A human respiratory syncytial virus fusion f protein variant, vaccines based thereon, and uses thereof

Abstract

The present application relates to a human respiratory syncytial virus (hRSV) fusion F protein variant, a vaccine based thereon and applications thereof. The hRSV fusion F protein variant of the present application (in particular the mRNA vaccine based thereon) can induce the body to produce high levels of neutralizing antibodies against respiratory syncytial virus, while being capable of activating cellular immune response, achieving dual activation of humoral immunity and cellular immunity, and having cross-protection efficacy against each subtype of hRSV. Therefore, the vaccine product based on the hRSV fusion F protein variant of the present application has important application value in the clinical treatment and prevention and control of respiratory syncytial virus.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically 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. The infection is more common in infants and young children under 2 years old, especially 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 development has long been hampered by the virus's complex pathogenic mechanisms and the immunopathological responses it induces. The F protein plays a crucial role in viral membrane fusion and host cell invasion, and its conserved epitopes are key targets for inducing neutralizing antibodies.

[0004] mRNA vaccines are a new type of vaccine that has emerged in recent years. Their basic principle is to introduce mRNA expressing an antigen target into the body through a specific delivery system, where it expresses the immunogenic protein, thereby activating the immune system and stimulating the body to produce a specific immunological response, thereby providing immune protection. Compared with other vaccines, mRNA vaccines have unique advantages: First, after entering the cell, mRNA can be translated and expressed in the cytoplasm of the cell without entering the cell 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 and do not rely on cell or bacterial culture. Their R&D cycle is short, production costs are low, and production processes and procedures 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. The use of vectors to deliver mRNA can also achieve efficient mRNA expression. The adjuvant effect of mRNA vaccines can also activate both humoral and cellular immunity.

[0005] Current RSV mRNA vaccine development targets the viral fusion protein (F protein) as its core antigenic target. While the development and design of RSV mRNA vaccines has become a research focus in this field, no safe and effective RSV mRNA vaccine has yet received regulatory approval. Therefore, there is an urgent need 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] Purpose of the Invention

[0007] In response to the defects or needs in the prior art, the object of the present invention is to provide a human respiratory syncytial virus (hRSV) fusion F protein variant that can efficiently stimulate a specific immune response to human respiratory syncytial virus (hRSV), a vaccine based thereon, and its application.

[0008] Solution

[0009] To achieve the purpose of the present invention, the present invention provides the following technical solutions:

[0010] In a 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 as shown in SEQ ID NO: 1, the human respiratory syncytial virus fusion F protein variant comprises the following amino acid mutations:

[0011] 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.

[0012] Optionally, the human respiratory syncytial virus fusion F protein variant has the amino acid sequence shown in SEQ ID NO:2.

[0013] Further feasible, the C-terminus of the human respiratory syncytial virus fusion protein variant is connected to a trimerization domain and / or a tag sequence; and / or the N-terminus of the human respiratory syncytial virus fusion protein variant further comprises a signal peptide sequence.

[0014] In a second aspect, the present invention provides a polynucleotide encoding the human respiratory syncytial virus fusion F protein variant as described in the first aspect above.

[0015] The polynucleotide can be DNA or mRNA.

[0016] In a preferred embodiment, the polynucleotide is an mRNA molecule comprising an open reading frame having the nucleotide sequence shown in SEQ ID NO: 3, or a nucleotide sequence having at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 3.

[0017] By "having at least 90% identity" is meant having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9% or 100% identity.

[0018] In a feasible embodiment, the mRNA molecule further comprises one or more elements selected from the group consisting of: a 5' untranslated region, a 3' untranslated region, a signal peptide, a 5' cap structure, and a 3' poly(A) tail.

[0019] In a possible embodiment, the mRNA molecule further comprises a chemical modification.

[0020] 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;

[0021] Preferably, the 3' poly(A) tail has a length of 50-150 nucleotides.

