F protein with stable pre-fusion conformation, nano-lipid particles and application

Abstract

The invention discloses F protein with stable pre-fusion conformation, nano-lipid particles and application of the F protein and the nano-lipid particles. The amino acid sequence of the respiratory syncytial virus F protein is subjected to point mutation, so that the F protein is stabilized at the F protein before fusion, the expression level of the obtained mutant F protein is high, and the humoral immunity and cellular immunity response level of a vaccine in an animal body is further improved.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to application number CN202311125642.X, filed on September 1, 2023, entitled “Pre-fusion conformationally stabilized F protein and nanolipid particles and uses”, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to the field of antiviral therapy, and in particular to F protein with stable pre-fusion conformation and nano lipid particles and uses thereof. Background Art

[0004] Respiratory syncytial virus (RSV) is a negative-sense, single-stranded RNA virus of the genus Pneumovirus. It is a common respiratory virus that infects the lungs and respiratory tract, usually causing mild cold symptoms. The main antigenic target for RSV vaccine development is its surface protein, the F protein, which is initially expressed as a single polypeptide precursor, named F0. F0 undergoes trimerization in the endoplasmic reticulum and is cleaved by furin at two conserved sites to produce F1, F2, and pep27 polypeptides. The pep27 polypeptide is removed and does not form part of the mature F protein. The F2 polypeptide is linked to the F1 polypeptide by two disulfide bonds to form a heterodimer. The three protomers of the F2-F1 heterodimer assemble into the mature F protein. The F protein exists in two conformations: prefusion and postfusion. The prefusion conformation is a metastable conformation, and RSV F promotes the fusion of the virus and host cell membranes by refolding from the unstable prefusion conformation to the stable postfusion conformation. Prefusion F and postfusion F share antigenic epitopes I-IV, but the two strongest epitopes that produce neutralizing antibodies and V only exist on the surface of pre-fusion F, while the level of neutralizing antibodies produced by post-fusion F is very low. Therefore, pre-fusion F is widely considered to be the main target for vaccine design.

[0005] The existing pre-fusion F design scheme can stabilize the F protein in the pre-fusion conformation. Immunizing animals or humans with soluble pre-fusion F (DS-Cav1) can significantly increase the level of serum neutralizing antibodies and produce stronger protection against RSV infection. However, there are reports that soluble DS-Cav1 pre-fusion F is still unstable and converts to the post-fusion F form during storage. The instability of DS-Cav1 F may have a negative impact on the immune response of the F protein.

[0006] In summary, there are still new strategies to stabilize the F protein in the prefusion conformation and generate a stronger immune response.

[0007] The information in the background technology is only for illustrating the general background of the present invention and should not be regarded as admitting or suggesting in any form that such information constitutes the prior art known to a person skilled in the art. Summary of the invention

[0008] In order to solve at least some of the technical problems in the prior art, the present invention provides an F protein with a stable pre-fusion conformation and its application. Specifically, the present invention includes the following contents.

[0009] In a first aspect of the present invention, there is provided an F protein with a stable pre-fusion conformation, which is optimized based on the F protein of respiratory syncytial virus A strain as a skeleton so as to stabilize it in a pre-fusion conformation.

[0010] In certain embodiments, according to the mutated F protein described in the first aspect of the present invention, the optimization refers to making the F protein contain a first mutation and a second mutation, the first mutation is located in the β2-α1 loop, and the second mutation is located in the α4-α5 hinge region.

[0011] In certain embodiments, according to the mutated F protein of the first aspect of the present invention, the first mutation and / or the second mutation are each selected from at least one of N67F, K68I, R213P and N216P.

[0012] In certain embodiments, the mutant F protein according to the first aspect of the present invention, wherein its amino acid sequence is as shown in SEQ ID NO: 1-4.

[0013] The second aspect of the present invention provides a nucleic acid molecule, wherein the nucleic acid molecule is capable of encoding the F protein with a stable pre-fusion conformation according to the first aspect; preferably, the nucleic acid molecule is mRNA.

[0014] The third aspect of the present invention provides a vector, wherein the vector comprises the nucleic acid molecule according to the second aspect.

[0015] The fourth aspect of the present invention provides a cell, wherein the cell comprises the nucleic acid molecule according to the second aspect or the vector according to the third aspect.

