WO2022188784A1 - Construction d'une souche chimérique atténuée de rougeole à remplacement du gène h - Google Patents

Construction d'une souche chimérique atténuée de rougeole à remplacement du gène h Download PDF

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WO2022188784A1
WO2022188784A1 PCT/CN2022/079777 CN2022079777W WO2022188784A1 WO 2022188784 A1 WO2022188784 A1 WO 2022188784A1 CN 2022079777 W CN2022079777 W CN 2022079777W WO 2022188784 A1 WO2022188784 A1 WO 2022188784A1
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virus
strain
measles virus
measles
chimeric
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安祺
朱凤才
田大勇
解丽霞
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上海青赛生物科技有限公司
北京赛尔富森生物科技有限公司
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    • C12N2760/18411—Morbillivirus, e.g. Measles virus, canine distemper
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    • C12N2760/18411—Morbillivirus, e.g. Measles virus, canine distemper
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Definitions

  • the invention belongs to the technical field of bioengineering, and in particular relates to an H gene-replaced chimeric measles attenuated strain and a preparation method and application thereof.
  • Measles is an acute systemic exanthematous infectious disease caused by Measles Virus (MV) infection.
  • MV Measles Virus
  • the main clinical manifestations of measles patients are fever, conjunctival congestion, runny nose, cough, maculopapular rash on the skin and measles mucosal spots on the oral mucosa, and may even have complications: diarrhea, deafness, blindness, encephalitis, myocarditis and severe malnutrition.
  • measles was a disease that seriously endangered the life and health of infants and young children. With the successful preparation of live attenuated vaccines, the incidence of measles has been kept at extremely low levels. However, in the past ten years, the incidence of measles in many countries in Europe, East Asia and South Asia has repeatedly risen. It is a typical feature that the genotypes of the epidemic strains in different regions are different. At the same time, more and more adults are infected with the measles virus. Symptoms of measles are usually more severe in adults.
  • the only measles vaccine product currently on the market is the live attenuated vaccine. Inactivated measles vaccine causes severe antibody-dependent enhancement.
  • H protein is the main immunogenic protein of measles virus and contains multiple neutralizing epitopes, and the neutralizing epitopes of H protein of different genotypes of MV are slightly different; this may be the reason that different genotype MV strains cannot achieve complete Reasons for cross protection.
  • Subunit vaccines based on H protein can induce significant immune responses in vivo, but there are no marketed products.
  • the purpose of the present invention is to provide an effective and highly safe means of preventing measles virus.
  • a chimeric measles virus attenuated strain is provided, and the attenuated strain is the measles virus rMV/H(H1a) with a deposit number of CCTCC NO: V202074.
  • the chimeric measles virus attenuated strain is replaced by the H gene sequence corresponding to SEQ ID NO: 6 in the wild-type A genotype measles virus genome with SEQ ID NO: 3 shows the nucleotide sequence of the H1a genotype measles virus H gene, obtained.
  • the genome of the chimeric measles virus attenuated strain has the nucleotide sequence shown in SEQ ID NO: 5 from the N gene to the L gene.
  • a strain derived from the attenuated chimeric measles virus as described in the first aspect of the present invention having one or more of the following properties:
  • (a) high replication ability the replication ability is equivalent to the in vitro replication ability of the measles virus rMV/H (H1a) whose deposit number is CCTCC NO: V202074; wherein, the replication ability is equivalent to that its replication rate is that the deposit number is CCTCC NO : ⁇ 80% of the replication rate of the measles virus rMV/H(H1a) of V202074, preferably ⁇ 90%, more preferably ⁇ 100%;
  • the immunogenicity is equivalent to the immunogenicity of the measles virus rMV/H(H1a) with the deposit number CCTCC NO: V202074; wherein, the immunogenicity is equivalent to its induced neutralization
  • the antibody titer is ⁇ 80%, preferably ⁇ 90%, more preferably ⁇ 100% of the neutralizing antibody titer induced by the measles virus rMV/H(H1a) whose deposit number is CCTCC NO: V202074;
  • the H gene in the genome is replaced by the H gene derived from the H1a genotype measles virus compared to the wild type A genotype measles virus vaccine strain.
  • the H gene derived from the H1a genotype measles virus includes: the coding sequence of the H1a genotype measles virus H protein, and the 5'UTR and 3 in the H1a genotype measles virus H gene 'UTR.