[0022] Preferably, the 5' end of the mRNA molecule may be methylated to improve its stability.

[0023] The mRNA of the present invention can be prepared by conventional methods well known to those skilled in the art. For example, DNA corresponding to the mRNA sequence can be obtained and then transcribed in vitro (optionally, the 5' end of the DNA is modified after transcription) to obtain the mRNA. Preferably, the preparation method further comprises purifying the transcribed mRNA.

[0024] In other preferred embodiments, the polynucleotide is a DNA molecule comprising a DNA sequence as shown in SEQ ID NO:4.

[0025] In a third aspect, the present invention provides an expression vector comprising the polynucleotide described in the second aspect above.

[0026] In a fourth aspect, the present invention provides a host cell transformed or transfected with the polynucleotide as described in the second aspect or the expression vector as described in the third aspect.

[0027] 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.

[0028] In a sixth aspect, the present invention provides a vaccine or immunogenic composition, comprising 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.

[0029] In some preferred embodiments, the vaccine or immunogenic composition is a human respiratory syncytial virus recombinant protein vaccine, which includes the human respiratory syncytial virus fusion F protein variant as described in the first aspect above and an adjuvant.

[0030] In other preferred embodiments, the vaccine or immunogenic composition is a human respiratory syncytial virus DNA vaccine, and the DNA vaccine comprises:

[0031] (1) eukaryotic expression vector; and

[0032] (2) A DNA sequence encoding the human respiratory syncytial virus fusion F protein variant as described in the first aspect above is constructed and incorporated into the eukaryotic expression vector.

[0033] Preferably, the DNA sequence encoding the human respiratory syncytial virus fusion F protein variant as described in the first aspect above is shown as SEQ ID NO: 4.

[0034] In other preferred embodiments, the vaccine or immunogenic composition is a human respiratory syncytial virus mRNA vaccine, and the mRNA vaccine comprises:

[0035] (I) an mRNA molecule encoding the human respiratory syncytial virus fusion F protein variant as described in the first aspect above; and

[0036] (II) mRNA delivery vector.

[0037] Preferably, the mRNA molecule comprises 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.

[0038] Preferably, the mRNA delivery vector may be a lipid- or polymer-based nanoparticle, or other delivery system (eg, a squalene-based cationic nanoemulsion), preferably a lipid nanoparticle.

[0039] The mRNA molecules can be encapsulated in liposomes or other mRNA delivery vehicles.

[0040] Preferably, the particle size distribution of the mRNA vaccine is in the range of 70-120 nm.

[0041] In a feasible embodiment, the mRNA vaccine can be administered via mucosal routes, intramuscular injection, subcutaneous injection, and intravenous injection.

[0042] In other preferred embodiments, the vaccine or immunogenic composition is a human respiratory syncytial virus-viral vector vaccine comprising:

[0043] (1) viral backbone vectors; and

[0044] (2) A DNA sequence encoding the human respiratory syncytial virus fusion F protein variant as described in the first aspect is constructed and incorporated into the viral backbone vector.

[0045] Preferably, the DNA sequence encoding the human respiratory syncytial virus fusion F protein variant as described in the first aspect is shown as SEQ ID NO: 4.

[0046] In possible embodiments, the vaccine or immunogenic composition is in the form of a nasal spray, oral formulation, suppository, or parenteral formulation.

[0047] Further preferably, the nasal spray is selected from aerosols, sprays and powder mists, the oral preparation is selected from tablets, powders, pills, granules, soft / hard capsules, film-coated preparations and ointments, and the parenteral preparation is a transdermal preparation, an ointment, a plaster, an external liquid, an injectable preparation (e.g., intramuscular injection, subcutaneous injection, intravenous injection preparation).

[0048] In the 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 thereof a preventive 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.

[0049] The "preventively and / or therapeutically effective amount" may vary depending on the subject of administration, the subject organ, symptoms, the method of administration, etc., and can be determined based on the doctor's judgment, taking into account the type of dosage form, the method of administration, the patient's age and weight, the patient's symptoms, etc.

