CN117304281B - Recombinant RSV F protein and application thereof - Google Patents
Recombinant RSV F protein and application thereof Download PDFInfo
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- CN117304281B CN117304281B CN202311594790.6A CN202311594790A CN117304281B CN 117304281 B CN117304281 B CN 117304281B CN 202311594790 A CN202311594790 A CN 202311594790A CN 117304281 B CN117304281 B CN 117304281B
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- 241000712461 unidentified influenza virus Species 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- GVJHHUAWPYXKBD-IEOSBIPESA-N α-tocopherol Chemical compound OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-IEOSBIPESA-N 0.000 description 1
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Abstract
The invention belongs to the technical field of biological medicine, and particularly relates to a recombinant RSV F protein and application thereof in preparation of respiratory syncytial virus vaccine. The recombinant RSV F protein is provided in the form of a combination mutation without engineering the natural furin cleavage site and pep27 of the wild-type RSV F protein. The recombinant RSV F protein has high expression quantity and good stability, and can be used for preparing products such as vaccine, respiratory syncytial virus antibody, respiratory syncytial virus resisting serum, diagnostic antigen and the like.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to a recombinant RSV F protein, a preparation method thereof and application thereof in preparation of respiratory syncytial virus vaccine.
Background
Human respiratory syncytial virus (Human Respiratory Syncytial Virus, hRSV) is one of the main causes of severe respiratory diseases in infants and elderly, and since hRSV was found in the 50 th century, scientific researchers at home and abroad have largely developed hRSV vaccines, which have been faced with great challenges of insufficient safety or poor immunogenicity, various hRSV vaccines entering the clinical trial stage have been declared "failed". In 2013, pre-fusion F protein (prefusion F protein, pre-F) conformation successfully resolved, and Pre-F-based hRSV vaccine showed good application prospect. Despite extensive research on the conformational stability of RSV F protein prior to fusion, the safety and efficacy of its use as a vaccine remains a significant challenge.
Accordingly, it is an urgent need for a vaccine comprising such a protein to provide RSV Pre-F protein having high safety, high expression level, enhanced immunogenicity and improved stability.
Disclosure of Invention
In order to solve the technical problems, the invention discloses a recombinant RSV F protein, a preparation method thereof and application thereof in preparing respiratory syncytial virus vaccine. The recombinant RSV F protein incorporates a mutation in the amino acid sequence relative to the amino acid sequence of a corresponding wild-type RSV F protein, the mutation comprising an amino acid substitution, deletion or addition.
In one aspect, the invention provides a recombinant Respiratory Syncytial Virus (RSV) protein comprising a soluble F protein polypeptide comprising at least one modification selected from the group consisting of:
(a) At least one pair of amino acid residues is substituted with cysteine;
(b) At least one cavity filling abrupt change;
(c) Some or all of the fusion peptide is replaced by an artificial linker sequence comprising proline; and
(D) Amino acid sequences comprising a heterotrimeric domain are added.
In some embodiments, the at least one modification comprises substitution of at least one pair of amino acid residues in the F1 subunit and/or the F2 subunit of the F protein polypeptide with cysteine.
In some embodiments, the cysteine-substituted amino acid residues comprise at least one pair of positions 151+288, 159+291, 176+190, 89+231, and 54+151 of the amino acid sequence of the wild-type RSV F protein.
In some embodiments, the at least one cavity filling mutation is an asparagine mutation at position 67 of the amino acid sequence of wild-type RSV F protein, said mutation being a substitution of a hydrophobic amino acid, utilizing steric filling of the multiple side chain hydrophobic amino acids to maintain the epitope Φ pre-fusion conformation.
In some embodiments, the asparagine at position 67 is mutated to phenylalanine or tryptophan.
In some embodiments, the portion of the fusion peptide that is substituted with the artificial linker sequence includes at least amino acid residues 137-144 of the wild-type respiratory syncytial virus F protein.
In some embodiments, the amino acid sequence of the artificial junction sequence is selected from GGPGGS, GGPGPGSE, GAPGS, GAPEPGE or GKPAPGE.
In some embodiments, the recombinant RSV F protein is mutated as compared to the wild-type respiratory syncytial virus F protein by:
(a) At least one pair of amino acid residues in the F1 subunit and/or F2 subunit of the recombinant RSV F protein compared to the wild-type respiratory syncytial virus F protein are substituted with cysteine;
(b) Asparagine at position 67 is mutated to phenylalanine or tryptophan; and
(C) Some or all of the fusion peptide is replaced by an artificial linker sequence comprising proline;
the amino acid sequence of the F protein of the wild respiratory syncytial virus is shown as SEQ ID NO. 1.
In some embodiments, the cysteine substitutions result in formation of a non-native disulfide bond linkage between the F1 subunit and the F2 subunit, or within the F1 subunit, of the recombinant RSV F protein, including disulfide bonds other than Cys69-Cys212 and Cys37-Cys439 formed between the F1 subunit and the F2 subunit.
In the pre-fusion conformation of the RSV F protein, the β2 and β4 chains are close to each other, facilitating the spatial folding of two discrete epitopes of the Φ epitope; the space position between the beta 2 chain and the beta 4 chain can be relatively stabilized by adding an unnatural disulfide bond, and the structure of the Pre-F protein can be well stabilized.
In some embodiments, the cysteine substitutions are selected from one or more of H159c+i291C, K c+s190C and a89c+l231C.
In some embodiments, the cysteine substitutions are a89c+l231C, a89c+l231C such that an interchain disulfide bond is formed between positions 89 and 231 of the recombinant RSV F protein.
In some embodiments, the cysteine substitutions are K176c+s190C and a89c+l231C such that an intrachain disulfide bond is formed between positions 176 and 190 and an interchain disulfide bond is formed between positions 89 and 231 of the recombinant RSV F protein.
