KR19990022028A - Method for producing influenza hemagglutinin multivalent vaccine - Google Patents

Abstract

A method for producing a recombinant influenza vaccine using DNA technology is provided. The resulting vaccine is based on a mixture of recombinant hemagglutinin antigens cloned from infectious influenza virus, preferably a trivalent influenza vaccine. Recombinant hemagglutinin antigen is a full length non-cleavage (HAO) glycoprotein produced from baculovirus expression vectors in cultured insect cells and purified under non-denaturing conditions. In a preferred embodiment, the cloned HA gene is modified by deleting the native hydrophobic signal peptide sequence and replacing it with the novel baculovirus chitinase signal peptide. A general approach for efficient extraction and purification of recombinant HA proteins generated in insect cells has also been described for the purification of rHA proteins from influenza A and B viruses.

Description

Method for producing influenza hemagglutinin multivalent vaccine

Infectious influenza is a cause of hemp hemorrhage and is a cause of significant disease rates and mortality worldwide. The movements have a very high attack rate and are responsible for the spread of the influenza virus in each community. Older people and those who are at risk for health problems are at increased risk for complications and hospitalization from influenza infection. In the United States alone, more than 10,000 deaths occurred during each of the seven influenza seasons between 1956 and 1988, resulting in pneumonia and influenza, and more than 40,000 deaths occurred during each of the two seasons (Update: Influenza Activity-United States and Worldwide, and Composition of the 1992-1993 Influenza Vaccine, Morbidity and Mortality Weekly Report, US Department of Health and Human Services, Public Health Service, 41 / No, 18: 315, 1992). Influenza viruses are highly polymorphic particles composed of two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). HA mediates the fusion of the virus-cell membrane during attachment of the virus to the host cell and during cell invasion of the virus. The influenza virus genome consists of eight naturally occurring antisense RNA fragments, and encodes the fourth major fragment HA gene. Influenza viruses are classified as type A, type B, and type C based on differences in antigen. Influenza A viruses are described by, for example, A / Beijing / 353/89 by nomenclature including subtype or type, geographic origin, strain number and isolation date. There are at least 13 HA subtypes (H1-H13) and 9 NA subtypes (N1-N9). All subtypes are found in algae, but H1-H3 and N1-N2 are found in humans, pigs and horses (Reference: Murphy and Webster, Orthomyxoviruses, in Virology, ed. Fields, B. N., Knipe, D. M., Chanock, R. M., 1091-1152 (Raven Press, New York, (1990)

Antibodies to HA neutralize the virus and form the basis for natural immunity against infection by influenza [Clements, Influenza Vaccines, in Vaccines: New Apporaches to Immunological Problems, ed. Ronald W. Ellis, pp. 129-150 (Butterworth-Heinmann, Stoneham, MA 1992)]. The antigenic diversity in the HA molecule is responsible for the frequent occurrence of influenza and the regulation of the limited infection by immunization.

The tertiary structure of HA and its interaction with sialic acid, a cellular receptor, have been extensively studied (Wilson, et al, Structure of the hemagglutinin membrane glycoprotein of influenza virus at 3A ° resolution Nature 289: 366-378 (1981 ); Weis, et al., Structure of the influenza virus hemagglutinin complexed with its receptor, sialic acid Nature, 333: 426-431 (1988); Murphy and Webster, 1990] HA molecules are present as trimer in virions. Each monomer is present with two chains HA1 and HA2 connected by a single disulfide bond. The infected host cell produces a glycosylated polypeptide precursor with a molecular weight of about 85,000, which is then cleaved with HA1 and HA2.

The presence of neutralizing IgG and IgA antibodies specific for influenza HA is associated with resistance to infection and disease (Clements, 1992). Inactivated whole virus and partially purified (cleaved into subunits) influenza vaccine are associated with HA . The influenza vaccine usually contains 7-25 μg of HA per each strain of influenza.

The role of NA, the other major surface glycoprotein in the protective immunity of antibodies or T-cells that respond to influenza, has not been elucidated. Neuraminidase is very unstable in the process of purification and storage [Murphy and Webster, 1990] and the quantification of NA within the current influenza vaccine has not yet been standardized. The purified HA vaccine, rather than NA, inhibits disease in animals challenged with influenza [Johansson, et al., Purified influenza virus hemagglutinin and neurominidase in eqivalent in stimulation of antibody response but inducible constrasting types of immunity to infection J. Virology, 63: 1239-1246 (1989)]. Experimental vaccines based on neuraminidase antigens have not been found to be protective in human trials [Reference: Orga et al., J. Infect. Dis. 135: 499-506 (1977)).

A licensed influenza vaccine consists of a formalin-inactivated whole, or a chemically cleaved subunit made from two influenza A subtypes (H1N1 and H3N2) and one influenza B subtype virus. Prior to each influenza season, the US Food and Drug Administration (FDA) vaccine and related biotechnology committee recommended a trivalent influenza vaccine composition for the coming season. The 1992-1993 vaccine contained A / Texas / 36/91 (HIN1), A / Beijing / 353/89 (H3N2) and B / Panama / 45/90 virus. The FDA advised that the 1993-1994 influenza vaccine should contain the same Texas and Panama and Panama strains and the new influenza A Beijing strain (A / Beijing / 32/92).

Prior to the influenza season, vaccination of high-risk people every year is the most effective measure to reduce the impact of influenza. The limitations of currently available vaccines are low utilization; Low efficiency in the elderly and children; Production in an egg; Antigenic diversity and adverse reactions.

The Centers for Disease Control (CDC) estimates that less than 30% of individuals at high risk for influenza are vaccinated each year [MMWR, 1992]. Current inactive vaccines have achieved high protection against disease among normal healthy adults when vaccine antigens and circulating influenza virus antigens are highly involved. The protection rate for diseases among the elderly is very low, especially for those who are not institutionalized [Clements, 1992]. Recent studies [References: Powers and Belshe, J. Inf. Dis. 167: 584-592 (1993)], significant antibodies reacting against a trivalent sub virion influenza vaccine were obtained in less than 30% of subjects over 65 years of age.

Seed viruses for influenza A and B vaccines are natural strains that replicate at high rates in the allantoin lining of eggs. Alternatively, the strain for the influenza A composition is a rear somatotype virus with the correct surface antigen gene. Rear sootton virus is due to the viral genome fragment having the characteristics of each parent strain. When one or more influenza virus strains infect the cells, these viral fragments are mixed to form a post-contrast leon containing a wide variety of genes from both parent strains.

Protection against the current full or split long influenza vaccine has a short lifespan and is attenuated as antigen drift occurs within the influenza strain. Influenza viruses undergo antigenic drift as a consequence of the immunological selection of the virus as well as the amino acid changes in the hemagglutinin molecule. Ideally, the vaccine strain matches an influenza virus strain that causes the disease. However, current manufacturing methods of influenza vaccines are limited by the propagation of the virus within the fetal chick egg. Not all influenza virus strains are replicated well within the egg; Therefore, the virus should be coordinated or a virus-reverse sentant should be created. A wide range of heterogeneity occurs within the hemagglutinin of the influenza virus cultured in the egg compared to the one isolated from infected individual groups growing in mammalian cells (Wang, et al., Virol. 171: 275-279 (1989); Rajakumar, et al., Proc. Nata. Acad Sci. USA 87: 4154-4158 (1990). Changes in HA during the selection and manufacture of influenza vaccines may result in a complex of antigenically distinct subgroups of virus. Thus, the virus in a vaccine may differ from the manifold in infectious strains, which may result in a suboptimal level of protection.

Immediate hypersensitivity reactions can occur due to residual egg protein in vaccinees in people with severe allergies. The 1976 swine influenza vaccine is associated with an increased frequency of Guillain-Barré syndrome. Subsequent vaccines made from other influenza strains have so far not been observed to increase the incidence of these rare diseases.

The method of producing an influenza vaccine that does not require propagation in the egg will produce a product of higher purity, which is less likely to cause an adverse immune response. In addition, higher purity vaccine manufacturing methods avoid the need for viral inactivation or organic extraction of viral membrane components, thereby avoiding denaturation of antigenic epitopes and safety concerns due to residual chemicals in the vaccine.

Moreover, the influenza vaccine produced in the absence of egg propagation will avoid genetic heterogeneity that occurs during adaptation and transfusion through the egg. This will result in improved efficiency due to better matching of vaccine and influenza strain.

Accordingly, the present invention provides a method of producing an influenza vaccine that does not require replication in an egg.

It is a further object of the present invention to provide a method of producing a fast, cost-effective and highly purified influenza vaccine and to permit the production of the vaccine from influenza one-dimensional.

Summary of the Invention

Is expressed in recombinant influenza hemagglutinin protein by expression in insect cells using a baculovirus expression system. The resulting protein is useful for making a multivalent influenza vaccine based on a complex of recombinant hemagglutinin antigens cloned from infectious influenza virus. The recombinant hemagglutinin protein is a full length unchallenged (HAO) glycoprotein containing HA1 and HA2 subunits (HAO) mode under non-denaturing conditions with a purity of 95% or more, preferably 99%

Methods for cloning influenza hemagglutinin genes from influenza A and B using a specially designed oligonucleotide probe and a polymerase chain reaction (PCR) method have also been disclosed. In a preferred embodiment, the cloned HA gene is modified by deletion of a nucleotide encoding a naturally occurring hydrophobic signal peptide sequence and substitution with a new baculovirus signal peptide to yield a sequence encoding a signal peptide immediately adjacent to hemagglutinin do. This chimeric gene is introduced into a baculovirus expression vector, which directs the baculovirus polyhedrin promoter to express the recombinant HA protein in infected insect cells. The 18 amino acid baculovirus signal peptide directs detoxification of rHA to the insect cell glycosylation pathway and not on mature rHA glycoprotein. In a preferred embodiment, the vector is designed not to encode any amino acid interposed between the signal peptide and the hemagglutinin protein.

This method can be extended to all types of influenza viruses and includes influenza viruses that infect humans, infecting the ubiquitous A (H1N1) subtype, A (H3N2) subtype and B type and other mammals and birds But is not limited to.

A general approach for efficient extraction and purification of recombinant HA proteins produced in insect cells is disclosed for the purification of rHA proteins from A subtype and B type influenza viruses. Recombinant vaccines can be developed from the primary source of influenza, e. G., Non-segregation from individual infected individuals, rather than adapted and cultured viruses in eggs. This allows the rapid development of the vaccine directly from infected strains of influenza, avoiding problems arising from adaptation of the virus for in-vivo culture and from patient response to egg contamination in the resulting vaccine.

Examples include formulations of the vaccine in an immunodominant dose and clinical efficacy including purified rHA antigens from influenza virus strains recommended by the FDA for the 1993/1994 and 1994/1995 influenza infection season . Functional immunity is measured using an assay that binds to influenza hemagglutinin or quantifies antibodies that block the ability of the influenza virus to aggregate red blood cells or neutralize influenza viruses. Protective immune responses to rHA vaccines are measured in animal or human challenge studies sensitive to influenza infection.

The present invention relates generally to the field of recombinant influenza vaccines.

Figure 1 is a schematic diagram of the cloning of the HA gene from influenza A strain, the purification of expressed rHA and the biological properties of rHA from purified viral RNA preparation.

Abbreviation: FDA-Food and Drug Administration; MDCK-Madin Darby Canadian Kidney; TPCK-tosylphenylalanyl chloromethyl ketone; RNA-ribonucleic acid; cDNA-complementary deoxy nucleic acid; HA-hemagglutinin; FBS-calf serum; PCR-polymerase chain reaction; BV-baculovirus.

Figure 2 is a more detailed schematic diagram of the method of Figure 1 applied to the cloning and expression of the HA gene of the influenza A / Texas / 36/91 strain. The influenza HA gene was obtained from purified RNA using reverse transcriptase and universal primer (SEQ ID NO: 1) from MDCK cells infected with influenza A / Texas / 36/91, followed by two rounds of PCR amplification and cloning. As shown, in one PCR reaction, the 5'-end primer SEQ ID NO: 2 and the 3'-end primer SEQ ID NO: 3 are used. The baculovirus rebauda vector is constructed to include a polyhedrin promoter and a signal peptide sequence from the Baculovirus 61K gene (a baculovirus gene encoding a signal peptide having a molecular weight of about 61,000), and a complete Followed by an encryption sequence. This recombinant vector is then used to generate a baculovirus expression vector that produces HA from these viral strains.

Figure 3 is a graph of the anti-HA immune response in five mice at 42 days, with the administration of 0.5 占 퐂 (black bars), 0.1 占 퐂 (shaded bars), 0.02 占 퐂 (spot stick) and 0.004 占 퐂 , RHAO-neat; A graph of antibody titer for Fluzone ^R vaccine and rHAO-alum.

Figures 4A, 4B and 4C are graphs of anti-HA immune responses in mice immunized with rHA or a licensed trivalent vaccine. HAI A / Texas / 36/91 (FIG. 4A), HAI, A / Chandon / 9/93 (FIG. 4B) and HAI B / Panama / 45/90 4C), attenuated rHA (diamonds) cultured in an egg (square) and FLUVIRON ^R.

Methods for producing recombinant influenza vaccines are described. The full length from influenza virus, unknotted (HAO) hemagglutinin antigen, is produced with the baculovirus expression vector in cultured insect cells and is purified under non-denaturing conditions.

Two or more purified hemagglutinin antigens obtained from influenza A and / or influenza B strains are mixed with each other to produce a multivalent influenza vaccine. Recombinant antigens may be combined with an auxiliary carrier to increase efficiency.

The use of recombinant DNA technology to produce an influenza vaccine offers several advantages: the recombinant DNA influenza vaccine can be produced under safer and more stringent control conditions; Propagation to infectious influenza in the egg is not required; The recombinant HA protein can be very highly purified, substantially eliminating side effects due to contaminating proteins; Since the purification process of recombinant HA does not involve the viral inactivation or the organic extraction of viral membrane components, the denaturation of the antigen and additional safety concerns due to residual chemicals in the vaccine can be avoided; The production of HA via recombinant DNA technology provides an opportunity to avoid genetic heterogeneity in application and through-inoculation periods through the egg, resulting in improved efficiency by allowing better match between vaccine strains and influenza infectious strains ; In addition, the recombinant approach allows time for selection based on more reliable infectious data because it allows for strain selection after one year.

