394 results about "F protein" patented technology

The invention discloses a modified bordetella pertussis strain and an application thereof. The modified Bordetella pertussis strain is characterized in that in the Bordetella pertussis strain BAA-589, the BP2992 protein is not expressed, or the inactive BP2992 protein is expressed, the pldA protein is not expressed, or the inactive pldA protein is expressed, and the mla F protein is not expressed, or the inactive mla F protein is expressed. When the Bordetella pertussis strain is treated by combining an ultrasonic method, compared with wild Bordetella pertussis BAA-589, the Bordetella pertussis strain has a remarkable high-foaming phenotype, the OMV secretion amount is remarkably increased, and a good technical foundation is laid for developing a Bordetella pertussis OMV vaccine.

The invention relates to the technical field of biology, in particular to a multivalent pneumococcal polysaccharide-protein conjugate vaccine for preventing pneumococcal infection and respiratory syncytial virus infection as well as a preparation method and application of the multivalent pneumococcal polysaccharide-protein conjugate vaccine. According to the multivalent pneumococcal polysaccharide-protein conjugate vaccine disclosed by the invention, carrier protein is RSV recombinant protein with immunogenicity; the pneumococcal polysaccharide is connected with the RSV recombinant protein through a covalent bond; the RSV recombinant protein is selected from Pre-F protein, and the amino acid sequence of the RSV recombinant protein is shown as SEQ ID NO: 2 or SEQ ID NO: 3. The conjugate vaccine provided by the invention is an innovative vaccine, two common diseases, namely pneumonia and respiratory syncytial virus infection, can be simultaneously prevented by using one vaccine product, and the number of vaccination times of the vaccine is greatly reduced. According to the invention, the carrier protein in the conjugate vaccine is subjected to mutation modification, so that the carrier protein can maintain structural stability and antigen cluster function in different environments.

The invention belongs to the field of biological medicine, and particularly relates to a respiratory syncytial virus F protein mutant and application thereof. The respiratory syncytial virus F protein mutant is characterized in that at least one pair of amino acid residues in an F1 subunit and / or an F2 subunit of F protein polypeptide are substituted by cysteine, and / or an F protein signal peptide part is substituted by a human immunoglobulin light chain signal peptide, and / or alanine A at the 107 site of a multi-base sequence is mutated into arginine R, lysine K at the 134 site is mutated into arginine R, and the amino acid residues in the F1 subunit and / or the F2 subunit of the F protein polypeptide are mutated into arginine. According to the invention, more neutralizing antibody epitopes can be exposed, the stable pre-fusion conformation is maintained, effective neutralizing antibody reaction and antibody binding reaction to the RSV subtypes A and B can be ensured, and the vaccine is suitable for various vaccine forms taking the RSV F protein as the antigen.

The invention relates to the field of prevention and treatment of respiratory diseases, and discloses a preparation method of a human respiratory syncytial virus preventive mRNA vaccine. The main components of the mRNA vaccine comprise mRNA for coding an antigen and lipid nanoparticles. According to the mRNA sequence provided by the invention, an independently researched and developed 5 'UTR / 3' UTR sequence and a codon optimization mode are adopted, and efficient translation of target protein can be realized. According to the mRNA vaccine provided by the invention, RSV F protein is selected as an antigen, and the RSV F protein sequence is optimally designed, so that stable pre-fusion conformation RSV F protein can be expressed, and after a mouse is immunized, the mouse can be induced to generate high-level RSV F protein specific binding antibody titer and neutralizing antibody titer.

The present disclosure provides inter alia, an RSV-F protein comprising at least two mutations relative to SEQ ID NO: 1 or 3 within a region of the protein corresponding to positions 474-523 of SEQ ID NO: 1 or 3; wherein the at least two mutations introduce, through substitution or insertion, a pair of C residues into the region, which form a disulphide bond.

The invention provides a respiratory syncytial virus F protein mutant and application thereof, and relates to the technical field of biology. The respiratory syncytial virus F protein mutant contains at least one recombinant F2-F1 polypeptide, each recombinant F2-F1 polypeptide independently contains a mutated fragment of an F0 polypeptide, and mutation comprises the following steps: (I) deleting positions 104-144 and substituting the positions 104-144 with a connecting peptide; (II) a 155C substitution, a 289C substitution, a 149C substitution and a 458C substitution; a disulfide bond is formed between 155C and 289C, and a disulfide bond is formed between 149C and 458C; and, (III) a 373R substitution. The respiratory syncytial virus F protein mutant has excellent immunogenicity, antigenicity, stability and tolerance.