In some embodiments, the cysteine substitution is h159c+i291C such that an intrachain disulfide bond is formed between position 159 and 291 of the recombinant RSV F protein.
In some embodiments, to maintain the spatial conformation and stability of the two discrete epitope peptides aa62-69 and aa196-209 prior to epitope Φfusion, hydrophobic amino acid substitutions are made in the region between the β1 and β2, and/or β3 and β4 structures near the epitope, with steric filling of the multiple side chain hydrophobic amino acids, to maintain the pre-epitope Φfusion conformation.
In some embodiments, the recombinant RSV F protein has at least one cavity filling mutation compared to the wild-type respiratory syncytial virus F protein, the cavity filling mutation comprising at least an asparagine mutation at position 67 to phenylalanine (i.e., N67F) or tryptophan (i.e., N67W).
In some embodiments, the abrupt cavity filling change is preferably N67F.
In other embodiments, the abrupt cavity filling change is preferably N67W.
In some embodiments, the portion of the fusion peptide that is substituted with the artificial linker sequence includes at least amino acid residues 137-144 of the wild-type respiratory syncytial virus F protein.
In some embodiments, the amino acid sequence of the artificial junction sequence is selected from GGPGGS, GGPGPGSE, GAPGS, GAPEPGE or GKPAPGE.
In some embodiments, the cysteine substitution is a89c+l231C, the asparagine at position 67 of the F protein amino acid sequence is mutated to tryptophan, and amino acid residues 137-144 of the F protein are substituted with the artificial linker sequence GAPGS.
In some embodiments, the cysteine substitution is G151c+i288C, the asparagine at amino acid sequence 67 of the F protein is mutated to tryptophan, and amino acid residues 137-144 of the F protein are substituted with the artificial linker sequence GAPGS.
In some embodiments, the cysteine substitution is G151c+i288C, asparagine at amino acid sequence 67 of the F protein is mutated to phenylalanine, and amino acid residues 137-144 of the F protein are substituted with artificial linker sequence GAPGS.
In some embodiments, the C-terminus of the recombinant RSV F protein comprises a trimerization domain selected from foldon or GCN 4.
In some embodiments, the trimerization domain comprised at the C-terminus of the recombinant RSV F protein is preferably foldon.
In some embodiments, the trimerization domain may be linked to the F1 subunit or a functional fragment thereof via a linker (such as an amino acid linker, e.g., sequence GG, GS, or SAIG). The linker may also be a longer linker, e.g. comprising the repeat sequence GG.
The F1 subunit of the recombinant RSV F protein can have the same length as the full-length F1 polypeptide of the corresponding wild-type RSV F protein; however, it may also have deletions, such as 1, 2, 3, 5, 10, 20 amino acid residues, up to 60 amino acid residues from the C-terminus of the full-length F1 polypeptide. The full length F1 polypeptide of the recombinant RSV F protein corresponds to amino acid positions 137-574 of the natural RSV F0 precursor and includes (from N-terminus to C-terminus) the extracellular domain (residues 137-524), the transmembrane domain (residues 525-550) and the cytoplasmic domain (residues 551-574).
In some embodiments, the F1 subunit of the recombinant RSV F protein lacks the entire cytoplasmic domain. In other embodiments, the F1 subunit lacks a cytoplasmic domain and a portion or all of a transmembrane domain. In some embodiments, the recombinant RSV F protein comprises an F1 domain in which the amino acid residue from position 510, 511, 512, 513, 514, 515, 520, 525, or 530 to 574 is absent. Typically, amino acids 514 through 574 may not be present for recombinant RSV F protein linked to trimerization domain. Thus, in some embodiments, amino acid residues 514 through 574 are absent from the F1 subunit of the recombinant RSV F protein. In other specific embodiments, the F1 subunit of the recombinant RSV F protein comprises amino acid residues 137-513 of the native F0 polypeptide sequence.
The sequence of the recombinant RSV F protein of the present invention includes a protease cleavage site sequence, such as the thrombin cleavage site (LVPRGS); protein tags, such as the 6×His-tag (HHHHHH) and the strepII tag (WSHPQFEK); or linker sequences (such as GG, GS, SAIG) that are not necessary for the function of the RSV F protein (e.g., to induce an immune response). Those skilled in the art will recognize such sequences and understand that, where appropriate, these sequences are not included in the disclosed recombinant RSV F proteins.
The "foldon" or "foldon domain" according to the present application generally refers to the residue at the C-terminal end of bacteriophage T4 fibrin. In the present application, the foldon domain may be 27 residues of the C-terminal end of phage T4 fibrin or a mutant thereof, an example of which is a sequence having GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 6). In the present application, the foldon domain may be a truncated or increased N-terminal or C-terminal 1, 2, 3, 4, 5, 6 or 10 amino acid truncated or increased 27 residues from the C-terminal end of bacteriophage T4 fibrin. As used herein, a mutant generally refers to a sequence that differs from a reference sequence by the inclusion of one or more differences (mutations). The difference may be a substitution, deletion or insertion of one or more amino acids.
In some embodiments, the amino acid sequence of the recombinant RSV F protein is set forth in SEQ ID NO: 13. 15 and 16.
In another aspect, the invention provides a recombinant nucleic acid comprising a polynucleotide sequence encoding the recombinant RSV F protein described above. In certain embodiments, the recombinant nucleic acid is codon optimized for expression in a selected prokaryotic or eukaryotic host cell. Host cells comprising nucleic acids encoding recombinant RSV F proteins are also a feature of the invention. Advantageous host cells include prokaryotic (i.e., bacterial) host cells, such as E.coli (E.coli), as well as many eukaryotic host cells, including fungal (e.g., yeast) cells, insect cells, and mammalian cells (such as CHO, VERO, HEK293 and expi293F cells).
