Cold-adapted influenza virus (18-Mar-2008)

This application is a continuation of application Ser. No. 08/082,846, filed Jun. 29, 1993 now abandoned. The contents of U.S. Ser. No. 08/082,846, are hereby incorporated by reference into the present disclosure.

		DATE OF
VIRUS	ACCESSION NO.	DEPOSIT
Wild type A/Ann Arbor/6/60	ATCC VR 2408	Jun. 10, 1993
(H2N2) egg passage 2(3)
Cold-adapted “Master Strain”	ATCC VR 2409	Jun. 10, 1993
A/Ann Arbor/6/60 7PI (H2N2)

Work on this invention has been supported since 1976 by the contract office of the National Institute of Allergy and Infectious Diseases with Contract Nos. 1-AI-72521, 1-AI-52564, and 1-AI-05053; by Public Health Service Research Grant AI-20591 from the National Institute of Allergy and Infectious Diseases; by Cancer Center Support (CORE) Grant CA-21765; by American Lebanese Syrian Associated Charities (ALSAC) of St. Jude Children's Research Hospital; and Pittsburgh Supercomputing Centers through the National Institutes of Health Division of Research Resources cooperative agreement U41RR04154. The United States Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to cold-adapted influenza virus and, more particularly, to a cold-adapted influenza virus vaccine and methods of preventing and treating influenza by employing the vaccine.

BACKGROUND OF THE INVENTION

The tremendous impact of influenza virus infections on the public health is widely recognized. Control of influenza has relied primarily on the use of inactivated influenza vaccines. More current approaches, however, have moved towards the use of live attenuated vaccine. Kilbourne, E. D. “Influenza” (Plenum Publishing Corp. New York), p. 291-332 (1987). The most promising efforts in the development of an effective live vaccine have centered on adapting the virus to grow at suboptimal temperatures. Maassab, H. F., et al., Vaccine 3:355-369 (1985). Using this approach, cold-adapted attenuated influenza viruses have been developed in both the former Soviet Union and the United States. Alexandrova, G. I., et al., Rev. Roum. Inframicrobil. 2:179-189 (1965); Maassab, H. F. Nature (London) 213: 612-614 (1967).

In particular, cold adaptation (ca) has permitted the A/Ann Arbor/6/60 (H2N2) (A/AA/6/60) virus of the present invention to grow as well at 25° C. as it does at 33° C. Maassab, H. F. Nature (London) 213:612-614 (1967); Maassab, H. F. “Biology of Large RNA Viruses” (Academic Press, New York), p. 542-565 (1970). The ca A/AA/6/60 virus is also temperature-sensitive (ts), a property that impedes replication at higher temperatures in the lungs and thus is highly desirable for live vaccines. Maassab, H. F., “Biology of Large RNA Viruses” (Academic Press, New York), p. 542-565 (1970); Mulder, J., et al., “Influenza” (Wolters-Noordhoff, Amsterdam), 1-6:78-80 (1972). Single-gene studies of this cold-adapted virus in a background of A/Korea/1/82 (H3N2) have identified the genes responsible for the ca and ts phenotypes and for attenuation in that gene constellation. Snyder, M. H., et al., J. Virol. 62(2):488-495 (1988).

Live attenuated vaccines are produced by reassorting the six internal genes of the cold-adapted A/Ann Arbor/6/60 influenza virus with the two surface genes of the currently circulating wild type (wt) virus, thereby producing a reassortant strain. Maassab, H. F. “Negative Strand Viruses” (Academic Press, New York), p. 755-763 (1975); Davenport, F. M., et al., J. Infect. Dis. 136:17-25 (1977). Vaccines prepared from ca A/AA/6/60 have proven both non-reactogenic and non-transmissible in preliminary field trials at six different medical centers involving over 20,000 people. Couch, R. B., et al., “Options for the Control of Influenza” (Alan R. Liss, New York), p. 223-241 (1986); Wright, P. F., et al., “Options for the Control of Influenza” (Alan R. Liss, New York), p. 243-253 (1986). These vaccines also provide higher IgA levels than the killed vaccines and afford longer-lasting protection in children. Murphy, B. R., et al., Infect. Immun. 36(3):1102-1108 (1982); Johnson, P. R., et al., J. Infect. Dis. 154(1):121-127 (1986). Currently, the ca A/AA/6/60 7PI (plaque-purified seven times) master strain preparation is under development for use as a live vaccine in children and other live virus vaccines are being developed using the live ca influenza vaccine as a model.

Cold-adapted reassortant vaccines have thus been shown to have the proper level of attenuation, immunogenicity, and non-transmissibility combined with proven genetic stability and are produced in acceptable tissue culture substrates. In general, live cold-adapted reassortant vaccines offer several advantages over the existing inactivated vaccine. These include the possible use of a single dose, and administration by the natural route of infection, i.e. intranasally. In addition, ca vaccines stimulate a wide range of antibody responses, and result in induction of both local and humoral immunity. Furthermore, these vaccines are cost-effective and can be rapidly produced and updated in the event of antigenic changes. In addition, laboratory guidelines are available for the assessment of virulence (reactogenicity in ferrets) and attenuation can be reproducibly achieved. Moreover, the presence of two phenotypic markers (the temperature-sensitive and cold-adapted phenotypes) allows for the evaluation of virulence and monitoring of the vaccine in the field.

However, despite the above-described advantages, until now virtually nothing has been known about the molecular basis of cold adaptation. Published information indicates that cold adaptation has produced one or more mutations in each of the genes encoding the internal proteins of the A/AA/6/60 master strain. Cox, N. J., et al., “Genetic Variation Among Influenza Viruses” (Academic Press), p. 639-652 (1981). However, all of the work has been done on viruses passaged 28 to 32 times in eggs in parallel with the virus passaged in primary chick kidney cells during cold adaptation. Cox, N. J., et al., Virol. 167:554-567 (1988). Studies, however, have shown a gradual buildup of mutations in the RNA1 of sequential 35° C. egg passages 2 through 28 of wild type viruses, and recent findings have shown the influence of host cell variation on influenza viruses passaged in chicken eggs. Katz, J. M., et al., Virol. 156:386-395 (1987). Thus, the mutations leading to cold adaptation and attenuation have heretofore been unknown.

It would thus be desirable to isolate and provide the wild type A/Ann Arbor/6/60 progenitor virus and determine the accurate nucleic acid sequence of its genome. It would further be desirable to identify the mutations leading to cold adaptation, thus accurately characterizing the nucleic acid sequence of the ca master strain. It would also be desirable to produce and provide cold-adapted influenza strains through reassortment with currently circulating wild type strains. It would also be desirable to produce and use a cold-adapted influenza vaccine to prevent and/or treat influenza.