The invention provides a respiratory syncytial virus antigenic polypeptide, nucleic acid and a vaccine. Compared with the respiratory syncytial virus wild type Pre-F protein, the respiratory syncytial virus antigenic polypeptide or the immunogenic fragment of the respiratory syncytial virus antigenic polypeptide has the following changes: the 104th to 144th regions of the Pre-F protein are replaced with GS linker; two groups of disulfide bonds are introduced into S155C, S290C, A149C and Y458C; the S190F and the V207L are used for realizing cavity filling; and P102A, L373R, I379V, M447V, P102A, L373R, the C terminal of the respiratory syncytial virus antigenic polypeptide or the immunogenic fragment thereof comprises a cytoplasm tail region fragment, and the cytoplasm tail region fragment is a fragment derived from 3-10 continuous amino acid residues of a PreF protein cytoplasm tail region. According to the present invention, the pre-F immunogenicity and / or stability can be improved, and the protein expression level can be easily improved.

The present invention relates to a respiratory syncytial virus subtype B (RSV-B) recombinant F protein, a polynucleotide encoding the same, a nucleic acid construct comprising the polynucleotide, an expression vector comprising the nucleic acid construct, a host cell wherein the polynucleotide, the nucleic acid construct or the expression vector is transformed or transfected, a stabilized trimer formed therefrom, and a method for preparing the same. An immunogenic composition comprising any of the foregoing, and its use in the preparation of a vaccine for the prevention and / or treatment of respiratory syncytial virus infection. The RSV-B recombinant F protein comprises at least one specific epitope of the pre-fusion F protein, a stable F protein tripolymer with pre-fusion conformation can be formed, the expression is stable, the form is uniform, and the yield is greatly improved; the formed F protein trimer has good immunogenicity, can stimulate an organism to generate high-level antibody titer, and has great significance in clinical treatment and prevention and control of the respiratory syncytial virus.

Provided are a respiratory syncytial virus antigenic polypeptide, a vaccine, and a use. First, a respiratory syncytial virus antigenic polypeptide or an immunogenic fragment thereof is provided. Compared with the amino acid sequence of a respiratory syncytial virus wild-type pre-F protein, the amino acid sequence of the respiratory syncytial virus antigenic polypeptide or the immunogenic fragment thereof has one or more mutation sites of amino acid residues at the following positions: positions 174-176, positions 181-184, positions 210-213, and positions 160-161. The improvement of pre-F immunogenicity can be achieved and / or the protein expression level can be improved.

The invention relates to an anti-RSV polypeptide membrane fusion inhibitor as well as a preparation method and application thereof. The invention discloses a compound, a pharmaceutical salt or a derivative thereof, a pharmaceutical composition and a preparation product containing the compound, and application of the compound and the pharmaceutical salt or the derivative in preparation of medicines for preventing and / or treating diseases caused by RSV or other paramyxoviridae viruses. The polypeptide disclosed by the invention shows the anti-RSV activity and safety which are obviously superior to those of positive control polypeptide. Molecular interaction experiments show that the polypeptide acts on the NHR region of the RSV F protein, and the fusion of the RSV virus and a target cell is prevented by forming an inactive six-helix structure with NHR, or by destroying the RSV NHR structure and inhibiting the process of forming a six-helix bundle by NHR and CHR in the F protein in the key step of RSV infection.

The invention provides a subunit vaccine for resisting poultry clostridium perfringens, which is characterized in that CPA and NetB proteins are truncated, and three gene sequences of auxiliary protease Fba are connected in series and fused through Linker to obtain recombinant protein. Immunogenicity detection on the N-C-F recombinant protein finds that the N-C-F recombinant protein can induce SPF chicken to generate antibodies. Immune protection test results show that the subunit vaccine prepared from the N-C-F protein and the adjuvant can achieve 100% immune protection effect when being used for immunizing 100 [mu] g / chicken and 200 [mu] g / chicken, and the subunit vaccine can be used as a subunit vaccine for resisting various toxin types.

The present invention is generally related to modified or mutated respiratory syncytial virus fusion (F) proteins and methods for making and using them, including immunogenic compositions such as vaccines for the treatment and / or prevention of RSV infection.