The invention also provides a method for preparing the recombinant RSV F protein, which comprises the following steps:
S1, synthesizing a DNA sequence corresponding to the recombinant RSV F protein, and cloning the DNA sequence into a plasmid vector;
s2, transfecting a host cell with a recombinant plasmid vector and expressing the host cell, wherein the host cell is a eukaryotic cell;
s3, purifying the culture product to obtain the recombinant RSV F protein.
In another aspect, the invention also provides the use of any of the recombinant RSV F proteins described above in the preparation of a vaccine for the prevention of RSV infection.
The invention further provides a vaccine comprising any of the recombinant RSV F proteins described above and a pharmaceutically acceptable carrier or excipient.
In some embodiments, the single dose of human vaccine comprises 60-120 μg of the recombinant RSV F protein. In some embodiments, the single dose of human vaccine contains preferably 60 μg of the recombinant RSV F protein. In some embodiments, the single dose of human vaccine comprises preferably 120 μg of the recombinant RSV F protein.
In some embodiments, the carrier or excipient comprises a buffer. Pharmaceutically acceptable carriers and excipients are well known and may be selected by those skilled in the art. Optionally, the pharmaceutically acceptable carrier or excipient further comprises at least one component that increases solubility and/or stability. Examples of solubilizing/stabilizing agents include detergents, such as laurel sarcosine and/or tween. Other examples of solubilizing agents/stabilizers include arginine, sucrose, trehalose, and the like. Thus, one skilled in the art can select suitable excipients and carriers to produce a formulation suitable for administration to a subject by a selected use. Suitable excipients include, but are not limited to, glycerol, polyethylene glycol, KCl, and salts related to calcium, magnesium, manganese, zinc and other divalent cations, and the like.
The vaccine provides protection against infection by at least one of RSV subtypes a and B. The amino acid sequences of the F proteins of subtypes a and B are about 90% identical. An example sequence of a F0 precursor polypeptide of subtype A is provided in SEQ ID NO.1 (strain A2; genBank: AAB 86664.1), and an example sequence of a F0 precursor polypeptide of subtype B is provided in SEQ ID NO.2 (strain 18537; swiss-Prot: P13843.1). Both are 574 amino acid sequences, of which the signal peptide sequence has also been reported as amino acids 1-25. In both sequences, the TM domain is approximately amino acids 530 to 550, but is alternatively reported as 525-548. Cytoplasmic tail starts at amino acid 548 or 550 and ends at amino acid 574, wherein the palmitoylated cysteine residue is at amino acid 550.
In some embodiments, the vaccine further comprises an adjuvant.
In some embodiments, the adjuvant comprises at least one of aluminum adjuvant, squalene, tocopherol, MPL, LPA, cpG, poly (I: C), and QS-21.
The adjuvant should generally be capable of enhancing a Th 1-biased immune response in a subject or population of subjects receiving vaccine administration and be safe and effective in the subject or population of subjects at the time of selection.
Optionally, the vaccine may further comprise at least one other antigen of a pathogenic organism different from RSV, for example, the pathogenic organism is a virus different from RSV, such as herpes zoster virus, human papilloma virus, hepatitis b virus, coronavirus or influenza virus. Alternatively the pathogenic organism may be a bacterium such as diphtheria, tetanus, pertussis, haemophilus influenzae or pneumococcus.
The invention also provides a method for preparing the vaccine for preventing RSV infection, which comprises the following steps:
s1, culturing a mammalian host cell to express the recombinant RSV F protein;
s2, purifying the expressed recombinant RSV F protein;
s3, packaging the purified recombinant RSV F protein and the adjuvant according to a proportion or fully mixing.
In some embodiments, the method further comprises additional steps of gene synthesis, construction of expression vectors, lyophilization of purified proteins, and the like. The expressed recombinant RSV F protein can be recovered and purified from recombinant cell culture by any one or several of a number of methods well known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, filtration, ultrafiltration, centrifugation, anion/cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. Finally, high performance liquid chromatography HPLC can be employed in the final purification step.
Compared with the prior art, the invention has the following beneficial effects:
The invention obtains the pre-fusion conformation of the RSV F protein capable of exposing more neutralizing antibody epitopes by carrying out various amino acid substitutions, deletions and additions on the wild type RSV F protein, and ensures that the pre-fusion conformation is maintained stably and simultaneously can cause effective neutralizing antibody reaction and binding antibody reaction on the RSV subtypes A and B. More particularly, the recombinant RSV F protein is used as an immunogen combined adjuvant, so that stronger immunity induction effect is obtained, humoral immunity response is improved, th1 type immunity is stimulated effectively, and immunogenicity of RSV antigen is improved greatly. The mutation mode disclosed by the invention is applicable to other virus strains of human RSV; is suitable for various vaccine forms which take RSV F protein as antigen, such as protein vaccine, nucleic acid vaccine, virus-like particle vaccine and carrier vaccine.
Detailed Description
The invention will be further illustrated by the following non-limiting examples, which are well known to those skilled in the art, that many modifications can be made to the invention without departing from the spirit thereof, and such modifications also fall within the scope of the invention. The following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention as embodiments are necessarily varied. The terminology used in the description is for the purpose of describing particular embodiments only and is not intended to be limiting, the scope of the present invention being defined in the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Preferred methods and materials of the invention are described below, but any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention. The following experimental methods are all methods described in conventional methods or product specifications unless otherwise specified, and the experimental materials used are readily available from commercial companies unless otherwise specified.