[0050] Beneficial effects

[0051] The human respiratory syncytial virus (hRSV) fusion F protein variant of the present invention (particularly 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 cellular immune responses 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 process, while also clearing latently infected cells. Compared with a single antibody-mediated immune strategy, it significantly improves the cross-protection efficacy against various hRSV subtypes (type A / B). Therefore, the vaccine based on the hRSV fusion F protein variant of the present invention is of great significance for the clinical treatment and prevention and control of respiratory syncytial virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] One or more embodiments are exemplarily illustrated by the accompanying figures, and these exemplary illustrations do not limit the embodiments. The word "exemplary" is used herein to mean "serving as an example, example, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0053] Figure 1 Schematic diagram of the structure of the hRSV mRNA molecule construct of the present invention.

[0054] Figure 2 This is the gel electrophoresis identification result of the respiratory syncytial virus mRNA molecule obtained by in vitro transcription in Example 1.

[0055] Figure 3 This is the particle size test result of the respiratory syncytial virus mRNA vaccine measured in Example 2.

[0056] Figure 4 The results of the specific IgG antibody titer test of the serum of mice immunized with the respiratory syncytial virus mRNA vaccine described in Example 4 are shown.

[0057] Figure 5 The results of the neutralizing antibody level test in the serum of mice immunized with the respiratory syncytial virus mRNA vaccine described in Example 5 are shown.

[0058] Figure 6 The cytokine activation in spleen cells of mice immunized with the respiratory syncytial virus mRNA vaccine described in Example 6 is shown.

[0059] Figure 7 The CD4+ T cell (Figure A) and CD8+ T cell (Figure B) immune responses induced by the respiratory syncytial virus mRNA vaccine described in Example 7 are shown, wherein the abscissa represents the percentage of positive cells. DETAILED DESCRIPTION

[0060] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0061] In addition, in order to better illustrate the present application, a large number of specific details are given in the specific embodiments below. Those skilled in the art should understand that the present application can also be implemented without some specific details. In some embodiments, the raw materials, elements, methods, means and the like which are well known to those skilled in the art are not described in detail, so as to highlight the main idea of the present application.

[0062] Unless otherwise explicitly indicated, throughout the specification and claims, the term "comprise" or its variants such as "contain" or "include" and the like will be understood to include the stated element or component, but not to exclude the presence of other elements or components.

[0063] Example 1: Design and synthesis of respiratory syncytial virus mRNA molecule

[0064] In this embodiment, an hRSV mRNA molecule was designed and prepared as a representative example of the present application.

[0065] The specific procedure is as follows:

[0066] (1) Design of hRSV F protein variant sequence

[0067] The following 24 epidemic mutation sites: 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 were introduced into the hRSV A2 strain F protein sequence as shown in SEQ ID NO: 1 to obtain the F protein variant sequence, and the amino acid sequence thereof is shown in SEQ ID NO: 2.

[0068] (2) Obtaining of DNA coding sequence of hRSV F protein variant

[0069] The amino acid sequence of the obtained F protein variant was codon-optimized to obtain its DNA coding sequence, which is shown in SEQ ID NO: 4.

[0070] (3) Preparation of DNA transcription template for hRSV mRNA

[0071] The above DNA coding sequence together with other necessary expression control elements (e.g., Figure 1 The hRSV mRNA molecular construct (shown in the figure) was introduced into the mRNA vaccine expression vector ABOP-028 (Zhang NN, et.al. AThermostable mRNA Vaccine against COVID-19) to construct a DNA transcription template for hRSV mRNA.