[0016] The fifth aspect of the present invention provides a nanolipid particle, wherein the nanolipid particle comprises the nucleic acid molecule according to the second aspect.

[0017] The sixth aspect of the present invention provides a pharmaceutical composition, which comprises the pre-fusion conformationally stabilized F protein or its encoding nucleic acid according to the first aspect, or comprises the nucleic acid molecule according to the second aspect, or comprises the nanolipid particle according to the fifth aspect; preferably, the pharmaceutical composition is a vaccine.

[0018] The seventh aspect of the present invention provides a method for preparing nanolipid particles, which comprises the step of encapsulating the nucleic acid molecule according to the second aspect with a lipid composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of an exemplary RSV prefusion F protein design (SEQ ID NO: 1).

[0020] Figure 2 Schematic diagram of another exemplary RSV prefusion F protein design (SEQ ID NO: 2).

[0021] Figure 3 Schematic diagram of another exemplary RSV prefusion F protein design (SEQ ID NO: 3).

[0022] Figure 4 Schematic diagram of another exemplary RSV prefusion F protein design (SEQ ID NO: 4).

[0023] Figure 5 Flow cytometry was used to detect the expression ratio of pre-fusion F in A549 cells. Among them, the mRNA of Moderna's F protein was designed on the backbone of RSV A using the same design concept as mRNA-1777, and tri0219-1 to tri0219-4 were the expression ratios of sequences 1-4 of the present invention. AM14 is an antibody that recognizes trimer pre-fusion F. The results showed that the expression ratios of pre-fusion F of sequences 1-4 of the present invention were higher than the mRNA expression ratios of wild-type F and Moderna F proteins.

[0024] Figure 6 BALB / c mouse immunization protocol.

[0025] Figure 7 Binding antibody detection results in BALB / c mice.

[0026] Figure 8 Neutralizing antibody detection results in BALB / c mice. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0030] F protein is an ideal target for RSV vaccine design. When RSV infects the human body, F protein will spontaneously convert from pre-fusion to post-fusion conformation. Wild-type F is a natural F protein. In the human body after RSV infection, both pre-fusion F and post-fusion F exist, but because pre-fusion F is unstable in energy (or structure), the ratio of converting to post-fusion F will be higher and higher. Therefore, post-fusion F protein will be more and more in the body. And post-fusion F has almost no neutralizing activity, and the human body cannot produce effective antibodies to neutralize the virus, so that RSV can replicate and proliferate in the body, escaping the immune response of the human body. In order to induce effective neutralizing antibodies and T cell immune response, vaccine antigens should select pre-fusion F.

[0031] There are five major neutralization sites on the surface of RSV F before fusion ( II, III, IV, V), the strongest neutralization sensitive site is located at the top of F, which is the site and V. Studies have shown that for sites The amino acids of the F protein are modified to stabilize the structure of the F protein before fusion. The present invention uses the F protein of the RSV A strain as the skeleton (see SEQ ID NO: 5) and designs a total of 4 full-length F sequences containing point mutations. The specific structures of sequences 1-4 are as follows: Figure 1-4 shown.

[0032] Sequence 1: N67F, R213P;

[0033] Sequence 2: N67F, N216P;

[0034] Sequence 3: K68I, R213P;

[0035] Sequence 4: K68L, R213P;

[0036] Among them, N is aspartic acid, F is phenylalanine, R is arginine, P is proline, K is lysine, I is isoleucine, and L is leucine.

[0037] N67 and K68 are located in the β2-α1 loop, and R213 and N216 are located in the α4-α5 hinge region. These four sites are all located at the top of RSV away from the cell membrane. The five secondary structural elements α2, α3, α4 and β3 / β4 hairpin structures on the F1 subunit are rearranged and fused with the α5 helix to form a long post-fusion helix α5post. Therefore, amino acid modification in these secondary structural regions may stabilize the pre-fusion structure of F. The β2-α1 loop can affect the movement of α4, and enhancing the interaction between α1 and α4 can stabilize the top region. Therefore, N67 or K68 can be used as a mutation site to enhance the interaction between α1 and α4. In order to increase the hydrophobic interaction between α1 and α4, three hydrophobic amino acids, phenylalanine, leucine or isoleucine, were introduced. In addition, in order to stabilize the α4-α5 hinge loop, R213 or N216 was mutated to proline. Prolines were introduced because their primary dihedral angles are restricted and their addition can reduce the flexibility of the α4-α5 hinge loop, thereby stabilizing the α4-α5 structure.