  • the replication ability of the derived virus strain is significantly higher than that of the wild-type A genotype measles virus vaccine strain.
  • the "replication ability is significantly higher" means that the replication rate of the derived virus strain is ⁇ 1.5 times that of the wild-type A genotype measles virus vaccine strain, preferably ⁇ 2 times, more preferably ⁇ 3 times, more preferably ⁇ 10 times, more preferably ⁇ 50 times, and optimally ⁇ 100 times.
  • the replication rate of the derived virus strain is ⁇ 10 times, preferably ⁇ 50 times, more preferably ⁇ 100 times that of the A genotype measles virus vaccine strain.
  • the replication rate of the derived virus strain is ⁇ 1.5 times, preferably ⁇ 2 times, more preferably ⁇ 3 times that of the A genotype measles virus vaccine strain.
  • the replication includes in vivo and in vitro replication of the virus.
  • the replication is in a host cell.
  • the host cell is selected from the group consisting of Vero cells, Vero-Slam cells, and CEF cells; preferably Vero-Slam cells.
  • the immunogenicity of the derived virus strain is significantly higher than that of the wild-type A genotype measles virus vaccine strain.
  • the "significantly higher immunogenicity" means that the neutralizing antibody titer induced by the derived virus strain is the same as the neutralizing antibody titer induced by the wild-type A genotype measles virus vaccine strain ⁇ 2 times, preferably ⁇ 2.5 times, more preferably ⁇ 5 times.
  • the neutralization ability of the derived virus strain to H1a virus is higher than or equal to the neutralization ability of the H1a genotype virus to the H1a genotype virus;
  • the neutralization titer is ⁇ 90%, preferably ⁇ 100%, more preferably ⁇ 120% of the neutralization titer of H1a genotype virus to H1a genotype virus.
  • the genome of the derived virus strain contains the following nucleotide sequences: the sequence identity to the nucleotide sequence shown in SEQ ID NO: 5 is ⁇ 85%, preferably ⁇ 90%, More preferably ⁇ 95% (eg, ⁇ 85%, ⁇ 86%, ⁇ 87%, ⁇ 88%, ⁇ 89%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98% or ⁇ 99%).
  • sequence identity to the nucleotide sequence shown in SEQ ID NO: 5 is ⁇ 85%, preferably ⁇ 90%, More preferably ⁇ 95% (eg, ⁇ 85%, ⁇ 86%, ⁇ 87%, ⁇ 88%, ⁇ 89%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98% or ⁇ 99%).
  • the H gene sequence corresponding to the H gene shown in SEQ ID NO: 6 in the wild-type A genotype measles virus genome is replaced with the H1a genotype measles virus shown in SEQ ID NO: 3 Nucleotide sequence of the H gene.
  • a virus-like particle containing the chimeric measles virus attenuated strain according to the first aspect of the present invention or a derivative according to the second aspect of the present invention virus strain is provided.
  • a vaccine composition comprising:
  • the carrier is a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier contains liquid, preferably water, saline or buffer.
  • the carrier further contains auxiliary substances, preferably fillers, lubricants, glidants, wetting agents or emulsifiers, pH buffer substances and the like.
  • the carrier further contains a cell transfection reagent.
  • the vaccine composition is a double vaccine or a multiple vaccine.
  • the vaccine composition further contains vaccine components derived from one or more pathogens selected from the group consisting of mumps, rubella, Japanese encephalitis, hepatitis A, chickenpox, polio inflammation or a combination thereof.
  • the vaccine components include inactivated strains, attenuated strains, or proteins, nucleic acids, and the like.
  • the vaccine composition further contains an adjuvant.
  • the adjuvant includes: particulate adjuvant and non-particulate adjuvant.
  • the particulate adjuvant is selected from the group consisting of aluminum salts, water-in-oil emulsions, oil-in-water emulsions, nanoparticles, microparticles, liposomes, immunostimulatory complexes, or a combination thereof.
  • the non-particulate adjuvant is selected from the group consisting of muramyl dipeptide and its derivatives, saponins, lipid A, cytokines, derived polysaccharides, bacterial toxins, microorganisms and their products such as branches Bacillus (Mycobacterium tuberculosis, BCG), Bacillus pumilus, Bacillus pertussis, Propolis, or a combination thereof.
  • the dose of virus in each dose of the vaccine composition is not less than 3.51g CCID 50 .