SUMMARY OF THE INVENTION

The cold-adapted A/Ann Arbor/6/60 7PI (H2N2) influenza strain (“master strain”) has been isolated and deposited, and its genome accurately sequenced and compared to its progenitor temperature-sensitive wild type E2(3) (wt 2(3)) virus. The A/Ann Arbor/6/60 virus is a single-stranded RNA virus having eight gene segments. During investigation of the virus leading to the vaccines of the present invention, unexpected deviations from previously reported sequences of the ca and wt were also identified. In particular, in the ca master strain sequences, seven nucleotide differences were found, occurring in the nucleoprotein gene (NP), the gene encoding an acidic polymerase protein (PA) and the gene encoding a basic polymerase polypeptide (PB2).

In comparing the cold-adapted master strain to the wt progenitor strain, four nucleotide differences encoding two amino acid differences were found in three gene segments. Computer-predicted RNA folds projected different secondary structures between the cold-adapted and wild type molecules based on the two silent differences between them. Genes coding for the PA, matrix (M), and non-structural (NS) proteins were identical between the two viruses. The differences suggest that cold adaptation may serve to provide conformational changes in the RNA structure advantageous to growth at 25° C. and provide a new form of genetic stability to the highly variable RNA genome.

With the identification of the correct nucleotide sequence of the ca master strain and its deposit, reassortant strains can now be produced which can be used as vaccines, to prophylactically and therapeutically treat influenza. Reassortant strains are produced by genetically combining the ca master strain with a variety of epidemic wild type viruses to yield reassortants which contain the hemagglutinin (HA) and neuraminidase (NA) gene segments of the wild type virus and the other six genome segments of the ca master strain. The reassortants thus contain the epidemic wild type strain genes that code for immunizing antigens found on the surface of the virus particle and the ca master strain genes that are responsible for the attenuated phenotype in humans and animals. To produce the vaccines of the present invention, a cold-adapted reassortant vaccine strain is passed once to prepare a virus seed lot which is used to produce vaccine pools.

In practicing the present invention, the amount of vaccine to be used or administered, alone or in combination with other agents, may vary with the patient being treated and may be monitored on a patient-by-patient basis by the physician. The vaccines of the present invention may also be administered in combination with other vaccines. Generally, a therapeutically effective amount of the vaccine will be administered for a therapeutically effective duration. By “therapeutically effective amount” and “therapeutically effective duration” is meant an amount and duration to achieve the desired therapeutic or prophylactic result in accordance with the present invention with medically acceptable side effects, which can be determined by those skilled in the medical arts.

The vaccines of the present invention may comprise the reassortant virus as well as a pharmaceutical formulation, together with a pharmaceutically acceptable carrier therefor. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Formulations include those suitable for oral, nasal, topical (including transdermal, buccal and sublingual), parenteral (including subcutaneous) and pulmonary administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.

It will be appreciated that administration of the vaccines of the present invention will also be by procedures well-established in the pharmaceutical arts, e.g. preferably intranasally or orally, and most preferably intranasally. Intramuscular, intravenous and intradermal administration is also contemplated by the present invention, either alone or in combination.

The present invention thus comprises isolated nucleic and amino acids with sequences corresponding to the ca master and wild type strain sequences set forth in Sequence ID Listings 1-40. By “isolated” is meant substantially purified from the natural state through chemical, biochemical, immunological or other means, or obtained in substantially pure form by other methods known to those skilled in the art. By “substantially pure” is meant substantially free from undesirable contaminants such as other proteins. Thus, these terms are not meant to exclude synthetic and recombinant nucleic and amino acids which are contemplated within the scope of the present invention. These terms are also not meant to exclude nucleic and amino acids which are linked, bound or intentionally combined with other moieties such as transgenes, labels, flanking amino acid sequences and the like. It will also be appreciated that although the viruses of the present invention are RNA viruses, the present invention further includes DNA sequences corresponding and complementary thereto.

The present invention further comprises isolated or substantially pure ca master strain and wild type E2(3) A/AA/6/60 virus. By “isolated” or “substantially pure strain” is meant the viral strain substantially free from other contaminants such as other viruses, bacteria, and the like.

The present invention further comprises reassortant viruses produced by combining the cold-adapted master strain with a variety of epidemic wild type viruses. The two surface protein genes of an epidemic wild type virus are operatively-linked to the six internal genes of the cold-adapted master strain. By “operatively-linked” is meant attached or assembled in a manner which allows for expression of the surface and internal genes. In the context of reassortant viruses, operative linkage will allow for the packaging of the reasserted RNA into virions. It will also be appreciated that the term “gene” is used comprehensively to include all polynucleotide sequences coding for the gene product or protein, and is not limited to naturally occurring coding and regulating elements.

In addition, the present invention comprises the production and use of cold-adapted influenza vaccines to prevent and/or treat influenza.

Additional objects, advantages, and features of the present invention will become apparent from the following description and appended claims, taken in conjunction with the accompanying drawings and Sequence ID Listings.

BRIEF DESCRIPTION OF THE DRAWINGS

The various advantages of the present invention will become apparent to one skilled in the art by reading the following specification and subjoined claims and by referencing the following drawings in which:

FIG. 1 shows the derivation of the progenitor wild type and cold-adapted master strain A/AA/6/60 in PCK cells; and

FIG. 2 shows the computer-projected RNA fold of cold-adapted and wild type 2(3) RNA1's (PB2's).

DETAILED DESCRIPTION OF THE INVENTION

Overview

The nucleic and amino acid sequences for the eight genes of the cold-adapted master strain A/Ann Arbor/6/60 7PI (H2N2) are set forth in Sequence ID Listings 1-20. The nucleic and amino acid sequences for the eight genes of the wild type A/Ann Arbor/6/60 (H2N2) Egg Passage 2(3) are set forth in Sequence ID Listings 21-40.