[0038] In the present invention, the lipid composition used to encapsulate the nucleic acid molecule includes compound C2, DSPC, CHOL, and DMG-PEG2000. The above components can be purchased from commercial products or prepared by themselves. For example, the compound C2 and its preparation method can be found in CN115957187A, the content of which is incorporated herein by reference in its entirety.

[0039] Example

[0040] In A549 (1x 10 5 ) cells were transfected with 1 and 2 μg of different F protein mRNAs, respectively. After 24 hours, the cells were collected and the expression of F protein was detected by flow cytometry. The results are shown in Figure 5 As shown. Figure 5 As shown, the four pre-fusion F antigens designed by the present invention have a higher proportion of pre-fusion F protein than the wild-type F protein when the cell expression is verified by flow cytometry in vitro, indicating that the design of the present invention can not only stabilize the F protein in the pre-fusion state, but also significantly increase the expression amount. Even compared with the wild type, the amount of pre-fusion F protein is increased. At present, the existing technology will cause the overall expression of F protein to decrease when the wild-type F protein is modified, and the amount of the corresponding stable fusion state F protein is also reduced. This technical effect of the present invention is completely unexpected. Among them, the expression ratio of the pre-fusion F protein recognized by the AM4 antibody of Moderna mRNA is 22%, and the expression ratios of the pre-fusion F protein of tri0219-1 to tri0219-4 of the present invention are 58%, 57%, 60% and 64% respectively. The codons used by all mRNAs of the present invention are the same.

[0041] Next, the inventors further conducted animal immunization experiments. For specific immunization schemes, see Figure 6 and Table 1. Specifically, the mice were vaccinated on days 0 and 21 according to the groups shown in Table 1 using different F proteins, blood was collected on days 14 and 25 to detect the binding antibody titer, and mice were killed on day 49 to collect blood and spleen.

[0042] Table 1 Grouping of tested vaccines

[0043]

[0044] Figure 7 The results of the BALB / c mouse binding antibody test are shown in Table 1. Figure 7 As shown, the wild-type F mRNA vaccine and Moderna's mRNA-1777 were used to prepare the control mRNA vaccine using the same lipid nanoparticle (LNP) process as sequences 1-4 of the present invention.

[0045] The preparation method of mRNA vaccine is as follows:

[0046] Accurately weigh compound C2 (Qi Chensheng self-developed cationic lipid), DSPC, CHOL, and DMG-PEG2000, and dissolve each lipid in an appropriate container with anhydrous ethanol for later use. The specific molar ratio of each lipid is: C2: DSPC: CHOL: DMG-PEG2000 = 45: 15: 38.5: 1.5; mix the lipid solution evenly according to the proportion as the organic phase, and configure the mRNA encoding the F protein into the aqueous phase (using citric acid buffer as the solvent, pH = 5.8).

[0047] The volume ratio of the organic phase to the aqueous phase is 1:3 on a microfluidic preparation instrument (e.g., PNI Ignite) to prepare a lipid nanoparticle suspension. The obtained lipid nanoparticle suspension is centrifuged and filtered through a 100kDa ultrafiltration centrifuge tube, purified and concentrated, and the concentrated liquid is packaged. The packaged samples are freeze-dried in a freeze dryer to obtain freeze-dried powder of the mRNA vaccine.

[0048] The particle size, PDI, and Zeta potential of the prepared lipid nanoparticle liquid sample and the reconstituted sample of the lyophilized powder were measured using a laser nanoparticle size analyzer, and the encapsulation efficiency (%) was measured using an ultraviolet spectrophotometer combined with a RiboGreen RNA kit.