  • the vaccine composition is in the form of injection.
  • a method for preparing a chimeric measles virus attenuated strain comprising the steps of:
  • the recombinant plasmid contains a nucleotide sequence selected from the group consisting of: a nucleus corresponding to the wild-type A genotype measles virus genome as shown in SEQ ID NO:6
  • the H gene of the nucleotide sequence is replaced with the nucleotide sequence shown in SEQ ID NO: 3; or the sequence identity with the nucleotide sequence shown in SEQ ID NO: 5 is ⁇ 85%, preferably ⁇ 90 %, more preferably ⁇ 95% (eg, ⁇ 85%, ⁇ 86%, ⁇ 87%, ⁇ 88%, ⁇ 89%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94% , ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98% or ⁇ 99%) nucleotide sequences;
  • (iii) respectively co-transfecting the host cells with the recombinant plasmids obtained in (i) and the three helper plasmids, after culturing for 2-4 days (preferably 3 days), lysing the cells and inoculating them into new cells
  • the cells are cultured, and when cytopathic changes are observed, the attenuated chimeric measles virus strain is obtained.
  • the host cell is selected from the group consisting of BSR-T7 cells, 293T cells, Vero cells, Slam/Vero cells, or a combination thereof.
  • an attenuated chimeric measles virus strain as described in the first aspect of the present invention, a derivative virus strain as described in the second aspect of the present invention, or a chimeric measles virus as described in the third aspect of the present invention The use of the virus-like particle described above is used to prepare a vaccine composition for preventing measles.
  • the measles is H1a genotype or A genotype measles.
  • the measles is H1a genotype measles.
  • a method for preparing a vaccine composition comprising the steps of:
  • step (ii) mixing the attenuated vaccine strain prepared in step (i) with an immunologically acceptable carrier, thereby preparing a vaccine composition.
  • a method of inoculation for preventing measles comprising the steps of: inoculating the attenuated chimeric measles virus strain as described in the first aspect of the present invention, as described in the second aspect of the present invention, to the object in need.
  • the derivative virus strain described above, the virus-like particle according to the third aspect of the present invention, or the vaccine composition according to the fourth aspect of the present invention comprising the steps of: inoculating the attenuated chimeric measles virus strain as described in the first aspect of the present invention, as described in the second aspect of the present invention, to the object in need.
  • the subject is a measles susceptible person over 8 months old.
  • the inoculation method includes subcutaneous injection inoculation.
  • the dose of the inoculation is 0.3-0.7 mL (preferably 0.4-0.6 mL, more preferably 0.5 mL) L, and the amount of virus is not less than 3.51g CCID 50 .
  • Figure 1 shows the chimeric virus gene backbone map.
  • Figure 2 shows the restriction map of the chimeric virus.
  • Figure 3 shows the sequencing and alignment results of chimeric viruses.
  • Figure 4 shows the results of electron microscopy identification of chimeric viruses.
  • Figure 5 shows the multistep growth properties of chimeric viruses in Vero cells.
  • Figure 6 shows the multistep growth properties of chimeric viruses in Vero-Slam cells.
  • Figure 7 shows the multistep growth properties of chimeric viruses in Vero-Slam cells.
  • Figure 8 shows the results of immunogenicity analysis of chimeric viruses.
  • the inventors developed a chimeric measles virus attenuated strain and its derivative strain for the first time. Specifically, the inventors used the genome of the A genotype vaccine strain as a framework, replaced its H gene with the H gene of the H1a genotype measles virus, constructed and rescued a chimeric virus; the experimental results showed that the chimeric virus obtained by the present invention was obtained.
  • the virus can show the typical growth characteristics of H1a genotype measles virus, and can produce higher immunity to H1a genotype virus in the body.
  • live attenuated vaccines against measles viruses of various circulating genotypes can be rapidly prepared, which is more efficient and safer than the traditional weakening process.
  • the present invention has been completed on this basis.
  • the term “about” means that the value may vary by no more than 1% from the recited value.
  • the expression “about 100” includes all values between 99 and 101 and (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the terms "containing” or “including (including)” can be open, semi-closed, and closed. In other words, the term also includes “consisting essentially of,” or “consisting of.”
  • polynucleotide refers to a chain-like compound formed by the polymerization of nucleotides.