TABLE 1
Gene Products of Influenza A and B Viruses

RNA	Gene Product(s)	Function
1	PB2	Viral polymerase component involved in RNA
		transcription
2	PB1	Viral polymerase component with RNA
		transcription and replication activities
3	PA	Viral polymerase component involved in RNA
		replication
4	HA	Virion surface attachment and fusion
		glycoprotein, major antigenic determinant
5	NA	Virion surface glycoprotein with receptor-
		destroying enzyme activity, major antigenic
		determinant
6	NP	Major nucleocapsid structural component and
		type-specific antigen
	NB	Glycoprotein pututive membrane ion channel
		found only in type B
7	M1	Membrane matrix protein and type-specific
		antigen
	M2	Nonglycosylated membrane ion channel, found
		only in type A
8	NS1	RNA-binding non-structural protein of
		transport function
	NS2	Cellular and virion protein of unknown
		function

The A/Ann Arbor/6/60 virus contains six internal genes, NS, M, NP, PA, basic polymerases (PB1 and PB2), and two surface genes, HA and NA. Seven nucleotide differences were found between the sequences of the present invention and those previously published for cold-adapted A/Ann Arbor/6/60: three in the NP gene, one in the PA gene and three in the PB2 gene. The eight viral genes and the discrepancies in the previously published sequences can be summarized as follows:

NS. The non-structural (NS) gene is the smallest RNA segment of influenza virus, 890 nucleotides long, and codes for the two non-structural proteins (NS1 and NS2) (nucleic acid Sequence ID Listing 1 and 3; amino acid Sequence ID Listings 2 and 4). There were no errors in the previously published sequences for the ca A/AA/6/60 NS1 and NS2 genes.

M. The matrix gene (M) is a 1,027 base nucleic acid sequence (nucleic acid Sequence ID Listings 5 and 7; amino acid Sequence ID Listings 6 and 8). There were also no errors in the previously published sequences for the ca A/AA/6/60 M gene.

NP. The nucleoprotein gene (NP) (nucleic acid Sequence ID Listing 9) is 1566 nucleotides in length and encodes a basic structural protein of 498 amino acid residues (amino acid Sequence ID Listing 10) which specifically interacts with RNA molecules to form ribonucleoprotein complexes and has sequences that direct its migration into the nuclei of infected cells. Despite previous reports, nucleotide 627 of NP is actually cytosine not adenine, and nucleotide 909 is guanine, not cytosine. In addition, nucleotide 113 was previously published as an adenine, although in GenBank it is reported as a cytosine. Cox, N. J. et al., Virol. 167:554-567 (1988). Regardless of this discrepancy, it is now known that nucleotide 113 is actually a cytosine.

PA. The polymerase acidic protein gene (PA) RNA sequence (nucleic acid Sequence ID Listing 11) is 2233 nucleotides in length and encodes an acidic polymerase protein 716 amino acids in length (amino acid Sequence ID Listing 12). Although previous publications indicate thymine at nucleotide 75 of PA, guanine is actually present at that position.

PB1. The polymerase basic 1 gene (PB1) RNA sequence (nucleic acid Sequence ID Listing 13) is 2341 nucleotides in length and encodes a basic polymerase protein 757 amino acids in length (amino acid Sequence ID Listing 14). No errors in the previously published sequence were found.

PB2. The polymerase basic 2 gene (PB2) RNA sequence (nucleic acid Sequence ID Listing 15) is 2341 nucleotides in length and encodes a basic polymerase polypeptide of 759 amino acids (amino acid Sequence ID Listing 16). There are three errors in the previously published sequence for PB2: thymine at 714 instead of the previously published cytosine at that position; guanine at 936 instead of adenine; and cytosine instead of thymine is the predominant base at 1933, with thymine as the secondary base.

HA and NA. The hemagglutinin gene (HA) and neuraminidase gene (NA) code for surface receptors. HA is 1773 nucleotides long and codes for a 562 amino acid sequence (nucleic acid Sequence ID Listing 17; amino acid Sequence ID Listing 18). See Schäfer, J. R. et al. Virol. 194:781-788 (1993). NA is 1467 nucleotides long and codes for a 469 amino acid sequence (nucleic acid Sequence ID Listing 19; amino acid Sequence ID Listing 20).

Results from previous studies indicate that cold adaptation causes mutations in every gene of the A/AA/6/60 master strain, thus ensuring the genetic stability of the virus. There are actually, however, four base differences in three of the internal genes of A/AA/6/60 after 28 passages in primary chicken kidney (PCK) cells and four passages in eggs. Two of the substituted bases are silent and two result in single amino acid differences in two of the genes. Moreover, the wt 2(3) progenitor virus is attenuated in ferrets. Hence, the stability and immunogenicity of the ca A/AA/6/60 vaccine appears to reflect inherent properties of the wt A/AA/6/60 E2(3) virus selected as the progenitor for the master strain. This interpretation is supported by the large number of amino acids unique to both wt 2(3) and ca viruses (see Table 3), some of which may be attenuating.

By attempting to identify changes arising from cold adaptation using the ca master strain and the wt 2(3) virus, there is at least one further critical variable—passage of the virus in different host tissues. It has been shown that the host cell influences the selection of antigenic variants of influenza viruses. Katz, J. M., et al., Virol. 156:386-395 (1987). In studies of the HA gene of H3N2 viruses, passage in Madin-Darby canine kidney (MDCK) cells and in primary chick kidney (PCK) cells selected populations that were homogeneous and true to the original isolate for this gene whereas passage in eggs selected heterogeneous populations. Katz, J. M., et al., J. Gen. Virol. 73:1159-1165 (1992). Thus, the changes observed could relate to the number of passages of each virus. The wild type 2(3) virus, with only two egg passages, is the only virus among all of those listed in GenBank to have isoleucine encoded by base 1276 of RNA2 and asparagine encoded by base 113 of NP. The positions of those two amino acids in the cold-adapted virus, with 29 PCK passages and 4 egg passages, are the same as those of all other viruses listed in GenBank. This finding suggests that the valine encoded by base 1276 in the cold-adapted PB1 is a host adaptation change rather than a cold adaptation change; the same holds for the threonine encoded by base 113 of the cold-adapted NP gene.

Differences between the wt 2(3) sequence as set forth herein and the wt 28-32 previously sequenced reflect mutations acquired during high passage in eggs at 35° C. Cox, N. J., et al., Virol. 167:554-567 (1988). These mutations may be the result of host adaptation in the egg or simply selection of the highly variable RNA population with the highest relative fitness. Clarke, D. K., et al., J. Virol. 67:222-228 (1993).

Since only the ca RNA1 has guanine (G) at position 141 and cytosine (C) at position 1933, by comparison with all other human RNA1's in GenBank, the two base changes between the wt 2(3) and ca RNA1's may in fact be cold-adapted changes. No wild type human viruses, including the wt 2(3) progenitor, have G at 141 or C at 1933. This suggests that cold adaptation may operate at the RNA level. Recent findings indicate that unique RNA structures in influenza viruses may have common regulatory functions. Parvin, J. D., et al., J. Virol. 63:5142-5149 (1989). The more stable conformation of the ca molecule predicted by base pairing might provide a growth advantage over the predicted conformation of the wt 2(3) molecule. The importance of RNA structure to biological function has been well documented for poliovirus. Racaniello, V. R., et al., Virol. 155:498-507 (1986). The presence of a hairpin structure at the 5′ noncoding end has been shown to be necessary for the ts phenotype of the virus.