Definition of terms
Reference throughout this specification to "one embodiment" means that a particular parameter, step, or the like described in that embodiment is included in at least one embodiment according to the present application. Thus, references to "one embodiment according to the present application," "in an embodiment," and the like are not intended to be interpreted as referring to the same embodiment, and references to "in another embodiment," "in a different embodiment according to the present application," "in another embodiment," and the like are not intended to mean that the recited feature is included in only a specific different embodiment. It will be appreciated by those of skill in the art that the specific parameters, steps, etc. disclosed in one or more of the embodiments of the application can be combined in any suitable manner.
In the present application, the term "cavity filling mutation" refers to the substitution of amino acid residues in the wild-type RSV F protein by amino acids that are expected to fill the internal cavity of the mature RSV F protein. In one application, such cavity filling mutations help stabilize the pre-fusion configuration of the F protein. The cavities in the pre-fusion configuration of the RSV F protein can be identified by methods known in the art, such as by visual inspection of the crystal structure of RSV F in the pre-fusion configuration, or by using computational protein design software.
In the present application, the term "DS-Cav1" refers to a form of the RSV F protein having the amino acid sequence described in McLellan et al, science, 342 (6158), 592-598, 2013.
In the present application, the term "pXCS847" refers to a form of the RSV F protein having the amino acid sequence shown in SEQ ID NO:19 described in the Condui CN 201680075615.8.
In the present application, the term "D25" refers to an antibody described in CN200880023301.9, which has the amino acid sequences of the heavy and light chain CDRs shown in fig. 11D of the specification, respectively.
In the present application, the term "101F" refers to an antibody described in US11261356, which has the amino acid sequences of the heavy and light chain CDRs of anti-RSV mAb 101F shown in figures 2a and 2b, respectively, of the specification of this application.
In the present application, the term "Mota" or "motavizumab" refers to an antibody described in WO2007002543A2, which has the amino acid sequences of the heavy and light chain CDRs of "antibody A4B4L1FR-S28R (aka motavizumab)" shown in table 2 of the specification, respectively.
In the present application, the term "comprising" generally means containing, summarizing, containing or comprising. In some cases, the meaning of "as", "consisting of … …" is also indicated.
EXAMPLE 1 Single mutant design and Gene Synthesis
The recombinant RSV F protein of the present invention is designed and prepared based on the amino acid sequence of Wild Type (WT) RSV F protein described in SEQ ID NO. 1. This example illustrates the design of various recombinant RSV F proteins, including a foldon domain and introduced amino acid mutations.
The template amino acid sequence before introducing the amino acid mutation is:
MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIATVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHSAWSHPQFEK（SEQ ID NO: 7）.
the amino acid sequences designed as follows were subjected to total gene synthesis according to codon optimisation of host Expi293F cells to determine the nucleic acid sequences.
TABLE 1 design of cysteine substituted mutants
| Mutant ID | Mutation |
| CM-1 | G151C+S287C |
| CM-2 | G151C+I288C |
| CM-3 | Y53C+G151C |
| CM-4 | Y53C+L303C |
| CM-5 | V56C+T189C |
| CM-6 | T58C+T189C |
| CM-8 | W52C+S150C |
| CM-11 | T54C+V301C |
| CM-12 | T54C+Q302C |
| CM-13 | T54C+L303C |
| CM-14 | T54C+G151C |
| CM-15 | A177C+T189C |
| CM-16 | A177C+S190C |
| CM-17 | A177C+L188C |
| CM-18 | H159C+I291C |
| CM-19 | A89C+L231C |
| CM-20 | A177C+T189C, T174P |
| CM-21 | A177C+S190C, T174P |
| CM-22 | A177C+L188C, T174P |
| CM-23 | I57C+S190C |
| CM-24 | T58C+V192C |
| CM-25 | I59C+V192C |
| CM-26 | I59C+L193C |
| CM-27 | G151C+Q302C |
| CM-28 | K176C+S190C |
| CM-29 | N175C+K191C |
| CM-30 | A177C+T189C |
| CM-31 | A89C+234C |
| CM-32 | F505C+K508C |
| CM-33 | G143C+S405C |
| CM-34 | A102C+I148C |
| CM-35 | L142C+N371C |
| CM-36 | S146C+Y457C |
TABLE 2 mutant design of cavity filling mutations
| Mutant ID | Mutation |
| VM-1 | S46L |
| VM-2 | V56F |
| VM-3 | I57F |
| VM-4 | N67F |
| VM-5 | N67W |
| VM-6 | K68H |
| VM-7 | G184I |
| VM-8 | V185E |
| VM-9 | V187F |
| VM-10 | T189F |
| VM-11 | A177K |
| VM-12 | L160K |
| VM-13 | G162I |
| VM-14 | V178K |
| VM-15 | F137W, F140W |
| VM-16 | V56F, N67F |
TABLE 3 design of mutants with substitution of proline containing artificial junction sequences
| Mutant ID | Mutation |
| LM-1 | M105GGPGGSM137 |
| LM-2 | M105GGPGPGSEM137 |
| LM-3 | M105GAPGSM137 |
| LM-4 | M105GAPEPGEM137 |
| LM-5 | M105GAPEPAPGEM137 |
| LM-6 | M103GGPGGSM145 |
| LM-7 | M103GGPGPGSEM145 |
| LM-8 | M103GAPGSM145 |
| LM-9 | M103GAPEPGEM145 |
| LM-10 | M103GAPEPAPGEM145 |
| LM-11 | M103GAPAPAPKPGEM145 |
| LM-12 | M103GKPAPGEM147 |
| LM-13 | M103GAPEPGEM147 |
| LM-14 | M103GAPEPAPGEM147 |
| LM-15 | L160P, M105GAPEPGEM137 |
| LM-16 | L160P, E161S, M105GAPEPGEM137 |
| LM-17 | L160P, E161S, M103GAPEPGEM145 |
| LM-18 | L160P, E161S, M103GAPEPGEM147 |
| LM-19 | G162P, M105GAPEPGEM137 |
| LM-20 | E161A, G162P, M105GAPEPGEM137 |
| LM-21 | E161A, G162P, M103GAPEPGEM145 |
| LM-22 | E161A, G162P, M103GAPEPGEM147 |
Note that: m 103、M105、M137、M145、M147 et al represent the mutation site of the amino acid residue in the F protein, for example, M 105GGPGGSM137 represents that the amino acid residues at positions 105-137 in the F protein are replaced by GGPGGS, and the same applies below.
