Human respiratory syncytial virus fusion F protein variant, vaccine based on human respiratory syncytial virus fusion F protein variant and application thereof

Abstract

The invention relates to a human respiratory syncytial virus (hRSV) fusion F protein variant, a vaccine based on the human respiratory syncytial virus fusion F protein variant and application of the human respiratory syncytial virus fusion F protein variant. The hRSV fusion F protein variant (especially an mRNA vaccine based on the hRSV fusion F protein variant) can induce an organism to generate a high-level neutralizing antibody aiming at the respiratory syncytial virus, meanwhile, cellular immune response can be activated, dual activation of humoral immunity and cellular immunity is achieved, and the hRSV fusion F protein variant has cross protection efficacy on all subtypes of the hRSV. Therefore, a vaccine product based on the hRSV fusion F protein variant has important application value in clinical treatment and prevention and control of the respiratory syncytial virus.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a human respiratory syncytial virus fusion F protein variant, a vaccine based thereon, and applications thereof. Background Art

[0002] Respiratory syncytial virus (RSV) is one of the primary pathogens causing lower respiratory tract viral infections. Its infection is prone to occur in infants and young children under 2 years old, especially showing a high incidence trend in winter and spring. The typical clinical features are severe respiratory infections such as interstitial pneumonia.

[0003] Since RSV was discovered more than half a century ago, vaccine research and development has been long restricted by the complex pathogenic mechanism of the virus and the immunopathological reactions induced after infection. The F protein plays a decisive role in the viral membrane fusion and host cell invasion processes, and its conserved antigenic epitopes are the key targets for inducing neutralizing antibodies.

[0004] mRNA vaccines are a newly emerging form of vaccine in recent years. Its basic principle is to introduce mRNA expressing antigen targets into the body through a specific delivery system, express immunogenic proteins in the body, thereby activating the immune system, stimulating the body to produce specific immunological reactions, and enabling the body to obtain immune protection. Compared with other vaccines, mRNA vaccines have their unique advantages: First, after entering the cell, mRNA can be translated in the cytoplasm to express the target protein without entering the nucleus, so there is no risk of integration into the host genome, and the safety is good; Second, mRNA vaccines can be synthesized in vitro, do not rely on cell or bacterial culture, have a short R & D cycle, low production cost, and the production process and flow can be standardized, which is conducive to large-scale production and quality control; In addition, by modifying mRNA, the stability and translation efficiency of mRNA can be improved; Using a vector to deliver mRNA can also achieve high-efficiency expression of mRNA; The adjuvant effect of mRNA vaccines can also activate both humoral immunity and cellular immunity simultaneously.

[0005] The current research and development of RSV mRNA vaccines take the viral fusion protein (F protein) as the core antigen target. Although the current R & D design of RSV mRNA vaccines has become the research focus in this field, no safe and effective RSV mRNA vaccine has obtained regulatory approval. Therefore, there is an urgent need in this field to develop an mRNA vaccine that can effectively prevent and treat RSV infection to meet the diverse immune needs of patients. Summary of the Invention

[0006] Object of the Invention In view of the defects or requirements existing in the prior art, the object of the present invention is to provide a human respiratory syncytial virus fusion F protein variant, a vaccine based on the same, and their applications, which can efficiently stimulate the specific immune response of human respiratory syncytial virus (hRSV).

[0007] Solution To achieve the object of the present invention, the present invention provides the following technical solutions: In the first aspect, the present invention provides a human respiratory syncytial virus fusion F protein variant, wherein, compared with the wild-type human respiratory syncytial virus F protein shown in SEQ ID NO: 1, the human respiratory syncytial virus fusion F protein variant contains the following amino acid mutations: L4P, A8T, T12I, T16A, A / V17I, F20L, G25S, K66E, Q101P, T / A103V, N105S, A122T, K124N, V152I, L172Q, S173L, K191R, K / N201S, I206M, K / Q209R, N276S, V384I, T529A, S540A.

[0008] Feasibly, the human respiratory syncytial virus fusion F protein variant has the amino acid sequence shown in SEQ ID NO: 2.

[0009] Further feasibly, a trimerization domain and / or a tag sequence are connected to the C-terminus of the human respiratory syncytial virus fusion F protein variant; and / or, a signal peptide sequence is further included at the N-terminus of the human respiratory syncytial virus fusion F protein variant.

[0010] In the second aspect, the present invention provides a polynucleotide encoding the human respiratory syncytial virus fusion F protein variant described in the first aspect above.