Baculovirus expression system

Baculovirus is a DNA virus with Baculoviridae. These viruses are known to have a narrow host-range limited primarily to insects (butterflies and mosquitoes) of the Lepidopteran species. Baculovirus Autographa California Neuclear Polyhedrosis Virus (AcNPV) with baculovirus phenotype replicates efficiently in sensitive cultured insect cells. AcNPV has about 130,000 base pairs of double-stranded circular DNA genomes and is well characterized for host range, molecular biology and genetics.

Many baculoviruses, including AcNPV, form large protein crystal stores in the nucleus of infected cells. The single polypeptide referred to as the polyhedrin is responsible for the protein weight of approximately 95% of this occlusion. The gene for the polyhedrin is present in a single copy within the AcNPV virus genome. Since the polyhedrin gene is not essential for viral replication in cultured cells, it can be easily modified to express a heterologous gene. The heterologous gene sequence is inserted into the AcNPV gene and is located 3 ' after the polyhedrin promoter sequence, so that it is under transcriptional control of the polyhedrin promoter.

Recombinant baculovirus expressing a heterologous gene is constructed by homologous recombination between a baculovirus DNA and a chimera plasmid containing the gene sequence. Recombinant viruses can be detected by their distinct plaque form and plaque-tablet homogeneity.

Baculovirus is particularly suitable for use as a eukaryotic cloning and expression vector. They are generally safe by a narrow host range limited to arthropods. U.S.A. The Environmental Protection Agency (EPA) has approved the use of three baculoviruses to control insect pests. AcNPV has been applied to crops for many years under the permissive use of EPA.

AcNPV wild-type and recombinant viruses are replicated in a variety of insect cells, including continuous cell lines derived from the autumn armyworm, Spodoptera (Noctuidae), Lepidoptera (Noctuidae). S. prugifera cells are groups of cells with a doubling time of 18-24 hours and can be propagated in a single layer or free suspension culture.

The recombinant HA protein may be produced in, but is not limited to, cells derived from the < RTI ID = 0.0 > Lefodopteran spodoptera frugiperda. ≪ / RTI > Other insect cells that can be infected by baculoviruses, such as, for example, Bombyx mori, Gallerian melanoma, Tricofulciano or Raman triadis, can also be used as appropriate substrates for producing recombinant HA proteins.

The most preferred host cell line for protein production from recombinant baculovirus is Sf900 +. Another preferred host cell line for protein production from recombinant baculovirus is Sf9. Sf900 + and Sf9 are non-transformed, non-tumor continuous cell lines derived from the fall armyworm Spodoptera frugiperda (lepidoptera; Sf900 + and Sf9 cells are propagated at 28 ± 2 ° C without supplementation of carbon dioxide. The culture medium used for Sf9 cells is TNMFH, supplemented with 10% fetal calf serum in a simple mixture of salts, vitamins, sugars and amino acids. No animal derived products (ie, trypsin, etc.) other than calf serum are used for cell proliferation. Serum free culture medium (available as Sf900 culture medium, Gibco BRL, Gaithersburg, MD) is also used for the growth of Sf9 cells and is preferred for propagation of Sf900 + cells.

Sf9 cells have a cell population doubling time of 18-24 hours and can be propagated in a single layer or free suspension culture. S. prugifera cells have not been reported to support replication of known mammalian viruses.

Those skilled in the art will appreciate that the expression vector is not limited to the baculovirus expression system. The recombinant HA protein is expressed in other expression vectors, such as insect cells transformed with consecutive expression of entomopoxvirus (insect poxvirus), cytosolic polyhedrosis virus (CPV), and recombinant HA gene (s) .

Isolation of influenza strains

One or more influenza strains have been isolated from diseased organisms. Preferably, influenza strains are identified by the Food and Drug Administration (FDA) or the CDC and have the potential for transmission during the next influenza season. An advantage of the methods described herein is that clinical samples obtained from patients infected with influenza, such as parenteral secretions, can be used as direct viral sources. Alternatively, they can be obtained from the FDA or CDC.

Propagation of influenza strains

The strain is then propagated in a virus that produces a high virus titre, such as Maddibia canina kidney (MDCK) cells (available from American Type Culutre Collction under accession number ATCC CCL34). For example, MDCK cells are infected in the presence of an optimal amount of fetal calf serum to produce high titers of tosylphenylalaninyl chloromethyl ketone (TPCK), some inactive trypsin and first subvirulent virus. MDCK cells are infected with influenza strains at low multivariate infections (0.1-0.5), as determined by standard HA assays [Reference: Rosen, Hemagglutination with Animal Viruses in Fundamental Tecnhique in Virology, ed. K. Habel and N.P. Salzman, pp. 276-28 (Academic Press, New York 1969), the teaching of which are incorporated herein. Infected cells are cultured at 33 ° C for 48 hours and the medium is analyzed for virus production using hemagglutination activity assay. The conditions to obtain the highest HA activity are used to prepare a large stock solution of influenza virus.

Purification of virus

The viral particles produced from the first subculture are purified from the medium by a known purification method such as sucrose density gradient centrifugation. For example, viruses are harvested after infection for 24-48 hours by centrifugation of the medium of MDCK cells infected with influenza. The obtained virus pellet is resuspended in the buffer and centrifuged through a buffered sucrose gradient. The influenza virus band is collected from a sucrose gradient range of 40-45%, diluted with buffer and centrifuged at 100,000 x g to obtain pellets. The purified virus pellet is resuspended in buffer and stored at -70 ° C.

Cloning of the influenza hemagglutinin gene

The outline of the method for cloning the HA gene is shown in Fig. Basically, the cells are infected with influenza strains to be cloned. Virus is collected from the cell culture medium and the viral RNA for influenza A strain or mRNA for influenza B strain is isolated. Viral RNA (-RNA) is extracted from purified virions and analyzed on formaldehyde agarose gels using standard procedures. CDNA is synthesized using a universal primer system for RNA from influenza A strain or using a random primer for mRNA from influenza B strain. Standard complementary DNA (cDNA) was generated using a universal oligonucleotide primer (5'-AGCAAAAGCAGG-3 '(SEQ ID NO: 1) homologous to all the hemagglutinin RNA fragments in influenza A and B [Reference: Davis (HON1) of influenza virus Gene, 10: 205-218 (1980)]. The primers include 5 'and 3' of the influenza hemagglutinin gene '5' and 3 'primers also have restriction sites at the ends not found in the hemagglutinin gene.

Hemagglutinin gene fragments are amplified using standard PCR procedures by mixing appropriate influenza A or B primers and influenza cDNA. The resultant double stranded DNA fragment obtained contains the complete mature hemagglutinin coding sequence. Polymerase chain reaction (PCR) is used to amplify the total HA gene, which is then cloned into a suitable bacterial host such as this cola. To identify the signal peptide of the HA gene, PCR is used to amplify the HA gene by sequencing the 5 'end and subtracting the next signal peptide. And then subcloned into a plasmid transfer vector containing the AcNPV polyhedrin promoter. The resultant transfer vector includes in the 5 'to 3' direction: baculovirus A. polyhedrin promoter from California NPV, ATG detoxification initiation codon, 61K baculovirus signal peptide, coding sequence for mature hemagglutinin , Natural hemagglutinin detoxification codon, polyhedrin RNA polyadenylation signal and flanking baculovirus DNA.

Next, the purified chimeric transfer plasmid DNA containing the cloned hemagglutinin gene is mixed with AcNPV wild-type DNA, co-precipitated with calcium, and transfected into S. prigi perda cells. Recombinant baculovirus is screened based on plaque type and further purified by further plaque-purification. The cloned recombinant baculovirus is screened for hemagglutinin expression and a single baculovirus expression vector is selected to produce a master virus bank.

Influenza A strain:

The HA gene from the influenza A strain is cloned from the purified virus RNA preparation. Viral RNA is extracted from purified influenza A virion containing 100 to 200,000 influenza hemagglutinin units (HAU) to 100 to 200 mu l. One HAU refers to the amount of virus that aggregates 50% of red blood cells in a standard flocculation assay [Rosen, 1969]. In order to decompose the protein, virion is treated with proteolytic enzyme K, then viral RNA is extracted using the same amount of phenol and chloroform and precipitated with ethanol in the presence of tRNA carrier. The viral RNA is resuspended in the buffer and decomposed into RNAse-free DNAse to remove any contaminating DNA, and then the extraction and precipitation steps are repeated. Next, viral RNA (vRNA) is analyzed using formaldehyde (Maniatis, et al., Molecular Cloning: A Laboratory Manual. pp. 86-96 and 366-367 (Cold spring Harbor Lab., Cold Spring N.Y. 1982).

Influenza B strain:

The HA gene of influenza B strain is cloned from total messenger RNA (mRNA) extracted from cells infected with influenza B strain. Next, total RNA is extracted from infected cells. The collected cells are lysed in the presence of guanidinium thiocyanate and the total cellular RNA is purified, for example, using an RNA extraction kit (from Pharmacia Biotech Inc. Piscataway, NJ). Total mRNA is extracted from cellular RNA using an oligo- (dT) -cellulose sponge column and using an mRNA purification kit (form Pharmacia Biotech Inc.).

Expression and processing of recombinant hemagglutinin in insect cells

A high level of recombinant hemagglutinin antigen is expressed in S. prugi peruvida cells infected with AcNPV-hemagglutinin vector. The primary gene product is unprocessed, full length hemagglutinin (rHAO), which is not secreted but remains bound to the surrounding membrane of infected cells. This recombinant HAO is a 68,000 molecular weight protein that is glycosylated by N-linkage to glycans that are distinct from the glycans produced by the expression of viral proteins in mammalian or avian cells, such as high-mannose type glycans. After decoding, there is evidence that rHAO forms an accumulating trimer in the cell membrane.

Vectors for the expression of HAO and other proteins

HAO is a better vector due to its superior stability in comparison to HA1 / HA2 complexes and maintains correct folding during purification and storage. Superior stability is particularly evident in strain B, resulting in a titer greater than five times that obtained by commercially available attenuated strain B.

As described in the Examples below, when the HA gene is cloned into pMGS12 through a restriction enzyme site, the HA mature signal peptide is removed and replaced with a baculovirus tDaNa signal peptide referred to as the 61 kD signal peptide. Since the HA gene is linked to the chitinase signal peptide through the cleavage site, there are three to five amino acids between the mature HAO protein and the 61 kD signal peptide, depending on the selected restriction enzyme site. Although it is not a problem in the influenza A strain, the B strain HAO expressing the additional amino acid does not fold properly.

Two methods have been developed to overcome this problem. The first is to use the novel vector pMGS3 which does not encode the 61 kD signal peptide. HAO with native signal peptide is cloned into a vector for expression. When confirmed by SDS-PAGE, B-strain HAO expressed in these vectors exhibits better glycosylation and processing than when expressed in pMGS12. HAO folds well and can be quantitatively converted to HA1 / HA2. Unfortunately, as determined by Western blotting, the yield is not high. The second method is to increase the yield using the pMGS 12 to 61 kD signal peptide to guide expression at the site where the HAO gene is inserted without using restriction enzymes. A novel vector containing the 61 kD signal peptide and the HAO gene without the sequence coding for the heterologous interfering amino acid is designated pMGS27.

pMGS27 can be used for cloning and expression of any gene in a baculovirus expression system. Instead of being cloned into vectors by restriction enzymes and linkages, the target gene is cloned into the vector by annealing. Tissues are available from Clontech as their PCR-directed cloning system. pMGS27 was constructed to be able to be linearized at the end of the coding region of the chitinase signal peptide and two long strands of Japanese chain are generated by treating linearized pMGS27 using dATP and T4 DNA polymerase.

The target gene was amplified using polymerase chain reaction (PCR) or reverse transcriptase-PCR (RT-PCR) using a pair of constructed oligonucleotides to create a Japanese chain tail tail complementary to the tail of pMGS27 , The PCR fragment is treated with T4 polymerase and dTTP. The two molecules can then be ligated by simple annealing into a circular plasmid to transform the host. In addition to being faster and simpler than the conventional restriction enzyme-linked method of cloning the HA gene into pMS12, pMGS27 did not obtain extra amino acids encoded by the restriction enzyme region created between the chitinase signal peptide and mature HA protein It has a great advantage. Such extra amino acids can sometimes cause differences such that the signal peptide is unable to cleave the signal or that the encoded protein is not folded correctly, as in the case of the B strain HA.

Purification of recombinant HAO

After several days of infection, any other method known to those of skill in the art for purification of recombinant proteins from non-denaturing, non-ionic detergents or insect cells (including, but not limited to, affinity or gel chromatography and antibody binding) To selectively extract rHAO from the surrounding membrane of ACNPV-hemagglutinin infected cells. DEAE ion exchange and lentile lectin affinity chromatography or other equivalent methods known to those skilled in the art can be used to further purify detergent soluble rHAO.