The invention relates to a mutant of an RSV pre-fusion conformation F protein, the mutant comprises amino acid mutation relative to an amino acid sequence of a wild type RSV F protein, the amino acid mutation is that amino acids at positions 392 and 493 or near the 493 are substituted by C, and a disulfide bond is formed between mutated positions 392C and 493C or near the 493C. The invention also provides a related DNA (deoxyribonucleic acid) coding sequence, a recombinant plasmid, an mRNA (messenger ribonucleic acid) sequence and a corresponding RSV vaccine. By introducing disulfide bonds at proper positions and combining with cavity filling, electrostatic mutation, proline mutation and the like, the expression and stability of the F protein with conformations before and after fusion and trimerization conformations are remarkably improved, and the F protein is remarkably inhibited from being converted into conformations after fusion. The mutant is also applied to preparation of RSV vaccines and detection of respiratory syncytial virus infection.

The invention relates to the technical field of virology, immunology and biomedicine, in particular to a human metapneumovirus F protein mutant and application thereof. The human metapneumovirus F protein mutant provided by the invention has stable prefusion conformation and high expression quantity, and can form a stable trimer, so that the human metapneumovirus F protein mutant can be used for HMPV vaccine development. The F protein mutant provided by the invention has excellent immunogenicity, and can induce excellent immune response when being used together with an HA201 composite adjuvant (QS21 + MPL liposome).

The invention provides a bovine parainfluenza virus type 3 QL3 strain, and the preservation number of the bovine parainfluenza virus type 3 QL3 strain is CGMCC No.46469. The invention also provides a bovine parainfluenza inactivated vaccine containing the bovine parainfluenza virus type 3 QL3 strain and purified F protein and HN protein of the QL3 strain. The novel bovine parainfluenza virus type 3 inactivated vaccine is obtained by optimizing the antigen components of the bovine parainfluenza virus type 3 inactivated vaccine, and high-level neutralizing antibodies can be generated after immunizing cattle; the safety of the vaccine is improved through process optimization. Safety and potency test results show that after the inactivated vaccine is used for immunizing cattle, no adverse reaction exists, and high-level neutralizing antibodies are generated. Results show that the immune effect of the bovine parainfluenza virus type 3 inactivated vaccine can be improved.

The invention belongs to the technical field of protein purification, and particularly relates to a recombinant human metapneumovirus pre-fusion F protein purification method which comprises the following steps: filtering fermentation liquor by using a deep filter to obtain fermentation clear liquor; capturing the recombinant hMPV F protein from the fermentation clarified liquid by using hydroxyapatite to obtain a purified liquid 1; carrying out virus inactivation on the purified solution 1 by adopting a low-pH method to obtain an inactivated solution; carrying out anion exchange chromatography on the inactivated solution to obtain a purified solution 2; carrying out composite weak anion chromatography on the purified solution 2 to obtain a purified solution 3; performing UF / DF on the purified liquid 3 to obtain an ultrafiltrate; and carrying out virus removal nanofiltration and sterile filtration on the ultrafiltrate to obtain a recombinant hMPV F stock solution. The method is simple and convenient, has good repeatability, is suitable for large-scale production, and can be effectively used for producing the recombinant human metapneumovirus pre-fusion F protein vaccine.

The present disclosure provides a human metapneumovirus (hMPV) vaccine comprising an hMPV F protein antigen, and methods of eliciting an immune response by administering said vaccine.

Provided is a respiratory syncytial virus (RSV) vaccine. The nucleic acid comprises a polynucleotide for encoding a mutant of an RSV F protein. Compared with a wild-type RSV F protein, the mutant at least comprises one or more of the following mutations: a disulfide bond mutation, a cavity-filling mutation and an electrostatic mutation.

The invention discloses a specific monoclonal antibody for resisting a Nipah virus Post-F protein, the monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3; wherein the HCDR1, the HCDR2 and the HCDR3 respectively comprise the HCDR1, the HCDR2 and the HCDR3 in a heavy chain variable region of which the amino acid sequence is any one of SEQ ID NO: 4, 13, 20, 25, 30, 34 or 42, or respectively have at least 85% sequence identity with the HCDR1, the HCDR2 and the HCDR3; and / or the LCDR1, the LCDR2 and the LCDR3 respectively comprise LCDR1, LCDR2 and LCDR3 in a light chain variable region with the amino acid sequence of any one of SEQ ID NO: 9, 16, 23, 28, 32, 37 or 46, or respectively have at least 85% sequence identity with the LCDR1, the LCDR2 and the LCDR3. The anti-Nipah virus Post-F protein monoclonal antibody disclosed by the invention can be specifically combined with a Post-F conformation protein, and has the application potential of being developed into a Nipah virus antibody detection kit or a virus neutralizing antibody drug.

