JP4031478B2 - Method for producing influenza hemagglutinin multivalent vaccine - Google Patents

Description

  The present invention is generally in the area of recombinant influenza vaccines.

  Methods for producing influenza vaccines that do not require breeding in eggs result in a purer product that would be less likely to cause an undesirable immune response. In addition, purer vaccine propagation will not require virus inactivation or organic extraction of viral membrane components, thereby raising the concern of denaturation of antigenic epitopes and residual compounds in the vaccine. It is something to avoid.

  Additionally, influenza vaccines produced without egg breeding will avoid genetic heterogeneity that occurs during adaptation and passage through eggs. This will result in a vaccine that is more compatible with influenza infectious disease strains and will have improved efficiency.

  Accordingly, it is an object of the present invention to provide a method for producing an influenza vaccine that does not require replication in eggs.

  It is a further object of the present invention to provide a method for producing an influenza vaccine that allows for the production of a vaccine from a primary source of influenza that is rapid, cost effective and highly purified.

  Provided is a method for preparing recombinant influenza hemagglutinin protein by expression in insect cells using a baculoylus expression system. The resulting protein is useful for making multivalent influenza vaccines based on a mixture of recombinant hemagglutinin antigens cloned from influenza viruses that have the potential to be infectious. Recombinant hemagglutinin antigen is full-length, has both HA1 and HA2 subunits (HA0), and is undegraded purified to a purity of greater than 95%, preferably 99%, under non-denaturing conditions (HA0) is a glycoprotein.

  A method for preparing a recombinant influenza vaccine is described. A full-length, undegraded (HA0) hemagglutinin antigen from influenza virus was produced in insect cells by the baculovirus expression system and purified under non-denaturing conditions. Two or more purified hemagglutinins from influenza A and / or influenza B strains were mixed together to form a multivalent influenza vaccine. The recombinant antigen may be combined with an adjuvant carrier to increase efficiency.

  SEQ ID NO: 6 is a nucleic acid sequence comprising a polynucleotide sequence encoding A / Beijing / 32/92 HA linked to a polynucleotide sequence encoding a baculovirus 61K signal peptide. SEQ ID NO: 7 is the corresponding amino acid sequence, which shows the amino acid sequence of HA bound to the baculovirus 61K signal peptide (amino acids 1-18).

  SEQ ID NO: 8 is a nucleic acid sequence comprising a polynucleotide sequence encoding A / Texas / 36/91 HA linked to a polynucleotide sequence encoding a baculovirus 61K signal peptide. SEQ ID NO: 9 is the corresponding amino acid sequence and shows the amino acid sequence of HA bound to the baculoyls 61K signal peptide (amino acids 1-18).

  SEQ ID NO: 10 is a nucleic acid sequence comprising a polynucleotide sequence encoding B / Panama / 45/90 HA. SEQ ID NO: 11 is the corresponding amino acid sequence.

  SEQ ID NO: 12 is a nucleic acid sequence comprising a polynucleotide sequence encoding B / Netherlands / 13/94 HA linked to a polynucleotide sequence encoding a baculovirus 61K signal peptide. SEQ ID NO: 13 is the corresponding amino acid sequence and shows the amino acid sequence of HA bound to the baculoyls 61K signal peptide (amino acids 1-18).

  SEQ ID NO: 14 is a nucleic acid sequence comprising a polynucleotide sequence encoding A / Shandong / 9/93 HA linked to a polynucleotide sequence encoding a baculovirus 61K signal peptide. SEQ ID NO: 15 is the corresponding amino acid sequence and shows the amino acid sequence of HA bound to the baculoyls 61K signal peptide (amino acids 1-18).

  SEQ ID NO: 16 is a nucleic acid sequence comprising a polynucleotide sequence encoding B / Shanghai / 4/94 HA linked to a polynucleotide sequence encoding a baculovirus 61K signal peptide. SEQ ID NO: 17 is the corresponding amino acid sequence and shows the amino acid sequence of HA bound to the baculoyls 61K signal peptide (amino acids 1-18).