In a preferred embodiment, rHAO is purified using a procedure that is softer and results in higher yields of rHAO from influenza B strains. This procedure is generally as follows:

Separates the HAO protein that forms an integral part of the insect cell membrane from soluble proteins, peripheral membrane proteins and most of the DNA and RNA by cell extraction in a relatively viscous alkaline solution (alkaline pH = 9.5-10.5). The viscosity is increased through the sucrose incorporation at a concentration of approximately 250 mM. In order to inhibit disulfide linkage of the protein in the mixture, for example, a disulfide-reducing agent such as? -Mercaptoethanol is contained at an effective concentration. The cells are suspended in the extraction buffer and centrifuged. In a low ionic strength buffer containing alkaline (conductivity generally less than 1 mS, pH 10.5) disulfide-reducing agent, the pellet is washed by homogenization and the pellet is centrifuged. Then, an amount of 0.3 to 1.5% of a detergent such as triton under an alkaline pH (preferably 9.5), and a quantity effective to prevent complex formation due to charge interactions such as betaine or persine of 0.3 to 1.0 M HAO is extracted from the pellet in a buffer containing de-coagulant. The HAO in the supernatant is then purified by anion exchange chromatography followed by cation exchange chromatography. After equilibrating the column in buffer containing approximately 1/10 of the detergent and disulphide-reducing agent concentration, it is possible to equilibrate the extract in the same buffer diluted at least 1: 2 with an additional buffer, for example DEAE or Q-Sepharose ^R (An agarose bead column having a quaternary amine group). The HAO is then eluted by reducing the pH to about 8.5. The eluted HAO is applied to the cation exchange column in essentially the same buffer. After the contaminants are eluted by reducing the pH to about 7.4, the HAO is eluted by increasing the salt concentration to 0.15M.

These preferred purification methods are described in detail below.

Preparation of recombinant HA-containing membrane fraction.

Recombinant HA expressing cells (6.2 g of cells from 0.34 L of culture) were mixed with 100 mg / mL in 100 mM sodium pyrophosphate, 100 mM sodium chloride, 250 mM sucrose, 0.1% beta-mercaptoethanol under pH 10.5 Suspend it. Cells are cleaved at a setting of 4 for 2 minutes using a Polytron ^R homogenizer (Brinkman Instrument Inc., Westbury, NY). In order to increase the solubility of contaminating proteins and increase the purity of the membrane preparation, the alkaline pH of the homogenization medium is required. Centrifuge the equal volume to 9,200 g for 30 minutes. Discard the supernatant and collect the pellet. After the low-ionic strength washing step, a membrane fraction is prepared. The pellet is resuspended to the original volume in cold 0.1% beta-mercaptoethanol (10.5) and homogenized at 4 settings for 2 minutes using a Polytron ^R homogenizer. Centrifuge the homogenate at 9,200 g for 30 minutes. Discard the supernatant and collect the pellet. A low-ionic strength wash removes the additional peri-membrane protein portion. The production of membrane fractions results in significant enhancement in recombinant HA and removal of contaminating nucleic acids.

Extraction of recombinant HA

Under conditions that do not denature the antigen, the recombinant HA is selectively extracted from the membrane pellet. Membrane pellets were resuspended in 41 mL of cold 10 mM ethanolamine (pH 9.5), 1% Triton N101, 0.1% beta-mercaptoethanol, 25 mM NaCl, 400 mM betaine with a setting of 4 for 2 minutes using Polytron homogenizer Homogenize. After incubation at 23 ° C for 40 minutes, the mixture is centrifuged at 9,200 g for 30 minutes. The supernatant containing the recombinant HA is dispensed and diluted twice with the same buffer.

Proteins are analyzed by SDS polyacrylamide gel electrophoresis. The sample was cleaved in a breaking water bath for 10 minutes in the presence of 2% sodium dodecyl sulphide (SDS) and 5% [beta] -mercaptoethanol and then electrophoresed on an 11% polyacrylamide gel in the presence of 0.1% SDS And then stained with Coomassie blue.

Purification by chromatography

Purification of the cholestate HA by chromatography and affinity chromatography on high-cost Lentil Lectin Sepharose is performed to produce a highly purified recombinant HA antigen suitable as a component of the non-denatured and human influenza vaccine It is excluded from the process by replacing it with a two-step chromatographic purification process. Chromatography gel matrices used are Pharmacia Q-Sepharose ^R Fast Flow and CM-Sepharose Fast Flow ^R.

Anion exchange chromatography

All chromatography is carried out at room temperature. The recombinant HA-containing extracts prepared as described above were mixed with 10 mM ethanolamine (pH 9.5), 0.1% Triton ^R N101, 0.01% beta-mercaptoethanol, 25 mM NaCl, Pharmacia Q-Sepharose Fast Flow ^R equilibrated with 400 mM betaine (5 mL in a C10 / 10 Pharmacia column) at 1 mL / min. The column is then washed with an equilibration buffer until the UV absorption of the effluent returns to the baseline. Under these conditions, the recombinant HA is bound to the column while allowing a portion of the contaminant to pass through. The partially purified recombinant HA is then eluted with 30 mM diethanolamine pH 8.5, 0.1% Triton ^R N101, 0.01% beta-mercaptoethanol, 25 mM NaCl, 400 mM betaine.

Cation exchange chromatography

Dilute Q-Sepharose eluate (23 mL) twice with 30 mM diethanolamine (pH 8.5), 0.1% Triton ^R N101, 0.01% β-mercaptoethanol, 10 mM NaCl, 400 mM betaine. The column is then washed with 35 mL of 10 mM sodium phosphate, pH 7.4, 0.1% Triton ^R N101, 0.01% beta-mercaptoethanol, 10 mM NaCl, 400 mM betaine. This treatment elutes contaminants from the column with the recombinant HA bound to CM Sephrose. The detergent is then removed by washing the column with 10 mM sodium phosphate (pH 7.4), 10 mM NaCl until the UV absorption of the effluent returns to the baseline. Purified recombinant HA is eluted with phosphate buffered saline (pH 7.5) (PBS).

Purified rHAO is resuspended in isotonic buffer. After removal of detergent, purified rHAO will efficiently aggregate red blood cells.

Structural and biological properties of recombinant HAO

The rHAO is purified to at least 95% purity, preferably 99% purity. It migrates primarily as the only major polypeptide of 68,000 molecular weight on SDS-polyacrylamide gel. The quaternary structure of recombinant HAO antigens purified by electron microscopy, trypsin resistance, density precipitation analysis, and ability to coagulate chicken erythrocytes is examined. These data show that recombinant HAO forms trimer assemblies into rosettes.

The purified rHAO does not aggregate the cells prior to removal of the detergent, suggesting that the antigen forms rosettes to form crosslinks with the red blood cells. The quantitative ability of the purified rHAO to aggregate cells is used as a measure of the lot-to-lot viscosity of the antigen. One hemagglutinin unit is defined as the amount of antigen required to achieve 50% aggregation in a standard hemagglutinin assay using chicken erythrocytes. Comparison data shows that the purified rHAO antigen efficiently aggregates erythrocytes compared to observations using whole influenza virions.

Recombinant HAO is divided into disulfide bonds and is prepared as described in Carr, CM and Kim, PS, Spring-loaded Mechanism for the Conformational Change of Influenza Hemagglutin, Cell 73: 823-832 (1993) Can lead to structural changes that together form chains HA1 and HA2. The cleavage of the recombinant HAO is described in more detail in Example 6 below. By dividing the natural HAO into HA1 and HA2, it is believed that the chain becomes infectious by acquiring a fusion with the cell, thereby creating an enhanced immune response. The process of antigens, such as influenza hemagglutinin, occurs by binding antigenic peptides to major histocompatibility (MHC) molecules. Antigen / MHC complexes are recognized by T cells as initiating an immune response as described in Hardening and Geuze, Current Opinion in Cell Biology 5: 596-605 (1993), which is incorporated herein by reference. However, rHAO produced in baculovirus is very stable and immunogenic as an intact molecule. Comparison of sugar molecules on HAO expressed in insect cells shows that HAO of glycans is different from when expressed in mammalian or avian cells.

Production of fusion protein

A fusion protein that constitutes HAO fused to a secondary antigen protein can be generated where the antigenicity of the secondary protein is low or has the advantage of eliminating the immunogenic response to multiple antigens. An example of a preferred secondary antigen is neuraminidase produced by influenza. The antigen may constitute an antigenic portion comprising a cell, virus or bacterial protein or at least five to eight amino acids thereof. Other antigens include hepatitis B antigens, HIV antigens and cancer-born antigens. The term " immune response " as used herein means a cellular response measured by antibody production to an antigen or by induction of a T cell interposed antigen response. In some cases, a linker of a non-antigenic amino acid may be inserted between the HA and the antigen to further enhance the antigenicity of the antigen as compared to HA. This process involves the construction of a DNA plasmid to fuse the target antigen gene to the fragment or all of the influenza virus HA gene using oligonucleotide probes and polymerase chain reaction (PCR) methodology.

The HA-targeted antigen fusion gene is modified by deletion of a native hydrophobic signal peptide sequence and replacement with a new baculovirus signal peptide for proper expression in insect cells. The fusion gene is introduced into a baculovirus expression vector, directing the transcription of the fusion protein in insect cells infected with the baculovirus polyhedron promoter. The 18 amino acid baculovirus signal peptide directs transcription into the insect cell glycosylation pathway of the HA-target antigen fusion polypeptide and is absent from the mature fusion protein.

For example, plasmid pA9440 containing the A / Beijing / 32/92 strain HA gene in the following pMGS12 baculovirus transfer plasmid was amplified using a Gene Amp PCR cloning kit (Perkin Elmer Cetus) It is used as a template for HA gene amplification by polymerase chain reaction (PCR). The PCR reaction mixture (100 占 퐇) contains 20 pmol of a primer designed to be added to the HA gene site. The 5 'and 3' primers were designed to have a restriction endonuclease region at the end not found in the HA gene. The 5 'PCR primer (0-567) for the HA0 and HA1 fragments starts at 52 basepairs downstream from the 5' end of the native HA gene coding sequence and removes the native signal peptide sequence and inserts SmaI Area. The 5 'PCR primer (0-651) for the HA2 fragment starts at nucleotide 1108 of the native HA gene following the codon encoding the arginine residue that is cleaved during HA1 and HA2 splitting of HA0. The 3 'PCR primer (0-680) for the HA0 and HA2 fragments was designed to remove the natural stop codon and add the KpnI region immediately after the HA coding sequence. The 3 'PCR primer for HA1 (0-679) was designed to cleave the gene immediately before the arginine residue removed during HA0 splitting. The amplification of the HA gene fragment is performed for 30 cycles each consisting of denaturation at 94 ° C for 1 minute, primer annealing at 55 ° C for 2 minutes, and elongation at 72 ° C for 2 minutes. The resulting amplified HA gene fragment is electrophoresed on an agarose gel, purified from the gel using a GeneClean kit (Bio 101, Inc.), and ligated into a plasmid designed to accommodate PCR-producing fragments. Thus, plasmids pB142, pB144 and pB330 containing the HA0, HA1 and HA2 gene fragments, respectively, are obtained.

The HA gene fragment is removed from the plasmids pB142, pB144 and pB330 using SmaI and KpnI restriction enzymes and subcloned into the AcNPV transfer plasmid pMGS12 by standard recombinant DNA technology (Sambrook et al., 1989). The pMGS12 plasmid contains the AcNPV polyhedron promoter from 5 'to 3', the ATG start codon, the sequence for the segmented signal peptide from the 61,000 molecular weight baculovirus glycoprotein (61K), the SmaI and KpnI restriction enzyme cloning regions, and the TAA universal stop Lt; / RTI > sequence. These regulatory regions flank the DNA from the EcoRI I fragment from the AcNPV genome (Summers and Smith, A Manual of Method for Baculovirus vectors and nisect ce culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555 (1987)). The cloned HA PCR fragment is cleaved from pCRII cloning vector using SmaI and KpnI, purified using agarose gel electrophoresis and GeneClean kit, and ligated into pMGS12 already digested with SmaI and KpnI. The resulting AcNPV transfer plasmids pB879, pB1201, and pB1205 contain coding regions for HA0, HA1 and HA2, respectively, linked to the baculovirus signal peptide, which is divisible from the 61K gene and the polyhedron promoter. pB879, pB1201 and pB1205 AcNPV transfer plasmids can be used to fuse HA0, HA2 or HA2 to any desired gene.

The second step in the construction of the HA-CEA fusion gene transfer plasmid is to insert the CEA coding sequence into the HA-encoding construct. Restriction endonuclease recognition / cleavage sites for SmaI and KpnI are located at both ends of the CEA gene through PCR amplification of plasmid pA9080. The 5 'PCR primer O-649 starts at the 82 base pairs from the 5' end of the gene lacking the native CEA signal peptide sequence. The 3 'PCR primer O-650 was designed to delete the last 72 base pairs from the 3' end of the gene encoding the hydrophobic C-terminal region sequence. Amplification of the CEA gene fragment is performed for 30 cycles consisting of 1 minute denaturation at 94 DEG C, 2 minutes retraction at 55 DEG C, and 2 minutes elongation at 72 DEG C, respectively. The resulting amplified CEA gene fragment is electrophoresed on an agarose gel, purified using the GeneClean method, and ligated into pCRII (Invitrogen) according to the manufacturer's instructions. The resulting plasmid pB806 contains the CEA gene with no natural signal peptide, no C-terminal hydrophobic region or stop codon, but with SmaI and KpnI regions at both ends of the gene.

Large-scale plasmid production is performed using pB806 plasmid, and DNA is digested with SmaI or KpnI. The CEA-encoding fragment is purified using agarose gel electrophoresis and GeneClean kit, and the purified fragment is ligated into three degraded HA-encoding constructs (pB879, pB1201 or pB1205) using the same restriction enzymes. For example, a CEA-encoding fragment with SmaI-cleaving ends was ligated into the HA0-, HA1- and HA2- encoding constructs cleaved with SmaI (pB879, pB1201 and pB1205, respectively) and ligated with plasmids pB1205, pB1555 and pB1584 Respectively. The CEA-encoding fragment with the KpnI-cleaving end is ligated into the HA0-, HA1 and HA2- encoding constructs cleaved with KpnI to generate pB1264, pB1564, and pB1593. The CEA gene insertion in the SmaI region locates the CEA coding sequence downstream of the HA coding sequence. For all constructs, the PCR primers were designed such that the EA gene was inserted into the HA and the fusion gene decoding was terminated in the universal decode termination signal TAATTAATTAA (SEQ ID NO: 4) in the pMGS12 vector sequence downstream of the KpnI region.