[0072] The specific method is as follows:

[0073] First, PCR primers were designed for amplifying the DNA coding sequence (abbreviated as "mF") and its vector fragment (abbreviated as "V-mF"), respectively, as shown in Table 1 below);

[0074] Table 1

[0075] ;

[0076] Then, using the synthesized DNA molecule encoding hRSV mRNA (whose sequence is shown in SEQ ID NO: 4) as a template, the primer pair mF-F and mF-R in Table 1 were used, and a high-fidelity DNA polymerase premix (Q5® High-Fidelity 2X Master Mix, purchased from NEB) was used for PCR amplification to obtain the DNA encoding fragment of hRSV mRNA (abbreviated as "mF"). Then, using the synthesized expression vector ABOP-028 as a template, the primer pair V-mF-F and V-mF-R in Table 1 were used, and a high-fidelity DNA polymerase premix (Q5® High-Fidelity 2X Master Mix, purchased from NEB) was used for PCR amplification 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.

[0077] Table 2

[0078] ;

[0079] Table 3

[0080] ;

[0081] Finally, the amplified fragment mF was ligated with the vector fragment V-mF in vitro using the In-Fusion seamless cloning kit (purchased from TAKARA). The ligation product was transformed into Stbl3 competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.), and the correct clone was picked to obtain the recombinant expression plasmid of hRSV mRNA, which can be used as a DNA transcription template for hRSV mRNA.

[0082] (4) In vitro transcription reaction

[0083] The DNA template was linearized using BsaI restriction endonuclease (purchased from NEB). The resulting linearized plasmid was used as a template for in vitro transcription using T7 RNA polymerase (purchased from Suzhou Nearshore Protein Biotechnology Co., Ltd.) and unmodified natural nucleoside triphosphates (ATP, CTP, UTP, and GTP, purchased from Shanghai Zhaowei Biotechnology Development Co., Ltd.) as substrates. After the in vitro transcription reaction, DNase (purchased from Suzhou Nearshore Protein Biotechnology Co., Ltd.) was added to digest the linearized DNA template. The synthesized mRNA was purified by adding 7.5 M LiCl solution (purchased from Invitrogen). The 5' end of the mRNA was capped using a capping enzyme (purchased from Suzhou Nearshore Protein Biotechnology Co., Ltd.), and the cap structure was methylated using a cap structure 2'-O-methyltransferase (purchased from Suzhou Nearshore Protein Biotechnology Co., Ltd.). Finally, the mRNA stock solution was purified by adding 7.5 M LiCl solution (purchased from Invitrogen) to obtain mRNA molecules encoding respiratory syncytial virus antigens.

[0084] The obtained mRNA molecules were subjected to 1% agarose gel electrophoresis experiment to identify the results. Figure 2 ;Depend on Figure 2 It can be seen that, judging by the molecular weight, the mRNA molecule obtained through the above steps is the target mRNA molecule.

[0085] Example 2: Preparation of respiratory syncytial virus mRNA vaccine and particle size determination

[0086] The mRNA molecule stock solution encoding the respiratory syncytial virus F protein variant obtained in Example 1 was quickly mixed with a lipid excipient combination (purchased from Suzhou Aibo Biotechnology Co., Ltd.) to encapsulate the mRNA in the lipid nanosphere preparation; after dialyzing overnight at 4°C in a PBS solution (purchased from Beijing Solebold Technology Co., Ltd.), it was concentrated and filtered for sterilization to obtain a finished respiratory syncytial virus mRNA vaccine preparation (abbreviated as "RSV-mF+LNP").

[0087] The particle size of the obtained mRNA vaccine preparation product was detected by DLS (Dynamic Light Scattering) method, and the results are shown in Table 1. Figure 3 .

[0088] Figure 3 It was shown that the particle size of the obtained mRNA vaccine preparation was in the range of 70-120 nm, and the size and morphology were uniform, which met the relevant standards of mRNA vaccine.

[0089] Example 3: Immunization of experimental animals and sample collection

[0090] In this example, 6-week-old female mice of BALB / c strain were used for immunization experiment; the experimental groups were divided into mRNA vaccine immunization group and placebo control group (as negative control), wherein the placebo control group was LNP immunization group.

