc gggucgcgcgcgcgcgc 840 uugccguggu aguggcugcg gcaccugacg cgggaccugg gggacucgcu cugguucacg 900 ugggacuucu cgaaguggca ccucuucccg uagaugucucu ggucguugaa gucccacguc 960 ggguggcucu cguagcacuc cagggguug uagugguugg acacggggaa gccgcuccac 1020 aaguugcggu guccaagcg gucgcacaug cggaccuug ccucuccua gucguugacg 1080 caccggcuga ugucgcacga cauguugcg cgucgaug cguggaaguu cacgaugccg 1140 cacucgggggu gguucgacuu gcuggacacg aagugguugc acaugcggcu gucgaagcac 1200 uaguccccgc ugcuccacuc cgucuagcgg gggccggucu ggccguucua gcggcugaug 1260 uugauguucg acgggcugcu gaaguggccg acgcacuagc ggaccuuguc guuguuggac 1320 cugucguucc acccgccguu gauguugaug gacauguccg acaaguccuu cucguuggac 1380 1440 ccgcaccucc cgaaguugac gaugaagggg gacgucucga ugccgaaggu cgggugguug 1500 1560 cgguggcaca cgccgggguu cuucucgugg uuggaccacu ucuuguucac gcacuugaag 1620 uugaaguugc cggacuggcc guggccgcac gacuggcucu cguuguucuu caaggacggg 1680 aaggucguca agccgucccu guagcggcug ugguggcugc ggcacucccu gggggucugg 1740 gaccucuagg accuguagug ggggacgucg aagccgccgc acucgcacua guggggccg 1800 ugguuuguggu cguuggucca ccggcacgac augguccugc acuugacgug gcuccacggg 1860 1920 1980 acgcuguagg gguagccgcg gccguagacg cggucgaugg ucugggucug guugucgggg 2040 uccucccggu ccucgcaccg gucgguccg uaguagcgga uguggguacuc ggacccgcgggg 2100 cucuugucgc accggauguc guuguugucg uagcgguagg ggugguugaa gugguagucg 2160 2220 2280 gucgacuugu cccgggacug gccguagcgg caccucgucc uguucuguug gguccuccac 2340 aagcgggucc acuucgucua gauguucugg gggggguagu uccugaagcc gccgaaguug 2400 2460 gacaaguugu uccacuggga ccggcugcgg ccgaaguagu ucgucaugcc gcugacggac 2520 ccgcuguagc ggcggucccu ggacuagacg cgggucuuca aguugccgga cuggcacgac 2580 ggggggggacg acuggcugcu cuacuagcgg gucauguggu cgcgggacga ccggccgugg 2640 uaguggucgc cgaccuggaa gccgcggccg cggcgggacg ucuaggggaa gcgguacguc 2700 uaccggaugu ccaaguugcc guagccgcac ugggucuugc acgacaugcu cuuggucuuc 2760 gacuagcggu uggucaaguu gucgcgguag ccguucuagg uccugucgga cucgucgugg 2820 cggucgcggg acccguucga cguccugcac cauuggucu ugcggguccg gcauugugg 2880 gaccacuucg ucgacucguc guugaagccg cgguagucgu cgcacgacuu gcuguaggac 2940 ucguccgacc uguucccaccu ccggcuccac gucuagcugu ccgacuagug gccguccgac 3000 gugucggacg ucuggugca cugggucguc gacuaguccc ggcggcuua gucccggucg 3060 cgguuggucc ggcggugguu cuacucgcuc acgcacgacc cggucucgu cuccccaccug 3120 aagacgccgu ucccgauggu ggacuacucg aagggggcucu cgcgggggu gccgcaccac 3180 aaggaacgugc acuggaugca cgggcgguc cucuucuuga agugguggcg ggggcgguag 3240 acggugcugc cguuccgggu gaaggggucc cucccgcaca agcacucguu gccgugggug 3300 accaagcacu gggucuccuu gaagaugcuc ggggucuagu agugguggcu guuguggaag 3360 cacucgccgu ugacgcugca ccacuagccg uagcacuugu uguggcacau gcugggggac 3420 gucgggcucg accugucgaa guuccuccuc gaccuguuca ugaaguucuu gguguggucg 3480 gggcugcacc uggacccgcu guagucgccg uaguugcggu cgcaccacuu guaggucuuc 3540 cucuagcugu ccgacuugcu ccaccgguuc uuggacuugc ucucggacua gcuggacguc 3600 cucgacccgu ucaugcucgu cauguaguuc accgggacca uguagaccga cccgaaguag 3660 cggccggacu agcgguagca cuaccacugg uaguacgaca cgacguacug gucgacgacg 3720 ucgacggacu ucccgacgac gucgcggaug cacuccuucc ugccgcucac ccacaaggac 3780 acu 3783 <210> 4 <211> 3783 <212> RNA <213> Artificial Sequence <220> <223> mRNA encoding the pre-fusion conformation S protein sequence <400> 4 aagcacaagg accacgacga cggagaccac ucgucgguca cgcacuugga cugguggucc 60 ugggucgacg gaggacggau gugguugucg aagugguccc cgcacaugau gggacuguuc cacaaguccu cgucgcacga cgugucgugg cacaaguccu aggacggaa gaagucguug cacuggacca aggugcggua ggugcacucg ccgugguugc cgugguucuc caagcuguug 300. ggacacgacg gaaaguugcu gccgcacaug aagcggucgu ggcucuucuc guuguaguag 360. uccccgaccu agaagccgug gugggaccug ucguucuggg ucucggacga cuagcacuug uugcgguggu ugcaccacua guuccacacg cucaagguca agacguugcu gggaaaggac ccgcacauga covered covered accused cgcucauga ccacaugucg 540. ucgcgguugu ugacgugga gcucaugcac ucggucgga aggacuaccu ggaccucccg 600. uucgucccgu ugaaguucuu ggacucccuc aagcacaagu ucuuguagcu gccgaugaag 660. uucuagaugu cguucgugug gggauaguug gaccacuccc uggacggagu cccgaagucg cgggaccucg gagaccaccu