The present invention is based on the discovery that a high titer reassortant influenza virus is produced in mammalian cell culture by replacing the NS gene of the A/PuertoRico/3/24 master strain with the NS gene of the A/England/1/53 strain. The invention provides influenza viruses and vaccines generated in mammalian cells as well as methods for producing such. The invention further provides an influenza virus master strain and kits for generating reassortant influenza viruses in mammalian cell culture and methods of making and using the master strain.

SUMMARY OF THE INVENTION

The present invention discloses methods for producing influenza virus and vaccine compositions by transfecting into a host cell the PB2, PB1, PA, NP and M genes from the A/PuertoRico/8/34 influenza strain, the NS gene from the A/England/1/53 influenza strain and the HA and NA genes from an influenza virus of interest to produce a reassortant influenza virus capable of high titer growth in the host cell. The influenza NS gene encodes two different proteins, NS1 and NS2.

In an embodiment of the present invention, the NS gene has the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment of the present invention the NS1 and NS2 proteins have the sequence set forth in SEQ ID NO: 3 or 4. In a preferred embodiment the NS1 and NS2 proteins have the sequence set forth in SEQ ID NO: 5 or 6. In a preferred embodiment of the present invention NS1 of the original A/England/1/53 protein has amino acid changes at one or more of the following positions relative to SEQ ID NO: 5: 21, 58, 60, 127, 174 or 189 and the deletion of amino acids 231 through 238; and one or more amino changes to positions 16, 31, 86 or 107 of SEQ ID NO: 6 in the NS2 protein. In a most preferred embodiment the amino acid changes are Gln-21 to Arg, Thr-58 to Ile, Val-60 to Ala, Asn-127 to Ser, Val-174 to Ile, Asp-189 to Asn and the deletion of amino acids 231 through 238 in the NS1 protein (FIG. 1), and Met-16 to Ile, Met-31 to Ile, Lys-86 to Arg or Phe-107 to Leu in the NS2 protein (FIG. 2).

These methods utilize mammalian host cells, preferably, mammalian cells which have been approved for human vaccine production, more preferably, Vero cells.

As an influenza virus of interest, these methods may utilize human, avian, swine or equine influenza viruses. In a preferred embodiment of the present invention the influenza virus of interest is an influenza A virus other than A/England/1/53. The HA and NA genes from any virus may be transfected into cells with the master strain of the present invention to produce a virus strain that grows more efficiently in the host cell.

Furthermore, this invention discloses influenza virus and vaccine compositions produced by transfecting into a host cell the PB2, PB1, PA, NP and M genes from the A/PuertoRico/8/34 influenza strain, the NS gene from the A/England/1/53 influenza strain and the HA and NA genes from an influenza virus of interest to produce a reassortant influenza virus capable of high titer growth in the host cell.

The present invention also discloses a kit containing expression plasmids which contain the PB2, PB1, PA, NP and M genes from the A/PuertoRico/8/34 influenza strain and the NS gene from the A/England/1/53 influenza strain. The expression plasmids from such a kit may be transfected with expression plasmids containing the HA and NA genes from any virus of interest into cells in order to generate a virus capable of high titer growth in a cell line approved for vaccine manufacture.

The present invention also discloses methods for producing viruses and vaccines which incorporate a modified A/PuertoRico/8/34 master strain. In the modified A/PuertoRico/8/34 master strain, the NS gene of the original virus is replaced with the NS gene of the A/England/1/53 virus, thus producing a virus master strain capable of high titer growth in host cells. A host cell is infected with the virus master strain and an influenza virus strain of interest to allow the two strains to reassort, thus producing a different virus subtype that exhibits high growth in the host cell. Alternatively, expression plasmids containing the PB2, PB1, PA, NP and M genes from A/PuertoRico/8/34, the NS gene from A/England/1/53 and the HA and NA genes from an influenza virus of interest are transfected into a host cell to produce a high growth strain of virus.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an influenza virus master strain that produces high titer virus in mammalian cells. Such master strain consists of expression plasmids that contain the PB2, PB1, PA, NP and M genes from the A/PuertoRico/8/34 virus strain and the NS gene from the A/England/1/53 virus strain. The HA and NA proteins from any virus of interest may be combined with such master strain to produce a high titer virus.