[0091] All mice were immunized with one 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 contained in the mRNA vaccine per mouse per injection.

[0092] The serum samples of the mice were collected on day 14 and day 28, respectively, for detecting the specific IgG antibody titers and neutralizing antibody titers in the immune serum. The collected immune mouse serum was inactivated at 56°C for 30 min and stored at -80°C for use.

[0093] In addition, the spleen tissues of the mice were also collected on day 28, and the spleen cells were separated from the spleen tissues, and the specific method was as follows: the spleen of the mouse was taken and placed in RPMI1640 culture medium containing 2% FBS (purchased from Invitrogen company) on ice; the cells were ground on a 70 μm filter into a 50 mL centrifuge tube, and centrifuged at 500g for 5 min; 5 mL of red blood cell lysis solution (purchased from Invitrogen company) was added to the cell precipitate, and the red blood cells were lysed at room temperature for 5 min; centrifuged at 500g for 5 min, washed the cells once with RPMI1640 culture medium containing 10% FBS, resuspended the cell precipitate with 1 mL of cryopreservation solution (purchased from Stem cell company), and stored the cells in liquid nitrogen for subsequent detection of cytokine activation and T cell immunity.

[0094] Example 4: Detection of pre-F protein-specific IgG antibody titers in immune mouse serum by ELISA method

[0095] In this embodiment, the mouse respiratory syncytial virus pre-F antibody (IgG) detection kit (purchased from Nanjing Novi Zhan Biotechnology Co., Ltd.) was used to detect the pre-F specific IgG titer of the serum samples collected from the mice on day 14 and day 28 in Example 3.

[0096] Specifically, the sample to be tested was first diluted by 1:100 with a sample diluent, and then diluted by 2-fold gradient, a total of 5-10 gradients; 100 μl of the sample to be tested was added to each well; after sealing the plate with a sealing film, it was placed in a 37°C incubator for 60 min; after incubation, the sealing film was removed, and the liquid in the well was discarded, at least 300 μl of 1x washing solution was added to each well, and after standing for 30 s, the washing solution was discarded, and the plate was washed for 4 times in succession; 100 μl of enzyme-labeled reagent was added to each well; the incubation and washing steps were repeated; 100 μl of color developing solution was added to each well, and after sealing with a sealing film, it was placed in a 37°C incubator for 15 min in the dark; the sealing film was removed, 50 μl of stop solution was added to each well, and mixed gently, and then the OD values at 450 nm and 630 nm of each well were detected by an enzyme-labeled instrument. The absorbance value was calculated by subtracting the OD value at 630 nm from the OD value at 450 nm of the same well. The end-point titer was defined as the serum dilution factor corresponding to the situation that the absorbance value (as described above, the absorbance at 450 nm minus the absorbance at 630 nm) of the serum produced by the immunized mice was 2.1 times greater than that of the placebo group. The antibody titer below the detection limit was defined as one-third of the detection limit.

[0097] The antibody titer detection results of the serum of the immunized mice, Figure 4 It is shown that: compared with the placebo group, the serum after the first immunization and the second immunization of the mRNA vaccine of the application contains a high level of pre-F specific IgG antibody, and the antibody titer of the serum after the second immunization is 1000 times higher than that after the first immunization, indicating that the mRNA vaccine has a good IgG antibody activation effect, and the IgG antibody titer after the second immunization is higher.

[0098] The above results show that: the hRSV mRNA vaccine of the application can induce the mice to produce a high level of specific IgG antibody against the preF protein.

[0099] Example 5: Micro-neutralization experiment for detecting the neutralizing antibody titer in the serum of the immunized mice

[0100] In this embodiment, the neutralizing antibody titer in the serum samples collected from the mice on day 28 in Example 3 was determined by a micro-neutralization experiment.