Although RNA viruses have notoriously high mutation rates and have been referred to as “quasi-species,” Holland, J. J., et al., Cur. Topics Microbiol. Immunol. (Springer-Verlag) 176:1-20 (1992), the ca A/AA/6/60 virus showed unusual stability after cold adaptation in PCK cells. In 33 passages there were only four sequence changes in the six internal genes, yielding a mutation rate of 2×10⁻⁶. Compared to expected chance mutation rates calculated for the NS gene in MDCK cells, one would have expected 21 sequence changes. Parvin, J. D., et al., J. Virol. 59(2):377-383 (1986). Since RNA viruses have not been shown to have proofreading functions, this low mutation rate may be an inherent property of the wild type polymerases or a result of the cold adaptation process, or both. Suarez has shown that wild type viruses comprise subgroups with different mutation rates. Suarez, P., et al., J. Virol. 66(4):2491-2494 (1992). The wt A/AA/6/60 may have a dominant population with a more error-free polymerase. In addition, certain positions may simply be difficult for the polymerase to read, owing to conformation of the RNA molecule. Lowering the growth temperature by 10° C. slows the whole replicative process including the speed at which the polymerase unit is moving. Thermus aquaticus (Taq) polymerase is notorious for its high error rate due in part to the high temperature of its use, and it has been shown that a 5° C. reduction in temperature increases the fidelity of Tub polymerase. Kainz, P., et al., Anal. Biochem. 202:46-49 (1992). The lower temperature may provide a slowed-down environment conducive to faithful copying even in areas with conformational bends and twists. Thus the A/AA/6/60 polymerase might exhibit greater fidelity at 25° C. than at 35° C.

Single gene cold-adapted reassortants, constructed to identify the genetic basis of the ca and ts phenotypes and of attenuation, should be interpreted with care. For instance, in the study by Snyder et al., conducted in a background of A/Korea/1/82 genes, both PA and M were implicated in attenuation. Snyder, M. H., et al., J. Virol. 62(2):488-495 (1988). Neither gene showed sequence differences from its wt 2(3) counterpart in the present analysis. This would suggest that single gene wt 2(3) PA or wt 2(3) M in an A/Korea background would react similarly to the ca PA and M single genes. From the sequence data, one would also expect that RNA1 encoding PB2 would contribute to the ca phenotype in single gene studies and yet only PA was involved. Snyder, M. H., et al., J. Virol. 62(2):488-495 (1988). Gene constellation studies suggest that single gene studies in one wild type may be applicable to only that wild type. Subbarao, E. K., et al., Virus Res. 25:37-50 (1992). In a different wild type background, the assignment of phenotype to specific ca genes might change because other wild type genes might be dominant or carry natural extragenic suppressor mutations. This emphasizes the need for the presence of six genes from the ca virus rather than five in ca reassortants to ensure maximum stability. Maassab, H. F., et al., J. Infect. Dis. 146(6):780-790 (1982).

SPECIFIC EXAMPLE 1

Sequencing

A. Materials and Methods

Viruses. All viruses were supplied by Professor H. F. Maassab at the University of Michigan and the ca master strain and wild type progenitor strain viruses have now been deposited with the ATCC as previously set forth. Steps in the preparation of the ca master strain A/AA/6/60 7PI (H2N2) live influenza virus and the wt A/AA/6/60 (H2N2) egg passage 2(3) virus are shown in FIG. 1. In FIG. 1, PCK cells refers to primary chick kidney cells, SPAFAS refers to specific pathogen-free eggs and PI refers to plaque-purified. To guard against any possibility of mix-up in the two viruses, the passage history of both viruses was carefully traced and their separate identities were verified. Moreover, the two viruses were grown in different institutions and sequenced separately. The authenticity of the wt A/AA/6/60 E2(3) virus is supported by sequence differences between the HA's and NA's of the cold-adapted and wild type viruses. Viruses grown in 11-day old embryonated chicken eggs and virion RNA were prepared as previously described. Bean, W. J., et al., Anal. Biochem. 102:228-232 (1980).

Growth and Infectivity of Viruses. Plaque titrations were performed with both viruses in PCK cells at 25° C., 33° C., and 39° C., and in MDCK cells at 33° C. and 39° C. Mills, J., et al., J. Infect Dis. 123:145-157 (1971). Plaque counts obtained at each of the three temperatures were compared to assess the ca and ts phenotypes of both viruses.

Ferret Studies. One week before infection with virus, 4 female ferrets were bled and screened for influenza antibody against A/Taiwan/1/86 (H1N1), A/Beijing/353/89 (H3N2), wt A/AA/6/60 (H2N2) E2(3) and B/Victoria/2/87. The animals' temperatures were taken twice a day for 1 week preceding their inoculation with 1×10⁹ EID₅₀ of wt E2(3), and then until they were sacrificed at either 3 or 8 days after infection. Lungs and turbinates of the ferrets were examined by previously reported methods. Maassab, H. F., et al., J. Infect Dis. 146(6):780-790 (1982).

Gene Cloning. Double-stranded cDNA was prepared as previously described. Huddleston, J. A., et al., Nucleic Acids Res. 10:1029-1039 (1982). Full-length double-stranded copies of genes 4 through 8 (HA, NA, NP, M, NS) were blunt-end ligated into the Pvu II site of vector Pvu II, obtained from C. Naeve at St. Jude Children's Research Hospital.

For the polymerase genes (PB1, PB2, PA), the first-strand cDNA was amplified by the polymerase chain reaction (PCR) using phosphorylated primers. “Gene-cleaned” PCR product was blunt-end ligated into the Pvu II site of pATX.

Nucleic Acid Sequencing. Nucleotides of all eight cloned genes of each virus were sequenced by the method of Chen and Seeburg using alkali-denatured DNA templates. Chen, E. Y., et al., DNA 4:165-170 (1985). Due to the extreme heterogeneity of RNA viruses, several clones of each gene were sequenced to avoid reporting the sequence of a minor mutant population. Clones of each orientation were sequenced for each gene. If the two clones differed at any position, as many as 7 clones of each gene were sequenced and the consensus sequence was reported. Compressions were resolved by the addition of 42% formamide to the gels.

Differences between the cold-adapted virus and the wild type E2(3) virus were confirmed by direct sequencing of the virion RNA, a method which would expose any mutations introduced by use of the Taq polymerase. Air, G. M. Virol. 97:468-472 (1979).