The present invention also provides reassortant influenza viruses and methods for generating such viruses by transfecting into a host cell expression plasmids that contain the PB2, PB1, PA, NP and M genes from the A/PuertoRico/8/34 virus strain, the NS gene from the A/England/1/53 virus strain, and the HA and NA genes from an influenza virus of interest.

Based on the observation of Govorkova EA, et al., J. Infect. Dis. 172(1):250-253, 1995, which described a master strain that produced high titer influenza virus in mammalian cells, applicants undertook studies to identify the molecular changes responsible for the A/England/1/53 vero-adapted (A/England/1/53/v-a) virus' high-yielding phenotype in Vero cells. The goal was to produce an altered A/PuertoRico/8/34 vaccine master strain adapted for optimal efficiency of viral rescue in the reverse genetics system for growth in Vero cells. Contrary to what was reported in the Govorkova et al. manuscript, sequence analysis of the complete genome of A/England/1/53/v-a showed that this high-yielding virus contained not only the HA and NA genes from A/England/1/53, but also the NS gene from this same strain. The remaining A/England/1/53/v-a proteins (PB2, PB1, PA, MP and M) had more than 99% nucleotide identity to those of A/PuertoRico/8/34. The NS gene from A/England/1/53/v-a had only 90% identity to the corresponding A/PuertoRico/8/34 gene, the HA gene had 98% identity and the NA gene 97% identity. Therefore, the greatest number of genetic changes between A/England/1/53/v-a and A/PuertoRico/8/34 was in the NS gene.

As used herein, a "reassortant" virus is a virus in which gene segments encoding antigenic proteins from a virus strain of interest (e.g. hemagluttinin and neuraminidase genes) are combined with gene segments encoding viral polymerase complex (PB2, PB1 and PA genes) or other similar genes (e.g., non-glycoprotein genes, including M genes and NS genes, and nucleoprotein (NP) genes) from viruses adapted for growth in culture (or attenuated viruses). The reassortant virus thus carries the desired antigenic characteristics in a background or master strain that permits efficient production in a host cell. Such a reassortant virus is a desirable "virus seed" for production of virions to produce vaccine (see Furminger, In: Nicholson, Webster and May (eds.), Textbook of Influenza, Chapter 24, pp. 324-332). The reassortant virus is preferably purified by a process that has been shown to give consistent results, before being inactivated or attenuated for vaccine production (see, e.g., World Health Organization TRS No. 673, 1982). In the present invention the master strain contains PB2, PB1, PA, NP and M genes from the A/PuertoRico/8/34 virus, NS from the A/England/1/53 virus, and the HA and NA genes from any virus strain.

As used herein, the NS gene originates from the A/England/1/53 virus, an influenza A virus strain obtained from the repository at St. Jude Children's Research Hospital, Memphis, Tenn., USA. The virus may contain one or more modifications that have been introduced in the noncoding region(s) and/or one or more modifications that have been introduced in the coding region(s). In a preferred embodiment, the NS gene of A/England/1/53 consists of the sequence of SEQ ID NO: 1. In a most preferred embodiment, the NS gene of A/England/1/53 consists of the sequence of SEQ ID NO:2. The NS1 protein encoded by SEQ ID NO: 1 has the amino acid sequence of SEQ ID NO: 3. The NS1 protein encoded by SEQ ID NO: 2 has the amino acid sequence of SEQ ID NO. 5.