ggacggauag ccguaguugu agugguccaa ggucugggac gaccgggacg uguccucgau ggacugggga ccgcugucgu cgucgccgac cuggcggccg 780 cggcggcgga cggccccccccccccccccccccccccqccqcccl 840 uugccguggu aguggcugcg gcaccugacg cgggaccugg gagacucgcu cugguucacg 900 ugggacuucu cgaaguggca ccucuucccg uagaugucucu ggucguugaa gucccacguc 960 ggauggcucu cguagcacuc cagggauug uagugguugg acacgggaaa gccgcuccac 1020 aaguugcggu guccaagcg gucgcacaug cggaccuug ccucuccua gucguugacg 1080 caccggcuga ugucgcacga cauguugcg cgucgaug cguggaaguu cacgaugccg 1140 cacucgggau gguucgacuu gcuggacacg aagugguugc acugcggcu gucgaagcac 1200 uaguccccgc ugcuccacuc cgucuagcgg ggaccgucu ggccguucua gcggcugaug 1260 uugauguucg acggcugcu gaaguggccg acggcuagc ggcuugc 1320 cugucgucc accgccguu gaguugaug cugucguccg acaaguccuu cucguuggac 1380 uucggaaagc ucucccugua gucguggcuc uagauggucc ggccgucgug gggaacguug 1440 ccgcaccucc cgaagugac gagaaggga gagacucga ugccgaaggu cggauggug 1500 1560 cgguggcaca cgccgggauu cuucucgugg uuggaccacu ucuuguucac gcacuugaag 1620 uugaaguugc cggacuggcc guggccgcac gacuggcucu cguuguucuu caaggacgga 1680 aaggucguca agccgucccu guagcggcug ugguggcugc ggcacucccu gggagucugg 1740 gaccucuagg accuguagug gggaacgucg aagccgccgc acucgcacua guggggaccg 1800 ugguuuguggu cguuggucca ccggcacgac augguccugc acuugacgug gcuccacgga 1860 caccgguagg ugcggcuggu cgacuggga uggaccucc acaugucgug gccgucguug 1920 1980 acgcuguagg gauagccgcg gccguagacg cggucgaugg ucugggucug guugucggga 2040 uccucccggu ccucgcaccg gucgguccg uaguagcgga uguggguacuc ggacccgcgggg 2100 cucuugucgc accggauguc guuguugucg uagcgguagg gaugguuugaa gugguagucg 2160 2220 uagacgccgc ugucguggcu cacgucguug gacgacgacg ucaugccguc gaagacgugg 2280 gucgacuugu cccgggacug gccguagcgg caccucgucc uguucuugug gguccuccac 2340 aagcgggucc acuucgucua gauguucugg ggaggauagu uccugaagcc gccgaaguug 2400 aagucggucu aggacggacu gggaucguuc ggaucguucu ccucgaagua gcuccuggac 2460 gacaaguugu uccacuggga ccggcugcgg ccgaaguagu ucgucaugcc gcugacggac 2520 ccgcuguagc ggcggucccu ggacuagacg cgggucuuca aguugccgga cuggcacgac 2580 ggaggagacg acuggcugcu cuacuagcgg gucauguggu cgcgggacga ccggccgugg 2640 uaguggucgc cgaccuggaa gccgcggccg cggcgggacg ucuagggaaa gcgguacguc 2700 uaccggaugu ccaaguugcc guagccgcac ugggucuugc acgacaugcu cuuggucuuc 2760 gacuagcggu uggucaaguu gucgcgguag ccguucuagg uccugucgga cucgucgugg 2820 cggucgcggg acccguucga cguccugcac cacuuggucu ugcggguccg ggacuugugg 2880 gaccacuucg ucgacucguc guugaagccg cgguagucgu cgcacgacuu gcuguaggac 2940 ucguccgacc uguucccaccu ccggcuccac gucuagcugu ccgacuagug gccguccgac 3000 gugucggacg ucuggugca cugggucguc gacuaguccc ggcggcuua gucccggucg 3060 cgguuggucc ggcggugguu cuacucgcuc acgcacgacc cggucucgu cuccccaccug 3120 aagacgccgu ucccgauggu ggacuacucg aagggagucu cgcggggagu gccgcaccac 3180 3240 acggugcugc cguuccgggu gaagggaucc cucccgcaca agcacucguu gccgugggug 3300 accaagcacu gggucuccuu gaaugcuc ggagucuagu agugguggcu guuguggaag 3360 3420 gucggacucg accugucgaa guuccuccuc gaccuguuca ugaaguucuu gguguggucg 3480 ggacugcacc uggacccgcu guagucgccg uaguugcggu cgcaccacuu guaggucuuc 3540 3600 cucgaccgu ucaugcucgu cauguaguuc accggaacca ugaagaccga cccgaaguag 3660 cggccggacu agcgguagca cuaccacugg uaguacgaca cgacguacug gucgacgacg 3720 ucgacggacu ucccgacgac gucgcggaug cacuccuucc ugccgcucac ccacaaggac 3780 acu 3783 <210> 5 <211> 3783 <212> RNA <213> Artificial Sequence <220> <223> mRNA encoding the pre-fusion conformation S protein sequence <400> 5 aagcacaagg accacgacga cggagaccac ucgucgguca cacacuugga cuguuguucu 60 ugugucgacg gaggacgaau guguuugucg aaguguucuc cucacaugau gggacuguuc 120 cacaagucuu cgucgcacga cgugucgugu guccuggaca aggacggaaa gaagucguug 180 cacuguacca aggugcgaua ggugcacucg ccuuguuugc cuuguuucuc uaagcuguug 240 ggacacgacg gaaaguugcu gccucacaug aagcgaucgu gucucuucuc guuguaguag 300 ucuccuaccu agaagccuug uugugaccug ucguucugug ucucggacga cuagcacuug 360 uugcgauguu ugcaccacua guuccacaca cucaagguca agacauugcu gggaaaggac 420 ccucacauga ugguguucuu guuguucucg accuaccucu cgcucaaguc ucacaugucg 480 540 uucguccuu ugaaguucuu ggacucucuuc