[0011] The polynucleotide can be DNA or mRNA.

[0012] In a preferred embodiment, the polynucleotide is an mRNA molecule, which contains an open reading frame having the nucleotide sequence shown in SEQ ID NO: 3, or a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 3.

[0013] The "having at least 90% identity" means having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9% or 100% identity.

[0014] In feasible embodiments, the mRNA molecule further comprises one or more elements selected from the following: 5' untranslated region, 3' untranslated region, signal peptide, 5' cap structure, 3' polyadenylate tail.

[0015] In feasible embodiments, the mRNA molecule further comprises a chemical modification.

[0016] Preferably, the 5' cap structure is a 5' guanosine cap, which is selected from any one of the following: m7Gppp(2'OMeA)pG, m7GpppApA, m7GpppApC, m7GpppApG, m7GpppApU; Preferably, the 3' polyadenylate tail has a length of 50 - 150 nucleotides.

[0017] Preferably, the 5' end of the mRNA molecule may have a methylation modification to improve its stability.

[0018] The mRNA of the present invention can be prepared by conventional methods well-known to those skilled in the art. For example, the DNA corresponding to the mRNA sequence can be obtained first, and it is transcribed in vitro (optionally, its 5' end is modified after transcription) to obtain mRNA. Preferably, its preparation method further includes: purifying the mRNA obtained by transcription.

[0019] In other preferred embodiments, the polynucleotide is a DNA molecule, and the DNA molecule contains the DNA sequence shown in SEQ ID NO: 4.

[0020] In a third aspect, the present invention provides an expression vector, which contains the polynucleotide as described in the second aspect above.

[0021] In a fourth aspect, the present invention provides a host cell, which is transformed or transfected with the polynucleotide as described in the second aspect above or the expression vector as described in the third aspect above.

[0022] In a fifth aspect, the present invention provides the use of the human respiratory syncytial virus fusion F protein variant as described in the first aspect above, the polynucleotide as described in the second aspect above, the expression vector as described in the third aspect above, or the host cell as described in the fourth aspect above in the preparation of a vaccine for preventing and / or treating human respiratory syncytial virus infection.

[0023] In a sixth aspect, the present invention provides a vaccine or immunogenic composition, which contains the human respiratory syncytial virus fusion F protein variant as described in the first aspect above, the polynucleotide as described in the second aspect above, the expression vector as described in the third aspect above, or the host cell as described in the fourth aspect above, and a physiologically acceptable vehicle, adjuvant, excipient, carrier, and / or diluent.

[0024] In some preferred embodiments, the vaccine or immunogenic composition is a recombinant protein vaccine against human respiratory syncytial virus, which comprises the human respiratory syncytial virus fusion F protein variant as described in the first aspect above and an adjuvant.

[0025] In some other preferred embodiments, the vaccine or immunogenic composition is a DNA vaccine against human respiratory syncytial virus, and the DNA vaccine comprises: (1) A eukaryotic expression vector; and (2) A DNA sequence encoding the human respiratory syncytial virus fusion F protein variant as described in the first aspect above, which is constructed into the eukaryotic expression vector.

[0026] Preferably, the DNA sequence encoding the human respiratory syncytial virus fusion F protein variant as described in the first aspect above is as shown in SEQ ID NO: 4.

[0027] In some other preferred embodiments, the vaccine or immunogenic composition is an mRNA vaccine against human respiratory syncytial virus, and the mRNA vaccine comprises: (I) An mRNA molecule encoding the human respiratory syncytial virus fusion F protein variant as described in the first aspect above; and (II) An mRNA delivery vector.

[0028] Preferably, the mRNA molecule contains an open reading frame, and the open reading frame has a nucleotide sequence as shown in SEQ ID NO: 3, or a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO: 3.

[0029] Preferably, the mRNA delivery vector can be a lipid- or polymer-based nanoparticle, or other delivery systems (for example, a cationic nanoemulsion based on squalene), and preferably a lipid nanoparticle.

[0030] The mRNA molecule can be encapsulated in liposomes or other mRNA delivery vectors.

[0031] Preferably, the particle size of the mRNA vaccine is distributed in the range of 70 - 120 nm.

[0032] In feasible embodiments, the mRNA vaccine can be administered by mucosal route, intramuscular injection, subcutaneous injection, intravenous injection and other routes.

[0033] In some other preferred embodiments, the vaccine or immunogenic composition is a human respiratory syncytial virus - viral vector vaccine, which comprises: (1) A viral backbone vector; and (2)Construct a DNA sequence encoding the human respiratory syncytial virus fusion F protein variant as described in the first aspect and incorporated into the viral backbone vector.