Such constructs can be promoted to enhance folding and immunogenicity by deletion of the intervening amino acids between the signal peptide and HAO or between the HAO and the fusion gene, as described below.

Manufacture and packaging of vaccines

rHA can be manufactured and packaged alone or in combination with other influenza antigens known to those of ordinary skill in the influenza vaccine industry. In a preferred embodiment, HA proteins from both strains A and B bind to form a multivalent vaccine.

In a particularly preferred embodiment, the HA can be combined with an effective amount of adjuvant to enhance the immunogenic response to the HA protein. Here, the only adjuvant widely used in humans is alum (aluminum phosphate or aluminum hydroxide) saponin and its purified ingredients Quil A, Freund's adjuvant and other adjuvants used for research purposes, and livestock has limited potential use in human vaccines Toxicity. However, muramyldipeptide, monophosphoryl lipid A, Goodman-Sniktoff et al. J. Immunol. 147: 410-415 (1991), which is incorporated herein by reference, as disclosed in Miller et al., J. Exp. Med. 176: 1739-1744 (1992), and encapsulation of proteins in lipid vesicles such as Novcasome ^TM lipid vesicles (Micro Vescular System, Inc., Nashura, NH) .

In a preferred embodiment, the vaccine is packaged in a single dose for parenteral (i. E., Intramuscular, transdermal or subcutaneous) administration or immunization by nasopharyngeal (i. Effective doses are determined as described in the Examples below. The carrier is generally water or buffered saline with or without preservatives. The antigen may be lyophilized for resuspension in solution at the time of administration.

The carrier may also be a delayed-release polymer system. Synthetic polymers are particularly useful for the production of vaccines which will result in controlled release of the antigen. An initial example of this is the one micron method for forming so-called fine particles as reported by Kreuter, J., [Microcapsules and Nanoparticles in Medicine and Pharmacology, M. Donbrow (Ed), CRC Press, Lt; RTI ID = 0.0 > of less < / RTI > diameter. Antibody responses as well as protection against infection with influenza viruses are also significantly better every time the antigen is administered with aluminum hydroxide. Experiments with other particles have demonstrated that the adjuvant effect of these polymers depends on particle size and hydrophobicity.

Microencapsulation has been applied to the injection of microencapsulated drugs to produce controlled release. A number of factors contribute to the selection of a particular polymer for microencapsulation. The repeatability of the polymer synthesis and microencapsulation process, the cost of the microencapsulated material and process, the toxicological profile, the requirement of various release dynamics and the physicochemical compatibility of the polymer, and the antigen are all factors to be considered. Examples of useful polymers are polycarbonates, polyesters, polyurethanes, polyorthoesters and polyamides, especially those which are biodegradable.

A frequent choice of carrier for drugs and more recent antigens is poly (d, 10 lactide-co-glycolide) (PLGA). It is a biodegradable polyester with long-term medical use in temporary artificial organs where no corrosive structure, corrugated plates and any other toxicity are visible. A wide range of drugs, including peptides and antigens, have been produced in PLGA microcapsules. See, for example, Eldridge, J. H., et al., Current Topics in Microbiology and Immunology. 1989, 146: 59-66] have accumulated a number of data on PLGA variants for the controlled release of antigens. Capture of antigens in PLGA microspheres of 1 to 10 micron diameter has been shown to result in remarkable adjuvant effects when administered orally. The PLGA microcapsulation process utilizes phase separation of the water-in-oil emulsion. The desired compound is prepared as an aqueous solution and the PLGA is dissolved in an appropriate organic solvent such as methylene chloride and ethyl acetate. These two unmixed solutions are co-emulsified by rapid agitation. Then, a non-solvent for the polymer is added to cause precipitation of the polymer around the periphery, thereby forming a microcapsule. The microcapsules are collected and stabilized with one of reagents (polyvinyl alcohol (PVA), gelatin, aglynate, polyvinylpyrrolidine (PVP), methylcellulose) and the solvent is removed by vacuum drying or solvent extraction.

The invention will be further understood by the following non-limiting examples.

Example 1: Amplification and purification of influenza virus

The following influenza vaccine strains are obtained from the FDA in the eluent:

A / Basing / 353/89-like (H3N2)

A / Basing / 32/92-like (H3N2)

A / Texas / 36/91-like (H1N1)

B / Panama / 45/90

To amplify influenza virus stock obtained from FDA cells, MDCK cells were infected in the presence of TPCK-treated trypsin (Sigma Chemical Co., St. Louis, Mo.) and the fetal calf serum concentration was adjusted to the highest threshold first migrating virus Lt; / RTI > MDCK cells were measured by standard HA analysis (Rosen, Hemagglutination with Animal Viruses in Fundamental Techniques in Virology, edited by K. Habel and NP Salzman, pp. 276-28 (Academic Press, New York 1969) (Influenza strains are infected with 0.1 to 0.5.) Infected cells are cultured for 48 hours at 33 DEG C and the medium is assayed for virus production using hemagglutination assay activity assay. The optimal concentrations of TPCK trypsin and fetal bovine serum for the influenza viruses are shown in Table 1.

[Table 1]

The optimal concentration of TPCK trypsin and fetal calf serum

Purification of influenza virus:

After 24-48 hours of infection, the virus is harvested from 10 T175 tissue culture flasks by purifying the medium for influenza infected MDCK cells (1,000 x g for 10 minutes). The virus is pelleted from the medium for 1 hour at 100,000 x g. The resulting virus pellet is resuspended in pH 7.4 to 1 ml of phosphate buffered saline (PBS) and centrifuged through 20 ml 20-60% (w / v) sucrose gradient in PBS. The influenza virus band is harvested from the 40-45% sucrose region of the gradient, diluted with PBS and pelleted at 100,000 x g. The purified virus pellet is resuspended in 0.5 ml PBS stored at -70 < 0 > C.

Example 2: Cloning of influenza A / Tecgas / 36/91 HA gene

A specific example of the cloning step for one influenza HA gene is shown in Fig. Viral RNA is extracted from influenza A / Texas / 36/91 obtained from CDC as described above. Influenza cDNA is produced using a universal primer complementary to the 3 'end of the influenza RNA fragment 5'-AGCAAAAGCAGG-3' (SEQ ID NO: 1) together with the Maoney Leukemia virus (M-MuLV) reverse transcriptase of the mouse. Purified virus RNA or mRNA (5 μg) is used as a template to produce cDNA using the M-MuLV reverse transcriptase supplied by First Strand cDNA Synthesis by Pharmacia Inc. The primer used for the cDNA of the viral RNA from the influenza A strand is the synthetic oligonucleotide primer (5'-AGCAAAAGCAGG-3 ') (SEQ ID NO: 1) or homologous to the 3' end of the HA gene virion fragment.

HA gene amplification from cDNA is performed by polymerase chain reaction using standard reaction conditions (Gene Amp. Kits; Cetus / Perkin Elmer, Norwalk, Conn.). As shown in Table 2, the PCR reaction mixture (100 μl) contained 5 'and 3' residues of influenza A (H3) or A (H1) or influenza B strain when measured by the continuous sequence found in the GenBank DNA data file. And 20 pmol of a primer specific to the end. Amplification is carried out for 30 cycles consisting of 1 min denaturation at 94 [deg.] C, 2 min at 55 [deg.] C and 3 min elongation at 72 [deg.] C. In order to calibrate the size before cloning, the PCR product is analyzed on 0.8% agarose gel.

PCR primers from the 5 'end of the HA gene: 5'-GGG GGT ACC CCC GGG AGC AAA AGC AGG GGA AAA TAA AAA-3' (SEQ ID NO: 2) TCT TATA ACG / T TAG / T ACT CCA TGG CCC-3 '(SEQ ID NO: 3) is used in PCR to generate a complete HA gene.

The 5 ' end of the 5 ' -GGG GGT ACC CCC GGG GAC ACA ATA TGT ATA GGC TAC CAT-3 ' (SEQ ID NO: 4) gene without a signal sequence is designed from the 5 ' Terminal: 5'-GAAAC GTC ACG TCT TAT ACG / T TAG / T ACT CCA TGG CCC-3 '(SEQ ID NO: 5). These are used for PCR to generate an HA gene having no signal peptide sequence. Then, insert it into the TA vector divided by KpnI. The 61K signal peptide and bacteriophage promoter for baculovirus expression are inserted into the TA vector containing the HA gene without the influenza signal peptide sequence. As a result, the baculovirus recombinant vector contains the polyhedron promoter, the 61K baculovirus signal peptide, and the HA gene for influenza A / Texas / 36/91.

HA gene from Influenza B strain is cloned from total messenger RNA (mRNA) extracted from MDCK cells infected with influenza B-strain B / Panama / 45/90. Total RNA is prepared from 5 T175 flasks of infected cells. The harvested cells are lysed in the presence of guanidium thiocyanate and the whole cell RNA is purified as described above. The total mRNA is extracted from cellular RNA using oligo- (dT) -cellulose spun column as described above.

The primers used for mRNA from influenza B strains are arbitrary-oligonucleotide DNA primers (Pharmacia, Inc.).

[Table 2]

PCR amplification

An example of a cDNA synthesis product is influenza virus A / Texas / 36/91 viral RNA as a template. The position of the cDNA fragment encoding the influenza protein can be determined as follows. The purified viral RNA is ligated in a reaction mixture with the universal DNA chain DNA primer 5 'AGCAAAAGCAGG-3' (SEQ ID NO: 1). This primer is complementary to the 3 ' end of the influenza virion fragment as described above. The reaction also involves the addition of [? - ³² P] dCTP to visualize the cDNA product isolated on a 1.5% alkaline hydrolysis gel (Maniatis, et al, 1982) and exposed to X-OMAT-AR film.

Example 3 Cloning HA Gene into a Bacterial Plasmid

Using the TA cloning system (Invitrogen, Inc.), the PCR amplified rHA gene is cloned into a pUC-like plasmid vector. The presence of the HA gene is demonstrated by restriction enzyme digestion analysis of the plasmid DNA purified by standard methods (Maniatis et al, 1982). The 5 'end of the rHA gene is then analyzed by DNA sequencing, allowing the primer to remove the sequence coding for the signal hydrophobic peptide at the N-terminal HA protein. Then, the cDNA product was amplified by PCR using the specific 5 'and 3' oligonucleotide primers shown in Table 2, and cloned into E. coli TA plasmid vector (Invitrogen, Inc.) using standard cloning method do. The result is that DNA clones are contained in the coding sequence of mature HA.

The rHA gene from A / Texas / 36/91, A / Beijing / 353/89, A / Beijing / 32/92 and B / Panama / 45/90 was baculized by standard methods (Maniatis et al, 1982) Subcloning into a viral expression vector. The HA gene is removed from the TA cloning plasmid using appropriate restriction enzymes, and the purified HA DNA fragment is inserted into the baculovirus combination plasmid. The resulting bacterial clones are screened for ampicillin resistance and then cleaved with restriction enzymes to release the inserted HA gene to confirm its presence. The recombinant plasmid containing the HA gene is purified on a cesium chloride-bromide ethidium gradient (Maniatis et al, 1982). The 5 'end of the plasmid is sequenced to determine the presence of the correct baculovirus signal (AcNPV polyhedron promoter, ATG detoxification initiation signal and baculovirus signal peptide sequence) and appropriate HA coding sequence within the correct reading frame. The DNA sequence at the 5 'end of the HA gene and the flanking AcNPV polyhedron promoter and baculovirus signal peptide (the first 18 amino acids of the amino acid sequence, respectively) are shown in the sequence listing.

SEQ ID NO: 6 encodes the 5 'end sequence (sequence range 1-481) of the HA gene for A / Beijing / 32/92. SEQ ID NO: 7 is the corresponding amino acid sequence (starting at the start codon ATG [nucleotide 21] of SEQ ID NO: 6). The amino acid sequence of the 61K signal peptide is described as amino acids 1-18 in SEQ ID NO: 7.

SEQ ID NO: 8 encodes the 5 'end sequence (sequence range 1-481) of the HA gene for A / Texas / 36/91. SEQ ID NO: 9 is the corresponding amino acid sequence (starting at the start codon ATG [nucleotide 21] of SEQ ID NO: 8). The amino acid sequence of the 61K signal peptide is described as amino acids 1-18 in SEQ ID NO: 9.

SEQ ID NO: 10 encodes the 5 'end sequence (sequence range 1-434) of the HA gene for B / Panama / 45/90. SEQ ID NO: 11 is the corresponding amino acid (starting at the start codon ATG [nucleotide 21] of SEQ ID NO: 10). The amino acid sequence of the 61K signal peptide is described as amino acids 1-18 in SEQ ID NO: 11.

In SEQ ID NOs: 6, 8, and 10, nucleotides 1-20 are the 3'end of the polyhedron promoter, nucleotides 21-74 encode the 61K signal peptide, and nucleotide 75 encodes the 5'end of the HA gene.

Example 4: Recombinant HA expression in insect cells

The chimeric recombinant plasmid containing the cloned HA gene is purified and 2 을 are mixed with 1 Ac AcNPV wild-type DNA. The DNA is co-precipitated with calcium and transfected into S. prugifera cells using standard methods (Smith, Summers, and Fraser, Mol. And Cell. Biol. 3: 2156-2165 (1983)). Recombinant baculovirus is identified based on plaque phase and then further purified by additional plaque-purification. The plaque-purified recombinant baculovirus is screened for rHA expression and one baculovirus expression vector is selected for further development.

S. frugiperde cells are infected with a baculovirus vector containing the HA gene from the influenza strain: B / Panama / 45/90. At 24, 48 and 72 hours after infection, 1x10 ⁶ cells are pulsed with 25 μCi [ ³⁵ S] methionine for 15 min to synthesize the labeled protein. The cells are harvested and the proteins are separated on a 11% polyacrylamide gel in the presence of 0.1% SDS. The radiolabeled protein is detected by exposure to X-OMAT-AR film. The location and size (kd) of the standard protein indicates that the 85 kd recombinant HA protein is one of the major proteins synthesized within the cell at 48 and 72 hours post-infection.