Table 4 control design
| Control group | SEQ ID NO: |
| WT | 1 |
| DS-Cav1 | 3 |
| pXCS847 | 4 |
| Post-F | 5 |
EXAMPLE 2 construction, expression and Primary screening of recombinant plasmids
The synthesized target gene is inserted into eukaryotic plasmid PTT5 vector for transfection of the Expi293F cell for expression. The epi 293F cells in the logarithmic growth phase were passaged one day before transfection and inoculated into 96-well deep well plates at a cell density such that the cell density reached 2.5X10 6/ml the next day. Mu.g of antigen expression plasmid was diluted in 40. Mu.l Opti-MEMI and 2.4. Mu.l of transfection reagent was diluted in 40. Mu.l Opti-MEM. The DNA and transfection reagent diluted separately were mixed and incubated for 20min at room temperature. The DNA-liposome complex was added to the cells and incubated at 37℃on a 5% CO 2, 3mm orbital shaker at 1200 rpm. Cell supernatants were collected 3 days after expression, and a double antibody sandwich ELISA method was used, in which 101F (Site IV specific antibody binding to both pre-and post-fusion F) was used as a coating antibody, D25 (monoclonal antibody specifically recognizing the phi epitope of pre-fusion F protein) and motavizumab (Site II specific antibody simultaneously recognizing both pre-and post-fusion F, hereinafter abbreviated as Mota) were used as marker antibody detection pairs, and expression of each mutant on Site phi and Site II epitopes was detected as OD values.
The double antibody sandwich ELISA method comprises the following specific steps:
1) Solution preparation
Coating liquid: sodium bicarbonate is 1.59g/L, sodium bicarbonate is 2.94mg/L;
20mM PB solution: disodium hydrogen phosphate; sodium dihydrogen phosphate;
Sealing liquid: PB 0.02M, 1.5% BSA+5% sucrose, 0.1% Proclin300;
dilution liquid: PB 0.02M, 1.5% BSA, 0.1% Proclin300, aminopyrine.
2) Antibody coating: the 101F antibody is diluted to 4 mug/ml by coating liquid, 100 mug/well is added into the enzyme-labeled plate hole, and the mixture is coated for 16 hours at the temperature of 2-8 ℃.
3) Closing: plate washer 300. Mu.l/well, 2 wash beats, add sealing solution 150. Mu.l/well, 37℃and incubate for 2 hours.
4) Sample adding: the sample to be tested and the enzyme-labeled antibody (2000-fold dilution) were added, incubated at 37℃for 30 minutes at 100. Mu.l/well, and washed 5 times with a plate washer.
5) Color development: the reaction mixture (A+B) was added to 100. Mu.l/well, and the mixture was reacted at room temperature for 15 minutes in the absence of light.
6) Reading: the microplate reader is opened in advance, the color development is complete, 50 μl/hole of stop solution is added into the reaction hole of the microplate, and the microplate reader is placed for reading.
The primary screen gave a design with high expression for the next screening or evaluation, the results are shown in the following table.
TABLE 5 high throughput transfection expression preliminary screening of cysteine-substituted mutant 96 well plates
The results show that mutants CM-2, CM-5, CM-6, CM-8, CM-14, CM-15, CM-18, CM-19, CM-23, CM-26, CM-27, CM-28, CM-32, CM-33, CM-34 and CM-35 can stably maintain neutralizing antibody epitopes Site phi and Site II, and can be used for the next screening and evaluation.
TABLE 6 mutant 96 well plate high throughput transfection expression primary screen for cavity filling mutations
The results show that the mutants VM-3, VM-4, VM-5, VM-6, VM-8, VM-10, VM-11, VM-12, VM-14 and VM-15 can stably maintain neutralizing antibody epitopes Site phi and Site II and can be used for the next screening and evaluation.
TABLE 7 high throughput transfection expression preliminary screening of proline containing Artificial junction sequence substituted mutant 96 well plates
The results show that mutants LM-3, LM-4, LM-5, LM-6, LM-7, LM-8, LM-9, LM-10, LM-12, LM-13, LM-14, LM-17, LM-18, LM-19, LM-20, LM-21 and LM-22 can stably maintain neutralizing antibody epitope Site phi and can be used for the next screening and evaluation.
EXAMPLE 3 evaluation of Single mutant expression level and stability
The logarithmic growth phase of the Expi293F cells was passaged one day before transfection, and inoculated into 96-well deep well plates at a cell density of 1.2X10 6/ml until the cell density reached 2.5X10 6/ml the next day. Diluting the expression plasmid and the transfection reagent by using Opti-MEMI respectively, standing at room temperature for 5min, slightly dripping the transfection reagent PEI into diluted DNA, mixing, and incubating at room temperature for 20min. The DNA-PEI complex was added to the cells and incubated at 37℃on a 5% CO 2, 3mm orbital shaker at 1200 rpm. After the single mutant was expressed for three days, the expressed cell supernatants were collected by centrifugation at 10000rpm, the expression supernatants were separately stored at 4℃for 7 days and 14 days, and then a double antibody sandwich ELISA method was used, 101F was used as a coating antibody, D25 and motavizumab (hereinafter abbreviated as Mota) were used as a marker antibody detection pair, the expression levels of each mutant expressed by the recombinant plasmid were detected, and the conformational stability of the Site phi and Site II epitopes after standing at 4℃for different times, wherein the expression levels were expressed as epitope-specific neutralizing antibody detection activity concentrations, the conformational stability was expressed as the active concentration ratio of day14/day0, and the results were shown in the following table.