aagcaaagu ucuugaagcu gccuaugaag 600 660 cgagaccucg gagaccaccu ggacggauag ccuuaguugu aguguucuaa ggucugugac 720 780 840 uugccuuguu agugucugcg acaccugaca cgagaccugg gagacucgcu cuguuuaca 900 ugugacuucu cgaaguguca ccucuucccu uagauggucu guucguugaa gucucacguc 960 ggaugucucu cguagcacuc uaagggauug uaguguuugg acacaggaaa gccucuccac 1020 aaguugcgau guucuaagcg aucgcacaug cgaaccuugu cuuucucuua gucguugaca 1080 caccgacuga ugucgcacga cauguugucg cgaucgaagu cguguaaguu cacaaugccu 1140 cacucgggau guuucgacuu gcuggacaca aaguguuuugc acaugcgacu gucgaagcac 1200 1260 uugauguucg acggacugcu gaaguguccu acacacuagc gaaccuuguc guuguuggac 1320 cugucguucc acccucuuu gauguugaug gacaugucug acaagucuuu cuuguuggac 1380 uucggaaagc ucucucugua gucgugucuc uagauggucc gaccuucgug uggaacauug 1440 ccucaccucc cuaguugac aaugaaggga gacgucuucga ugccuaaggu cggauguuug 1500 1560 cgaugucaca caccuggauu cuucucgugu uuggaccacu ucuuguucac acacuugaag 1620 uugaaguugc cugacugucc uuguccucac gacugucucu cguuguucuu caaggacgga 1680 aaggucguca agccuucucu guagcgacug uguugucugc gacacucucu gggagucugu 1740 1800 uguuuuguu cguuggucca ccgacacgac augguccugc acuugacaug ucuccacgga 1860 Caccgauagg Ugggacugg Caccgauagg Uguaccucuc Acaugucgug Uccuuchugg 1920 cacaaggucu gucgacuc uacgacuag gucgacuc 1980 acacuguagg gauagccucg accuuagaca cgaucgaugg ucugucug uuugucggga 2040 ucuucucgau cucgcaccg aucgucucg uaguagcgaa uguguuacuc ggacccucga 2100 cucuugucgc accgaouguc guguagucg uagcgauagg gaguouga guguagucg 2160 cacuguuguc ucuaggacgg acacucguac uguuucuguu cgcaccugac auguacaug 2220 shaking oscillation storm shaking shaking shaking frequency 2280 gucgacuug cucgacug uccuuagcga cuccucgucc ugucuug uguccuccac 2340 aagcgagucc accuacgucua gauguucugu ggaggauagu accugaagcc accuaguug 2400 shake shake shake shake shake shake shake 2460 ccuagugu uccacuguga ccgacugcga ccuaguagu uccucaugcc ucugacagac 2520 ccucuguagc gggaucucu ggacuagc cgagucucu aguugccuga cuguccgac 2580 ggaggagacg acugucugcu cuacuagcga gucauguguu cgcgagacga ccgaccuugu 2640 uaguguucgc cuaccuguaa gccucgaccu cgacgagacg ucuagggaaa gcgauacguc 2700 uaccgaaugu cuaaguugcc uuagccucac ugugucuugc acgacaugcu cuuggucuuc 2760 gacuagcgau uggucaaguu gucgcgauag ccuuucuagg uccugucgga cucgucgugu 2820 cgaucgcgag acccuuucga cguccugcac cacuuggucu ugcgaguccg agacuugugu 2880 gaccacuucg ucgacucguc guugaagccu cgauagucgu cgcacgacuu gcuguaggac 2940 ucgucugacc uguuccaccu ccgacuccac gucuagcugu cugacuagug uccuucugac 3000 gucucggacg ucuguaugca cugugucguc gacuagucuc gacgacucua gucucgaucg 3060 cgauuggacc gacgauguuu cuacucgcuc acacacgacc cugucucguu cucucaccug 3120 aagacaccuu ucccuauggu ggacuacucg aagggagucu cgcgaggagu gccucaccac 3180 aaggacgugc acuguaugca cggacgaguc cucuucuuga aguguugucg aggacgauag 3240 acagugcugc cuuuccgagu gaagggaucu cucccucaca agcacucguu gccuugugug 3300 accaagcacu gugucucuuu gaagaugcuc ggagucuagu aguguugucu guuguguaag 3360 cacucgccuu ugacacugca ccacuagccu uagcacuugu ugugucacau gcugggagac 3420 gucggacucg accugucgaa guuccuccuc gaccuguuca ugaaguucuu gguguguucg 3480 ggacugcacc uggacccucu guagucgccu uaguugcgau cgcaccacuu guaggucuuc 3540 cucuagcugu cugacuugcu ccaccgauuc uuggacuugc ucucggacua gcuggacguc 3600 cucgacccuu ucaugcucgu cauguaguuc accggaacca uguagaccga cccuaaguag 3660 cgaccugacu agcgauagca cuaccacugu uaguacgaca caacauacug uucgacaaca 3720 ucgacagacu ucccuacaac aucgcgaaug cacucuuucc ugccucucac ccacaaggac 3780 acu 3783 <210> 6 <211> 72 <212> RNA <213> Artificial Sequence <220> <223> Adjuvant sequence <400> 6 uaccgccacu accgcggcgc gugggaccac gacgacgacu cgccgcgcga ccgcgacugg 60 gucuggaccc gc 72 <210> 7 <211> 60 <212> RNA <213> Artificial Sequence <220> <223> Adjuvant sequence <400> 7 uaccgcuaau accgcggcgc gugggaccac gacgacgacu cgccgcgcga ccgcgacugg 60 <210> 8 <211> 72 <212> RNA <213> Artificial Sequence <220> <223> Adjuvant sequence <400> 8 uacgcgcacu ggcgcggcgc guggcacgac gacgacgacu cgcgccgcga ccgcgacugg 60 cuuuggaccc gc 72 <210> 9 <211> 45 <212> RNA <213> Artificial Sequence <220> <223> Adjuvant sequence <400> 9 uaccuaaccu