As used here in PR8/Eng-NS represents the virus produced when expression plasmids containing 7 genes from A/PuertoRico/8/34 (PB2, PB1, PA, NP, M, HA and NA) were combined with the expression plasmid containing the NS gene from A/England/1/53/v-a in a virus rescue experiment. Virus rescue is described in Hoffmann et al., Proc. Natl. Acad. Sci. USA, 97:6108-6113, 2000. The NS gene sequence in A/England/1/53/v-a and PR8/Eng-NS are identical (SEQ ID NO:2).

Sequences for the A/PuertoRico/8/34 virus are found in GenBank Accession Nos. NC004518-NC004525.

A "virus of interest" is any influenza A virus. It can be a virus that produces disease in an animal or a strain of virus a person may wish to study in the laboratory. The strains of viruses include, but are not limited to, human, avian, swine and equine.

"Expression plasmid" is a DNA vector comprising an "inner transcription unit" and an "outer transcription unit". Expression plasmids may be used to generate any type of RNA virus, preferably positive or negative strand RNA viruses, segmented or unsegmented genome RNA viruses or double stranded RNA viruses. Expression plasmids of the present invention may be generated by, but are not limited to, methods disclosed in Neumann et al., Proc. Natl. Acad. Sci. USA 96:9345, 1999 and U.S. patent application Ser. No. 20030035814 or Hoffmann et al., Proc. Natl. Acad. Sci. USA 97:6108-6113, 2000 and U.S. patent application Ser. No. 20020164770.

The term "transfection" or "transfecting" means the introduction of a foreign nucleic acid into a cell so that the host cell will express the introduced gene or sequence to produce a desired polypeptide, coded by the introduced gene or sequence. The introduced gene or sequence may also be called a "cloned" or "foreign" gene or sequence, may include regulatory or control sequences, such as start, stop, promoter, signal, secretion, or other sequences used by a cell's genetic machinery. The gene or sequence may include nonfunctional sequences or sequences with no known function. A host cell that receives and expresses introduced DNA or RNA has been "transformed" and is a "transformant" or a "clone." The DNA or RNA introduced to a host cell can come from any source, including the same genus or species as the host cell, or from a different genus or species.

The present invention contemplates isolation or synthesis of genes encoding influenza viral proteins to be used in the invention, including a full length, or naturally occurring form of an influenza viral protein, and any antigenic fragments thereof from any influenza viral source. As used herein, the term "gene" refers to an assembly of nucleotides that encode a protein or proteins, and includes cDNA and viral genomic DNA nucleic acids.

An influenza gene of interest, whether viral genomic DNA or cDNA, can be isolated from any subtype of influenza virus. Methods for obtaining an influenza viral hemagglutinin gene, for example, are well known in the art (Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratoiy Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Techniques for isolating viral RNA and transcribing it into cDNA are well known in the art (Hoffmann, et al., Proc. Natl. Acad. Sci. USA 97:6108-6113, 2000; Hoffmann, et al., Arch. Virology 146: 2275-2290, 2002).

Any influenza virus potentially can serve as the source for the HA and NA genes of interest. The DNA may be obtained by standard procedures known in the art from cloned DNA by chemical synthesis, by cDNA cloning, or by the cloning of genomic influenza viral DNA, or fragments thereof, purified from the desired cell (See, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratoiy Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Glover, D. M., DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II, 1985).

Cell Lines

According to methods for reassorting viruses of the present invention, suitable host cells include embryonated egg cells, Vero cells or other mammalian cells, preferably ones approved and certified according to the WHO requirements for vaccine production (Mizrahi, ed., Viral Vaccines, Wiley-Liss, N.Y., pp. 39-60, 1990). Non-limiting examples of cell lines that can be suitable for methods, viruses and compositions used in the present invention, include, but are not limited to, mammalian fibroblast or cultured epithelial cells as continuous cell lines. Further non-limiting examples include Vero, MDCK, 293T, BK-21 and CV-1 cells, readily available from commercial sources (e.g., ATCC, Rockville, Md.).