  • vaccine refers to biological products prepared against various pathogenic microorganisms for prophylactic or therapeutic vaccination. Vaccines are roughly classified into live attenuated vaccines, inactivated vaccines, recombinant protein vaccines, vector vaccines, nucleic acid vaccines, etc.; herein, live attenuated vaccines are preferred.
  • serotype refers to a classification of pathogens: in general, the immunogenicity of a certain virus or bacterium, eg, if the serotype is the same, then generally the immunogenicity of different strains of the virus of the same serotype is consistent.
  • the term "genotype” refers to the classification of viruses or bacteria at the genetic level, which is more granular than serotyping.
  • cross-protection insufficiency refers to pathogens of different serotypes, one of which as a vaccine does not fully protect against challenge with other serotypes of pathogens, which is referred to as cross-protection insufficiency.
  • viruses such as measles virus, mumps virus, etc.
  • there is only one serotype but there is also a certain phenomenon of incomplete cross-protection between different genotypes.
  • MV Measles Virus
  • MV belongs to the genus Morbillivirus of the family Paramyxoviridae, and its virions are spherical with a diameter of about 120 nm to 250 nm.
  • the MV genome is a single-stranded negative-stranded RNA with a total length of about 16kb.
  • the genome is N-P/C/V-M-F-H-L from the 3' end to the 5' end, encoding 6 structural and functional proteins: nuclear protein (N), phosphorylated protein (P), membrane protein (M), fusion protein (F) , hemagglutinin glycoprotein (H) and RNA-dependent RNA polymerase (L).
  • MV has only one serotype, there are many genotypes.
  • WHO recently confirmed that there are currently 24 genotypes of measles virus.
  • the genotypes of the measles epidemic strains in different regions are different.
  • the genotypes of the measles epidemic strains in my country are mainly H1a subtypes, while those in Europe are mainly G genotypes.
  • Existing studies have also proved that the immunogenicity of MVs of different genotypes is slightly different, and cross-protection cannot be achieved.
  • H protein is the main immunogenic protein of measles virus and contains multiple neutralizing epitopes, and the neutralizing epitopes of H protein of different genotypes of MV are slightly different; this may be the reason that different genotype MV strains cannot achieve complete Reasons for cross protection.
  • H proteins In the measles virus of A genotype and H1a genotype, there are respective H proteins.
  • the H proteins of the two have certain homology. Its amino acid sequences are shown in SEQ ID NOs: 1 and 2, respectively.
  • chimeric virus As used herein, “chimeric virus”, “chimeric measles virus”, “chimeric measles virus attenuated strain”, “attenuated strain of the present invention”, “measles virus rMV/H(H1a)” are used interchangeably , all refer to the measles virus rMV/H(H1a) with the deposit number CCTCC NO: V202074 described in the first aspect of the present invention.
  • the provided chimeric measles virus attenuated strain is derived from the wild-type A genotype measles virus vaccine strain, and on the basis of which, the H gene in the genome is derived from the H gene of the H1a genotype measles virus replace.
  • the post-replacement region (or newly inserted sequence) in the attenuated chimeric measles virus strain not only includes the H1a genotype measles virus H protein encoding gene, but also includes the 5'UTR of the H gene in the H1a genotype measles virus and 3'UTR sequence for better expression of its H protein in recombinant viruses.
  • the genome sequence from the N gene to the L gene of the wild-type A genotype measles virus is shown in SEQ ID NO: 4, and the genome of the chimeric measles virus of the present invention has the following nucleotides from the N gene to the L gene Sequence: the sequence shown in SEQ ID NO:5, wherein the H gene sequence shown in SEQ ID NO:6 in the wild-type A genotype measles virus genome is replaced by the H1a gene shown in SEQ ID NO:3 The nucleotide sequence of the measles virus H gene.
  • the present invention provides derivative strains derived from the attenuated chimeric measles virus strains of the present invention.