The present invention provides engineered soluble F proteins of paramyxoviruses, such as respiratory syncytial virus (RSV), human metapneumovirus (hMPV), and human parainfluenza virus (hPIV). These engineered proteins are stabilized by specific modifications in the wild-type soluble F sequence, such as substitutions in the 023 chain and / or the introduction of engineered disulfide bonds in the 0 hairpin in the F1 subunit. The present invention also provides a nanoparticle vaccine comprising an engineered soluble F immunogen displayed on a self-assembled nanoparticle. The invention also provides methods of using such vaccine compositions in a variety of therapeutic applications, e.g., methods for preventing or treating viral infections, such as RSV, MPV, and PIV infections.

Provided is a mutant of a respiratory syncytial virus (RSV) pre-fusion conformational F protein. The mutant comprises an amino acid mutation relative to the amino acid sequence of a wild-type RSV F protein, the amino acid mutation being amino acids at a position 392 and a position 493 or a position near the position 493 being substituted by C, and a disulfide bond being formed between a position 392C and a position 493C or a position near the position 493C after the mutation. Also provided are a related DNA coding sequence, recombinant plasmid and mRNA sequence and a corresponding RSV vaccine. By introducing a disulfide bond at a suitable position, and combining same with cavity filling, electrostatic mutation, proline mutation and the like, the expression and stability of an F protein having pre- and post-fusion conformations and a trimerized conformation are significantly improved, and the conformation of the F protein is significantly inhibited from being converted into the post-fusion conformation. The mutant is also applied to preparation of an RSV vaccine and RSV infection detection.

The invention discloses a reconstructed respiratory syncytial virus F protein trimer, relates to the technical field of genetic engineering vaccines, and designs an RSV F protein trimer, a monomer of the trimer does not contain a p27 sequence, and a furin enzyme digestion peptide fragment aa110-aa136 is deleted; the F2 structural domain and the F1 structural domain are connected through a GS connexon; the invention also designs point mutation combinations, and the point mutation combinations can form an inter-chain disulfide bond, so that covalent connection of the trimer is realized, and the problem of depolymerization of the trimer is fundamentally solved; the trimer is stabilized in a pre-F state, a post-F sample structure does not exist, and an ELISA result shows that a neutralizing epitope is completely reserved; the trimer antigen structure is challenged by adopting a thermal acceleration experiment method, and a result shows that the purity of the trimer antigen reaches 95% or above after the trimer antigen is placed at 37 DEG C for 28 days, and the trimer antigen is very good in stability; when the trimer is used for preparing the vaccine, the standard required by laws and regulations can be met without freeze-drying.

The present invention relates to the technical field of biomedicine. Specifically provided are a respiratory syncytial virus (RSV) Pre-F recombinant protein nanoparticle vaccine and a preparation method therefor. By performing amino acid mutation on an RSV Pre-F protein, and performing fusion expression on the mutated Pre-F protein and ferritin particles in eukaryotic cells, PreF-Ferritin fusion protein nanoparticles, in which eight Pre-F protein trimers are densely displayed on the surface of each ferritin particle, are obtained. By stabilizing and exposing antigen epitopes required to be displayed and disrupting or masking unrequired antigen epitopes, the PreF-Ferritin fusion protein nanoparticles effectively improve the immunogenicity and the production stability of the antigen. Experiments show that: when the PreF-Ferritin fusion protein is injected into mice, high-titer protective sera can be acquired, and the mice sera can yield relatively high neutralizing titers against RSV.

The present invention relates to RSV F protein mutants and their applications, belonging to the field of vaccines. Based on the amino acid sequence of wild-type RSV F protein, the present invention has carried out modifications in multiple aspects and in various ways, including disulfide bond mutations, proline mutations, cavity filling mutations, electrostatic mutations, p27 sequence modifications, and / or trimerization domain modifications, and designed a variety of RSV F protein mutants. Then, through gene synthesis, expression, and ELISA detection, the present invention tested the pre-fusion conformation (pre-F) ratio and stability under high and low temperature conditions of the RSV F protein mutants, and screened out several RSV F protein mutants with a relatively high pre-fusion conformation ratio and stable structure. Finally, candidate antigen representatives were selected for immunogenicity detection, which proved that they have good immunogenicity and can be used for the development of RSV vaccines, the preparation of RSV-specific antibodies, or RSV detection reagents.