WHO certified, or certifiable, continuous cell lines are preferred for producing influenza virus vaccines of the present invention. The requirements for certifying such cell lines include characterization with respect to at least one of genealogy, growth characteristics, immunological markers, virus susceptibility tumorigenicity and storage conditions, as well as by testing in animals, eggs, and cell culture. Such characterization is used to confirm that the continuous cell lines are free from detectable adventitious agents. In some countries, karyology may also be required. In addition, tumorigenicity is preferably tested in cells that are at the same passage level as those used for vaccine production.

It is preferred to establish a complete characterization of the continuous cell line to be used, so that appropriate tests for purity of the final product can be included. Data that can be used for the characterization of a continuous cell line to be used in the present invention includes (a) information on its origin, derivation, and passage history; (b) information on its growth and morphological characteristics; (c) results of tests of adventitious agents; (d) distinguishing features, such as biochemical, imunological, and cytogenetic patterns which allow the cells to be clearly recognized among other cell lines; and (e) results of tests for tumorigenicity. Preferably, the passage level, or population doubling, of the cell line used is as low as possible.

Vaccines

The present invention provides vaccines and methods for production of influenza vaccines for treating or preventing influenza viral infections. For the production of an influenza vaccine in Vero cells, five plasmids containing the non glycoprotein segments (e.g., PB2, PB1, PA, NP and M) from the high yield strain A/PuertoRico/8/34 and the NS gene from the A/England/1/53 influenza strain are co-transfected with two expression plasmids containing the HA and NA genes of an influenza virus of interest. Virus produced in accordance with the invention can be used in traditional or new approaches to vaccination (see Bilsel and Kawaoka, In: Nicholson, Webster and May (eds.), Textbook of Influenza, Chapter 32, pp. 422-434). In particular, the present invention overcomes defects of current technology, with respect to low influenza virus titers generated in Vero cells.

The present invention permits the rapid development of a high titer desired reassortment virus in Vero cells. Thus, it advantageously positions a vaccine manufacturer to generate a sufficient quantity of vaccine to meet public health needs and ensure standardization, which is an important requirement in the event of a pandemic when the rapid production of a vaccine is required. Because the vaccines of the invention permit production in an approved cell culture system, they avoid non-specific pathogens, bacteria, and allergenic proteins that may be present in vaccines prepared in embryonated eggs.

Adjuvants have been used with vaccines (e.g., influenza vaccines) (see Wood and Williams, In: Nicholson, Webster and May (eds.), Textbook of Influenza, Chapter 23, pp. 317-323). The term "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen. An adjuvant can serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator that non-specifically enhances the immune response (Hood, et al., Immunology, Second Ed., Benjamin/Cummings: Menlo Park, Calif., p. 384, 1984). Often, a primary challenge with an antigen alone, in the absence of an adjuvant, is that the antigen will fail to elicit a humoral or cellular immune response. Adjuvants include, but are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. An example of a preferred synthetic adjuvant is QS-21. Alternatively, or in addition, immunostimulatory proteins can be provided as an adjuvant or to increase the immune response to a vaccine. Preferably, the adjuvant is pharmaceutically acceptable.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.

Inactivated Vaccines

Inactivated virus vaccines are well established for vaccinating against RNA viral infection (e.g., influenza) (see Nichol, In: Nicholson, Webster and May (eds.), Textbook of Influenza, Chapter 27, pp. 358-372). To prepare inactivated virus, the transfected virus is grown either in cell culture or in embryonated eggs. Virus can be inactivated by treatment with formaldehyde, beta-propiolactone, ether, ether with detergent (such as Tween-80), cetyl trimethyl ammonium bromide (CTAB) and Triton N101, sodium deoxycholate and tri(n-butyl) phosphate (Furminger, supra; Wood and Williams, supra). Inactivation can occur after or prior to clarification of allantoic fluid (from virus produced in eggs); the virions are isolated and purified by centrifugation (Furminger, supra, see p. 326). To assess the potency of the vaccine, the single radial immunodiffusion (SRD) test can be used (Schild et al, Bull. World Health Organ., 52:43-50 and 223-31, 1975; Mostow et al., J. Clin. Microbiol., 2:531, 1975). The inactivated vaccine can be administered intramuscularly by injection.