  • the derivative strains of the chimeric attenuated measles virus strains of the present invention have one or more of the following properties:
  • (a) high replication ability the replication ability is equivalent to the in vitro replication ability of the measles virus rMV/H (H1a) whose deposit number is CCTCC NO: V202074; wherein, the replication ability is equivalent to that its replication rate is that the deposit number is CCTCC NO : ⁇ 80% of the replication rate of the measles virus rMV/H(H1a) of V202074, preferably ⁇ 90%, more preferably ⁇ 100%;
  • the immunogenicity is equivalent to the immunogenicity of the measles virus rMV/H(H1a) with the deposit number CCTCC NO: V202074; wherein, the immunogenicity is equivalent to its induced neutralization
  • the antibody titer is ⁇ 80%, preferably ⁇ 90%, more preferably ⁇ 100% of the neutralizing antibody titer induced by the measles virus rMV/H(H1a) whose deposit number is CCTCC NO: V202074;
  • the H gene in the genome is replaced by the H gene derived from the H1a genotype measles virus compared to the wild type A genotype measles virus vaccine strain.
  • the replication ability of the derived virus strain provided by the present invention is significantly higher than that of the wild type A genotype measles virus vaccine strain.
  • the "replication ability is significantly higher” means that the replication rate of the derived virus strain is ⁇ 1.5 times that of the wild-type A genotype measles virus vaccine strain, preferably ⁇ 2 times, better ⁇ 3 times, more preferably ⁇ 10 times, more preferably ⁇ 50 times, and optimally ⁇ 100 times.
  • the replication is performed in Vero-slam cells, and the replication rate of the derived virus strain is ⁇ 10 times that of the A genotype measles virus vaccine strain, preferably ⁇ 50 times, more preferably ⁇ 100 times.
  • the replication is performed in Vero cells, and the replication rate of the derived virus strain is ⁇ 1.5 times, preferably ⁇ 2 times, more preferably ⁇ 3 times that of the A genotype measles virus vaccine strain .
  • the replication includes in vivo and in vitro replication of the virus.
  • the replication is in a host cell.
  • the host cells are selected from the group consisting of Vero cells, Vero-Slam cells, and CEF cells; preferably Vero-Slam cells.
  • the derived virus strain is significantly more immunogenic than the wild type A genotype measles virus vaccine strain.
  • the "significantly higher immunogenicity" means that the neutralizing antibody titer induced by the derived virus strain is ⁇ 2 times that induced by the wild-type A genotype measles virus vaccine strain, which is more than Preferably ⁇ 2.5 times, more preferably ⁇ 5 times.
  • the neutralization ability of the derived virus strain of the present invention to H1a virus is higher than or equivalent to that of H1a genotype virus to H1a genotype virus; preferably, the neutralization titer of the derivative virus strain to H1a virus is H1a gene
  • the neutralization titer of H1a genotype virus against H1a genotype virus is ⁇ 90%, preferably ⁇ 100%, more preferably ⁇ 120%.
  • the genome of the derived virus strain contains the following nucleotide sequences: the sequence identity to the nucleotide sequence shown in SEQ ID NO: 5 is ⁇ 85%, preferably ⁇ 90%, more preferably ⁇ 95% (eg, ⁇ 85%, ⁇ 86%, ⁇ 87%, ⁇ 88%, ⁇ 89%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98% or ⁇ 99%).
  • the sequence identity to the nucleotide sequence shown in SEQ ID NO: 5 is ⁇ 85%, preferably ⁇ 90%, more preferably ⁇ 95% (eg, ⁇ 85%, ⁇ 86%, ⁇ 87%, ⁇ 88%, ⁇ 89%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94%, ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98% or ⁇ 99%).
  • the H gene sequence corresponding to the H gene shown in SEQ ID NO:6 in the wild-type A genotype measles virus genome is replaced with the H1a genotype measles virus H gene shown in SEQ ID NO:3. Nucleotide sequence.
  • a method for preparing the chimeric measles virus attenuated strain of the present invention comprising the steps of:
  • the recombinant plasmid contains a nucleotide sequence selected from the group consisting of: a nucleus corresponding to the wild-type A genotype measles virus genome as shown in SEQ ID NO:6
  • the H gene of the nucleotide sequence is replaced with the nucleotide sequence shown in SEQ ID NO: 3; or the sequence identity with the nucleotide sequence shown in SEQ ID NO: 5 is ⁇ 85%, preferably ⁇ 90 %, more preferably ⁇ 95% (eg, ⁇ 85%, ⁇ 86%, ⁇ 87%, ⁇ 88%, ⁇ 89%, ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93%, ⁇ 94% , ⁇ 95%, ⁇ 96%, ⁇ 97%, ⁇ 98% or ⁇ 99%) nucleotide sequences;
  • (iii) respectively co-transfecting the host cells with the recombinant plasmids obtained in (i) and the three helper plasmids, after culturing for 2-4 days (preferably 3 days), lysing the cells and inoculating them into new cells
  • the cells are cultured, and when cytopathic changes are observed, the attenuated chimeric measles virus strain is obtained.