[0034] Preferably, the DNA sequence encoding the human respiratory syncytial virus fusion F protein variant as described in the first aspect is as shown in SEQ ID NO: 4.

[0035] In a feasible embodiment, the vaccine or immunogenic composition is in the form of a nasal spray, oral preparation, suppository or parenteral preparation.

[0036] Further feasibly, the nasal spray is selected from aerosols, sprays and powder aerosols, the oral preparation is selected from tablets, powders, pills, granules, soft / hard capsules, film coatings and ointments, and the parenteral preparation is a transdermal agent, ointment, plaster, topical liquid, injectable preparation (e.g., intramuscular injection, subcutaneous injection, intravenous injection preparation).

[0037] In a seventh aspect, the present invention provides a method for preventing and / or treating human respiratory syncytial virus infection, the method comprising: administering to a subject in need a prophylactically and / or therapeutically effective amount of the human respiratory syncytial virus fusion F protein variant as described in the first aspect above, the polynucleotide as described in the second aspect above, the expression vector as described in the third aspect above, the host cell as described in the fourth aspect above, or the vaccine or immunogenic composition as described in the sixth aspect above.

[0038] The "prophylactically and / or therapeutically effective amount" may vary depending on the administration object, target organ, symptoms, administration method, etc., and can be determined according to the doctor's judgment considering the type of dosage form, administration method, age and weight of the patient, symptoms of the patient, etc.

[0039] Beneficial effects The human respiratory syncytial virus (hRSV) fusion F protein variant of the present invention (especially the mRNA vaccine based thereon) can not only induce the body to produce high levels of neutralizing antibodies against respiratory syncytial virus, but also activate the production of cytokines involved in the cellular immune response and activate specific CD4+ / CD8+ T cell responses, achieving dual activation of humoral immunity and cellular immunity. This synergistic immune protection mechanism can effectively block the hRSV virus adsorption and cell fusion processes, and can also clear latently infected cells, significantly enhancing the cross-protection efficacy against various subtypes (A / B types) of hRSV compared to a single antibody-mediated immune strategy. Therefore, the vaccine based on the hRSV fusion F protein variant of the present invention has great significance for the clinical treatment and prevention and control of respiratory syncytial virus. Description of the drawings

[0040] One or more embodiments are illustrated by way of example in the accompanying drawings, and such illustrative descriptions do not constitute a limitation on the embodiments. The special term "exemplary" here means "serving as an example, embodiment or illustration". Any embodiment illustrated as "exemplary" here does not have to be construed as superior or better than other embodiments.

[0041] Figure 1 This is a schematic diagram of the structure of the hRSV mRNA molecular construct of the present invention.

[0042] Figure 2 This is the gel electrophoresis identification result of the respiratory syncytial virus mRNA molecule obtained by in vitro transcription in Example 1.

[0043] Figure 3 This is the particle size detection result of the respiratory syncytial virus mRNA vaccine measured in Example 2.

[0044] Figure 4 This shows the detection result of the specific IgG antibody titer in the serum of mice immunized with the respiratory syncytial virus mRNA vaccine described in Example 4.

[0045] Figure 5 This shows the detection result of the neutralizing antibody level in the serum of mice immunized with the respiratory syncytial virus mRNA vaccine described in Example 5.

[0046] Figure 6 This shows the activation of cytokines in splenocytes of mice immunized with the respiratory syncytial virus mRNA vaccine described in Example 6.

[0047] Figure 7 This shows the immune responses of CD4+ T cells (Figure A) and CD8+ T cells (Figure B) induced by immunization with the respiratory syncytial virus mRNA vaccine described in Example 7, where the abscissa represents the percentage of positive cells. Detailed implementation manners

[0048] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0049] In addition, for a better illustration of the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be implemented without some of these specific details. In some embodiments, raw materials, components, methods, means, etc. well-known to those skilled in the art are not described in detail in order to highlight the gist of the present invention.

[0050] Unless otherwise clearly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "having" etc. will be understood to include the stated elements or components, without excluding other elements or other components.

[0051] Example 1: Design and synthesis of respiratory syncytial virus mRNA molecule In this example, an hRSV mRNA molecule was designed and prepared as a representative example of the present invention.