Attenuated Live Virus

Live, attenuated influenza virus vaccines, using reassortant virus of the invention, can also be used for preventing or treating influenza virus infection, according to known method steps: Attenuation is performed by any method well know in the art, preferably achieved through the use of reverse genetics.

Attenuating mutations can be introduced into influenza virus genes by site-directed mutagenesis to rescue infectious viruses bearing these mutant genes. Attenuating mutations can be introduced into non-coding regions of the genome, as-well as into coding regions. Such attenuating mutations can also be introduced into genes other than the HA or NA, e.g., the PB2 polymerase gene (Subbarao et al., J. Virol. 67:7223-7228, 1993). Thus, new viruses can also be generated bearing attenuating mutations introduced by site-directed mutagenesis, and such new viruses can be used in the production of live attenuated reassortants.

It is preferred that such attenuated viruses maintain the genes from the reassortant virus that encode antigenic determinants substantially similar to those of the original virus of interest. This is because the purpose of the attenuated vaccine is to provide substantially the same antigenicity as the original virus of interest, while at the same time lacking infectivity to the degree that the vaccine causes minimal chance of inducing a serious pathogenic condition in the vaccinated mammal.

Thus, the reassortant virus can be attenuated or inactivated, formulated and administered, according to known methods, as a vaccine to induce an immune response in a mammal. Methods are well-known in the art for determining whether such attenuated or inactivated vaccines have maintained similar antigenicity to that of the original virus of interest. Such known methods include the use of antisera or antibodies to eliminate viruses expressing antigenic determinants of the donor virus; chemical selection (e.g., amantadine or rimantidine); HA and NA activity and inhibition; and DNA screening (such as probe hybridization or PCR) to confirm that genes encoding the antigenic determinants (e.g., HA or NA genes) are not present in the attenuated viruses. See, e.g., Robertson et al., Giornale di Igiene e Medicina Preventiva 29.4-58, 1988; Kilbourne, Bull. M2 World Health Org. 41:643-645, 969; Aymard-Henry et al., Bull. World Health Org. 481:199-202, 1973; Mahy et al., J. Biol. Stand. 5:237-247, 1977; Barrett et al., Virology: A Practical Approach, Oxford IRL Press, Oxford, pp. 119-150, 1985; Robertson et al., Biologicals 20:213-220, 1992.

The vaccines of the present invention may be administered topically, parenterally, transmucosally, e.g. orally, nasally, or rectally, or transdermally. Administration that is parenteral, e.g., via intravenous injection, also includes, but is not limited to, intra-arteriole, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial.

The subject of the present invention is also a kit containing reagents according to the invention for the generation of influenza viruses and vaccines. Contents of the kit include, but are not limited to, expression plasmids containing the A/PuertoRico/8/34 genes that encode the PB2, PB1, PA, NP and M genes and an A/England/1/53 NS gene. The kit may contain expression plasmids that contain the HA and NA genes from a particular virus. Expression plasmids containing no virus genes may also be included so that the individual user is capable of incorporating the HA and NA genes from any influenza virus of interest. Mammaliam cell lines may also be included with the kit, including but not limited to, Vero and MDCK. Other components such as buffers, controls, and the like, known to those of ordinary skill in art, may be included in such kits.
 

Claim 1 of 16 Claims

1. A method for producing a reassortant influenza virus comprising transfecting host cells with expression plasmids containing PB2, PB1, PA, NP and M genes from A/PuertoRico/8/34 influenza strain, an NS gene encoding an NS1 protein having an amino acid sequence comprising SEQ ID NO:5 and an NS2 protein having an amino acid sequence comprising SEQ ID NO:6, and HA and NA genes from an influenza virus of interest other than A/England/1/53, to obtain a reassortant influenza virus.
 

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