Sequence Analysis. The IntelliGenetics software package (Palo Alto, Calif.) was used to analyze nucleotide sequence data. Chou-Fasman two-dimensional protein structure predictions were made with programs available at the St. Jude Molecular Biology Computing Center. The reliability of protein folding by this method is predicted to be approximately 60%. Fasman, G. D. “Prediction of Protein Structure and the Principals of Protein Confirmation” (Plenum, New York), p. 417-467 (1986).

The Zuker Fold program on the Cray Y-MP supercomputer at the Pittsburgh Supercomputing Center was used to study the folding of RNA molecules. Optimal foldings were obtained using the Zuker algorithm which calculates the structure exhibiting minimal free energy. Zuker, M., et al., Nucleic Acids Res. 9:133-148 (1981). This program calculates the structure that is energetically most favorable and has a predicted accuracy of 80%, although the structure with the lowest free energy may not represent all biologically active structures. Zuker, M., et al., Nucleic Acids Res. 9:133-148 (1981).

B. Results

Biological Properties. The ca and ts characteristics of the viruses in PCK cells was first examined. The ca master strain reached essentially the same titer at 25° C. (3.0×10⁸) as it did at 33° C., but failed to grow at 39° C. (see Table 2), fulfilling accepted criteria for cold adaptation and temperature sensitivity. By contrast, on day 6, the wt E2(3) virus had produced fewer than 1.0×10⁵ plaques at 25° C., although by day 8 it had generated 5.0×10⁶ plaques, indicating a subpopulation of virus capable of growth at low temperatures. The 4-log reduction in growth at 39° C. compared with that at 33° C. demonstrates the ts phenotype of the wt 2(3) virus. Similar results were obtained in MDCK cells at 33° C. and 30° C. (data not shown).

The pathogenicity of the wild type 2(3) virus was studied in ferrets. The virus was not recovered from lung tissue in any of the 4 animals examined, and it was recovered from turbinates in only the 2 animals sacrificed on day 3 (data not shown). None of the ferrets showed physical signs of illness, such as coryza, lethargy or sneezing. Rises in temperature ranging from 1° C. to 1.5° C. were observed, but they persisted for only several hours and were not considered significant since normal temperatures fluctuated by 1° C. These results, which correspond to findings with the ca virus, indicate that the wt 2(3) virus was attenuated before cold adaptation. Maassab, H. F., et al., J. Infect. Dis. 146(6):780-790 (1982).

TABLE 2
Infectivity Titers of A/AA/6/60 (H2N2)

Number of Plaques in Primary Chick Kidney Cellsa

Virus	33° C.b	39° C.b	25° C.
ca Master Strain	6.0 × 108	<1.0 × 104	5.0 × 107 on day 6c
A/AA/6/60 (H2N2)			8.0 × 107 on day 7
7PI (SE4)			3.0 × 108 on day 8
wt A/AA/6/60	1.5 × 108	2.0 × 104	<1.0 × 105 on day 6
(H2N2) E2(3)			8.0 × 105 on day 7
			5.0 × 106 on day 8
aSimilar results were obtained in MDCK cells at 33° C. and 39° C.
bInfectivity titers at 33° C. and 39° C. were determined on post-infection day 4
cPost-infection days.

Tests were also performed employing ferrets to determine whether the cold-adapted vaccine would interfere with or block growth of the influenza virus. The experimental protocol and results of this study are set forth in U.S. Pat. No. 5,149,531, issued Sep. 22, 1992 to Younger et al., hereby incorporated by reference.

Sequencing. Table 3 compares sequencing results of the ca master strain with wt E2(3) virus. The data represent consensus DNA sequencing of multiple clones. If the clone consensus indicated a difference between the two viruses, RNA sequence data were used to support the findings. Positions reported as mixed populations in Table 3 show the distribution of the clones.

Between the internal genes of the ca and the wt 2(3) viruses, no differences were found in the genes coding for PA, M or NS, even though PA and M were previously reported to be important for attenuation of the ca master strain and cold adaptation was attributed to PA. Snyder, M. H., et al., J. Virol. 62(2):488-495 (1988). Differences were found in the genes coding for PB2, PB1 and NP.

TABLE 3
Sequence Differences between wt 2(3) and ca A/Ann Arbor/6/60 Viruses

wt A/AA/6/60 E2

ca A/AA/6/60

	Base	Amino		Amino		Amino
Gene	No.	Acid No.	Base	Acid	Base	Acid

PB2	141		A/g(4/2)		G (5)
	1933		T/c(4/2)		C/t(4/1)
PB1	1276	418	A (5)	Ile	G/a(4/3)	Val
PA			—	—	—	—
HA	144	34	A (2)	Asn	T (2)	Ile
	455	138	G (2)	Ala	A (2)	Thr
	729	229	A (2)	Lys	C (2)	Thr
NA	394		C (2)		T (4)
	604		A (2)		T (4)
NP	113	23	A/c(2/1)	Asn	C/a(3/1)	Thr
M			—	—	—	—
NS			—	—	—	—

In Table 3 above, in positions with mixed bases, the capital letter represents the dominant base. The distribution of the clones representing the positions with differences between the wt 2(3) and the ca internal genes are shown next to the bases.

RNA1 (PB2). Two nucleotide differences, in bases 141 and 1933, were found between the ca and wt 2(3) RNA1 genes, which encode a basic polymerase protein 759 amino acids in length. Called PB2, this protein is part of the transcriptase complex and has been identified as recognizing and binding the cap structure of the host-cell primer RNA. Plotch, S. J., et al., Cell 23:847-858 (1981). Both changes are in the coding region but are silent. Moreover, bases 141 and 1933 of the ca RNA1 are unique among all other human RNA1 sequences in GenBank. Position 1933 in the wt 2(3) and ca RNA1 segments is a mixed population of two bases; however, the darker band in the RNA sequence (thymine (T) in wt 2(3) and cytosine (C) in ca) conforms with the consensus DNA sequence reported in Table 3.

To assess the potential functional significance of the two nucleotide sequence differences between the ca and the wt 2(3) viruses, the Zuker RNA-fold algorithm and computer modeling techniques were used to predict RNA secondary structures. As shown in FIG. 2, the difference at base 141 does not impinge on the predicted structure of RNA1 because it is part of an unpaired loop in both molecules; however, the change at nucleotide 1933, T in wt 2(3) to C in ca (shown by arrows in FIG. 2), does affect the predicted fold of RNA1. The RNA fold of the ca virus has greater stability than the analogous fold of wt 2(3), as judged by its lower free energy of −736.2 compared to −733.6 for the wt 2(3) molecule. Both folds were pivoted −25° at pair 1068/1381 and 180° at pair 1675/1861 to better visualize the area of difference between the two molecules. The single base change at 1933 causes a cascade of 163 pairing differences, from base 1888 to base 2151, and thus might constitute a true cold adaptation. Similar RNA1 sequencing results were obtained for a wt A/AA/6/60 E3(4) passage virus.