ggaccuaaga caaagaccac cgccgccgcu gggcg 45 <210> 10 <211> 3783 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence encoding the pre-fusion conformation S protein sequence <400> 10 aagcacaagg accacgacga cggggaccac tcgtcggtca cgcacttgga ctggtgggcc 60 tgggtcgacg gggggcggat gtggttgtcg aagtgggccc cgcacatgat ggggctgttc 120 cacaaggcct cgtcgcacga cgtgtcgtgg gtcctggaca aggacgggaa gaagtcgttg 180 cactggacca aggtgcggta ggtgcactcg ccgtggttgc cgtggttcgc caagctgttg 240 gggcacgacg ggaagttgct gccgcacatg aagcggtcgt ggctcttctc gttgtagtag 300 gccccgacct agaagccgtg gtgggacctg tcgttctggg tctcggacga ctagcacttg 360 ttgcggtggt tgcaccacta gttccacacg ctcaaggtca agacgttgct ggggaaggac 420 ccgcacatga tggtgttctt gttgttctcg acctacctct cgctcaaggc ccacatgtcg 480 tcgcggttgt tgacgtggaa gctcatgcac tcggtcggga aggactacct ggacctcccg 540 ttcgtcccgt tgaagttctt ggacgccctc aagcacaagt tcttgtagct gccgatgaag 600 ttctagatgt cgttcgtgtg ggggtagttg gaccacgccc tggacggggt cccgaagtcg 660 cgggacctcg gggaccacct ggacgggtag ccgtagttgt agtgggccaa ggtctgggac 720 gaccgggacg tggcctcgat ggactggggg ccgctgtcgt cgtcgccgac ctggcggccg 780 cggcggcgga tgatgcaccc gatggacgtc ggggcctgga aggacgactt catgttgctc 840 ttgccgtggt agtggctgcg gcacctgacg cgggacctgg gggactcgct ctggttcacg 900 tgggacttct cgaagtggca cctcttcccg tagatggtct ggtcgttgaa ggcccacgtc 960 gggtggctct cgtagcacgc caaggggttg tagtggttgg acacggggaa gccgctccac 1020 aagttgcggt gggccaagcg gtcgcacatg cggaccttgg ccttcgccta gtcgttgacg 1080 caccggctga tgtcgcacga catgttgtcg cggtcgaagt cgtggaagtt cacgatgccg 1140 cactcggggt ggttcgactt gctggacacg aagtggttgc acatgcggct gtcgaagcac 1200 taggccccgc tgctccacgc cgtctagcgg gggccggtct ggccgttcta gcggctgatg 1260 ttgatgttcg acgggctgct gaagtggccg acgcactagc ggaccttgtc gttgttggac 1320 ctgtcgttcc acccgccgtt gatgttgatg gacatggccg acaaggcctt ctcgttggac 1380 ttcgggaagc tcgccctgta gtcgtggctc tagatggtcc ggccgtcgtg ggggacgttg 1440 ccgcacctcc cgaagttgac gatgaagggg gacgtctcga tgccgaaggt cgggtggttg 1500 ccgcacccga tggtcgggat ggcccaccac cacgactcga agctcgacga cgtgcggggg 1560 cggtggcaca cgccggggtt cttctcgtgg ttggaccact tcttgttcac gcacttgaag 1620 ttgaagttgc cggactggcc gtggccgcac gactggctct cgttgttctt caaggacggg 1680 aaggtcgtca agccggccct gtagcggctg tggtggctgc ggcacgccct gggggtctgg 1740 gacctctagg acctgtagtg ggggacgtcg aagccgccgc actcgcacta gtgggggccg 1800 tggttgtggt cgttggtcca ccggcacgac atggtcctgc acttgacgtg gctccacggg 1860 caccggtagg tgcggctggt cgactggggg tggaccgccc acatgtcgtg gccgtcgttg 1920 cacaaggtct gggcccggcc gacggactag ccgcggctcg tgcacttgtt gtcgatgctc 1980 acgctgtagg ggtagccgcg gccgtagacg cggtcgatgg tctgggtctg gttgtcgggg 2040 gccgcccggg cctcgcaccg gtcggtctcg tagtagcgga tgtggtactc ggacccgcgg 2100 ctcttgtcgc accggatgtc gttgttgtcg tagcggtagg ggtggttgaa gtggtagtcg 2160 cactggtggc tctaggacgg gcactcgtac tggttctggt cgcacctgac gtggtacatg 2220 tagacgccgc tgtcgtggct cacgtcgttg gacgacgacg tcatgccgtc gaagacgtgg 2280 gtcgacttgg cccgggactg gccgtagcgg cacctcgtcc tgttcttgtg ggtcctccac 2340 aagcgggtcc acttcgtcta gatgttctgg ggggggtagt tcctgaagcc gccgaagttg 2400 aagtcggtct aggacgggct ggggtcgttc gggtcgttcg cctcgaagta gctcctggac 2460 gacaagttgt tccactggga ccggctgcgg ccgaagtagt tcgtcatgcc gctgacggac 2520 ccgctgtagc ggcgggccct ggactagacg cgggtcttca agttgccgga ctggcacgac 2580 gggggggacg actggctgct ctactagcgg gtcatgtggt cgcgggacga ccggccgtgg 2640 tagtggtcgc cgacctggaa gccgcggccg cggcgggacg tctaggggaa gcggtacgtc 2700 taccggatgg ccaagttgcc gtagccgcac tgggtcttgc acgacatgct cttggtcttc 2760 gactagcggt tggtcaagtt gtcgcggtag ccgttctagg tcctgtcgga ctcgtcgtgg 2820 cggtcgcggg acccgttcga cgtcctgcac cacttggtct tgcgggtccg ggacttgtgg 2880 gaccacttcg tcgactcgtc gttgaagccg cggtagtcgt cgcacgactt gctgtaggac 2940 tcggccgacc tgttccacct ccggctccac gtctagctgg ccgactagtg gccggccgac 3000 gtctcggacg tctggatgca ctgggtcgtc gactaggccc ggcggctcta ggcccggtcg 3060 cggttggacc ggcggtggtt ctactcgctc acgcacgacc cggtctcgtt cgcccacctg 3120 aagacgccgt tcccgatggt ggactactcg aagggggtct cgcggggggt gccgcaccac 3180 aaggacgtgc actggatgca