Example 5 Production and Purification of Recombinant HA

Using a baculovirus expression vector containing the gene for influenza A / Beijing / 353/89 produced for A / Beijing / 32/92 hemagglutinin under the control of a polyhedron promoter as described above, S. prugifera cells. Cells were grown at a density of 1 × 10 ⁶ cells / mL in TNMFH medium (Gibco BRL, Gaithersburg, MD) supplemented with 10% fetal calf serum at 27 ° C. and infected with A8611 recombinant baculovirus (MOI) 1. During infection, influenza A / Beijing / 353/89 hemagglutinin is produced under transcriptional control of the baculovirus polyhedron promoter. The cells are harvested at 72 hours after infection by centrifugation at 3,400 x G for 15 minutes, resuspended in serum-free TNMFH medium and then washed by centrifugation at 10,400 x g for 30 minutes. The supernatant is removed and the infected cell pellet is stored at -70 ° C.

Methods have been developed to selectively extract recombinant HA from infected cells under conditions that do not denature the antigen. Unless otherwise indicated, all extraction steps are carried out at 4 ° C. Using a Polytron ^TM homogenization machine (Brinkmann Instruments Inc. Westbury, NY) in 40 mL of cold 30 mM Tris-HCl, pH 8.4, 25 mM LiCl, 1% (v / v Tween-20, 1 mg / mL rupeptin) The cell pellet (approximately 5 x 10 ⁸ cells) from the 0.5 L culture is cleaved for 2 minutes, and the homogenate is centrifuged for 30 minutes at 9,200 x g. Discard the supernatant and collect the pellet. 10 mM ethanolamine, pH 11, 25 mM LiCl, 2% Tween-20, and 10 mM ethanolamine, to remove the soluble substance and the surrounding membrane protein from the insect cells without extracting the main membrane proteins such as rHA. The pellet is homogenized in a setting of 4 for 2 minutes, within 20 minutes. After incubation for 60 minutes on ice, the pH of the homogenate is adjusted to 8.4 with 1N HCl and the insoluble material is centrifuged at 9,200 x g for 30 minutes Remove the supernatant containing soluble rHA, measure the pH and it is necessary Is adjusted to 8.4 at room temperature. The insoluble material resuspended in 40mL of water for analysis. High -pH, under the residual water solution and Tween-20 detergent conditions and the pH lowered, to dissolve the HA protein, the major membrane.

Proteins are analyzed by SDS polyacrylamide gel electrophoresis. The sample was cleaved in a water bath for 10 minutes in the presence of 2% sodium dodecyl sulfate (SDS) and 5% beta-mercaptoethanol and then electrophoresed on 11% polyacrylamide gel in the presence of 0.1% SDS , And stained with Coomassie blue.

In order to purify recombinant HA to produce highly purified recombinant HA antigen suitable as a component of the non-denatured and human influenza vaccine, a chromatographic purification method was developed. Purification of A / Beijing / 353/89 HA from S. frugiperda cells infected with the recombinant virus A8611 is carried out using the following method.

The chromatographic gel matrix used to purify HA from 0.5 L of infected S. prigi perda was obtained from 30 mL Pharmacia DEAE Sepharose Fast Flow (in Parmacia C16 / 20 column) and 4 mL Pharmacia Lentil Lectin Sepharose 4B (Pharmacia C10 / 10 Column). The outlet of the DEAE column is connected to the inlet of the lentile lectin column and the S / N 2 cell extract prepared as described above is applied to the coupled column at a flow rate of 1 ml / min. The column is washed with 25 mM LiCl, 0.5% Tween-20 in 30 mM Tris-HCl, pH 8.4 until the UV absorption at 280 nm of the lentil lectin effluent returns to baseline. Under these conditions, most contaminating proteins bind DEAE but recombinant HA flows through the column. Residual contaminants pass through the lectin column and the glycosylated rHA binds to the lentil lectin affinity matrix. The DEAE column is separated and the lectin column is washed with 40 ml 30 mM Tris-HCl, 25 mM LiCl, 0.5% Tween-20, pH 8.4. At this stage the Tween-20 detergent is replaced with a detergent which can be removed from the protein by dialysis, such as DOC. The recombinant HA is then eluted with 40% of about 30 mM Tris-HCl, 25 mM LiCl, pH 8.4, 0.4% (v / v) sodium deoxycholate containing 0.3 M a-D-methylmannoside. The results are analyzed by 11% PAGE.

Due to the genetic variability of the influenza HA protein, the details of the purification method can be varied with each unique recombinant HA protein. For example, rHA can be coupled to a DEAE ion exchange column instead of passing through it. If this situation occurs, the rHA will be removed from the DEAE column by washing the column with a buffer containing high concentrations of LiCl, NaCl or other salts.

To remove the DOC detergent and other buffer components, the eluate from the lectin column containing purified rHA is dialyzed against phosphate buffered saline, pH 7.5 (PBS). The purified recombinant HA has a purity of at least 95% as determined by analysis on SDS polyacrylamide gel.

Example: Analysis of rHA protease resistance

Mature HA is assembled into a trimer structure that is resistant to degradation of HA monomers (Murphy and Webster, 1990) and a variety of proteases including trypsin. Therefore, tolerance to trypsin treatment is used as an assay for functional trimer formation. The following procedure is used to study the resistance of rHA to protease treatment.

Two aliquots of purified rHA

60 μg / ml of (A / Beijing / 353/89) is cultured on ice for 30 minutes in 30 mM Tris-HCl, pH 8.4, 150 mM NaCl in the presence and absence of 50 μg / ml TPCK-treated trypsin. The reaction is stopped by adding 57.4 mM phenylmethylsulfonyl fluoride in isoprene to a final concentration of 1 mM. An aliquot of each sample was denatured by constantly reducing conditions in 3% SDS, electrophoresed on 11.5% polyacrylamide gel, and transferred to a nitrocellulose filter using a standard Western blotting procedure. HA polypeptides are detected using guinea pig anti-HA sera prepared against purified rHA and goat anti-guinea pig IgG alkaline forwarder conjugates.

The untreated rHA migrates to the size of the HA precursor (HAO). The protease treatment generates two major bands that migrate to the expected size for influenza hemagglutinin HA1 and HA2. The results show that trypsin cleaves the rHA protein once to produce two polypeptides of the expected size for HA1 and HA2. No further protein-mediated processing takes place. These results indicate that the rHA purified by the method is resistant to degradation of the protease. This property is consistent with rHA in trimer form.

Example 7: Immunogenicity of rHA using standardized rat potency assay

One approach to measure the immunogenicity of an antigen is to determine the amount required to induce a detectable antibody response in the rat (rat potency assay). Standardized rat efficacy assays are used to determine the immunogenicity of the rHA0 vaccine. Five to ten groups of mice are immunized once with a vaccine containing a series of dilutions of rHA, e.g., 0.500, 0.1, 0.02, and 0.04 [mu] g purified rHA. After 28 days of immunization, sera are collected and antibodies against anti-rHA antigen are measured by standard enzyme-linked immunological solid-phase assay (ELISA) on 96-well microtiter plates. If the OD450 of the 1: 100 dilution of antiserum on day 28 is greater than three standard deviations above the mean of OD450 of the rat immunized sera, the mice are seroconverted. An effective dose of the vaccine (ED50) required to seroconvert 50% of the rats is the mechanism of measurement of the immunogenicity of the antigen.

For example, four groups of 10 rats are immunized once with 0.1 μg, 0.02 μg, 0.004 μg, or 0.0008 μg of rHAO vaccine (five-fold dilution). Serum is collected 28 days after immunization and assayed for each rHA0 antigen in the vaccine for seroconversion during ELISA analysis. Calculate the dose (ED ₅₀ ) needed to seroconvert 50% of the rats and set a minimum ED ₅₀ for each rHA0 antigen.

The preliminary data show that a single 0.004 microgram of rHAO will convert at least 50% of the serum of the rats.

Example 8: Administration of rHA combined with adjuvant and comparison with an influenza vaccine which is easy to obtain

A commercial and influenza vaccine containing the A / Beijing / 353/89 strain of influenza, FLUZONE ^R (Connauht Laboratories, Inc.), which was tested in alum or neat rat efficacy of purified rHA from influenza A / Beijing / Inc. Swiftwater, Pa.). The vaccine is administered at a dose of 0.5 [mu] g, 0.1 [mu] g, 0.02 [mu] g and 0.04 [mu] g. Rats are boosted at 28 days with the capacity of the purified rHA described above. Serum is collected at 42 days and titrated by ELISA analysis for IgG anti-HA antibody.

The results are shown in Fig. In the absence of adjuvant, a dose of 0.5 μg leads to the production of significant antibody titers (200,000). In the presence of adjuvant, a significant dose of as little as 0.004 ug of rHA0 produces significant antibody. An animal immunized with rHA (neat) produces approximately the same level of anti-HA antibody as the commercial vaccine. Alum increases the immunogenicity of rHA and produces an anti-HA titer that is 10-fold greater than without supplements.

In summary, a comparison between purified rHAO and influenza total virion vaccine ^R (FLUZONE, Connaught Laboratories, Inc., Swiftwater, Pa.) Indicates that rHA0 elicits a similar immune response in mice over 42 days. Adsorption of rHAO to alum significantly increases the immunogenicity of purified rHAO in rats, as determined by the assay described in Example 7. [ Combination with Alum results in a higher IgG hemagglutinin antibody than the Fluzone ^R influenza vaccine.

Example 9: Hemagglutination inhibition study.

Haemagglutination Inhibition (HAI) antibodies inhibit the ability of influenza to bind three of the four known epitopes on hemagglutinin to aggregate red blood cells (Wilson et al, Hemaglutinin of influenza virus at 3 A resolution Structure of membrane glycoprotein Nature, 289: 366-378 (1981)). These epitopes are clustered around the sialic acid receptor binding site on the hemagglutinin trimer. Antibodies to these sites will neutralize viral infectivity (Weis, et al., Structure of influenza virus hemagglutinin complexed with receptor, sialic acid, Nature 333: 426-431 (1988)). The potency and specificity of the HAI antibody is an important measure of potential for the influenza vaccine to protect against infection with influenza strains and related strains.

Studies are performed in mice to compare the ability of purified rHA0 and FLUZONE ^R (Connaught Laboratories, Inc., Swiftwater, PA) from A / Beijing / 353/89 to elicit HAI antibodies. Groups of 5 rats were injected three times with 0.5 μg, 0.1 μg, 0.02 μg or 0.004 μg of rHAO or FLUZONE ^R of this amount on days 0 and 28 and thus gave an equivalent level of recombinant or viral A / Beijing / 353 / 89 Administration of hemagglutinin. For example, mice in the highest dose group are immunized with 1.5 [mu] g of FLUZONE ^R hemagglutinin (0.5 [mu] g hemagglutinin from each strain) and 0.5 [mu] g of rHA0. Additional hemagglutinin antigens of FLUZONE ^R from the other two influenza strains can generate some cross-reactive antibodies.

The anti-hemagglutinin antibody (hemagglutinin IgG) is measured in a standard dilution ELISA against purified rHAO. The HAI antibody is measured for the 4 hemagglutinin units of the antigen (whole influenza A / Beijing / 353/89 virus (A / Bei), purified rHA A / Beijing / 353/89 antigen, and FLUZONE ^R ). HAI is a reciprocal of the highest dilution of antiserum that inhibits 50% aggregation of chicken erythrocytes.

Table 3 summarizes the serum hemagglutinin IgG and HAI titers in 42-day-old rats. High levels of anti-hemagglutinin antibodies are generated with recombinant rHA0 vaccine. These are about 10 times more potent than FLUZONE ^R. It is most important that rHA0 produces antibodies with good titers that inhibit the aggregation of red blood cells by the vaccine A / Beijing / 353/89 virus and the rHA0 antigen. Therefore, the rHA0 vaccine produces an HAI antibody that equally well recognizes the influenza A / Beijing virus as an immunogen. The anti-serum to inhibit the lower HAI titers ^R FLUZONE vaccine in H. aggregation of two different strains of non-Ruti mageul for ^R FLUZONE may be due to force. Conversely, FLUZONE ^R immunized mice produce high HAI antibodies when measured against itself. HAI translocation for influenza A / Beijing / 353/89 virus and rHA0 antigen is significantly reduced. A similar pattern was observed in rats in the lower dose group.

[Table 3]

HAI activity for rHA0 and FLUZONE ^R

These data also suggest that there is a genetic difference between the influenza A / Beijing / 353/89 strains in FLUZONE ^R and the same strains of influenza obtained from the FDA and passed once to the eggs prior to using the HAI assay. The fact that the antibodies generated in response to recombinant HA0 cloned from A / Beijing / 353/89 inhibited red blood cell aggregation by influenza strains and by themselves affect the sialic acid receptor binding sites on the conformational rHA0 antigen during cloning There is good evidence that there has been no genetic change.

Example 10: Formulation and clinical efficacy of 1993/1994 influenza vaccine

A series of human clinical trials are conducted to demonstrate human safety and immunogenicity characteristics of an experimental influenza vaccine containing recombinant HA and to obtain preliminary data on the protective efficacy of these vaccines against natural infections during the epidemic period. The results show that a vaccine containing recombinant influenza hemagglutinin (rHAO) produced according to the methods described herein is surprisingly comparable to a commercially available attenuated flu vaccine produced in an egg without experiencing a surprisingly near local reaction Indicating a superior protective immune response.