[0052] The specific procedure is as follows: (1) Design of hRSV F protein variant sequence Based on the hRSV A2 strain F protein sequence shown in SEQ ID NO: 1, the following 24 epidemic mutation sites were introduced: L4P, A8T, T12I, T16A, A / V17I, F20L, G25S, K66E, Q101P, T / A103V, N105S, A122T, K124N, V152I, L172Q, S173L, K191R, K / N201S, I206M, K / Q209R, N276S, V384I, T529A, S540A, to obtain the F protein variant sequence, whose amino acid sequence is shown in SEQ ID NO: 2.

[0053] (2) Obtaining the DNA coding sequence of hRSV F protein variant The amino acid sequence of the obtained F protein variant was codon-optimized to obtain its DNA coding sequence, as shown in SEQ ID NO: 4.

[0054] (3) Preparation of DNA transcription template for hRSV mRNA The above DNA coding sequence, together with other necessary expression regulatory elements (for example, referring to Figure 1 the structural schematic diagram of the hRSV mRNA molecule construct shown) was introduced into the mRNA vaccine expression vector ABOP-028 (Zhang NN, et.al. A Thermostable mRNA Vaccine against COVID-19) to construct the DNA transcription template for hRSV mRNA.

[0055] The specific method is as follows: First, design PCR primers for amplifying the DNA coding sequence (abbreviated as "mF") and its vector fragment (abbreviated as "V-mF") respectively, as shown in Table 1 below; Table 1 ; Then, using the synthesized DNA molecule encoding hRSV mRNA (whose sequence is shown in SEQ ID NO: 4) as a template, and the primer pairs mF-F and mF-R in Table 1, perform PCR amplification using a high-fidelity DNA polymerase premix (Q5® High-Fidelity 2X Master Mix, purchased from NEB) to obtain the DNA coding fragment of hRSV mRNA (abbreviated as "mF"); then, using the synthesized expression vector ABOP-028 as a template, and the primer pairs V-mF-F and V-mF-R in Table 1, perform PCR amplification using a high-fidelity DNA polymerase premix (Q5® High-Fidelity 2X Master Mix, purchased from NEB) to obtain the ABOP-028 vector fragment (abbreviated as "V-mF"); the specific amplification system for the above PCR amplification is shown in Table 2, and the PCR program is shown in Table 3; Table 2 ; Table 3 ; Finally, use the In-Fusion seamless cloning kit (purchased from TAKARA) to perform in vitro ligation of the amplified fragment mF and the vector fragment V-mF, transform the ligation product into Stbl3 competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.), pick the correct clones to obtain the recombinant expression plasmid of hRSV mRNA, which can be used as the DNA transcription template for hRSV mRNA.

[0056] (4) In vitro transcription reaction The above DNA transcription template was linearized using BsaI restriction endonuclease (purchased from NEB); using the obtained linearized plasmid as a template, T7 RNA polymerase (purchased from Novoprotein Scientific Inc., Suzhou) was used, and unmodified natural ribonucleoside triphosphates (ATP, CTP, UTP, and GTP, purchased from Shanghai Zhaowei Biotechnology Development Co., Ltd.) were used as substrates for in vitro transcription reaction; after the in vitro transcription reaction was completed, DNase (purchased from Novoprotein Scientific Inc., Suzhou) was added to digest the linearized DNA template; 7.5 M LiCl solution (purchased from Invitrogen) was added to purify the synthesized mRNA; a 5'-end capping reaction was carried out using a capping enzyme (purchased from Novoprotein Scientific Inc., Suzhou), and the cap structure was methylated by a cap structure 2'-O-methyltransferase (purchased from Novoprotein Scientific Inc., Suzhou); finally, 7.5 M LiCl solution (purchased from Invitrogen) was added to purify and harvest the mRNA stock solution, obtaining an mRNA molecule encoding the respiratory syncytial virus antigen; The obtained mRNA molecules were identified by 1% agarose gel electrophoresis experiment, and the results are shown in Figure 2 ; from Figure 2 it can be seen that, judged by the molecular weight, the mRNA molecules obtained through the above steps are the target mRNA molecules.

[0057] Example 2: Preparation and particle size determination of respiratory syncytial virus mRNA vaccine The mRNA stock solution of the respiratory syncytial virus F protein variant obtained in Example 1 was quickly mixed with a lipid excipient combination (purchased from Abb Biopharma Co., Ltd., Suzhou) to encapsulate the mRNA in a lipid nanoparticle preparation; after overnight dialysis at 4°C in PBS solution (purchased from Solarbio Science & Technology Co., Ltd., Beijing), it was concentrated, filtered, and sterilized to obtain the finished product of the respiratory syncytial virus mRNA vaccine preparation (abbreviated as "RSV-mF+LNP").