cgggcgggtc ctcttcttga agtggtggcg ggggcggtag 3240 acggtgctgc cgttccgggt gaagggggcc ctcccgcaca agcactcgtt gccgtgggtg 3300 accaagcact gggtcgcctt gaagatgctc ggggtctagt agtggtggct gttgtggaag 3360 cactcgccgt tgacgctgca ccactagccg tagcacttgt tgtggcacat gctgggggac 3420 gtcgggctcg acctgtcgaa gttcctcctc gacctgttca tgaagttctt ggtgtggtcg 3480 gggctgcacc tggacccgct gtagtcgccg tagttgcggt cgcaccactt gtaggtcttc 3540 ctctagctgg ccgacttgct ccaccggttc ttggacttgc tctcggacta gctggacgtc 3600 ctcgacccgt tcatgctcgt catgtagttc accgggacca tgtagaccga cccgaagtag 3660 cggccggact agcggtagca ctaccactgg tagtacgaca cgacgtactg gtcgacgacg 3720 tcgacggact tcccgacgac gtcgcggatg cacgccttcc tgccgctcac ccacaaggac 3780 act 3783 <210> 11 <211> 3783 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence encoding the pre-fusion conformation S protein sequence <400> 11 aagcacaagg accacgacga cggggaccac tcgtcggtca cgcacttgga ctggtggtcc 60 tgggtcgacg gggggcggat gtggttgtcg aagtggtccc cgcacatgat ggggctgttc 120 cacaagtcct cgtcgcacga cgtgtcgtgg gtcctggaca aggacgggaa gaagtcgttg 180 cactggacca aggtgcggta ggtgcactcg ccgtggttgc cgtggttctc caagctgttg 240 gggcacgacg ggaagttgct gccgcacatg aagcggtcgt ggctcttctc gttgtagtag 300 tccccgacct agaagccgtg gtgggacctg tcgttctggg tctcggacga ctagcacttg 360 ttgcggtggt tgcaccacta gttccacacg ctcaaggtca agacgttgct ggggaaggac 420 ccgcacatga tggtgttctt gttgttctcg acctacctct cgctcaagtc ccacatgtcg 480 tcgcggttgt tgacgtggaa gctcatgcac tcggtcggga aggactacct ggacctcccg 540 ttcgtcccgt tgaagttctt ggactccctc aagcacaagt tcttgtagct gccgatgaag 600 ttctagatgt cgttcgtgtg ggggtagttg gaccactccc tggacggggt cccgaagtcg 660 cgggacctcg gggaccacct ggacgggtag ccgtagttgt agtggtccaa ggtctgggac 720 gaccgggacg tgtcctcgat ggactggggg ccgctgtcgt cgtcgccgac ctggcggccg 780 cggcggcgga tgatgcaccc gatggacgtc gggtcctgga aggacgactt catgttgctc 840 ttgccgtggt agtggctgcg gcacctgacg cgggacctgg gggactcgct ctggttcacg 900 tgggacttct cgaagtggca cctcttcccg tagatggtct ggtcgttgaa gtcccacgtc 960 gggtggctct cgtagcactc caaggggttg tagtggttgg acacggggaa gccgctccac 1020 aagttgcggt ggtccaagcg gtcgcacatg cggaccttgt ccttctccta gtcgttgacg 1080 caccggctga tgtcgcacga catgttgtcg cggtcgaagt cgtggaagtt cacgatgccg 1140 cactcggggt ggttcgactt gctggacacg aagtggttgc acatgcggct gtcgaagcac 1200 tagtccccgc tgctccactc cgtctagcgg gggccggtct ggccgttcta gcggctgatg 1260 ttgatgttcg acgggctgct gaagtggccg acgcactagc ggaccttgtc gttgttggac 1320 ctgtcgttcc acccgccgtt gatgttgatg gacatgtccg acaagtcctt ctcgttggac 1380 ttcgggaagc tctccctgta gtcgtggctc tagatggtcc ggccgtcgtg ggggacgttg 1440 ccgcacctcc cgaagttgac gatgaagggg gacgtctcga tgccgaaggt cgggtggttg 1500 ccgcaccga tggtcgggat gtcccaccac cacgactcga agctcgacga cgtgcgggg 1560 cggtggcaca cgccggggtt cttctcgtgg ttggaccact tcttgttcac gcacttgaag 1620 ttgaagttgc cggactggcc gtggccgcac gactggctct cgttgttctt caaggacggg 1680 aaggtcgtca agccgtccct gtagcggctg tggtggctgc ggcactccct gggggtctgg 1740 gacctctagg acctgtagtg ggggacgtcg aagccgccgc actcgcacta gtggggccg 1800 tggttgtggt cgttggtcca ccggcacgac atggtcctgc acttgacgtg gctccacggg 1860 caccggtagg tgcggctggt cgactggggg tggacctccc acatgtcgtg gccgtcgttg 1920 cacaaggtct ggtcccggcc gacggactag ccgcggctcg tgcacttgtt gtcgatgctc 1980 acgctgtagg ggtagccgcg gccgtagacg cggtcgatgg tctgggtctg gttgtcgggg 2040 tcctcccggt cctcgcaccg gtcggtctcg tagtagcgga tgtggtactc ggacccgcgg 2100 ctcttgtcgc accggatgtc gttgttgtcg tagcggtagg ggtggttgaa gtggtagtcg 2160 cactggtggc tctaggacgg gcactcgtac tggttctggt cgcacctgac gtggtacatg 2220 tagacgccgc tgtcgtggct cacgtcgttg gacgacgacg tcatgccgtc gaagacgtgg 2280 gtcgacttgt cccgggactg gccgtagcgg cacctcgtcc tgttcttgtg ggtcctccac 2340 aagcgggtcc acttcgtcta gatgttctgg ggggggtagt tcctgaagcc gccgaagttg 2400 aagtcggtct aggacgggct ggggtcgttc gggtcgttct cctcgaagta gctcctggac 2460 gacaagttgt tccactggga ccggctgcgg ccgaagtagt tcgtcatgcc gctgacggac 2520 ccgctgtagc ggcggtccct ggactagacg cgggtcttca agttgccgga ctggcacgac 2580 gggggggacg actggctgct ctactagcgg gtcatgtggt cgcgggacga ccggccgtgg 2640 