RNA2 (PB1). The only nucleotide change found between the RNA2 genes of the ca and wt 2(3) viruses occurred at base 1276, resulting in a substitution of valine (ca) for isoleucine (wt 2(3)), both of which are hydrophobic and uncharged. RNA2 encodes a basic polymerase (PB1) that mediates transcription and elongation of the mRNA chain. Braam, J., et al., Cell 34:609-618 (1983). Analysis of protein secondary structures predicted by Chou-Fasman and Garnier-Osguthorpe methods, as well as computer-predicted RNA structures, failed to reveal differences between the ca and wt 2(3) RNA2's. Valine is not an amino acid unique to the ca virus because later passages of the wt A/AA/6/60 virus (both wt E6 and wt E28) also have valine at this position, as do all other RNA2's in GenBank. Both DNA clones and RNA sequencing show that base 1276 comprises a mixed population of adenine (A) and guanine (G) in the ca RNA2; however, the G predominates.

RNA6 (NP). The nucleoprotein gene (RNA6) encodes a basic protein 498 amino acids in length which specifically interacts with RNA molecules to form ribonucleoprotein complexes. Huddleston, J. A., et al., Nucleic Acids Res. 10:1029-1039 (1982). NP is necessary for transcription and is a major determinant of host range. Huang, T. S., et al., J. Virol. 64:5669-5 673 (1990); Scholtissek, C., et al., Virol. 147:287-294 (1985). There was one difference between the wt 2(3) and the ca NP molecules, at base 113 leading to substitution of threonine for asparagine, neither of which is hydrophobic or charged. The reverse change was reported in Cox, N. J., et al., Virol. 167:554-567 (1988).

Although having similar protein secondary structures by Chou-Fasman and Garnier-Osguthorpe predictions, the two RNA molecules showed a distinct difference in their predicted RNA structures. In wt 2(3) RNA6, base 113 creates a larger unpaired loop making the molecule less stable than ca RNA6 (structure not shown). DNA cloning and RNA sequencing revealed that base 113 is a mixed population of A and C in both the wt 2(3) and the ca RNA6's; however, in the wt 2(3) the consensus base is A and in the ca the consensus base is C.

The asparagine in the wt 2(3) virus is unique among all reported NP molecules (see Table 3), but not the threonine of the ca virus. The A/AA/6/60 (wt and ca) viruses are the only viruses in 54 GenBank sequences with an inserted A at base 1550 near the putative polyadenlyation signal.

RNA4 (HA) and RNA5 (NA). The sequences of ca RNA4 (HA) and ca RNA5 (NA) have not been previously reported, as neither molecule is included in ca reassortant vaccines. RNA4 encodes the hemagglutinin (HA) surface glycoprotein (562 amino acids in length), while RNA5, encodes the neuraminidase (NA) surface glycoprotein (469 amino acids in length). Two silent nucleotide differences were observed between ca RNA5 and wt 2(3) RNA5 at bases 394 and 604. Three additional differences seen at bases 144, 455, and 729 of ca RNA4 and wt 2(3) RNA4 coded for amino acid changes: asparagine to isoleucine (position 34), alanine to threonine (position 138) and lysine to threonine (position 229). The presence of clear differences in these two surface genes underscores the different passage histories of the two viruses and provides additional evidence for their separate identities.

SPECIFIC EXAMPLE 2

Sequence Comparisons

Sequence of Wild Type Progenitor. Table 4 presents positions for each gene where the ca and wt 2(3) viruses have unique amino acids, by comparison to previous GenBank sequences. Webster, R. G., et al., Microbiol. Rev. 56(1):152-179 (1992). In Table 5, a comparison to data previously published is shown and differences between the wt 2(3) and ca sequences as set forth herein, and the previously published sequences, are shown in bold type and bracketed. In positions with mixed bases, the capital letter represents the predominant base. Some of these amino acids found only in the two ts A/AA/6/60 viruses may be attenuating. However, many of the viruses reported in GenBank have been extensively passaged in the laboratory and will have accumulated mutations related to high relative fitness and host adaptation. Comparison to the A/AA/6/60 wt 28 virus previously sequenced provides further insight into attenuating lesions. Cox, N. J., et al., Virol. 167:554-567 (1988).

TABLE 4
Unique Amino Acid Differences between Temperature-sensitive
and Attenuated wt 2(3) and ca A/AA/6/60
Viruses and Other Influenza Viruses in GenBank

No. in

A/AA/6/60

GenBank

Gene	GenBank	Base No.	ca/wt 2(3)	wt 28	Virusesb

PB2a	27	821	Ser	Asn	Asn
		954	Glu	Glu	Asp
PB1	23	215	His	His	Pro
		1096	Lys	Lys	GIu
		1276	Val/Ile	Val	Val
		1395	Asp	Glu	Glu
		1660	Leu	Leu	Met
PA	21	599	His	His	Arg
		2167/8	Pro	Leu	Leu
NP	54	113	Thr/Asn	Thr	Thr
		1550	A	—	—
M1	44	453	Val	Val	Ala
		457	Leu	Leu	Phe
		678/9	Val	Val	Ala
M2	44	847	His	His	Arg
		969	Ser	Ala	Ala
NS1	73	35	Pro	Pro	Ser
		483	Thr	Ala	Glu
aFive other silent differences.
bSources of GenBank viruses for each gene used in phylogenetic analysis are reported in Webster R.G., et al., Microbiol. Rev. 56(1):152-179 (1992).

TABLE 5
Summary of Comparative Sequence Data for A/Ann Arbor/6/60
Wild Type and Cold-Adapted Viruses