[0058] The particle size of the obtained mRNA vaccine preparation was detected by the DLS (Dynamic Light Scattering) method, and the results are shown in Figure 3 .

[0059] Figure 3 It shows that the particle size of the obtained mRNA vaccine preparation is in the range of 70 - 120 nm, with uniform size and morphology, meeting the relevant standards of mRNA vaccines.

[0060] Example 3: Immunization of experimental animals and sample collection In this example, female mice of the BALB / c strain at 6 weeks of age were used for the immunization experiment. The experimental group was divided into an mRNA vaccine immunization group and a placebo control group (as a negative control), where the placebo control group was the LNP immunization group.

[0061] All mice were immunized with a single dose of the hRSV mRNA vaccine prepared in Example 2 on Day 0 and Day 14 respectively. The mice in the placebo control group were injected with the same amount of empty LNP at the same time. The inoculation method was intramuscular injection, and the inoculation dose was 5 μg of mRNA-containing vaccine or the same amount of empty LNP as the mRNA vaccine per mouse per injection.

[0062] Mouse serum samples were collected on Day 14 and Day 28 respectively to detect the specific IgG antibody titer and neutralizing antibody titer in the immune serum. The collected immune mouse serum was inactivated at 56 °C for 30 min and stored at -80 °C for later use.

[0063] In addition, mouse spleen tissues were collected on Day 28, and spleen cells were isolated from the spleen tissues. The specific method was as follows: Take the mouse spleen, place it in RPMI1640 medium containing 2% FBS (purchased from Invitrogen) and place it on ice; Grind the cells through a 70 μm filter into a 50 mL centrifuge tube and centrifuge at 500 g for 5 min; Add 5 mL of red blood cell lysate (purchased from Invitrogen) to the cell pellet, let it stand at room temperature for 5 min to lyse red blood cells; Centrifuge at 500 g for 5 min, wash the cells once with RPMI1640 medium containing 10% FBS, resuspend the cell pellet with 1 mL of cryopreservation solution (purchased from Stem cell), and freeze the cells in liquid nitrogen for subsequent detection of cytokine activation and T cell immunity.

[0064] Example 4: Detection of the pre-F protein-specific IgG antibody titer in the immune mouse serum by ELISA In this example, a mouse respiratory syncytial virus pre-F antibody (IgG) detection kit (purchased from Nanjing Novozymes Biotech Co., Ltd.) was used to measure the pre-F specific IgG titer of the mouse serum samples collected on Day 14 and Day 28 in Example 3.

[0065] Specifically, the test sample is first diluted at a ratio of 1:100 with the sample diluent, and on this basis, 2-fold serial dilutions are performed for a total of 5 - 10 dilutions; 100 μl of the test sample is added to each well; after sealing the plate with a sealing film, it is placed in a 37°C constant temperature incubator and incubated for 60 min; after incubation, the sealing film is removed, the liquid in the wells is discarded, at least 300 μl of 1× washing solution is added to each well, the washing solution is discarded after standing for 30 s, and the plate is washed 4 times continuously; 100 μl of the enzyme-labeled reagent is added to each well; the incubation and washing steps are repeated; 100 μl of the chromogenic solution is added to each well, after sealing the plate with a sealing film, it is placed in a 37°C constant temperature incubator and incubated in the dark for 15 min; the sealing film is removed, 50 μl of the termination solution is added to each well, and gently mixed, then the OD values of each well at 450 nm and 630 nm can be detected with an enzyme-linked immunosorbent assay (ELISA) reader. The absorbance value is calculated by subtracting the OD value at 630 nm from the OD value at 450 nm of the same well. The endpoint titer is defined as: the serum dilution multiple corresponding to when the absorbance value generated by the immune mouse serum (as described above, the absorbance at 450 nm minus the absorbance at 630 nm) is greater than 2.1 times the absorbance value of the placebo group. The antibody titer below the detection limit is defined as one-third of the detection limit.

[0066] Detection results of the antibody titer in the immune mouse serum Figure 4 Show that: compared with the placebo group, the sera of the mRNA vaccine of the present invention after the first and second immunizations both contain high levels of pre-F specific IgG antibodies. Among them, the serum antibody titer after the second immunization increased by 1000 times compared with that after the first immunization, indicating that the mRNA vaccine has a good IgG antibody activation effect, and the IgG antibody titer is higher after the second immunization.