tagtggtcgc cgacctggaa gccgcggccg cggcgggacg tctaggggaa gcggtacgtc 2700 taccggatgt ccaagttgcc gtagccgcac tgggtcttgc acgacatgct cttggtcttc 2760 gactagcggt tggtcaagtt gtcgcggtag ccgttctagg tcctgtcgga ctcgtcgtgg 2820 cggtcgcggg acccgttcga cgtcctgcac cacttggtct tgcgggtccg ggacttgtgg 2880 gaccacttcg tcgactcgtc gttgaagccg cggtagtcgt cgcacgactt gctgtaggac 2940 tcgtccgacc tgttccacct ccggctccac gtctagctgt ccgactagtg gccgtccgac 3000 gtctcggacg tctggatgca ctgggtcgtc gactagtccc ggcggctcta gtcccggtcg 3060 cggttggacc ggcggtggtt ctactcgctc acgcacgacc cggtctcgtt ctcccacctg 3120 aagacgccgt tcccgatggt ggactactcg aagggggtct cgcggggggt gccgcaccac 3180 aaggacgtgc actggatgca cgggcgggtc ctcttcttga agtggtggcg ggggcggtag 3240 acggtgctgc cgttccgggt gaaggggtcc ctcccgcaca agcactcgtt gccgtgggtg 3300 accaagcact gggtctcctt gaagatgctc ggggtctagt agtggtggct gttgtggaag 3360 cactcgccgt tgacgctgca ccactagccg tagcacttgt tgtggcacat gctgggggac 3420 gtcgggctcg acctgtcgaa gttcctcctc gacctgttca tgaagttctt ggtgtggtcg 3480 gggctgcacc tggacccgct gtagtcgccg tagttgcggt cgcaccactt gtaggtcttc 3540 ctctagctgt ccgacttgct ccaccggttc ttggacttgc tctcggacta gctggacgtc 3600 ctcgacccgt tcatgctcgt catgtagttc accgggacca tgtagaccga cccgaagtag 3660 cggccggact agcggtagca ctaccactgg tagtacgaca cgacgtactg gtcgacgacg 3720 tcgacggact tcccgacgac gtcgcggatg cactccttcc tgccgctcac ccacaaggac 3780 act 3783 <210> 12 <211> 3783 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence encoding the S protein sequence in the pre-fusion conformation <400> 12 aagcacaagg accacgacga cggagaccac tcgtcggtca cgcacttgga ctggtggtcc 60 tgggtcgacg gaggacggat gtggttgtcg aagtggtccc cgcacatgat gggactgttc 120 cacaagtcct cgtcgcacga cgtgtcgtgg gtcctggaca aggacggaaa gaagtcgttg 180 cactggacca aggtgcggta ggtgcactcg ccgtggttgc cgtggttctc caagctgttg 240 ggacacgacg gaaagttgct gccgcacatg aagcggtcgt ggctcttctc gttgtagtag 300 tccccgacct agaagccgtg gtgggacctg tcgttctggg tctcggacga ctagcacttg 360 ttgcggtggt tgcaccacta gttccacacg ctcaaggtca agacgttgct gggaaaggac 420 ccgcacatga tggtgttctt gttgttctcg acctacctct cgctcaagtc ccacatgtcg 480 tcgcggttgt tgacgtggaa gctcatgcac tcggtcggaa aggactacct ggacctcccg 540 ttcgtcccgt tgaagttctt ggactccctc aagcacaagt tcttgtagct gccgatgaag 600 ttctagatgt cgttcgtgtg gggatagttg gaccactccc tggacggagt cccgaagtcg 660 cgggacctcg gagaccacct ggacggatag ccgtagttgt agtggtccaa ggtctgggac 720 gaccgggacg tgtcctcgat ggactgggga ccgctgtcgt cgtcgccgac ctggcggccg 780 cggcggcgga tgatgcaccc gatggacgtc ggatcctgga aggacgactt catgttgctc 840 ttgccgtggt agtggctgcg gcacctgacg cgggacctgg gagactcgct ctggttcacg 900 tgggacttct cgaagtggca cctcttcccg tagatggtct ggtcgttgaa gtcccacgtc 960 ggatggctct cgtagcactc caagggattg tagtggttgg acacgggaaa gccgctccac 1020 aagttgcggt ggtccaagcg gtcgcacatg cggaccttgt ccttctccta gtcgttgacg 1080 caccggctga tgtcgcacga catgttgtcg cggtcgaagt cgtggaagtt cacgatgccg 1140 cactcgggat ggttcgactt gctggacacg aagtggttgc acatgcggct gtcgaagcac 1200 tagtccccgc tgctccactc cgtctagcgg ggaccggtct ggccgttcta gcggctgatg 1260 ttgatgttcg acggactgct gaagtggccg acgcactagc ggaccttgtc gttgttggac 1320 ctgtcgttcc acccgccgtt gatgttgatg gacatgtccg acaagtcctt ctcgttggac 1380 ttcggaaagc tctccctgta gtcgtggctc tagatggtcc ggccgtcgtg gggaacgttg 1440 ccgcacctcc cgaagttgac gatgaaggga gacgtctcga tgccgaaggt cggatggttg 1500 ccgcacccga tggtcggaat gtcccaccac cacgactcga agctcgacga cgtgcgggga 1560 cggtggcaca cgccgggatt cttctcgtgg ttggaccact tcttgttcac gcacttgaag 1620 ttgaagttgc cggactggcc gtggccgcac gactggctct cgttgttctt caaggacgga 1680 aaggtcgtca agccgtccct gtagcggctg tggtggctgc ggcactccct gggagtctgg 1740 gacctctagg acctgtagtg gggaacgtcg aagccgccgc actcgcacta gtggggaccg 1800 tggttgtggt cgttggtcca ccggcacgac atggtcctgc acttgacgtg gctccacgga 1860 caccggtagg tgcggctggt cgactgggga tggacctccc acatgtcgtg gccgtcgttg 1920 cacaaggtct ggtcccggcc gacggactag ccgcggctcg tgcacttgtt gtcgatgctc 1980 acgctgtagg gatagccgcg gccgtagacg cggtcgatgg tctgggtctg gttgtcggga 2040 tcctcccggt cctcgcaccg gtcggtctcg tagtagcgga