  SEQ ID NO: 18 is a nucleic acid sequence comprising a polynucleotide sequence encoding HA of B / Harbin / 7/94. SEQ ID NO: 19 is the corresponding amino acid sequence.

  SEQ ID NO: 20 is a nucleic acid sequence comprising a polynucleotide sequence encoding A / Johannesburg / 33 / 94HA linked to a polynucleotide sequence encoding a baculovirus 61K signal peptide. SEQ ID NO: 21 is the corresponding amino acid sequence and shows the amino acid sequence of HA bound to the baculoyls 61K signal peptide (amino acids 1-18).

Example 1
Influenza virus propagation and purification The following influenza vaccine strains were obtained from the FDA in chicken egg urine:
A / Beijing / 353/89 (H3N2)
A / Beijing / 32/92 (H3N2)
A / Texas / 36/91 (H1N1)
B / Panama / 45/90
Example 2
Cloning of the influenza A / Texas / 36/91 HA gene A specific example of a cloning procedure for one of the influenza HA genes is shown in FIG.

Example 3
Cloning the HA gene into a bacterial plasmid The PCR amplified rHA gene was cloned into a pUC-like plasmid vector using the TA cloning system (Invitrogen, Inc.).

Example 4
Expression of recombinant HA gene in insect cells A chimeric recombinant plasmid containing the cloned HA gene was purified and 2 μg was mixed with 1 μg of AcNPV wild type DNA.

Example 5
Production and purification of recombinant HA A baculoylus expression vector containing the gene for influenza A / Beijing / 353/89, A8611, A / Beijing / 32/92 hemagglutinin as described above for the polyhedrin promoter Made substantially the same, and Used to infect frugiperda cells.

Example 6
Analysis of rHA protease resistance Mature HA assembles into a tertiary structure that is resistant to enzymes containing trypsin that degrades HA monomers (Murphy and Webster, 1990).

Example 7
RHA immunogenicity using a standardized mouse potency assay. One method for measuring the immunogenicity of an antigen is to determine the amount required to induce a detectable antibody response in the mouse. Occasionally (mouse efficacy assay).

Example 8
Administration of rHA in combination with adjuvant and comparison with available influenza vaccines The mouse efficacy of purified rHA from influenza A / Beijing / 353/89 was tested with or without alum (commercially available) The vaccine was compared to Fluzone® (Connaught Laboratories, Inc. Swiftwater, PA) containing influenza A / Beijing / 353/89.

Example 9
Hemagglutinin inhibition studies Hemagglutinin inhibition (HAI) antibodies bind to three of the four known antibodies and interfere with the ability to aggregate influenza erythrocytes (Wilson et al., “Structure of the”. hemagglutinin membrane glycoprotein of influenza virus at 3Å resolution “Nature, 289: 366-378 (1981)).

Example 10
Systematic description and clinical efficiency of 1993/1994 influenza vaccines A series of human clinical trials have been conducted to characterize the safety and immunogenicity of experimental influenza vaccines containing recombinant HA in humans and the This was done to obtain preliminary data on the protective effect against natural infection during the season of infectious diseases.

Example 11
Method for making an improved HA0 cloning vector An improved cloning vector for expression of mature HA, wherein the gene encoding HA is located immediately after the sequence encoding the sequence of the chitinase signal peptide Things were designed.

Example 12
Preparation and effect of 1995-1996 influenza virus vaccine A and B trivalent forms Influenza virus vaccine, purified recombinant hemagglutinin, trivalent, A and B types (A / Texas / 36/92/1 (H1N1), A / Johannesburg / 33/94 (H3N2) and B / Harbin / 7/94) are non-infectious subunits derived from purified recombinant influenza hemagglutinin antigen (HA).

FIG. 1 shows the cloning of HA gene from influenza A strain from a viral RNA preparation, purification of expressed rHA, and biological activity of rHA.