TABLE 8 selection of expression levels and conformational stability of cysteine-substituted mutants
Note that: "/" indicates no reactivity detected or no assay
TABLE 9 conformational stability of cysteine-substituted mutants
Note that: "/" indicates no reactivity detected or no assay
The cysteine-substituted mutants CM-2, CM-18, CM-19, CM-28, CM-35, CM-33 and CM-14 perform better by comprehensively considering the expression level and the thermostability. The decrease of the D25 detection activity concentration is not obvious or occurs in comparison with the wild type after 14 days of storage at 4 ℃, and the retention rate of the conformation before fusion is obviously higher than that of the wild type F protein. And has stability comparable to or higher than DS-Cav 1.
TABLE 10 mutant expression level and conformational stability screening of cavity filling mutations
Note that: "/" indicates no reactivity detected or no assay
TABLE 11 mutant conformational stability of cavity filling mutations
The expression quantity and the thermal stability are comprehensively considered, and the mutants VM-4 and VM-5 have better performance. The decrease of the D25 detection activity concentration is not obvious or occurs in comparison with the wild type after 14 days of storage at 4 ℃, and the retention rate of the conformation before fusion is obviously higher than that of the wild type F protein. And has stability comparable to DS-Cav 1.
Table 12 selection of expression levels and conformational stability of proline containing Artificial linker substituted mutants
Note that: "/" indicates no reactivity detected or no assay
TABLE 13 conformational stability of proline containing artificial junction sequence substituted mutants
Note that: "/" indicates no reactivity detected or no assay
The mutants LM-6, LM-7, LM-8, LM-9 and LM-12 have better performance by comprehensively considering the expression quantity and the thermal stability. The D25 detection activity concentration is not obviously reduced or is not reduced after being stored for 14 days at the temperature of 4 ℃ compared with the wild type, and the conformational residue rate before fusion is obviously higher than that of the wild type F protein. And has stability comparable to or higher than DS-Cav 1. In further studies, it was found that when combined mutants were formed with other mutations, there was a higher expression level when the 137 th to 144 th amino acid residues were replaced with the artificial linker sequence, and thus mutants in which the 137 th to 144 th amino acid residues were replaced with the artificial linker sequence were mainly selected for the next screening and evaluation.
Example 4 evaluation of combinatorial mutant design, expression level and stability
The signal peptide of the template amino acid sequence was replaced with CD33 signal peptide (SEQ ID NO: 8), the linker between the foldon domain and F1 subunit was replaced with GGSGGSG, and the recombinant plasmid was constructed and expressed by the method of example 2, based on the above-selected single mutant with superior expression and thermal stability, by designing the amino acid sequence according to the following table and determining the nucleic acid sequence by codon optimization according to the host Expi293F cell.
Table 14 combinatorial mutant design
| Mutant ID | Mutation | SEQ ID NO: |
| RC169 | K176C,S190C，A89C,L231C | 9 |
| RC172 | H159C,I291C，N67W | 10 |
| RC176 | M137GGPGPGSEM144，H159C,I291C | 11 |
| RC177 | M137GGPGGSM144，H159C,I291C | 12 |
| RC200 | M137GAPGSM144，A89C,L231C，N67W | 13 |
| RC207 | M137GAPGSM144，G151C,I288C | 14 |
| RC208 | M137GAPGSM144，G151C,I288C,N67F | 15 |
| RC209 | M137GAPGSM144，G151C,I288C,N67W | 16 |
| RC223 | T54C,G151C，M137GAPGSM144 | 17 |
| RC236 | K176C,S190C，M137GGPGGSM144，N67F | 18 |
| RC237 | K176C,S190C，M137GGPGPGSEM144，N67W | 19 |
After three days of combined mutant expression, centrifugally collecting the expressed cell supernatant at 10000rpm, taking the expression supernatant, respectively incubating for 1 hour at 50 ℃ and 60 ℃, then adopting a double-antibody sandwich ELISA method, taking 101F as a coating antibody and D25 as a labeled antibody detection pair, and detecting the expression quantity and conformational stability of Site phi epitopes after each mutant expressed by the recombinant plasmid is incubated at 4 ℃ for 1h and at 50 ℃ for 1h and at 60 ℃ for 1h, wherein the expression quantity is expressed as the epitope specific neutralizing antibody detection active concentration, and the conformational stability is expressed as the active concentration ratio of 50 ℃/4 ℃ and 60 ℃/4 ℃, and the results are shown in the following table.
TABLE 15 expression level and conformational stability of combination mutants
Mutants RC200, RC208 and RC209 show higher expression levels than 4 control groups, have conformational stability equivalent to or better than that of the pXCS847 and DS-Cav1 control groups even at 50 ℃ and 60 ℃, and can be used as vaccine immunogen components to ensure temporary storage within 1 hour under the extreme temperature condition of 50-60 ℃.
Example 5 preparation of proteins
5.1 Large Scale expression of proteins
Recombinant proteins were expressed by transient transfection in Expi293F cells using PEI MAX. The day prior to transfection, the logarithmic growth phase of the Expi293F cells were passaged and seeded at a cell density of 2.5X10 6/ml in 500ml/1000ml shake flasks. On the day of transfection, the cell density was diluted to 2.5X10 6/ml, the expression plasmid DNA and the transfection reagent PEIMAX were diluted with Opti-MEM, respectively, and left standing for 5min at room temperature, the diluted transfection reagent PEIMAX was gently dripped into the diluted expression plasmid DNA and mixed well, and incubated for 10min at room temperature. The DNA-PEI complex was added to the cells and incubated in a carbon dioxide shaker at 37℃with 8% CO 2 at 120 rpm. Culture supernatants were collected on day 4 post-transfection and cell debris was removed by centrifugation twice at 4000rpm and 10000 rpm. The culture supernatant was sterile filtered and the protein purified by HIS TRAP HP and strep ii two-step affinity chromatography.