[0049] The physical and chemical quality control data of the prepared lipid nanoparticles are shown in the following table:

[0050] Table 2

[0051] Sample Storage format Particle size PDI Potential (mV) Encapsulation rate (%) Wild Type F Lyophilized reconstitution 157.7 0.17 46 97.2 Positive Control (Moderna) Lyophilized reconstitution 172.2 0.11 49 97 tri0219-1 Lyophilized reconstitution 165.8 0.16 49 97.5 tri0219-2 Lyophilized reconstitution 153.1 0.13 48 97.1 tri0219-3 Lyophilized reconstitution 167.6 0.12 54 97.1 tri0219-4 Lyophilized reconstitution 183 0.17 52 97.4

[0052] tir0219-1 to tir0219-4 are F mRNA vaccines of sequences 1-4 of the present invention. Blood was collected 14 days after the second injection of the vaccine (20 μg), and the titer of binding antibodies in the serum of BALB / c mice was detected by ELISA. The results showed that the binding antibody level produced by the mRNA vaccines of sequences 1-4 of the present invention was equivalent to or higher than that of wild-type F. The binding antibody level produced by Moderna mRNA was lower than that of wild-type F. Among them, the binding antibody serum titer of tri0219-1, tri0219-2, and tri0219-3 groups was 10 6 -10 8 ng / ml; Moderna sequence is between 10 5 -10 7 The binding antibody levels of tri0219-1, tri0219-2 and tri0219-3 were significantly higher than those of mRNA-1777.

[0053] Blood was collected 14 days after the second injection of the vaccine (20 μg), and the titer of binding antibodies of BALB / c mouse serum to RSV A2 and B 18537 was detected by fluorescence in HEp-2 cells. The results showed that the level of neutralizing antibodies against RSV A2 and B 1853 produced by the mRNA vaccine of sequence 1-4 of the present invention was higher than that of wild-type F and Moderna vaccine. Similar to the binding antibody situation, the level of neutralizing antibodies produced by Moderna mRNA was lower than that of wild-type F.

[0054] Among them, tri0219-1 group targets A2 strain in 2 8 -2 11 Moderna sequence is between 2 5 -2 9 The unmodified wild-type F sequence is between 2 6 -2 9 The neutralizing antibody titers produced by tri0219-1 and tri0219-3 against RSV A2 were significantly higher than those produced by mRNA-1777.

[0055] The tri0219-1 group targets strain B in 2 9 -2 12 Moderna sequence is between 2 7 -2 11 The unmodified wild-type F sequence is between 2 6 -2 10 The neutralizing antibody titers against RSV B produced by tri0219-1 and tri0219-2 were significantly higher than those produced by mRNA-1777.

[0056] The specific sequences of the corresponding primers or molecules are provided in this specification, and according to relevant regulations, this application also provides a sequence table in a computer-readable form. It should be noted that the sequences in the sequence table in a computer-readable form are for reference only. In the event that the sequences in this specification are inconsistent with the sequences in the sequence table in a computer-readable form, the sequence content in this specification shall prevail.

[0057] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims should be based on the broadest interpretation to cover all modifications and equivalent structures and functions.

Claims

1. A pre-fusion conformationally stable F protein, characterized in that: It is based on the F protein of respiratory syncytial virus A strain and is optimized to stabilize it in the pre-fusion conformation.

2. The F protein with a stable pre-fusion conformation according to claim 1, characterized in that The optimization refers to making the F protein contain a first mutation and a second mutation, wherein the first mutation is located in the β2-α1 loop, and the second mutation is located in the α4-α5 hinge region.

3. The F protein with a stable pre-fusion conformation according to claim 2, characterized in that The first mutation and / or the second mutation are each selected from at least one of N67F, K68I, R213P and N216P.

4. The F protein with a stable pre-fusion conformation according to claim 1, characterized in that Its amino acid sequence is shown in SEQ ID NO: 1-4.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule is capable of encoding and producing the pre-fusion conformationally stable F protein according to any one of claims 1 to 4; Preferably, the nucleic acid molecule is mRNA.

6. A carrier, characterized in that The vector comprises the nucleic acid molecule according to claim 5.

7. A cell, characterized in that The cell comprises the nucleic acid molecule according to claim 5 or the vector according to claim 6.

8. A nano lipid particle, characterized in that: It comprises the nucleic acid molecule according to claim 5.

9. A pharmaceutical composition, characterized in that Comprising the pre-fusion conformationally stabilized F protein or its encoding nucleic acid according to any one of claims 1 to 4, or comprising the nucleic acid molecule according to claim 5, or comprising the nanolipid particle according to claim 8; Preferably, the pharmaceutical composition is a vaccine.

10. A method for preparing nano lipid particles, characterized in that: The method comprises the step of encapsulating the nucleic acid molecule according to claim 5 with a lipid composition.

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