Abbreviations: FDA, Food and Drug Administration; MDCK, Madin Darby dog kidney; TPCK, tosylphenylalanyl chloromethyl ketone; RNA, ribonucleic acid; HA, hemagglutinin; FBS, fetal bovine serum; PCR replication chain reaction BV, baculoyls.
FIG. 2 is a more detailed view of the method of FIG. 1 applied to the cloning and expression of the HA gene of influenza A / Texas / 36/91. The influenza HA gene is derived from RNA purified from MDCK cells infected with influenza A / Texas / 36/91 strain, using reverse transcriptase and universal primer (SEQ ID NO: 1), followed by two rounds of PCR amplification and cloning. Was obtained. As shown, 5 ′ end primer SEQ ID NO: 2 and 3 ′ end primer SEQ ID NO: 3 were used in the first round of PCR reaction. In the second round of PCR reaction, 5 ′ end primer SEQ ID NO: 4 and 3 ′ end primer SEQ ID NO: 5 were used. The baculoyl recombinant vector is a signal peptide derived from the polyhedrin promoter and the baculoyls 61K gene (the baculoyls gene encoding a signal peptide having a molecular weight of about 61000) followed by the complete coding sequence of the mature HA protein. Constructed including sequences. This combined vector was then used to create a baculovirus expression vector that produced HA from this virus strain. FIG. 3 is a graph of anti-HA immune response in mice on day 42, n = 5, with antibody titers for rHA0-neat; Fluzone® vaccine and rHA-alum dose Illustrated at 0.5 μg (black bar), 0.1 μg (shaded bar), 0.02 μg (polka dot bar), and 0.004 μg (white bar). FIG. 4a shows the anti-HA immune response in mice immunized with rHA or a licensed trivalent vaccine (1994-1995 standard), with HIA titers for the period (weeks) after vaccination. Illustrated for rHA (HAI A / Texas / 36/91) (diamonds) and attenuated vaccine FLUVIRON® (squares) cultured in eggs. FIG. 4b shows the anti-HA immune response in mice immunized with rHA or approved trivalent vaccine (1994-1995 standard), with HIA titers for the period (weeks) after vaccination. Illustrated for rHA (HAI A / Shangdong / 9/93) (diamonds) and attenuated vaccine FLUVIRON® (squares) cultured in eggs. FIG. 4c shows the anti-HA immune response in mice immunized with rHA or approved trivalent vaccine (1994-1995 standard), with HIA titers for the period (weeks) after vaccination. Illustrated for rHA (HAI B / Panama / 45/90) (diamonds) and attenuated vaccine FLUVIRON® (squares) cultured in eggs.

Claims (7)

  A vaccine composition comprising a recombinant glycosylated influenza hemagglutinin protein produced by a baculovirus expression system in cultured insect cells and purified to a purity of greater than 95%, the recombinant glycosylated influenza hemagglutinin protein comprising: Operably linked to a baculovirus signal peptide having the amino acid position 1-18 of the sequence of SEQ ID NO: 7 or 9, wherein the hemagglutinin protein is immunogenic and protects when used as a vaccine A vaccine composition characterized by inducing an immune response.   The vaccine composition according to claim 1, further comprising an adjuvant.   The vaccine composition according to claim 1 or 2, wherein the influenza is selected from the group consisting of influenza A strain and influenza B strain.   Effectiveness of recombinant glycosylated influenza hemagglutinin protein produced by baculovirus expression system and purified to greater than 95% in cultured insect cells by vaccination of animals (except humans) against influenza The recombinant glycosylated influenza hemagglutinin protein is a baculovirus signal peptide having an amino acid at amino acid positions 1-18 of the sequence of SEQ ID NO: 7 or 9. A method characterized in that it is operably linked and the hemagglutinin protein is immunogenic and induces a protective immune response when used as a vaccine.   5. The method of claim 4, further comprising administering the protein to the animal in combination with an adjuvant.   6. The method according to claim 4 or 5, wherein the influenza is selected from the group consisting of influenza A strain and influenza B strain.   The method according to claim 4, wherein the animal is a mammal.

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