[0067] The above results show that: the hRSV mRNA vaccine of the present invention can induce mice to produce high levels of specific IgG antibodies against the preF protein.

[0068] Example 5: Detection of the neutralizing antibody titer in the immune mouse serum by the micro-neutralization assay In this example, the neutralizing antibody titer in the mouse serum samples collected on the 28th day in Example 3 was determined by the micro-neutralization assay.

[0069] The specific method is as follows: Dilute the epidemic strains ON1 and BA9 of respiratory syncytial virus to a dose of 100 CCID50 respectively; add 75 µL of cell maintenance medium to each well in the first row (A) of the serum test plate, and add 50 µL of maintenance medium to the remaining wells; take 25 µL of inactivated serum samples and add them to the first row respectively, with 2 wells for each serum, to make a 1 / 4 serum dilution. Use a micropipette to sequentially aspirate 50 µL of the dilution from A to G and add it to the subsequent wells to complete the two-fold serial dilution of all sera. After mixing the last well, discard 50 µL; add 50 µL of the target virus solution containing 100 CCID50 to each well of the 96-well culture plate with diluted sera, and supplement the blank control well with an equal amount of diluent. Place all culture plates in a 37 °C cell incubator for 2 h. After neutralization, add 100 µL of cell maintenance medium to each well and place it in a 37 °C incubator for 5 days; use the target virus solution of 100 CCID50 / 50 µL as the stock solution and perform a ten-fold serial dilution to 1 / 1000. Inoculate it into a cell culture plate, with 8 wells inoculated for each dilution, 50 µL / well. Supplement each well with 50 µL of cell culture medium (equivalent to the serum volume) and culture it in a 37 °C, CO2 incubator; perform a preliminary judgment and observation every day after virus inoculation, and finally determine the result on the fifth day; calculate the highest dilution of the serum that can neutralize 50% of virus infection (using the cytopathic effect (CPE) as an index) through the Karber method and based on the serum serial dilution data, and the reciprocal of this dilution is the neutralizing antibody titer NT 50 。

[0070] The results are shown in Figure 5 。 Figure 5 The results show that the mRNA vaccine of the present invention can effectively induce mice to produce a neutralizing antibody titer NT 50 against the epidemic strain ON1 of respiratory syncytial virus of 1 / 1810, and the neutralizing antibody titer NT 50 against BA9 is 1 / 678. This result shows that: after immunization with this vaccine, it can produce a high titer level of neutralizing antibodies, and it can play an effective antiviral role against respiratory syncytial viruses of subtypes A and B.

[0071] Example 6: Detection of cytokine activation in the spleens of immunized mice by ELISpot method The specific method is as follows: The splenocytes cryopreserved in Example 3 were thawed into a 96-well plate and cultured overnight in a 37°C, 5% CO2 cell culture incubator; the next day, IFN-γ, TNF-α, IL2, and IL4 ELISpot plates (purchased from Thermo Fisher Scientific, USA) were equilibrated at room temperature for 30 min, and 200 μL of RPMI1640 medium containing 10% FBS was added to each well and incubated at room temperature for 30 min to activate the plates; negative control groups, experimental groups, and positive control groups were set up, and 100 μL of RPMI1640 containing 10% FBS, respiratory syncytial virus F protein peptide pool (0.3 μg / well, the total sequences of each peptide segment in the peptide pool are shown in SEQ ID NO: 9, and in the peptide pool, each peptide segment is 15 amino acids long, and they overlap by 10 amino acids with each other, a total of 141 polypeptides), and coenzyme A (0.15 μg / well) were added respectively; 2.5×10 5 splenocytes were added to each well and incubated in a 37°C, 5% CO2 cell culture incubator for 48 h; the detection antibody (R4-6a2 biotin) was diluted to 1 μg / mL with PBS containing 0.5% FBS, 100 μL was added to each well, and incubated at room temperature for 2 h; Streptavidin-ALP was diluted 1000-fold with PBS containing 0.5% FBS, 100 μL was added to each well, and incubated at room temperature for 1 h; BCIP / NBT-plus was filtered through a 0.45 μm filter, and 100 μL was added to each well; when obvious spots appeared in the positive control group, the reaction was terminated with distilled water, the plates were dried, and the spots on the plates were photographed and counted.

[0072] The results are shown in Figure 6 . Figure 6 It shows that under the in vitro stimulation of the respiratory syncytial virus F protein peptide pool, the mRNA immunization group highly stimulated the production of cytokines such as IFN-γ, TNF-α, IL2, and IL4 in mouse splenocytes, suggesting that the mRNA immunization of the present invention can activate the production of cytokines involved in cellular immunity, thereby exerting a cellular immune response.