The present invention relates to a respiratory syncytial virus (RSV) F protein reconstructed polypeptide, a polynucleotide encoding the same, a nucleic acid construct comprising the polynucleotide, an expression vector comprising the nucleic acid construct, a host cell in which the polynucleotide, the nucleic acid construct or the expression vector is transformed or transfected, and a stabilized multimer formed from the reconstructed polypeptide, an immunogenic composition comprising any of the foregoing, and its use in the preparation of a vaccine for the prevention and / or treatment of respiratory syncytial virus infection. The RSV F protein reconstructed polypeptide has good immunogenicity, can stimulate an organism to generate high-level neutralizing antibody titer, and has great significance in clinical treatment and prevention and control of the respiratory syncytial virus.

The invention discloses a stable pre-fusion RSV F protein and application thereof, the RSV F protein comprises one or more amino acid mutations to stabilize the RSV F protein in a pre-fusion conformation, and the RSV F protein has high affinity and neutralizing activity. Also disclosed are fusion proteins or multimers, immunogens, virus-like particles or protein nanoparticles comprising the RSV F protein, as well as nucleic acid molecules encoding the same, vectors, host cells, and immunogenic compositions.

The invention discloses an RSV pre-F tripolymer protein combination and application thereof in preparation of bivalent RSV vaccines, and relates to the technical field of human vaccines. The vaccine is prepared by mixing an RSV-A subtype pre-F tripolymer protein and an RSV-B subtype pre-F tripolymer protein in equal mass, and matching with an aluminum hydroxide adjuvant with the concentration of 0.35 mg / 0.5 mL. A p27 fragment and a C-terminal esterification region of each of the two F proteins are deleted, the two F proteins are connected with F2 / F1 through GSGSGS, inter-chain disulfide bonds are introduced to S146C / N460C and A149C / Y458C sites respectively, intra-chain disulfide bonds are introduced to S155C / S290C sites respectively, and stable pre-F trimers are spontaneously assembled after secretion expression of CHO cells; the vaccine does not need to be freeze-dried, still keeps high purity and neutralizing epitope integrity after being stored at 37 DEG C for 28 days, can obviously induce neutralizing antibody and T cell immunity, and can be used for preventing various RSV-A / B infections.

Related to the field of prevention and treatment of respiratory diseases, and disclosed a method for preparing a preventive mRNA vaccine against human respiratory syncytial virus. In some embodiments, the mRNA vaccine comprises an antigen-encoding mRNA and lipid nanoparticles. In some embodiments, the mRNA sequence utilizes an improved 5'UTR / 3'UTR sequence and codon optimization methods for efficient translation of the target protein. In some embodiments, the mRNA vaccine expresses an RSV F protein that is used as an antigen, wherein the sequence of the RSV F protein is optimized to achieve stable RSV pre-F protein expression. After immunization, the mRNA vaccine induces high levels of RSV F protein-specific binding antibody titer and neutralizing antibody titer in mice.

The invention relates to an RSV F protein soluble fragment, an autotrimer structural domain and application thereof, and belongs to the technical field of recombinant protein. Based on an amino acid sequence of a wild RSV F protein, a soluble and rigid sequence is screened out, a soluble fragment is constructed, disulfide bond mutation pair modification is performed on the basis of the soluble fragment to obtain a self-derived trimer structural domain sequence, and the self-derived trimer structural domain sequence is used for replacing a transmembrane region and an intracellular region of the RSV F protein. Therefore, trimerization can be realized, stable pre-fusion conformation can be maintained, and soluble expression in cells can be realized at the same time. The invention also provides an RSV F protein mutant containing an autotrimer structural domain sequence, the generation and the stability of the pre-F conformation trimer are proved, a representative is selected from the pre-F conformation trimer for animal experiments, and it is proved that the pre-F conformation trimer has good immunogenicity and can be used for developing RSV vaccines and preparing RSV specific antibodies or RSV detection reagents.

The invention provides a respiratory syncytial virus (RSV) F protein comprising at least one modification or mutation selected from the group consisting of: (1) introducing one or more amino acid mutations into the RSV F protein for filling a hydrophobic cavity; (2) introducing one or more amino acid mutations for electrostatic modification into the RSV F protein; and (3) removing a p27 peptide fragment in the RSV F protein. As a recombinant protein antigen, the RSV F protein is high in stability and easy to store and transport, the expression quantity is increased, the yield is easy to increase, and the modified RSV F protein is higher in immunogenicity and can generate a better immune protection effect.