tgtggtactc ggacccgcgg 2100 ctcttgtcgc accggatgtc gttgttgtcg tagcggtagg gatggttgaa gtggtagtcg 2160 cactggtggc tctaggacgg acactcgtac tggttctggt cgcacctgac gtggtacatg 2220 tagacgccgc tgtcgtggct cacgtcgttg gacgacgacg tcatgccgtc gaagacgtgg 2280 gtcgacttgt cccgggactg gccgtagcgg cacctcgtcc tgttcttgtg ggtcctccac 2340 aagcgggtcc acttcgtcta gatgttctgg ggaggatagt tcctgaagcc gccgaagttg 2400 aagtcggtct aggacggact gggatcgttc ggatcgttct cctcgaagta gctcctggac 2460 gacaagttgt tccactggga ccggctgcgg ccgaagtagt tcgtcatgcc gctgacggac 2520 ccgctgtagc ggcggtccct ggactagacg cgggtcttca agttgccgga ctggcacgac 2580 ggaggagacg actggctgct ctactagcgg gtcatgtggt cgcgggacga ccggccgtgg 2640 tagtggtcgc cgacctggaa gccgcggccg cggcgggacg tctagggaaa gcggtacgtc 2700 taccggatgt ccaagttgcc gtagccgcac tgggtcttgc acgacatgct cttggtcttc 2760 gactagcggt tggtcaagtt gtcgcggtag ccgttctagg tcctgtcgga ctcgtcgtgg 2820 cggtcgcggg acccgttcga cgtcctgcac cacttggtct tgcgggtccg ggacttgtgg 2880 gaccacttcg tcgactcgtc gttgaagccg cggtagtcgt cgcacgactt gctgtaggac 2940 tcgtccgacc tgttccacct ccggctccac gtctagctgt ccgactagtg gccgtccgac 3000 gtctcggacg tctggatgca ctgggtcgtc gactagtccc ggcggctcta gtcccggtcg 3060 cggttggacc ggcggtggtt ctactcgctc acgcacgacc cggtctcgtt ctcccacctg 3120 aagacgccgt tcccgatggt ggactactcg aagggagtct cgcggggagt gccgcaccac 3180 aaggacgtgc actggatgca cggacgggtc ctcttcttga agtggtggcg gggacggtag 3240 acggtgctgc cgttccgggt gaagggatcc ctcccgcaca agcactcgtt gccgtgggtg 3300 accaagcact gggtctcctt gaagatgctc ggagtctagt agtggtggct gttgtggaag 3360 cactcgccgt tgacgctgca ccactagccg tagcacttgt tgtggcacat gctgggagac 3420 gtcggactcg acctgtcgaa gttcctcctc gacctgttca tgaagttctt ggtgtggtcg 3480 ggactgcacc tggacccgct gtagtcgccg tagttgcggt cgcaccactt gtaggtcttc 3540 ctctagctgt ccgacttgct ccaccggttc ttggacttgc tctcggacta gctggacgtc 3600 ctcgacccgt tcatgctcgt catgtagttc accggaacca tgtagaccga cccgaagtag 3660 cggccggact agcggtagca ctaccactgg tagtacgaca cgacgtactg gtcgacgacg 3720 tcgacggact tcccgacgac gtcgcggatg cactccttcc tgccgctcac ccacaaggac 3780 act 3783 <210> 13 <211> 3783 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence encoding the pre-fusion conformation S protein sequence <400> 13 aagcacaagg accacgacga cggagaccac tcgtcggtca cacacttgga ctgttgttct 60 tgtgtcgacg gaggacgaat gtgtttgtcg aagtgttctc ctcacatgat gggactgttc 120 cacaagtctt cgtcgcacga cgtgtcgtgt gtcctggaca aggacggaaa gaagtcgttg 180 cactgtacca aggtgcgata ggtgcactcg ccttgtttgc cttgtttctc taagctgttg 240 ggacacgacg gaaagttgct gcctcacatg aagcgatcgt gtctcttctc gttgtagtag 300 tctcctacct agaagccttg ttgtgacctg tcgttctgtg tctcggacga ctagcacttg 360 ttgcgatgtt tgcaccacta gttccacaca ctcaaggtca agacattgct gggaaaggac 420 cctcacatga tggtgttctt gttgttctcg acctacctct cgctcaagtc tcacatgtcg 480 tcgcgattgt tgacatgtaa gctcatgcac tcggtcggaa aggactacct ggacctccct 540 ttcgtccctt tgaagttctt ggactctctc aagcacaagt tcttgtagct gcctatgaag 600 ttctagatgt cgttcgtgtg tggatagttg gaccactctc tggacggagt ccctaagtcg 660 cgagacctcg gagaccacct ggacggatag ccttagttgt agtgttctaa ggtctgtgac 720 gaccgagacg tgtcttcgat ggactgtgga cctctgtcgt cgtcgcctac ctgtcgacct 780 cgacgacgaa tgatgcaccc tatggacgtc ggatcttgta aggacgactt catgttgctc 840 ttgccttgtt agtgtctgcg acacctgaca cgagacctgg gagactcgct ctgtttcaca 900 tgtgacttct cgaagtgtca cctcttccct tagatggtct gttcgttgaa gtctcacgtc 960 ggatgtctct cgtagcactc taagggattg tagtgtttgg acacaggaaa gcctctccac 1020 aagttgcgat gttctaagcg atcgcacatg cgaaccttgt ctttctctta gtcgttgaca 1080 caccgactga tgtcgcacga catgttgtcg cgatcgaagt cgtgtaagtt cacaatgcct 1140 cactcgggat gtttcgactt gctggacaca aagtgtttgc acatgcgact gtcgaagcac 1200 tagtctcctc tgctccactc tgtctagcga ggacctgtct gtcctttcta gcgactgatg 1260 ttgatgttcg acggactgct gaagtgtcct acacactagc gaaccttgtc gttgttggac 1320 ctgtcgttcc accctccttt gatgttgatg gacatgtctg acaagtcttt ctcgttggac 1380 ttcggaaagc tctctctgta gtcgtgtctc tagatggtcc gaccttcgtg tggaacattg 1440 cctcacctcc ctaagttgac aatgaaggga gacgtctcga tgcctaaggt cggatgtttg 1500 cctcacccta tggtcggaat