[0073] Example 7: Detection of T cell immune memory immune response induced by mRNA vaccine by flow cytometry The specific method is as follows: The splenocytes cryopreserved in Example 3 were thawed into a 96-well plate and cultured overnight in a 37°C, 5% CO2 cell culture incubator; the cells were counted, and 2.5×10 6Cells; negative control (treated with the same volume of DMSO), positive control (treated with phorbol ester PMA), and peptide pool stimulation group were set up, with 3 replicates in each group; among them, in the peptide pool stimulation group, a respiratory syncytial virus F protein peptide pool (such as that used in Example 6) at 3 μg / mL was used, and co-stimulators anti-CD28 (2 μg / mL) and anti-CD49d (2 μg / mL) were added; in the positive control group, 100 ng / mL PMA was added; in the negative control group, DMSO, the solvent of the peptide pool, was added; after incubation in a cell culture incubator at 37 °C and 5% CO2 for 1 h, 0.7 μg / mL golistop and 1 μg / mL goliplug were added, and incubation was continued in a cell culture incubator at 37 °C and 5% CO2 for 9 h; the cells were collected into 1.5 mL Eppendorf tubes, centrifuged at 500 g for 5 min, the culture medium was discarded, and after adding 1 mL of PBS for washing, the cell pellet was retained; 50 μL of flow antibodies (with fluorescent dye zombie aqua, anti-CD16 / 32, anti-CD3, anti-CD4, anti-CD8) were added to resuspend the cells, and incubation was carried out in the dark at 4 °C for 30 min; 100 μL of fixative was added to resuspend the cells, and incubation was carried out in the dark at 4 °C for 30 min; 1 mL of perm buffer was added to wash the cells, and the cell pellet was retained; 50 μL of intracellular cytokine antibodies (anti-IFN-γ, anti-TNF-α, anti-IL-2, anti-IL-4) were added, and incubation was carried out in the dark at 4 °C for 30 min; 200 μL of PBS was added to resuspend the cells, and the cells were detected by flow cytometry.

[0074] The detection results of the immune responses of CD4+ T cells and CD8+ T cells are shown respectively in Figure 7 Figure A and Figure B in

[0075] As can be seen from Figure 7 Figure A and Figure B in

[0076]

[0074] CD4+T cells and CD8+T cells immune response detection results are shown respectively in Figure 7 Figure A and Figure B in

[0075] As can be seen from Figure 7 Figure A and Figure B in

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

[0077] Sequences involved in this article: SEQ ID NO: 1 (Amino acid sequence of wild-type hRSV F protein): MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTQATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN; SEQ ID NO: 2 (Amino acid sequence of the hRSV F protein variant of the present invention): MELPILKTNAIITILAIVTLCFASSQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTSAVNSRARRELPRFMNYTLNNTKNTNVTLSKKRKRRFLGFLLGVGSAIASGNAVSKVLHLEGEVNKIKSALQLTNKAVVSLSNGVSVLTSRVLDLKNYIDNQLLPMVNRQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITAIIIVIIVILLALIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN; SEQ ID NO: 3 (mRNA sequence of the hRSV F protein variant of the present invention): SEQ ID NO: 4 (DNA sequence of the hRSV F protein variant of the present invention): SEQ ID NO: 5 (Primer mF-F) TCCCTGCCCCAGAGCTGCCAATCCTGAA; SEQ ID NO: 6 (Primer mF-R) GCTCCAGCCTAGAATTCTTATTAATTGCTGAAGGCGA; SEQ ID NO: 7 (Primer V-mF-F) ATTAATAAGAATTCTAGGCTGGAGCCTCGGTGGC; SEQ ID NO: 8 (Primer V-mF-R) TTGGCAGCTCTGGGGCAGGGAAGG; SEQ ID NO: 9 (Total sequence of each peptide segment in the respiratory syncytial virus F protein peptide pool) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPPTNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEINLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGMDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN.

Claims

1. A human respiratory syncytial virus fusion F protein variant, characterized in that, Compared with the wild-type human respiratory syncytial virus F protein shown in SEQ ID NO: 1, the human respiratory syncytial virus fusion F protein variant contains the following amino acid mutations: L4P, A8T, T12I, T16A, A / V17I, F20L, G25S, K66E, Q101P, T / A103V, N105S, A122T, K124N, V152I, L172Q, S173L, K191R, K / N201S, I206M, K / Q209R, N276S, V384I, T529A, S540A.