  • the host cell is selected from the group consisting of BSR-T7 cells, 293T cells, Vero cells, Slam/Vero cells, or a combination thereof.
  • a method for preparing a vaccine composition comprising the steps of:
  • step (ii) mixing the attenuated vaccine strain prepared in step (i) with an immunologically acceptable carrier, thereby preparing a vaccine composition.
  • the vaccine composition provided in the present invention comprises:
  • the carrier is a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier contains a liquid, preferably water, saline or buffer.
  • the carrier also contains auxiliary substances, preferably fillers, lubricants, glidants, wetting agents or emulsifiers, pH buffer substances and the like.
  • the carrier further contains a cell transfection reagent.
  • the vaccine composition of the present invention is a double vaccine or a multiple vaccine.
  • the vaccine composition further comprises a vaccine component derived from one or more pathogens selected from the group consisting of mumps, rubella, Japanese encephalitis, hepatitis A, chickenpox, polio, or a combination thereof .
  • the vaccine components include inactivated strains, attenuated strains, or proteins, nucleic acids, and the like.
  • the vaccine composition further contains adjuvant.
  • the adjuvants include: particulate and non-particulate adjuvants.
  • the particulate adjuvant is selected from the group consisting of aluminum salts, water-in-oil emulsions, oil-in-water emulsions, nanoparticles, microparticles, liposomes, immunostimulatory complexes, or combinations thereof .
  • the non-particulate adjuvant is selected from the group consisting of muramyl dipeptides and derivatives thereof, saponins, lipid A, cytokines, derived polysaccharides, bacterial toxins, microorganisms and products thereof Such as mycobacteria (Mycobacterium tuberculosis, BCG), Bacillus pumilus, Bacillus pertussis, propolis, or a combination thereof.
  • mycobacteria Mycobacterium tuberculosis, BCG
  • Bacillus pumilus Bacillus pertussis, propolis, or a combination thereof.
  • the dose of virus in each dose of the vaccine composition is not less than 3.51g CCID50.
  • the vaccine composition of the present invention is in the form of injection.
  • chimeric measles virus attenuated strain and “measles virus rMV/H(H1a)" of the present invention, which are used interchangeably, have been deposited in the China Center for Type Culture Collection (CCTCC) on October 31, 2020 ) at: Wuhan University, Wuhan, China.
  • CTCC China Center for Type Culture Collection
  • the preservation number of the virus strain of the present invention is CCTCC NO: V202074.
  • the attenuated chimeric measles virus strain described in the first aspect of the present invention, the derivative virus strain described in the second aspect of the present invention, or the virus-like particle described in the third aspect of the present invention all have the ability to prepare a vaccine composition use, the vaccine composition is used to prevent measles.
  • the measles is H1a genotype or A genotype measles, preferably H1a genotype measles.
  • the present invention provides an inoculation method for preventing measles, comprising the steps of: inoculating the attenuated chimeric measles virus strain described in the first aspect of the present invention, the derivative virus described in the second aspect of the present invention to a needy object strains, virus-like particles according to the third aspect of the invention, or vaccine compositions according to the fourth aspect of the invention.
  • the subject is a measles susceptible person over 8 months old.
  • the inoculation method includes subcutaneous injection.
  • the dose of the inoculation is 0.3-0.7mL (preferably 0.4-0.6mL, more preferably 0.5mL), and the amount of virus is not less than 3.5lg CCID 50 .
  • the chimeric virus has a better protective effect on the circulating H1a genotype measles virus.
  • Example 1 Construction and identification of chimeric virus full-length cDNA
  • FIG. 1 it is a chimeric measles virus backbone diagram.
  • the full-length cDNA of the chimeric virus was successfully cloned by restriction enzyme digestion (as shown in Figure 2).
  • pMV, pMV/H(H1a) full-length clones and helper plasmids were bulk extracted; co-transfected into cells for virus rescue.
  • 293T cells were seeded in six-well plates for overnight culture, and cell wells with 80-90% cell confluency were selected for virus rescue.