gtctcaccac cacgactcga agctcgacga cgtgcgagga 1560 cgatgtcaca cacctggatt cttctcgtgt ttggaccact tcttgttcac acacttgaag 1620 ttgaagttgc ctgactgtcc ttgtcctcac gactgtctct cgttgttctt caaggacgga 1680 aaggtcgtca agccttctct gtagcgactg tgttgtctgc gacactctct gggagtctgt 1740 gacctctagg acctgtagtg tggaacatcg aagcctcctc actcgcacta gtgtggacct 1800 tgtttgtgtt cgttggtcca ccgacacgac atggtcctgc acttgacatg tctccacgga 1860 caccgatagg tgcgactggt cgactgtgga tgtacctctc acatgtcgtg tccttcgttg 1920 cacaaggtct gttctcgacc tacagactag cctcgactcg tgcacttgtt gtcgatgctc 1980 acactgtagg gatagcctcg accttagaca cgatcgatgg tctgtgtctg tttgtcggga 2040 tcttctcgat cttcgcaccg atcggtctcg tagtagcgaa tgtgttactc ggaccctcga 2100 ctcttgtcgc accgaatgtc gttgttgtcg tagcgatagg gatgtttgaa gtgttagtcg 2160 cactgttgtc tctaggacgg acactcgtac tgtttctgtt cgcacctgac atgttacatg 2220 tagacacctc tgtcgtgtct cacatcgttg gacgacgacg tcatgccttc gaagacatgt 2280 gtcgacttgt ctcgagactg tccttagcga cacctcgtcc tgttcttgtg tgtcctccac 2340 aagcgagtcc acttcgtcta gatgttctgt ggaggatagt tcctgaagcc tcctaagttg 2400 aagtcggtct aggacggact gggatcgttc ggatcgttct cttcgaagta gctcctggac 2460 gacaagttgt tccactgtga ccgactgcga cctaagtagt tcgtcatgcc tctgacagac 2520 cctctgtagc gacgatctct ggactagaca cgagtcttca agttgcctga ctgtcacgac 2580 ggaggagacg actgtctgct ctactagcga gtcatgtgtt cgcgagacga ccgaccttgt 2640 tagtgttcgc ctacctgtaa gcctcgacct cgacgagacg tctagggaaa gcgatacgtc 2700 taccgaatgt ctaagttgcc ttagcctcac tgtgtcttgc acgacatgct cttggtcttc 2760 gactagcgat tggtcaagtt gtcgcgatag cctttctagg tcctgtcgga ctcgtcgtgt 2820 cgatcgcgag accctttcga cgtcctgcac cacttggtct tgcgagtccg agacttgtgt 2880 gaccacttcg tcgactcgtc gttgaagcct cgatagtcgt cgcacgactt gctgtaggac 2940 tcgtctgacc tgttccacct ccgactccac gtctagctgt ctgactagtg tccttctgac 3000 gtctcggacg tctgtatgca ctgtgtcgtc gactagtctc gacgactcta gtctcgatcg 3060 cgattggacc gacgatgttt ctactcgctc acacacgacc ctgtctcgtt ctctcacctg 3120 aagacacctt tccctatggt ggactactcg aagggagtct cgcgaggagt gcctcaccac 3180 aaggacgtgc actgtatgca cggacgagtc ctcttcttga agtgttgtcg aggacgatag 3240 acagtgctgc ctttccgagt gaagggatct ctccctcaca agcactcgtt gccttgtgtg 3300 accaagcact gtgtctcttt gaagatgctc ggagtctagt agtgttgtct gttgtgtaag 3360 cactcgcctt tgacactgca ccactagcct tagcacttgt tgtgtcacat gctgggagac 3420 gtcggactcg acctgtcgaa gttcctcctc gacctgttca tgaagttctt ggtgtgttcg 3480 ggactgcacc tggaccctct gtagtcgcct tagttgcgat cgcaccactt gtaggtcttc 3540 ctctagctgt ctgacttgct ccaccgattc ttggacttgc tctcggacta gctggacgtc 3600 ctcgaccctt tcatgctcgt catgtagttc accggaacca tgtagaccga ccctaagtag 3660 cgacctgact agcgatagca ctaccactgt tagtacgaca caacatactg ttcgacaaca 3720 tcgacagact tccctacaac atcgcgaatg cactctttcc tgcctctcac ccacaaggac 3780 act 3783

Claims

1. The protein as follows (a): (a) A protein consisting of the amino acid sequence shown in SEQ ID No.

1.

2. Use of the protein according to claim 1 as an antigen in the preparation of a vaccine.

3. A DNA encoding the protein according to claim 1, wherein the nucleotide sequence of the DNA comprises the sequence shown in SEQ ID No.

12.

4. An expression vector comprising the DNA according to claim 3.

5. An mRNA, whose nucleotide sequence includes the sequence shown in SEQ ID No.

4.

6. The mRNA according to claim 5, wherein the 5' end of the nucleotide sequence further comprises an adjuvant sequence, wherein the adjuvant sequence is a nucleotide sequence that promotes protein secretion and expression; The adjuvant sequence includes the sequence shown in SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 or SEQ ID No.

9.

7. The mRNA according to claim 6, wherein the mRNA comprises the mRNA shown by the sequence shown in SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 or SEQ ID No.9 combined with the sequence shown in SEQ ID No.

4.

8. A preventive vaccine comprising the mRNA according to any one of claims 5 to 7.

9. The vaccine according to claim 8, further comprising a PBS solution for dissolving mRNA.

Images