Data from Study

Data Previously Publisheda

			wt
wt A/AA/6/60 E2(3)	ca	ca	A/AA/6/60

Base

A/AA/6/60

A/AA/6/60

E28

Gene	No.	Base	AA	Base	AA	Base	AA	Base	AA
PB2	141+	A/g		G		G		A
	426	C		C		C		T
	714	T		[T]		[C]		[C]
	821	G	ser	G	ser	G	ser	A	asp
		265
	963	G		[G]		[A]		[A]
	1182	T		T		T		A
	1212	T		T		T		C
	1353	G		G		G		T
	1923	G		G		G		A
	1933~	T/c		[C]/t		[T]		T
PB1	123	G		G		G		A
	486	T		T		T		C
	1195	G	glu	G	glu	G	glu	A	lys
		391
	1276{circumflex over ( )}	A/g	ile	G/a	val	G	val	G	val
		418
	1395	T	asp	T	asp	T	asp	G	glu
		457
	1766	G	gly	G	gly	G	gly	A	glu
		581
	2005	A	thr	A	thr	A	thr	G	ala
		661
	2019	T		T		T		C
PA	20	C		C		C		T
	75	G		[G]		[T]		[T]
	1861	G	glu	G	glu	G	glu	A	lys
		613
	2167	C	pro	C	pro	C	pro	T	leu
		715
	2168	C		C		C		T
HA	144	A 34	asn	T	ile
	455	G	ala	A	thr
		138
	729	A	lys	C	thr
		229
NA	394	C		T
	604	A		T
NP	113<	A/c	asn	C/[a]	thr	[A]	asn	C	thr
		23
	146	G 34	gly	G	gly	G	gly	A	asp
	627	C		[C]		[A]		A
	909	G		[G]		[C]		C
	1550	A		A		A		—
M	969	T	ser	T	ser	T	ser	G	ala
NS	483	A	thr	A	thr	A	thr	G	ala
		153
	813	G		G		G		A
aCox, N. J., et al., Virol. 167: 554-567 (1988).
The distribution of the clones representing the positions with the differences between the wt 2(3) and the ca viruses are listed below:
+wt 2(3) PB2 141 four clones A, two clones G
ca PB2 141 five clones G
~wt 2(3) PB2 1933 four clones T, two clones C
ca PB2 1933 four clones C, one clone T
{circumflex over ( )}wt 2(3) PB1 1276 five clones A
ca PB1 1276 four clones G, three clones A
<wt 2(3) NP 113 two clones A, one clone C
ca NP 113 three clones C, one clone A

SPECIFIC EXAMPLE 3

Reassortant Schemes

A. Type A Reassortants

The following is a procedure for developing Type A 6/2 cold-adapted influenza virus vaccine (CAIV) reassortants.

Materials

Media. The media used in this sample were prepared using the following components: a) HBSS—500 ml HBSS (BioWhitaker 10-508); 0.5 ml gentamicin sulfate 50 mg/ml (BioWhitaker 17-518); and adjust pH to 7.0 using 0.5N NaOH; b) 2×Eagle's−500 ml HBSS (BioWhitaker 10-508); 10 ml BME amino acids (GIBCO 320-1051); 10 ml BME vitamins (GIBCO 320-1040); 10 ml L-glutamine (GIBCO 320-5030); and 0.5 ml gentamicin sulfate 50 mg/ml (BioWhitaker 17-518); adjust pH to 7.0 using 0.5N NaOH; c) 0.5N NaOH−2 g NaOH; 100 ml Type I deionized water; sterilize by autoclaving 250° C. for 15 min, liquid cycle.

Inoculum. Inocula were prepared as follows: Cold-adapted Master Strain Parent (A/Ann Arbor/6/60-7PI)— make a 10⁻² dilution in 2×Eagle's. Wild Type Parent—make a 10⁻¹ dilution in 2×Eagle's. Combine equal volumes of the two diluted:parents (1:1 dilution) and use this as the inoculum.

Cells. Use SPAFAS-derived primary chick kidney (SPF-PCK) cells grown in 16×125 mm tissue culture tubes on the fifth day after seeding.

Passages

SPF-CK1 Passage. SPF-CK1 passages were performed as follows: 1) remove growth media from ten SPF-PCK tubes; 2) wash SPF-PCK tubes with 1 ml of HBSS media; 3) inoculate with 0.3 ml of inoculum per tube; 4) adsorb at room temperature for 90 min while continuously rocking at low speed; 5) remove inoculum; 6) wash SPF-PCK tubes with 1 ml of HBSS media; 7) add 1 ml of 2×Eagle's media and incubate at 33° C.; 8) after 24 hr feed tubes with 0.3 ml of 2×Eagle's media; and 9) observe cells daily for cytopathic effect (CPE). When CPE is >75%, pass the tubes to CK2 (usually 48-72 hr).

SPF-CK2 Passage. SPF-CK2 passages were performed as follows: 1) remove growth media from the SPF-PCK tubes; 2) wash SPF-PCK tubes with 1 ml of HBSS media; 3) serially pass the CK1 passage with 0.3 ml of inoculum per tube; 4) adsorb at room temperature for 90 min while continuously rocking at low speed; 5) remove inoculum; 6) wash SPF-PCK tubes with 1 ml of HBSS media; 7) add 0.3 ml of ferret antisera against A/AA/6/60-7PI which has been treated by the trypsin-periodate method to remove nonspecific inhibitors which has been filter sterilized (0.22μ). Use a 1:32-1:56 final dilution of sera (note that the treated sera is a 1:8 dilution); 8) adsorb at room temperature for 15 min while continuously rocking at low speed; 9) add 1 ml of 2×Eagle's media and incubate at 33° C.; and 10) observe cells daily for CPE. When CPE is >75%, pass the tubes to CK3 (usually 48-72 hr).

SPF-CK3 Passage. The procedure for this passage was identical to the CK2 passage. When the CPE of this passage is >75%, plaque-purify the material in SPF-PCK cells.

Plaque Purification/Genotype Screening

1PI (1st) Plaque Purification. First plaque purification and genotype screening were performed as follows: 1) serially dilute the CK3 passage in 2×Eagle's media through a 10⁻⁴ dilution, one ml of each dilution is needed per flask infected; 2) plaque the 10⁻³ and 10⁻⁴ dilution of each tube at 33° C. following the procedure for plaquing in PCK cells; 3) pick several plaques for each tube. Using a sterile cotton plugged Pasteur pipet which has been bent to a 90° angle remove the agar and cells surrounding a well-isolated plaque. Draw a small volume of HBSS into the Pasteur pipet prior to picking the plaque to facilitate the expulsion of the plaque from the Pasteur pipet. Transfer the plaque material to a sterile capped tube containing 0.5 ml of 2×Eagle's media. One plaque from each tube is passed in SPAFAS eggs and the other plaques should be frozen at −70° C. as backup material; 4) pass one plaque in two SPAFAS eggs (0.2 ml of inoculum per egg) at 33° C. for 72 hr. Refrigerate eggs at 4° C. for at least one hr prior to harvesting the allantoic fluid. Determine the hemagglutinin titer (HA) of the egg pool to confirm the presence of virus and determine plaquing dilutions for the next purification. Two eggs will provide all the virus needed; and 5) genotype the 1 PI egg material following the genotype procedure to identify potential 6/2 candidates.