[0101] The specific method is as follows:

[0102] Respiratory syncytial virus epidemic strain ON1 and BA9 strain virus were diluted to a dose of 100 CCID50, respectively; 75 μL of cell maintenance solution was added to each well in the first row (A) of the serum test plate, and 50 μL of maintenance solution was added to the remaining wells; 25 μL of inactivated serum sample was added to the first row, and each serum was added to two wells to prepare a 1 / 4 serum dilution. Using a micro pipette, 50 μL of diluent was sequentially added to the subsequent wells from A to G, and 2-fold serial dilutions of all sera were completed. 50 μL of the last well was discarded after mixing; 50 μL of the target virus solution containing 100 CCID50 was added to each well of the 96-well culture plate after dilution of the serum, and the same amount of diluent was added to the blank control well. All culture plates were placed in a 37°C cell culture incubator for 2h. After neutralization, 100 μL of cell maintenance solution was added to each well, and the culture was placed in a 37°C incubator for 5 days; the target virus solution of 100 CCID50 / 50 μL was diluted 10 times in series to 1 / 1000 as a stock solution. It was inoculated in a cell culture plate, 8 wells for each dilution, 50 μL / well. 50 μL of cell culture solution (equivalent to the amount of serum) was added to each well, and the culture was placed in a 37°C, CO2 incubator; the culture was observed every day after virus inoculation, and the final result was determined on the fifth day; based on the serum dilution data, the highest dilution of serum that can neutralize 50% of virus infection (using cytopathic effect (CPE) as an indicator) was calculated by Karber method, and the reciprocal of the dilution was the neutralizing antibody titer NT 50 .

[0103] The results are shown in Figure 5 . Figure 5 The results show that the mRNA vaccine of the application can effectively induce mice to produce neutralizing antibody titers NT 50 1 / 1810 against respiratory syncytial virus epidemic strain ON1, and neutralizing antibody titers NT 50 1 / 678 against BA9, which indicates that the vaccine after immunization can produce high titer levels of neutralizing antibodies, which can effectively resist viruses against A and B subtypes of respiratory syncytial virus.

[0104] Example 6: Detection of cytokine activation in the spleen of immunized mice by ELISpot method

[0105] The specific method is as follows:

[0106] The frozen spleen cells in Example 3 were revived in a 96-well plate and cultured overnight in a cell culture incubator at 37°C and 5% CO2. 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 minutes, and 200 μL of RPMI1640 medium containing 10% FBS was added to each well, and the plate was incubated at room temperature for 30 minutes to activate the plate. A negative control group, an experimental group, and a positive control group were set up, and 100 μL of RPMI1640 containing 10% FBS, a respiratory syncytial virus F protein peptide pool (0.3 μg / well, the total sequence of each peptide in the peptide pool is shown in SEQ ID NO: 9, and each peptide in the peptide pool is 15 amino acids long, and there is a 10 amino acid overlap between them, for a total of 141 peptides), and coenzyme A (0.15 μg / well) were added to each well. 2.5×10 5 Splenocytes were incubated in a cell culture incubator at 37°C and 5% CO2 for 48 hours; the detection antibody (R4-6a2 biotin) was diluted to 1 μg / mL using 0.5% FBS in PBS, 100 μL was added to each well, and the cells were incubated at room temperature for 2 hours; Streptavidin-ALP was diluted 1000-fold using 0.5% FBS in PBS, 100 μL was added to each well, and the cells were incubated at room temperature for 1 hour; BCIP / NBT-plus was filtered using 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.

[0107] See the results Figure 6 . Figure 6 It was shown that under the in vitro stimulation of the respiratory syncytial virus F protein peptide pool, the mRNA immunization group efficiently stimulated the production of cytokines such as IFN-γ, TNF-α, IL2, and IL4 in mouse spleen cells, 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.