2. The human respiratory syncytial virus fusion F protein variant according to claim 1, wherein The human respiratory syncytial virus fusion F protein variant has the amino acid sequence shown in SEQ ID NO:

2.

3. The human respiratory syncytial virus fusion F protein variant according to claim 2, characterized in that, The C-terminus of the human respiratory syncytial virus fusion F protein variant is linked to a trimerization domain and / or a tag sequence; and / or, the N-terminus of the human respiratory syncytial virus fusion F protein variant further contains a signal peptide sequence.

4. A polynucleotide encoding the human respiratory syncytial virus fusion F protein variant according to any one of claims 1-3.

5. The polynucleotide according to claim 4, wherein The polynucleotide is DNA or mRNA.

6. The polynucleotide according to claim 5, wherein The polynucleotide is an mRNA molecule, which contains an open reading frame having the nucleotide sequence shown in SEQ ID NO: 3, or a nucleotide sequence having at least 90% identity with the nucleotide sequence shown in SEQ ID NO:

3.

7. The polynucleotide according to claim 6, wherein The mRNA molecule further contains one or more elements selected from the following: 5' untranslated region, 3' untranslated region, signal peptide, 5' cap structure, and 3' polyadenylate tail; and / or, the mRNA molecule further contains chemical modifications.

8. The polynucleotide according to claim 7, wherein The 5' cap structure is a 5' guanosine cap, which is selected from any one of the following: m7Gppp(2'OMeA)pG, m7GpppApA, m7GpppApC, m7GpppApG, m7GpppApU; and / or, the 3' polyadenylate tail has a length of 50-150 nucleotides; and / or, the 5' end of the mRNA molecule has a methylation modification.

9. The polynucleotide according to claim 5, wherein The polynucleotide is a DNA molecule having the DNA sequence shown in SEQ ID NO:

4.

10. An expression vector containing the polynucleotide according to any one of claims 4-9.

11. A host cell transformed or transfected with the polynucleotide according to any one of claims 4-9 or the expression vector according to claim 10.

12. Use of the human respiratory syncytial virus fusion F protein variant according to any one of claims 1-3, the polynucleotide according to any one of claims 4-9, the expression vector according to claim 10, or the host cell according to claim 11 in the preparation of a vaccine for preventing and / or treating human respiratory syncytial virus infection.

13. A vaccine or immunogenic composition comprising a human respiratory syncytial virus fusion F protein variant as described in any one of claims 1-3, a polynucleotide as described in any one of claims 4-9, an expression vector as described in claim 10, or a host cell as described in claim 11, and a physiologically acceptable vehicle, adjuvant, excipient, carrier, and / or diluent.

14. The vaccine or immunogenic composition according to claim 13, characterized in that, It is a recombinant protein vaccine against human respiratory syncytial virus, which comprises a human respiratory syncytial virus fusion F protein variant as described in any one of claims 1-3 and an adjuvant.

15. The vaccine or immunogenic composition according to claim 13, which is a DNA vaccine against human respiratory syncytial virus, and the DNA vaccine comprises: (1) A eukaryotic expression vector; and (2) A DNA sequence encoding a human respiratory syncytial virus fusion F protein variant as described in any one of claims 1-3, which is constructed into the eukaryotic expression vector.

16. The vaccine or immunogenic composition according to claim 13, which is an mRNA vaccine against human respiratory syncytial virus, and the mRNA vaccine comprises: (I) An mRNA molecule encoding a human respiratory syncytial virus fusion F protein variant as described in any one of claims 1-3; and (II) An mRNA delivery vector.

17. The vaccine or immunogenic composition according to claim 16, wherein The mRNA molecule comprises an open reading frame having a nucleotide sequence as shown in SEQ ID NO: 3, or a nucleotide sequence having at least 90% identity with the sequence shown in SEQ ID NO: 3; and / or, the mRNA delivery vector is a lipid nanoparticle.

18. The vaccine or immunogenic composition according to claim 13, which is a human respiratory syncytial virus - viral vector vaccine, and it comprises: (1) A viral backbone vector; and (2) A DNA sequence encoding a human respiratory syncytial virus fusion F protein variant as described in any one of claims 1-3, which is constructed into the viral backbone vector.

19. The vaccine or immunogenic composition according to any one of claims 13-18, characterized in that, The vaccine or immunogenic composition is in the form of a nasal spray, oral preparation, suppository, or parenteral preparation.

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