5.2 Protein purification
Starting an SCG chromatographic system and connecting with an instrument, regulating the system parameter pressure to be not more than 0.3Mpa, alarming to be high-sensitivity, centrifuging the cell supernatant to be purified at 10000rpm for 8min, and filtering for later use. The expressed immunogenic proteins were purified using a two-step chromatography.
(1) HIS TRAP HP (5 ml) chromatography: the column (HIS TRAP HP ml) was equilibrated with 50ml Buffer A (20 mM PB, 500 mM NaCl, pH 7.4) at a flow rate of 5ml/min for 10 minutes, and the filtered supernatant was pre-treated at a flow rate of 2ml/min after equilibration. After loading, the column was washed with 50ml Buffer A (20 mM PB, 500 mM NaCl, pH 7.4) at a flow rate of 5ml/min for 10 minutes until the UV was toward the baseline position, and then the immunogenic protein was eluted with Buffer B (20 mM PB, 500 mM NaCl, 500 mM Imidazole, pH 7.4) at a flow rate of 5 ml/min.
(2) Strep ii (1 ml) chromatography: the column (strep II purification column) was equilibrated to UV towards baseline position using 30ml Buffer C (100 mM Tris-HCl, 150 mM NaCl, 1mM EDTA, pH 8.0) at a flow rate of 1 ml/min; HIS TRAP HP (5 ml) chromatographic eluent is loaded at a flow rate of 1ml/min, loading is completed, and a chromatographic column is balanced to an ultraviolet UV (ultraviolet) towards a base line by a Buffer C at a flow rate of 1 ml/min; finally, buffer D (50 mM biotin in Binding Buffer) was used to elute the immunogenic protein at a flow rate of 1 ml/min. After washing with pure water, the NaOH (50 mM) caustic wash column was regenerated.
Quantification: and uniformly mixing the eluted proteins, quantifying the proteins at UV280nm, taking out a small amount of proteins for electrophoresis detection, and after the rest of proteins are packaged and frozen at-80 ℃.
EXAMPLE 6 evaluation of conformational stability of immunogens
The stability of the purified protein immunogen conformation was identified using Pre-F Pre-fusion conformation specific mab D25. The stability of the candidate immunogen in conformation under different conditions of temperature, osmotic pressure, repeated freezing and thawing and the like is examined, and the affinity of the candidate immunogen with the D25 monoclonal antibody is tested.
Table 16 examines conditions
The results show that although each recombinant RSV F protein contains a furin cleavage site and pep27 that are native to the wild-type RSV F protein, the mutations disclosed by the invention are sufficient to stabilize it in the pre-fusion conformation and the ratio of the concentration of neutralizing antibody detection activity to D25 before treatment is close to 1 with 1h at 50 ℃, high/low osmotic pressure, and repeated freeze thawing, especially after 1h at 60 ℃, the levels of RC200, RC208, and RC209 are comparable to the DS-Cav1 and pXCS847 levels of the control group, all significantly higher than wild-type WTs.
Example 7 animal test
Female BALB/C mice of 6-8 weeks of age are selected for immunization, 6 mice in each group are randomly grouped, and vaccine double-dot immunization is carried out on the mice by intramuscular injection on days 0 and 21, and 50 mu L of each of left and right legs comprises recombinant RSV F protein candidate antigens RC200, RC208 and RC209; WT, DS-Cav1, pXCS847 control antigen group; and a PBS control group. The antigen dose was 12 μg/mouse. Serum was collected on day 20 and day 35, respectively, and the collected serum was stored at-20 ℃ for subsequent detection of neutralizing antibodies and bound antibodies.
7.1 Neutralizing antibody detection:
The university of martial arts virology focus laboratory was commissioned to detect neutralizing antibodies using the stored RSV A2 strain. The method comprises the following specific steps:
(1) After immunization, the serum is incubated in a water bath at 56 ℃ for 30 minutes;
(2) The inactivated serum samples were diluted 2-fold in 96-well plates at a starting dilution of 1:8 using DMEM medium containing 2% fbs;
(3) Adding 50-100 PFU of RSV A2 strain into the hole, uniformly mixing, and incubating for 1 hour under the condition of 5% CO 2;
(4) Transferring the virus and serum mixed solution into a 96-well plate pre-inoculated with monolayer Vero cells, and culturing for 7 days at 37 ℃ under the condition of 5% CO 2;
(5) Cytopathy was observed, and neutralizing antibody titer, defined as the highest serum dilution with more than 50% of intact Vero cells, was calculated using Reed-Muench method.
The results show that the geometric average titers of neutralizing antibodies induced by candidate antigens RC200, RC208 and RC209 are equivalent to the levels of pXCS847 and DS-Cav1, and have no significant difference, and the geometric average titers of neutralizing antibodies induced by all the proteins are significantly higher than those of the WT and post-F proteins.
7.2 Detection of bound antibodies:
(1) Purified pre-F and post-F proteins were diluted to 3. Mu.g/mL coated 96-well plates, 100. Mu.L/well, incubated overnight at 4℃blocked with 1.5% BSA-PBS for 2 hours at 37 ℃, washed, and dried;
(2) The serum sample is subjected to 2-time serial dilution by using a sample diluent, 100 mu L of the sample is added to each hole by taking 1:200 as a detection initial point, and the sample is incubated for 30 minutes at 37 ℃;
(3) Horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (Abclonal, #as 003) was diluted 1:4000, 100 μl was added per well and incubated for 30min at 37 ℃;
(4) Color development was performed using 3,3', 5' -Tetramethylbenzidine (TMB), 100. Mu.L was added to each well, and incubated at room temperature for 15 minutes in the absence of light;
(5) The reaction was stopped by adding 50 μl of 2M H 2SO4 per well, and the OD values at 450 nm and 630 nm were measured for each well using a microplate reader (MD, spectromax I3X);
(6) The absorbance value of the negative control is 2.1 times of the Cut-off value, and the maximum dilution multiple of the absorbance value of the sample which is more than or equal to the Cut-off value is the titer of the sample.