[0108] Example 7: Flow cytometry detection of T cell memory immune response induced by mRNA vaccine

[0109] The specific method is as follows:

[0110] The spleen cells frozen in Example 3 were revived in 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 (equal volume DMSO treatment), positive control (phorbol ester PMA treatment), and peptide pool stimulation group were set up respectively, with 3 replicates for each group; among them, the peptide pool stimulation group used 3 μg / mL respiratory syncytial virus F protein peptide pool (as used in Example 6), and added co-stimulators anti-CD28 (2 μg / mL) and anti-CD49d (2 μg / mL); 100 ng / mL PMA was added to the positive control group; the negative control group was added with peptide pool solvent DMSO; after incubation in a cell culture incubator at 37°C and 5% CO2 for 1 hour, 0.7 μg / mL golistop and 1 μg / mL goliplug were added, and incubated in a cell culture incubator at 37°C and 5% CO2 for 9 hours; the cells were collected into a 1.5 mL EP tube, centrifuged at 500g for 5 minutes, the culture medium was discarded, and 1 mL PBS was added to wash the cell pellet; 50 μL flow cytometry antibody (with fluorescent dye zombie) was added Resuspend the cells with 100 μL of fixative and incubate at 4°C in the dark for 30 min; add 1 mL of perm buffer to wash the cells and retain the cell pellet; add 50 μL of intracellular factor antibodies (anti-IFN-γ, anti-TNF-α, anti-IL-2, anti-IL-4) and incubate at 4°C in the dark for 30 min; add 200 μL of PBS to resuspend the cells and incubate at 4°C in the dark for 30 min.

[0111] The results of CD4+ T cell and CD8+ T cell immune response detection were shown in Figure 7 Figures A and B in the figure.

[0112] Depend on Figure 7 As shown in Figures A and B, under the in vitro stimulation of the respiratory syncytial virus F protein peptide pool, the spleen cells of mice immunized with the mRNA of the present invention produced a Th1-biased CD4+ T cell response (expressing IFN-γ, TNF-α) and a significant antigen-specific multifunctional CD8+ T cell response (expressing IFN-γ); this indicates that the mRNA immunization of the present invention can activate specific CD4+ / CD8+ T cell responses and play a role in cellular immune protection.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the present invention.

[0114] Sequences covered in this article:

[0115] SEQ ID NO: 1 (amino acid sequence of wild-type hRSV F protein):

[0116] ;

[0117] SEQ ID NO: 2 (amino acid sequence of the hRSV F protein variant of the present invention):

[0118] MELPILKTNAIITILAIVTLCFASSQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTSAVNSRARRELPRFMNYTLNNTKNTNVTLSKKRKRRFLGFLLGVGSAIASGNAVSKVLHLEGEVNKIKSALQLTNKAVVSLSNGVSVLTSRVLDLKNYIDNQLLPMVNRQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITAIIIVIIVILLALIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN;

[0119] SEQ ID NO:3(本发明的hRSV F蛋白变体的mRNA序列):

[0120]

[0121] SEQ ID NO: 4 (DNA sequence of the hRSV F protein variant of the present invention):

[0122]

[0123] SEQ ID NO: 5 (primer mF-F)

[0124] TCCCTGCCCCAGAGCTGCCAATCCTGAA;

[0125] SEQ ID NO: 6 (primer mF-R)

[0126] GCTCCAGCCTAGAATTCTTATTAATTGCTGAAGGCGA;

[0127] SEQ ID NO: 7 (primer V-mF-F)

[0128] ATTAATAAGAATTCTAGGCTGGAGCCTCGGTGGC;

[0129] SEQ ID NO: 8 (primer V-mF-R)

[0130] TTGGCAGCTCTGGGGCAGGGAAGG;

[0131] SEQ ID NO: 9 (total sequence of each peptide segment in the respiratory syncytial virus F protein peptide pool)

[0132] 。

Claims

1. A human respiratory syncytial virus fusion F protein variant, characterized in that The amino acid sequence of the human respiratory syncytial virus fusion F protein variant is shown in SEQ ID NO:

2.