Materials and methods

vaccine. The recombinant HA vaccine used herein contains whole length non-dividing HA (HAO) glycoprotein from influenza A / Beijing / 32/92 (H3N2) virus. Recombinant HA0 (rHA0) is produced by cultivating in a respidor (insect) cell, followed by exposure to baculovirus containing a cDNA insert encoding the HA gene. The expressed protein is purified to 95% or less as determined by a quantitative scanning density meter of electrophoretic bulk antigen on sodium dodecyl sulfate-polyacrylamide gel under non-denaturing conditions. The identity of the peptide is confirmed by amino acid analysis, N-terminal sequencing and Western blot analysis with anti-influenza A / Beijing / 32/92 serum. The rHA0 vaccine containing a specific amount of the synthetic HA antigen is dissolved in a phosphate-buffered physiological saline solution or absorbed in an aluminum phosphate (alum) adjuvant in the form of a gel suspension. The approved tertiary subvirion vaccines used in this study were influenza A / Texas / 36/91 (N1N1), A / Beijing / 32/92 (H3N2) and B / Panama, 45, 15 [mu] g / dose of HA from FLUZONE ^TM weakened flu vaccine, Connaught Laboratories, Swiftwater, Pa.

Clinical studies. The same study protocol was approved by the Institute Review Boards of St. Louis and University of Rochester. Enroll in two associations of healthy adults between the ages of 18 and 45. (1) 15 μg rHAO, (2) ug rHAO plus alum, (3) 90 μg rHAO, (4) approved tertiary inactivated influenza vaccine or (5) saline placebo) in a double-blind fashion The subject is randomly assigned to receive one. The vaccine is administered by intramuscular injection in a volume of 0.5 ml. To calculate an initial assessment of the stability of the three vaccine preparations containing rHAO, the first 25 inoculated subjects were randomized independently for the remaining subjects (e.g., 5 for each study purpose) and the remaining vaccination was continued Monitor 48 hours prior to inoculation by phone contact. Have all subjects complete a diary report card for the adverse reaction, including local and systemic syndrome for the first 6 days after inoculation. The syndrome should be rated by itself as weak, moderate, or severe. If the temperature in the oral cavity is low and the fever is present, the participant should report it. If there is a local ridge or erythema at the injection site, the area is graded according to whether it is less than or equal to one quarter of its diameter. All inoculations are performed during the last week of November and the first week of December. Serum samples were obtained from each subject at the time of inoculation, 3 weeks after inoculation, and again at the end of March 1994 or April 1994, at least two to three weeks after the influenza virus was no longer circulating in the local system. Applicants from each association are instructed to contact the training center if they experience an influenza illness during the winter influenza epidemic. Influenza diseases have a scouring of the nose and pharynx obtained for viral culture and dynamics in subjects reporting myalgia or chill fever and / or systemic syndrome. The encrypted identification number is written in the clinical sample and processed by the blind method.

Serology. For each type of serum test assay, all samples from both associations are tested in one batch by a single laboratory. The unspecified inhibitor was removed by receptor-destroying enzyme and the cold agglutinin was removed by hemoadhesive adsorption at 4 ° C, followed by standard microtitration analysis to determine the presence or absence of serum (A), anti-coagulation (HAI) antibody versus A / Beijing / 32/93 (H3N2) . Reverse is defined as the highest serum dilution that completely inhibits hemagglutination by the virus 4 antigen unit using 1: 4 as the starting dilution. Serum HA-specific immunoglobulin G (IgG) antibodies are measured by enzyme-linked immunoadsorbent assay (ELISA) using purified rHAO from influenza A / Beijing / 32/92 (H3N2) as a coating antigen. The order of reconstitution from outside the solid phase consisted of (1) the total rHA0 antigen, (2) serum samples, (3) alkaline phosphatase-adhesive goat anti-human IgG, and (4) p- nitrophenyl phosphate disodium substrate . ELISA reverse transcripts are expressed as the highest dilutions of the optical density of the antigen-containing wells at least twice the optical density of the corresponding control wells without antigen. Neutralizing antibodies is described by Treanor, JJ, and Betts, RF, J. Infect. Dis, 168: 455-459 (1993). Briefly, serial dilutions of heat-inactivated serum are mixed at about TCID50 of influenza A / Beijing / 32/92 (H3N2) virus and incubated for 1 hour at 37 ° C. The virus-serum mixture is then absorbed into a fusogenic single layer of Madin-Darby canine kidney (MDCK) cells in 96-well plates at room temperature for 1 hour. To remove residual inoculum, re-fed serum-free Dulbecco's MEM, wash with 2 μg / ml trypsin and incubate at 33 ° C for 72 h in 5% CO ₂ . The cells were then fixed with methanol, viral replication was performed using a panel of mouse monoclonal antibodies specific for the matrix and nuclear protein of influenza A virus (Ceters for Disease Control, Atlanta, Ga.), And then the alkaline phosphatase- It is estimated by IgG. The end-point reversal of the serum is defined as the highest dilution that results in a reduction of more than 50% in the signal compared to the non-neutralized control well.

virology. Virus cultivation of corundum swatches is performed by each association by standard techniques. Samples are inoculated into MDCK or Rithus monkey kidney cells and cultured for 14 days at 33 ° C. Hemolysis of the cell monolayer is tested with 0.4% guinea pig red blood cells. Influenza viruses are identified in heme-positive cultures using H3-specific antiserum (Centers for Disease Control).

Statistical analysis. The reciprocal HAI, ELISA IgG and neutralizing antibody locus are algebraically converted in the statistical analysis. An important response to inoculation is defined as a 4-fold increase in antibody titer between serum samples before inoculation and 3 weeks after inoculation. Experimental evidence of influenza A (H3N2) viral infection was obtained by isolating the virus from the nasopharyngeal secretions and / or isolating the sera HAI antibody titer between the specimen after 3 weeks of inoculation collected in December and the epidemic climate- It is defined as an increase of more than twice. The differences between the vaccine groups were tested using Fisher's extract test to compare the proportion of subjects with adverse reactions, significant antibody responses or laboratory-proven influenza diseases or infections, and to compare the mean reciprocal log ₂ antibody titers after inoculation Analyze using analysis of variance (ANOVA). Apply the modified Bonferroni inequality and the Tukey-Kramer test suitable for describing the multiplexable comparisons.

result

Reaction origin. The rHA0 vaccine used in this study is safe and highly resistant. The frequency of the adverse reaction seems to be unaffected by changing the amount of rHA0 antigen from 15 μg to 90 μg, but may be slightly increased by the addition of the alum. The localized erythema, pain and eruption at the injection site are more frequently reported more frequently by the recipient of an approved subvirion vaccine than the recipient of 15 [mu] g to 90 [mu] g rHA0 in saline, respectively. After immunization with an approved vaccine, all syndromes are graded as being virtually mild, with the exception of one person who has experienced severe pain, emesis, and progression in the arms, and the duration is usually 1 to 2 days. If there is local erythema and / or hardening, it is less than one quarter of the size.

Immunogenicity. The baseline level of serum HAI antibody versus influenza A / Beijing / 32/92 (H3N2) virus was 1: 8 or less in 64 (50%) of the 127 registered subjects. Most of the subjects in each of the four vaccine groups had HA-specific serum responses measured by HAI and ELISA (Table 4). Serum HAI antibody titers were greater than 1: 32 in all vaccine recipients except two patients receiving 15 μg rHAO and one receiving an approved vaccine. Inoculation is also associated with the generation of most of the volunteers' neutralizing antibodies. The mean is elevated in antibody titer and the serum conversion rate tends to be slightly lower after immunization with 15 μg rHA0 than inoculated with an approved vaccine, but this difference is not statistically significant. Antibody response versus rHA0 is not improved by the addition of alum. Subjects immunized with 90 [mu] g rHA0 achieve an average HAI and ELISA IgG antibody titers 2 to 5 times higher than in any of the remaining three vaccine groups (differences in serum HAI titers are statistically significant).

Protective efficacy. During the supervision period, a total of 28 influenza diseases were reported by 26 subjects. Four of these (influenza A (H3N2) viruses isolated from nasopharyngeal cultures have viruses (three received placebo and one immunized with 15 μg rHAO). Significant increases in HAI antibody titer versus influenza A / Beijing / 32/92 (H3N2) between pre- and post-epidemic serum specimens were found not only in some other individuals reporting the disease but also in three of four culture- do. Subsequently, a single rHA0 recipient developing a laboratory-proven influenza illness has a positive culture 31 days after immunization and seroconverts to a 1: 32 HAI titer before inoculation or a post-inoculation (prevalence) titer of 1: 32. One additional volunteer immunized with two additional placebo recipients and an approved vaccine had serum evidence of infection with influenza A (H3N2) during the epidemic in the absence of clinical disease. Most placebo recipients have laboratory-proven influenza A (H3N2) disease (p.05) or infection (p.005) when compared to all inoculated subjects (or all subjects receiving rHA0) as a group .

The above findings indicate that influenza vaccines containing the purified rHA0 antigen prepared as described in the present patent application are highly resistant and capable of eliciting a protective immune response in a human subject. The rHA0 evaluated in this study is much less reactive than an approved tertiary subvirion vaccine containing half of the total HA antigen (eg, 45 μg), rather than a response to saline placebo above 90 μg Local reverse reaction occurs.

The neutralization, HA-specific antibody response versus 15 [mu] g rHA0 production is comparable to those derived by subvirion vaccines and is significantly improved by increasing the amount of rHAO to 90 [mu] g.

In the circulation of influenza A (H3N2) virus, the overall rate of infection and disease that can occur from natural exposure to the influenza strain is much lower among subjects vaccinated than placebo recipients. The data suggest that the protective immunity provided by rHAO is comparable or predominant to that induced by the current readily available vaccine when administered in particularly high dosages.

[Table 4]

A vaccine containing recombinant hemagglutinin (rHAO) purified from influenza A / Beijing 32/92 (H2N2), an approved tertiary subvirion containing 15 μg HA from A / Beijing 32/92 (H2N2) Or saline placebo, the serum antibody response of young adult subjects.

Example 11: Preparation of improved HAO cloning vector

An improved cloning vector for mature HA expression is designed in which the HA coding gene is located immediately downstream of the sequence encoding the chitinase signal peptide.

Generation of linear pMGS27 with Japanese chain tail

In the pMGS12 plasmid, HA is cloned into the Sma1 or Kpn1 site immediately downstream from the kitanase signal peptide. Nucleic acid and amino acid sequences are shown in SEQ ID NOS: 22 and 23, respectively.

The 5'-kittinase signal peptide Sma1Kpn1

This region is altered by an oligo-directed mutagenesis to produce pMGS27 (the changed base is underlined) (SEQ ID NO: 24):

Polymide pMGS27 is linearized with Pst1 cleavage (residues 6 to 35 of SEQ ID NO: 24 shown):

Linear pMGS27 is then treated with T4 DNA polymerase + dATP to produce a Japanese chain tail as shown (complement of residues 23 to 36 and residues 6 to 18 of SEQ ID NO: 24).

Cloning of the target HA gene into pMGS27

Step 1. Synthesis of PCR primers.

Forward raising (SEQ ID NO: 25):

(5 ' terminal 20 bases of mature HA)

Reverse oligonucleotide (complement of SEQ ID NO: 26):

(3 ' terminal 20 bases of mature HA)

PCR of HA gene

PCR of the target HA gene was performed using two oligos (SEQ ID NOS: 25 and 26): < RTI ID = 0.0 >

annealing of the target HA gene into pMGS27 and Coli transformation

T4 DNA polymerase-treated PCR fragments of linear pMGS27 and HA genes are mixed. The two molecules are mutually annealed, To form a cyclic plasmid to be directly used for coli transformation. The diagrams include SEQ ID NOs: 25 and 26, residues 23 to 36 and 6 to 18 of SEQ ID NO: 24.

As can be seen, there is no extra amino acid between the signal peptide and mature HA.

Example 12: Preparation and efficiency of trivalent A and B 1995-1996 influenza virus vaccines

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). The HA gene is cloned from the recommended strains of influenza A and B viruses from the Center for Disease Control / Food and Drug Administration, and the homology of each cloned gene is determined by DNA sequencing. A baculovirus expression vector containing the cloned HA gene from influenza virus strain A / Texas / 36/91 (H1N1), A / Johannesburg / 33/94 (H3N2), B / Harbin / 7/94 Recombinant HA antigen is generated in cultured insect cells. The recombinant HA protein is a full length unchicken hemagglutinin (rHAO) with a molecular weight of approximately 69,000. rHA0 is produced in Spodoptera frugiperda (refeedtran) cell line maintained in serum-free culture medium. The trivalent vaccine consists of two strains of influenza A mixed at the same rate and purified (more than 95%, more likely more than 99% purity) rHAO from one B strain. The vaccine is supplied for clinical use as purified A and B type rHA0 proteins in a phosphate buffered physiological saline solution without the addition of preservatives.

Animal studies using mono-, di-, and tri-valent rHA0 vaccines demonstrate that they are not significantly toxic. There are no detectable toxicants or contingencies in the vaccine. General stability and immunogenicity studies of A / Beijing / 32/92 and A / Texas / 36/91 rHA0 are performed in rats and guinea pigs. No adverse reactions were noted. In rats, single immunization with 15 μg of rHA0 antigen without adjuvant induces high levels of anti-HA IgG antibody, hemagglutinin inhibition (HAI) antibody and neutralizing antibody after 2-3 weeks.

In one study, groups of 10 rats were adapted to a medium containing purified rHAO A / Beijing / 32/92 (H3N2) or 10% TaSo serum prepared in cells adapted to medium containing 10% fetal calf serum Lt; RTI ID = 0.0 > rHA0 < / RTI > (rHA-SF) prepared in insect cells adapted to serum-free medium. Two weeks and three weeks after the injury, blood samples are prepared from rats and serum samples are prepared. Each serum is assayed for anti-HA IgG and HAI antibodies. Both rHA0 and rHA0-SF antigens yielded similar potencies of anti-HA and HAI antibodies. Both rHA0 and rHA0-SF antigens yielded similar potencies of anti-HA and HAI antibodies. After two weeks in a single area, most rats had significant titers of HAI antibody, and up to 3 weeks 8/10 of rats in each group had a HAI titer of 32 or higher. These and other biochemical and immunological studies demonstrate that rHAO produced in serum-free insect cell culture is indistinguishable from rHA0 produced under serum-containing fermentation conditions. 3 rHA0 influenza vaccine and a licensed purified virus surface antigen vaccine Fluvirin ^R (an attenuated influenza virus vaccine produced by incubation in an egg). Each vaccine contained 15 μg of rHAO or viral HA per 0.5 ml from A / Texas / 36/91 (H1 / N1), A / Shandong / 9/93 (H3N2), and B / Panama / 45/90 influenza strains Lt; / RTI > Both recombinant rHA0 and Fluvirin ^R vaccine are phosphate buffered saline. After 0, 2 and 3 weeks of infection, a group of 10 rats is collected and serum is prepared. Serum samples are measured for anti-HA IgG antibody-inhibiting antibodies against egg-growing influenza virus and against neutralizing antibodies against egg-growing influenza virus from each virus strain.