The results show that the geometric mean titers of Pre-F specific antibodies induced by candidate antigens RC200, RC208 and RC209 are about 2 times that of WT, 1.3 times that of pXCS847, comparable to DS-Cav1 levels.
In conclusion, the recombinant RSV F protein provided by the invention has higher stability and expression level, and the vaccine composition containing the recombinant RSV F protein can induce the generation of neutralizing antibodies with high titer against RSV, which indicates that the vaccine provides better protective effect during virus invasion and has important significance for the research and development of RSV vaccine candidates.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.
Claims (11)
1. A recombinant RSV F protein having a pre-fusion conformation and immunogenicity, wherein the mutation of the recombinant RSV F protein compared to a wild-type respiratory syncytial virus F protein consists of:
(a) The wild type respiratory syncytial virus F protein is subjected to substitution of G151C+I288C cysteine;
(b) The 67 th asparagine of the F protein of the wild respiratory syncytial virus is mutated into phenylalanine or tryptophan; and
(C) The part of fusion peptide of wild type respiratory syncytial virus F protein is replaced by artificial connecting sequence containing proline, the part of fusion peptide replaced by artificial connecting sequence is 137 th-144 th amino acid residue of wild type respiratory syncytial virus F protein, the amino acid sequence of artificial connecting sequence is GAPGS;
wherein the amino acid sequence of the wild type respiratory syncytial virus F protein is shown as SEQ ID NO.1, and the C-terminal of the recombinant RSV F protein comprises a foldon trimerization domain.
2. The recombinant RSV F protein of claim 1, wherein the recombinant RSV F protein has an amino acid sequence set forth in SEQ ID NO: 15 and 16.
3. A recombinant RSV F protein having a pre-fusion conformation and immunogenicity, wherein the mutation of the recombinant RSV F protein compared to a wild-type respiratory syncytial virus F protein consists of:
(a) Wild type respiratory syncytial virus F protein undergoes a K176C + S190C cysteine substitution;
(b) Mutation of asparagine at position 67 of wild type respiratory syncytial virus F protein to phenylalanine; and
(C) The part of fusion peptide of wild type respiratory syncytial virus F protein is replaced by artificial connecting sequence containing proline, the part of fusion peptide replaced by artificial connecting sequence is 137 th-144 th amino acid residue of wild type respiratory syncytial virus F protein, the amino acid sequence of artificial connecting sequence is GGPGGS;
wherein the amino acid sequence of the wild type respiratory syncytial virus F protein is shown as SEQ ID NO.1, and the C-terminal of the recombinant RSV F protein comprises a foldon trimerization domain.
4. The recombinant RSV F protein of claim 3, wherein the recombinant RSV F protein has an amino acid sequence set forth in SEQ ID NO: shown at 18.
5. A recombinant RSV F protein having a pre-fusion conformation and immunogenicity, wherein the mutation of the recombinant RSV F protein compared to a wild-type respiratory syncytial virus F protein consists of:
(a) Wild type respiratory syncytial virus F protein undergoes a K176C + S190C cysteine substitution;
(b) The 67 th asparagine of the F protein of the wild respiratory syncytial virus is mutated into tryptophan; and
(C) The part of fusion peptide of wild type respiratory syncytial virus F protein is replaced by artificial connecting sequence containing proline, the part of fusion peptide replaced by artificial connecting sequence is 137 th-144 th amino acid residue of wild type respiratory syncytial virus F protein, the amino acid sequence of artificial connecting sequence is GGPGPGSE;
wherein the amino acid sequence of the wild type respiratory syncytial virus F protein is shown as SEQ ID NO.1, and the C-terminal of the recombinant RSV F protein comprises a foldon trimerization domain.
6. The recombinant RSV F protein according to claim 5, wherein said recombinant RSV F protein has an amino acid sequence as set forth in SEQ ID NO: 19.
7. A recombinant RSV F protein having a pre-fusion conformation and immunogenicity, wherein the mutation of the recombinant RSV F protein compared to a wild-type respiratory syncytial virus F protein consists of:
(a) Wild type respiratory syncytial virus F protein undergoes A89C+L231C cysteine substitutions;
(b) The 67 th asparagine of the F protein of the wild respiratory syncytial virus is mutated into tryptophan; and
(C) The part of fusion peptide of wild type respiratory syncytial virus F protein is replaced by artificial connecting sequence containing proline, the part of fusion peptide replaced by artificial connecting sequence is 137 th-144 th amino acid residue of wild type respiratory syncytial virus F protein, the amino acid sequence of artificial connecting sequence is GAPGS;
wherein the amino acid sequence of the wild type respiratory syncytial virus F protein is shown as SEQ ID NO.1, and the C-terminal of the recombinant RSV F protein comprises a foldon trimerization domain.
8. The recombinant RSV F protein of claim 7, wherein the recombinant RSV F protein has an amino acid sequence set forth in SEQ ID NO: shown at 13.
9. Use of the recombinant RSV F protein of any one of claims 1-8 for the preparation of a vaccine for preventing RSV infection.
10. A vaccine comprising the recombinant RSV F protein of any one of claims 1-8 and a pharmaceutically acceptable carrier or excipient.
11. The vaccine of claim 10, further comprising an adjuvant.
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