  • the specific process is as follows: the full-length plasmid pMV or pMV/H(H1a) (4 ⁇ g), the helper plasmid pcDNA3.1-N (1.5 ⁇ g), pcDNA3.1-P (0.2 ⁇ g), pcDNA3.1-L (1.0 ⁇ g) ), pCAGGS-T7 (1.0 ⁇ g) and LipofectamineTM 2000 transfection reagent (12 ⁇ L) were mixed in 500 ⁇ L DMEM medium, incubated at 37°C for 20 min; cells were washed 3 times with PBS, and then the plasmid transfection reagent mixture was added to the cell wells, Incubate at 37°C for 6h.
  • Collect the cell supernatant with cell fusion and inoculate new cells to cell fusion lesions take 200ul cell supernatant to extract total RNA, then remove DNA with a DNA removal kit, and use specific primers H-C-F and H-C-R respectively (as shown in Table 1). ), specific fragments were amplified and sequenced.
  • the virus was expanded and cultured, the virus supernatant was collected, clarified by centrifugation and concentrated 100 times.
  • the virus particles of the chimeric virus were clearly observed by transmission electron microscopy, and the particle size was comparable to that of the parental virus rMV. The results are shown in Figure 4. .
  • Example 3 In vitro characterization of chimeric viruses
  • a cell suspension was prepared, the cell concentration was between 1.0 ⁇ 10 5 -5.0 ⁇ 10 5 (unit), and the cells were seeded into a 12-well plate at 2 ⁇ 10 5 (unit)/well.
  • the titers of chimeric viruses rMV/H(H1a) and rMV increased rapidly from the second day after virus infection, and reached the peak on the fourth day; There is no significant difference.
  • the titer of MV-1 was maintained at 2lgCCID 50 /ml, which was more than 1000 times lower than the other two strains; and the infectious virus titer could no longer be detected on the fifth day after infection.
  • This indicates that the replication properties of the chimeric virus rMV/H(H1a) are similar to rMV, but do not exhibit too many characteristics of the wild-type virus.
  • the results are shown in Figure 6.
  • the replication ability of the chimeric virus in Vero-Slam cells is still similar to that of rMV.
  • MV-1 was able to replicate rapidly on the 1st and 2nd day after infection, and the titer reached 5lgCCID 50 /ml, and the virus titer no longer increased and decreased slowly from the third day after infection.
  • the speed and degree of cytopathic changes caused by the chimeric virus after H protein replacement were also compared in Vero cells and Vero-Slam cells, respectively.
  • the evaluation was performed on the BALB/c mouse model.
  • mice 5-week-old female BALB/c mice were randomly divided into 6 groups with 10 mice in each group.
  • Each mouse in the immunization group was intraperitoneally injected with 500ul of 1.0 ⁇ 10 5 CCID 50 (unit)/ml virus suspension, and the control group was injected with an equal volume DMEM.
  • Each group was given secondary immunization 28 days after the first immunization, and orbital blood was collected 14 days after the second immunization. 4 degrees overnight, centrifuged at 3000 rpm for 10 minutes, and collected serum. Serum was inactivated at 56°C for 30 minutes.
  • Vero-SLAM cell suspension was prepared, the cell concentration was between 1.0 ⁇ 10 5 -5.0 ⁇ 10 5 (units), and 100 ⁇ L/well was added to a 96-well plate mixed with serum and virus.
  • the cytopathic changes were observed and recorded on the 7th day, and the ED50 was calculated by the Reed/Muench method on the 10th day.
  • chimeric virus rMVH(H1a) is a candidate vaccine strain against the H1a genotype measles virus.

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Abstract

La présente invention concerne un procédé de construction d'une souche chimérique atténuée de rougeole à remplacement du gène H et une application associée. La souche atténuée est un virus de la rougeole rMV/H(H1a) dont le numéro de dépôt est le CCTCCNO : V202074. La présente invention concerne également une composition vaccinale contenant la souche chimérique atténuée de la rougeole à remplacement du gène H ou une souche virale dérivée de cette dernière en tant que principe actif, et son procédé de préparation.
PCT/CN2022/079777 2021-03-10 2022-03-08 Construction d'une souche chimérique atténuée de rougeole à remplacement du gène h WO2022188784A1 (fr)

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CN113999824B (zh) * 2021-12-30 2022-11-11 北京赛尔富森生物科技有限公司 H1a基因型嵌合麻疹病毒减毒株和制备方法及用途
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