As evidenced in Figures 4a, 4b and 4c. Both recombinant rHAO and licensed influenza vaccines induce high levels of serum IgG antibodies to hemagglutinin from each of the three influenza strains in the vaccine. IgG antibody titers that are 4 to 10 times more potent than the rHA0 vaccine-licensed vaccine are induced. As shown in Table 5, antibodies against hemagglutinin of chicken erythrocytes were also generated with both influenza A and B strain viruses. The HAI registrar is equal or higher than the licensed vaccine for each of the three influenza virus strains in the rHA0 vaccine rats. The vaccine also induces high levels of neutralizing antibodies against specific influenza A and B virus strains as measured in standard microtiter virus neutralization assays. The thin geometric antibodies station licensure purified virus surface antigen vaccine, is about two times higher in mice immunized with rHA0 than was the Fluvirin ^R. These results indicate that the triad formulation of the 1994-1995 type A B influenza strain rHA0 vaccine is comparable or superior to the subunit influenza vaccine approved for functional HAI and neutralizing serum antibody induction against all three strains of influenza in the vaccine Prove it.

[Table 5]

3 is a comparison of vaccines and rHA0 Fluvirin ^R

Modifications and variations of the methods and compositions described herein for use in the manufacture and use of recombinant influenza vaccines will be apparent to those skilled in the art. Such variations and modifications are also within the scope of the appended claims.

Sequence List

(1) General information

(I) Applicant: Microgenesis Inc.

(Ii) Title of the invention: Method for producing influenza hemagglutinin multivalent vaccine

(Iii) SEQ ID NO: 32

(Iv) Communication address:

(A) Recipient: Patriarch. Fabst

(B) Distance: West Peachtree Street 1201 West Atlantic Center 2800

(C) City: Atlanta

(D) Note: Georgia

(E) Country: United States

(F) Postal Code: 30309-3450

(V) Computer readable form:

(A) Medium type: floppy disk

(B) Computer: IBM PC compatible model

(C) Operating system: PC-DOS / MS-DOS

(D) Software: PatentIn Release # 1.0, Version # 1.25

(Vi) Application data:

(A) Application No. PCT / US95 / 06750

(B) Filing date: May 5, 1995

(C) Classification:

(Ⅸ) Communication information:

(A) Tel: (404) -873-8794

(B) Fax: (404) -873-8795

(2) Information on SEQ ID NO: 1:

(I) Sequence characteristics:

(A) Length: 12 base pairs

(B) Type: Nucleic acid

(C) Single-sided type:

(D) Phase: Linear

(Ii) Molecular type: DNA (genome)

(Iii) Hypothesis: None

(Iv) Antisense: None

(Vi) Initial source:

(A) Organism: influenza virus

(X) Publication information

(A) Author: Davis, et al.

(B) Name: Construction and Characterization of a Bacterial Clone Containing the Hemagglutinin Gene of the WSN Strain (HON1) of Influenza Virus

(C) Journal: Gene

(D) Volume: 10

(F) Page: 205-218

(G) Year of publication: 1980

(xi) Sequence description: SEQ ID NO: 1:

(2) Information on SEQ ID NO: 2:

(I) Sequence characteristics:

(A) Length: 39 base pairs

(B) Type: Nucleic acid

(C) Single-sided type:

(D) Phase: Linear

(Ii) Molecular type: DNA (genome)

(xi) Sequence description: SEQ ID NO: 2:

(2) Information on SEQ ID NO: 3:

(I) Sequence characteristics:

(A) Length: 35 base pairs

(B) Type: Nucleic acid

(C) Single-sided type:

(D) Phase: Linear

(Ii) Molecular type: DNA (genome)

(xi) Sequence description: SEQ ID NO: 3:

(2) Information on SEQ ID NO: 4:

(I) Sequence characteristics:

(A) Length: 39 base pairs

(B) Type: Nucleic acid

(C) Single-sided type:

(D) Phase: Linear

(Ii) Molecular type: DNA (genome)

(xi) Sequence description: SEQ ID NO: 4:

(2) Information on SEQ ID NO: 5:

(I) Sequence characteristics:

(A) Length: 35 base pairs

(B) Type: Nucleic acid

(C) Single-sided type:

(D) Phase: Linear

(Ii) Molecular type: DNA (genome)

(xi) Sequence description: SEQ ID NO: 5:

(2) Information on SEQ ID NO: 6:

(I) Sequence characteristics:

(A) Length: 1793 base pairs

(B) Type: Nucleic acid

(C) Single-sided type:

(D) Phase: Linear

(Ii) Molecular type: DNA (genome)

(Iii) Hypothesis: None

(Iv) Antisense: None

(Vi) Initial source:

(A) Organism: influenza virus

(C) Separate individual: A / Beijing / 32/92 rHA

(ix) Features

(A) Name / Key: Polyhedrin mRNA leader (part)

(B) Location: 1 to 18

(ix) Features

(A) Name / Key: AcNPV 61K protein signal sequence Encoding region

(B) Location: 19 to 72

(ix) Features

(A) Name / Key: SmaI restriction site

(B) Location: 76 to 81

(ix) Features

(A) Name / Key: mature rHA encryption area

(B) Location: 73 to 1728

(ix) Features

(A) Name / Key: KpnI restriction site

(B) Location: 1771 to 1777

(ix) Features

(A) Name / Key: BglII restriction site

(B) Location: 1776 to 1782

(ix) Features

(A) Name / Key: Universal decode termination signal

(B) Location: 1783 to 1793

(xi) Sequence description: SEQ ID NO: 6:

(2) Information on SEQ ID NO: 7:

(I) Sequence characteristics:

(A) Length: 570 amino acids

(B) Type: Amino acid

(C) Single-sided type:

(D) Phase: Linear

(Ii) Molecular form: Peptide

(Iii) Hypothesis: None

(Iv) Antisense: None

(V) Fragment type: N-maltose

(Vi) Initial source:

(A) Organism: influenza virus

(C) Separate individual: A / Beijing / 32/92 rHA

(ix) Features

(A) Name / Key: AcNPV 61K protein signal sequence

(B) Location: 1 to 18

(ix) Features

(A) Name / Key: Maturity rHA

(B) Location: 19 to 552

(xi) Sequence description: SEQ ID NO: 7:

(2) Information on SEQ ID NO: 8:

(I) Sequence characteristics:

(A) Length: 1766 base pairs

(B) Type: Nucleic acid

(C) Single-sided type:

(D) Phase: Linear

(Ii) Molecular type: DNA (genome)

(Iii) Hypothesis: None

(Iv) Antisense: None

(Vi) Initial source:

(A) Organism: influenza virus

(C) Separate individual: A / Texas / 36/91 rHA

(ix) Features

(A) Name / Key: Polyhedrin mRNA leader (part)

(B) Location: 1 to 18

(ix) Features

(A) Name / Key: AcNPV 61K protein signal peptide coding region

(B) Location: 19 to 72

(ix) Features

(A) Name / Key: SmaI restriction site

(B) Location: 76 to 81

(ix) Features

(A) Name / Key: KpnI restriction site

(B) Location: 82 to 87

(ix) Features

(A) Name / Key: SmaI restriction site

(B) Location: 88 to 93

(ix) Features

(B) Location: 73 to 1734

(ix) Features

(A) Name / Key: KpnI restriction site

(B) Location: 1744 to 1749

(ix) Features

(A) Name / Key: BglII restriction site

(B) Location: 1750 to 1755

(ix) Features:

(A) Name / Key: Universal decode termination signal

(B) Location: 1756 to 1766

(xi) Sequence description: SEQ ID NO: 8:

(2) Information on SEQ ID NO: 9:

(I) Sequence characteristics:

(A) Length: 572 amino acids

(B) Type: Amino acid

(C) Single-sided type:

(D) Phase: Linear

(Ii) Molecular form: Peptide

(Iii) Hypothesis: None

(Iv) Antisense: None

(V) Fragment type: N-maltose

(Vi) Initial source:

(A) Organism: influenza virus

(C) Separate individual: A / Texas / 36/91 rHA

(ix) Features

(A) Name / Key: AcNPV 61K protein signal sequence

(B) Location: 1 to 18

(ix) Features

(A) Identification / Key: Maturity rHA

(B) Position: 19 to 554

(xi) Sequence description: SEQ ID NO: 9

(2) Information on SEQ ID NO: 10:

(I) Sequence characteristics:

(A) Length: 1799 base pairs

(B) Type: Nucleic acid

(C) Single-sided type:

(D) Phase: Linear

(Ii) Molecular type: RNA (genome)

(Iii) Hypothesis: None

(Iv) Antisense: None

(Vi) Initial source:

(A) Organism: influenza virus

(C) Individual separator: B / Panama / 45/90 rHA

(ix) Features

(A) Name / Key: Polyhedrin mRNA leader (part)

(B) Location: 1 to 18

(ix) Features

(A) Name / Key: HA signal peptide sequence Encryption domain

(B) Location: 19 to 69

(ix) Features

(A) Name / Key: SmaI restriction site

(B) Position: 22 to 27

(ix) Features

(A) Name / Key: mature rHA encryption area

(B) Location: 70 to 1773

(Ⅸ) Features

(A) Name / Key: KpnI restriction site

(B) Location: 1777 to 1782

(Ⅸ) Features

(A) Name / Key: BglII restriction site

(B) Location: 1783 to 1788

(Ⅸ) Features

(A) Name / Key: Universal decode termination signal

(B) Location: 1789 to 1799

(xi) Sequence description: SEQ ID NO: 10:

(2) Information on SEQ ID NO: 11:

(I) Sequence characteristics:

(A) Length: 585 amino acids

(B) Type: Amino acid

(C) Single-sided type:

(D) Phase: Linear

(Ii) Molecular form: Peptide

(Iii) Hypothesis: None

(Iv) Antisense: None

(V) Fragment type: N-maltose

(Vi) Initial source:

(A) Organism: influenza virus

(C) Individual separator: B / Panama / 45/90 rHA

(Ⅸ) Features

(A) Name / Key: HA signal peptide

(B) Location: 1 to 17

(xi) Features

(A) Name / Key: Maturity rHA

(B) Location: 18 to 568

(xi) Sequence description: SEQ ID NO: 11

(2) Information on SEQ ID NO: 12:

(I) Sequence characteristics:

(A) Length: 1811 base pairs

(B) Type: Nucleic acid

(C) Single-sided type:

(D) Phase: Linear

(Ii) Molecular type: DNA (genome)

(Iii) Hypothesis: None

(Iv) Antisense: None

(Vi) Initial source:

(A) Organism: influenza virus

(C) Individual separator: B / Netherlands / 13/94 rHA

(ix) Features

(A) Name / Key: Polyhedrin mRNA leader (part)

(B) Location: 1 to 18

(Ⅸ) Features

(A) Name / Key: AcNPV 61K protein signal sequence Encoding region

(B) Location: 19 to 72

(ix) Features

(A) Name / Key: SmaI restriction site

(B) Location: 76 to 81

(ix) Features

(A) Name / Key: mature rHA encryption area

(B) Position: 73 to 1785

(Ⅸ) Features

(A) Name / Key: KpnI restriction site

(B) Location: 1789 to 1794

(Ⅸ) Features

(A) Name / Key: BglII restriction site

(B) Location: 1795 to 1800

(xi) Features

(A) Name / Key: Universal decode termination signal

(B) Location: 1801 to 1811

(xi) Sequence description: SEQ ID NO: 12:

(2) Information on SEQ ID NO: 13:

(I) Sequence characteristics:

(A) Length: 589 amino acids

(B) Type: Amino acid

(C) Single-sided type:

(D) Phase: Linear

(Ii) Separation type: Peptide

(Iii) Hypothesis: None

(Iv) Antisense: None

(V) Fragment type: N-maltose

(Vi) Initial source:

(A) Organism: influenza virus

(C) Individual separator: B / Netherlands / 13/94 rHA

(Ⅸ) Features

(A) Name / Key: AcNPV 61K protein signal sequence

(B) Location: 1 to 18

(Ⅸ) Features

(A) Name / Key: Maturity rHA

(B) Location: 19 to 571

(xi) Sequence description: SEQ ID NO: 13

(2) Information on SEQ ID NO: 14:

(I) Sequence characteristics:

(A) Length: 1757 base pairs

(B) Type: Nucleic acid

(C) Single-sided type:

(D) Phase: Linear

(Ii) Molecular type: DNA (genome)

(Iii) Hypothesis: None

(Iv) Antisense: None

(Vi) Initial source:

(A) Organism: influenza virus

(C) Separate individual components: A / Shandong / 9/93 rHA

(Ⅸ) Features

(A) Name / Key: Polyhedrin mRNA leader (part)

(B) Location: 1 to 18

(Ⅸ) Features

(A) Name / Key: AcNPV 61K protein signal sequence Encoding region

(B) Location: 19 to 72

(Ⅸ) Features

(A) Name / Key: SmaI restriction site

(B) Location: 76 to 81

(Ⅸ) Features

(A) Name / Key: mature rHA encryption area

(B) Location: 73 to 1728

(Ⅸ) Features

(A) Name / Key: KpnI restriction site

(B) Location: 1735-1740

(Ⅸ) Features

(A) Name / Key: BglII restriction site

(B) Location: 1741 to 1746

(Ⅸ) Features

(A) Name / Key: Universal decode termination signal

(B) Location: 1747 to 1757

(xi) Sequence description: SEQ ID NO: 14:

(2) Information on SEQ ID NO: 15:

(I) Sequence characteristics:

(A) Length: 571 Amino acids

(B) Type: Amino acid

(C) Single-sided type:

(D) Phase: Linear

(Ii) Molecular form: Peptide

(Iii) Hypothesis: None

(Iv) Antisense: None

(V) Fragment type: N-maltose

(Vi) Initial source:

(A) Organism: influenza virus

(C) Separate individual components: A / Shandong / 9/93 rHA

(Ⅸ) Features

(A) Name / Key: AcNPV 61K protein signal sequence

(B) Location: 1 to 18

(A) Name / Key: Maturity rHA

(B) Location: 19 to 553

(xi) Sequence description: SEQ ID NO: 15:

(2) Information on SEQ ID NO: 16:

(I) Sequence characteristics:

(A) Length: 1814 base pairs

(B) Type: Nucleic acid

(C) Single-sided type:

(D) Phase: Linear

(Ii) Molecular type: DNA (genome)

(Iii) Hypothesis: None

(Iv) Antisense: None

(Vi) Initial source:

(A) Organism: influenza virus

(C) Individual separator: B / Shanghai / 4/94 rHA

(Ⅸ) Features

(A) Name / Key: Polyhedrin mRNA leader (part)

(B) Location: 1 to 18

(Ⅸ) Features

(A) Name / Key: AcNPV 61K protein signal sequence Encoding region

(B) Location: 19 to 72

(Ⅸ) Features

(A) Name / Key: SmaI restriction site

(B) Location: 76 to 81

(Ⅸ) Features

(A) Name / Key: KpnI restriction site

(B) Location: 82 to 87

(A) Name / Key: mature rHA encryption area

(B) Position: 73 to 1794

(Ⅸ) Features

(A) Name / Key: Universal decode termination signal

(B) Location: 1804 to 1814

(xi) Sequence description: SEQ ID NO: 16:

(2) Information on SEQ ID NO: 17:

(I) Sequence characteristics:

(A) Length: 592 Amino acids

(B) Type: Amino acid

(C) Single-sided type:

(D) Phase: Linear

(Ii) Molecular form: Peptide

(Iii) Hypothesis: None

(Iv) Antisense: None

(V) Fragment type: N-maltose

(Vi) Initial source:

(A) Organism: influenza virus

(C) Individual separator: B / Shanghai / 4/94 rHA

(Ⅸ) Features

(A) Name / Key: AcNPV 61K protein signal peptide

(B) Location: 1 to 18

(A) Name / Key: Maturity rHA

(B) Location: 19 to 574

(xi) Sequence description: SEQ ID NO: 17:

(2) Information on SEQ ID NO: 18

(I) Sequence characteristics:

(A) Length: 1802 base pairs

(B) Type: Nucleic acid

(C) Single-sided type:

(D) Phase: Linear

(Ii) Molecular type: DNA (genome)

(Iii) Hypothesis: None

(Iv) Antisense: None

(Vi) Initial source

(A) Organism: influenza virus

(C) Individual separator: B / Harbin / 7/94 rHA

(Ⅸ) Features

(A) Name / Key: Polyhedrin mRNA leader (part)

(B) Location: 1 to 18

(Ⅸ) Features

(A) Name / Key: HA signal peptide sequence Encryption domain

(B) Location: 19 to 69

(Ⅸ) Features

(A) Name / Key: SmaI restriction site

(B) Position: 22 to 27

(Ⅸ) Features

(A) Name / Key: mature rHA encryption area

(B) Location: 70 to 1776

(Ⅸ) Features

(A) Name / Key: KpnI restriction site

(B) Location: 1780 to 1785

(Ⅸ) Features

(A) Name / Key: BglII restriction site

(B) Location: 1786 to 1791

(Ⅸ) Features

(A) Name / Key: Universal decode termination signal

(B) Location: 1792 to 1802

(xi) Sequence description: SEQ ID NO: 18:

(2) Information on SEQ ID NO: 19

(I) Sequence characteristics:

(A) Length: 586 amino acids

(B) Type: Amino acid

(C) Single-sided type:

(D) Phase: Linear

(Ii) Molecular form: Peptide

(Iii) Hypothesis: None

(Iv) Antisense: None

(V) Fragment type: N-maltose

(Vi) Initial source:

(A) Organism: influenza virus

(C) Individual separator: B / Harbin / 7/94 rHA

(Ⅸ) Features

(A) Name / Key: HA signal peptide

(B) Location: 1 to 17

(A) Name / Key: Maturity rHA

(B) Location: 18 to 569

(xi) Sequence description: SEQ ID NO: 19:

(2) Information on SEQ ID NO: 20:

(I) Sequence characteristics:

(A) Length: 1757 base pairs

(B) Type: Nucleic acid

(C) Single-sided type:

(D) Phase: Linear

(Ii) Molecular type: DNA (genome)

(Iii) Hypothesis: None

(Iv) Antisense: None

(Vi) Initial source

(A) Organism: influenza virus

(C) Separate individual: A / Johannesburg / 33/94 rHA

(Ⅸ) Features

(A) Name / Key: Polyhedrin mRNA leader (part)

(B) Location: 1 to 18

(Ⅸ) Features

(A) Name / Key: AcNPV 61K protein signal peptide coding region

(B) Location: 19 to 72

(Ⅸ) Features

(A) Name / Key: SmaI restriction site

(B) Location: 76 to 81

(Ⅸ) Features

(A) Name / Key: mature rHA, encryption domain

(B) Position: 73 to 1731

(Ⅸ) Features

(A) Name / Key: KpnI restriction site

(B) Location: 1735-1740

(Ⅸ) Features

(A) Name / Key: BglII restriction site

(B) Location: 1741 to 1747

(Ⅸ) Features

(A) Name / Key: Universal decode termination signal

(B) Location: 1747 to 1757

(xi) Sequence description: SEQ ID NO: 20:

(2) Information on SEQ ID NO: 21:

(I) Sequence characteristics:

(A) Length: 571 Amino acids

(B) Type: Amino acid

(C) Single-sided type:

(D) Phase: Linear

(Ii) Molecular form: Peptide

(Iii) Hypothesis: None

(Iv) Antisense: None

(V) Fragment type: N-maltose

(Vi) Initial source:

(A) Organism: influenza virus

(C) Separate individual: A / Johannesburg / 33/94 rHA

(Ⅸ) Features

(A) Name / Key: AcNPV 61K protein signal sequence

(B) Location: 1 to 18

(A) Name / Key: Maturity rHA

(B) Location: 19 to 569

(xi) Sequence description: SEQ ID NO: 21:

(2) Information on SEQ ID NO: 22:

(I) Sequence characteristics:

(A) Length: 39 bases

(B) Type: Nucleic acid

(C) Single-sided type:

(D) Phase: Linear

(xi) Sequence description: SEQ ID NO: 22:

(2) Information on SEQ ID NO: 23:

(I) Sequence characteristics:

(A) Length: 13 amino acids

(B) Type: Amino acid

(C) Single-sided type:

(D) Phase: Linear

(Ii) Molecular form: Peptide

(xi) Sequence description: SEQ ID NO: 23:

(2) Information on SEQ ID NO: 24:

(I) Sequence characteristics:

(A) Length: 43 base pairs

(B) Type: Nucleic acid

(C) Single-sided type:

(D) Phase: Linear

(xi) Sequence description: SEQ ID NO: 24:

(2) Information on SEQ ID NO: 25:

(I) Sequence characteristics:

(A) Length: 18 base pairs

(B) Type: Nucleic acid

(C) Single-sided type:

(D) Phase: Linear

(xi) Sequence description: SEQ ID NO: 25:

(2) Information on SEQ ID NO: 26:

(I) Sequence characteristics:

(A) Length: 18 base pairs

(B) Type: Nucleic acid

(C type:

(D) Phase: Linear

(xi) Sequence description: SEQ ID NO: 26:

(2) Information on SEQ ID NO: 27:

(I) Sequence characteristics:

(A) Length: 39 bases

(B) Type: Nucleic acid

(C) Single-sided type:

(D) Phase: Linear

(xi) Sequence description: SEQ ID NO: 27:

(2) Information on SEQ ID NO: 28:

(I) Sequence characteristics:

(A) Length: 38 base

(B) Type: Nucleic acid

(C) Single-sided type:

(D) Phase: Linear

(xi) Sequence description: SEQ ID NO: 28:

(2) Information on SEQ ID NO: 29:

(I) Sequence characteristics:

(A) Length: 44 bases

(B) Type: Nucleic acid

(C) Single-sided type:

(D) Phase: Linear

(xi) Sequence description: SEQ ID NO: 29:

(2) Information on SEQ ID NO: 30:

(I) Sequence characteristics:

(A) Length: 47 bases

(B) Type: Nucleic acid

(C) Single-sided type:

(D) Phase: Linear

(xi) Sequence description: SEQ ID NO: 30:

(2) Information on SEQ ID NO: 31:

(I) Sequence characteristics:

(A) Length: 36 bases

(B) Type: Nucleic acid

(C) Single-sided type:

(D) Phase: Linear

(xi) Sequence description: SEQ ID NO: 31:

(2) Information on SEQ ID NO: 32:

(I) Sequence characteristics:

(A) Length: 50 bases

(B) Type: Nucleic acid

(C) Single-sided type:

(D) Phase: Linear

(xi) Sequence description: SEQ ID NO: 32:

Claims (26)

Recombinant influenza HAO hemagglutinin protein expressed in a baculovirus expression system in cultured insect cells. The protein of claim 1, further comprising a baculovirus signal peptide directly coupled to the HAO protein without an intervening amino acid. The protein of claim 1, further comprising a pharmaceutically acceptable carrier for administration as a vaccine. The protein of claim 1, further comprising a pharmaceutically acceptable carrier and adjuvant for administration as a vaccine. 4. The protein of claim 3 which is a polymeric filament system with a pharmaceutically acceptable carrier. The protein according to claim 1, wherein the influenza is selected from the group consisting of influenza A strain and influenza B strain. 7. The protein according to claim 6, wherein the influenza virus infects humans. The protein according to claim 1, further comprising a second protein fused to hemagglutinin. 9. The selected protein of claim 8, selected from the group consisting of hepatitis B virus protein, HIV protein, carinoembryonic antigen, and neuraminidase. The following 5 '→ 3' sequence: polyhedrin promoter from baculovirus, ATG detoxification initiation codon, signal peptide, mature hemagglutinin coding sequence from influenza strain, detoxification codon, and polyhedrin RNA polyadenylation signal A vector for producing a recombinant influenza HAO hemagglutinin protein. 11. A baculovirus protein vector according to claim 10, wherein the signal peptide has a molecular weight of about 61K and an amino acid sequence as set forth in the first 18 amino acids of SEQ ID NO: 11. The vector of claim 10, wherein the signal peptide is an influenza hemagglutinin protein promoter. 11. The vector of claim 10, wherein the signal peptide and the hemagglutinin coding sequence do not encode any interchangeable amino acids. 11. A vector according to claim 10, further comprising a second protein coding sequence which is expressed as a fusion protein with hemaglutinin. 11. The vector according to claim 10, which is transfected in cultured insect cells. The following 5 '→ 3' sequence: a hemagglutinin coding sequence from an influenza strain selected from the group consisting of a polyhedrin promoter from baculovirus, an ATG detoxification initiation codon, a signal peptide, an influenza A strain and an influenza B strain, Terminating codon, and a polyhedrin RNA polyadenylation signal, followed by culturing the cells in a nutrient medium. 17. The method of claim 16 further comprising isolating the HAO influenza hemagglutinin protein from the cells at least 95% purity. 18. The method of claim 17, wherein the hemagglutinin is separated from the non-membrane protein at an alkaline pH, the membrane-bound protein is washed to elute the hemagglutinin, Separating hemagglutinin from other proteins, and separating hemagglutinin from other proteins bound to the cation exchange resin by varying the salt concentration. Collecting the virus from the cell culture medium, isolating the mRNA for the viral RNA in the case of influenza A strain or the bacterium B strain; CDNA was synthesized from a strain of influenza A using a universal primer for viral RNA (5'-AGCAAAAGCAGG-3 '(SEQ ID NO: 1) or a random primer for mRNA from influenza B strain (wherein 5' and 3 'primers There is a restriction enzyme site at the end that is not found in the hemagglutinin gene); Amplifying an influenza A or B primer and influenza cDNA mixed with a hemagglutinin gene fragment to generate a double stranded DNA fragment containing a fully mature hemagglutinin coding sequence; The signal peptide of the hemagglutinin gene is identified to amplify the hemagglutinin gene without the signal peptide; Cloning a hemagglutinin gene without a signal peptide in an AcNPV polyhedrin promoter-containing vector. A hemagglutinin coding sequence from influenza strains selected from the group consisting of the following 5 '→ 3' sequences: a polyhedrin promoter from baculovirus, an ATG detoxification initiation codon, a signal peptide, an influenza A strain and an influenza B strain, A transcription termination codon, and a polyhedrin RNA polyadenylation signal. 20. The method of claim 19, wherein the hemagglutinin gene is cloned in a vector using PCR to encode a signal peptide directly coupled to hemagglutinin without the vector having any intervening amino acid. 20. The method of claim 19, further comprising the step of transfecting the vector in insect cells and screening cells for hemagglutinin expression. A method of vaccinating an animal against influenza comprising administering to the animal an effective amount of recombinant influenza HAO hemagglutinin protein expressed in a baculovirus expression system in a cultured insect cell. 23. The method of claim 22, further comprising administering the protein to a polymeric delivery system. 23. The method of claim 22, wherein the influenza is selected from the group consisting of influenza A strains and influenza B strains. 23. The method of claim 22, wherein the animal is selected from the group consisting of mammals and bird species. 23. The method of claim 22, wherein the animal is a human.