2. The human respiratory syncytial virus fusion F protein variant according to claim 1, characterized in that The C-terminus of the human respiratory syncytial virus fusion F protein variant is further connected 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 comprises a signal peptide sequence.

3. A polynucleotide encoding the human respiratory syncytial virus fusion F protein variant according to any one of claims 1-2.

4. The polynucleotide according to claim 3, wherein The polynucleotide is DNA or mRNA.

5. The polynucleotide according to claim 4, characterized in that The polynucleotide is an mRNA molecule, which comprises an open reading frame. The nucleotide sequence of the open reading frame is shown in SEQ ID NO:

3.

6. The polynucleotide according to claim 5, wherein The mRNA molecule further comprises one or more elements selected from the group consisting of a 5' untranslated region, a 3' untranslated region, a signal peptide, a 5' cap structure, and a 3' poly(A) tail; And / or, the mRNA molecule further comprises a chemical modification.

7. The polynucleotide according to claim 6, characterized in that The 5' cap structure is a 5' guanosine cap selected from any one of the following: m7Gppp(2'OMeA)pG, m7GpppApA, m7GpppApC, m7GpppApG, m7GpppApU; and / or, the 3' poly(A) tail has a length of 50-150 nucleotides; And / or, the 5' end of the mRNA molecule has a methylation modification.

8. The polynucleotide according to claim 4, wherein The polynucleotide is a DNA molecule, and the DNA sequence of the DNA molecule is shown in SEQ ID NO:

4.

9. An expression vector comprising the polynucleotide according to any one of claims 3 to 8.

10. A host cell transformed or transfected with the polynucleotide according to any one of claims 3 to 8 or the expression vector according to claim 9.

11. Use of the human respiratory syncytial virus fusion F protein variant according to any one of claims 1 to 2, the polynucleotide according to any one of claims 3 to 8, the expression vector according to claim 9 or the host cell according to claim 10 in preparing a vaccine for preventing and / or treating human respiratory syncytial virus infection.

12. A vaccine or immunogenic composition comprising the human respiratory syncytial virus fusion F protein variant according to any one of claims 1-2, the polynucleotide according to any one of claims 3-8, the expression vector according to claim 9 or the host cell according to claim 10, and a physiologically acceptable vehicle, adjuvant, excipient, carrier and / or diluent.

13. The vaccine or immunogenic composition according to claim 12, characterized in that It is a human respiratory syncytial virus recombinant protein vaccine, which comprises the human respiratory syncytial virus fusion F protein variant according to any one of claims 1 to 2 and an adjuvant.

14. The vaccine or immunogenic composition according to claim 12, which is a human respiratory syncytial virus DNA vaccine, comprising: (1) eukaryotic expression vector; and (2) A DNA sequence encoding the human respiratory syncytial virus fusion F protein variant according to any one of claims 1 to 2, constructed and incorporated into the eukaryotic expression vector.

15. The vaccine or immunogenic composition according to claim 12, which is a human respiratory syncytial virus mRNA vaccine, comprising: (I) an mRNA molecule encoding the human respiratory syncytial virus fusion F protein variant according to any one of claims 1 to 2; and (II) mRNA delivery vector.

16. The vaccine or immunogenic composition according to claim 15, characterized in that The mRNA molecule comprises an open reading frame, the nucleotide sequence of which is shown in SEQ ID NO: 3; And / or, the mRNA delivery vector is a lipid nanoparticle.

17. The vaccine or immunogenic composition according to claim 12, which is a human respiratory syncytial virus-viral vector vaccine comprising: (1) Viral backbone vector; and (2) A DNA sequence encoding the human respiratory syncytial virus fusion F protein variant according to any one of claims 1 to 2, constructed into the viral backbone vector.

18. The vaccine or immunogenic composition according to any one of claims 12 to 17, characterized in that The vaccine or immunogenic composition is in the form of a nasal spray, oral formulation, suppository or parenteral formulation.

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