2PI (2nd) Plaque Purification. Second plaque purification and genotype screening were performed as follows: 1) plaque the 1PI egg material in SPF-PCK cells at 33° C. following the procedure for plaquing in PCK cells, using the following appropriate dilutions to obtain well-isolated plaques:

TABLE 6
HA Titers	Approximate Dilutions
<1:32	10−3 and 10−4
≦1:128	10−4 and 10−5
≦1:512	10−5 and 10−6
>1:512	10−5 , 10−6 and 10−7

2PI plaques should be derived from the same material which is genotyped since the egg passage may exert selective pressure on the plaques; and 2) pick several plaques following the procedure previously described. One plaque from each tube will be replaqued in SPF-PCK cells and the other plaques should be frozen at −70° C. as backup material.

3PI (3rd) Plaque Purification. Third plaque purification and genotype screening were performed as follows: 1) plaque the 2PI plaques in SPF-PCK cells at 33° C. following the procedure for plaquing in PCK cells. The appropriate dilutions for this passage are 10⁻¹ and 10⁻²; 2) pick several plaques following the procedure previously described. At this time you should know which are potential 6/2's and non-candidates can be discarded. One plaque will be amplified in SPAFAS eggs at 33° C. and the others should be frozen at −70° C. as backup material; 3) genotype the 6/2 candidates to confirm that 3PI passages have the 6/2 gene configuration; and 4) characterize the phenotypic profile of the 6/2 vaccine candidates at 25° C., 33° C. and 39° C. to confirm the presence of the ca and ts markers.

B. Type B Reassortants

The following is a procedure for developing Type B 6/2 cold-adapted influenza virus vaccine (CAIV) reassortants.

Materials

Media. The media used in part B of this example were prepared as described in part A above.

Inocula. Inocula of the ca master strain parent and wild type are diluted to 10⁻².

Cells. SPAFAS primary chick kidney (SPF-PCK) were grown as described in part A above.

Passages

SPF-CK1 Passage. SPF-CK1 passages were performed as follows: 1) remove growth media from ten SPF-PCK tubes; 2) wash SPF-PCK tubes with 1 ml of HBSS media; 3) inoculate with 0.3 ml of inoculum per tube; 4) adsorb at room temperature for 90 min while continuously rocking at low speed; 5) remove inoculum; 6) wash SPF-PCK tubes with 1 ml of HBSS media; 7) add 0.3 ml of ferret antisera against B/AA/1/66 CL 4-1-7PI treated by the trypsin-periodate method to remove nonspecific inhibitors and filter sterilized (0.22μ). Use a 1:56 final dilution of sera (note that the treated sera is a 1:8 dilution); 8) adsorb at room temperature for 15 min while continuously rocking at low speed; 9) add 1 ml of 2×Eagle's media and incubate at 25° C.; and 10) observe cells daily for cytopathic effect (CPE). When CPE is >75%, pass the tubes to CK2 (usually 72-96 hr).

SPF-CK2 Passage. SPF-CK2 passages were performed as follows: 1) remove growth media from the SPF-PCK tubes; 2) wash SPF-PCK tubes with 1 ml of HBSS media; 3) serially pass the CK1 passage with 0.3 ml of inoculum per tube; 4) adsorb at room temperature for 90 min while continuously rocking at low speed; 5) remove inoculum; 6) wash SPF-PCK tubes with 1 ml of HBSS media; 7) add 0.3 ml of ferret antisera against B/AA/1/66 CL 4-1-7PI treated by the trypsin-periodate method to remove nonspecific inhibitors which has been filter sterilized (0.22μ). Use a 1:56 final dilution of sera (note that the treated sera is a 1:8 dilution); 8) adsorb at room temperature for 15 min while continuously rocking at low speed; 9) add 1 ml of 2× Eagle's media and incubate at 33° C.; and 10) observe cells daily for CPE. When CPE is >75%, pass the tubes to CK3 (usually 48-72 hr).

Plaque Purification/Genotype Screening

1PI (1st) Plaque Purification. First plaque purifications and genotype screening were performed as follows: 1) serially dilute the CK2 passage in 2×Eagle's media through a 10⁻⁴ dilution, one ml of each dilution is needed per flask infected; 2) plaque the 10⁻³ and 10⁻⁴ dilution of each tube at 33° C. following the procedure for plaquing in PCK cells; 3) pick several plaques for each tube. Using a sterile, cotton-plugged Pasteur pipet which has been bent to a 90° angle, remove the agar and cells surrounding a well-isolated plaque. Draw a small volume of HBSS into the Pasteur pipet prior to picking the plaque to facilitate the expulsion of the plaque from the Pasteur pipet. Transfer the plaque material to a sterile capped tube containing 0.5 ml of 2×Eagle's media. One plaque will be passed in SPAFAS eggs and the others should be frozen at −70° C. as backup material; 4) pass one plaque in two SPAFAS eggs (0.2 ml of inoculum per egg) at 33° C. for 72 hr. Refrigerate eggs at 4° C. for at least one hr prior to harvesting the allantoic fluid. Determine the hemagglutinin titer (HA) of the egg pool to confirm the presence of virus and determine plaquing dilutions for the next purification. Two eggs will provide all the virus needed; and 5) genotype the 1PI egg material following the genotype procedure to identify potential 6/2 candidates.

2PI (2nd) Plaque Purification. Second plaque purification and genotype screening were performed as follows: 1) plaque the 1PI egg material in SPF-PCK cells at 33° C. following the procedure for plaquing in PCK cells. Use the appropriate dilutions to obtain well-isolated plaques, such as the following:

TABLE 7
HA Titers	Approximate Dilutions
<1:32	10−3 and 10−4
≦1:128	10−4 and 10−5
≦1:512	10−5 and 10−6
>1:512	10−5 , 10−6 and 10−7

2PI plaques should be derived from the same material which is genotyped since the egg passage may exert selective pressure on the plaques; and 2) pick several plaques following the procedure previously described. One plaque will be replaqued in SPF-PCK cells and the others should be frozen at −70° C. as backup material.

3PI (3rd) Plaque Purification. Third plaque purification and genotype screening were performed as follows: 1) plaque the 2PI plaques in SPF-PCK cells at 33° C. following the procedure for plaquing in PCK cells. The appropriate dilutions for this passage are 10⁻¹ and 10⁻²; 2) pick several plaques following the procedure previously described. At this time you should know which are potential 6/2's and non-candidates can be discarded. One plaque will be amplified in SPAFAS eggs at 33° C. and the others should be frozen at −70° C. as backup material; 3) genotype the 6/2 candidates to confirm that 3PI passages have the 6/2 gene configuration; and 4) characterize the phenotypic profile of the 6/2 vaccine candidates at 25° C., 33° C. and 37° C. to confirm the presence of the ca and ts markers.

C. Influenza Virus

A number of cold-adapted reassortants and cold-adapted influenza vaccines (CAIV) have been produced and clinically tested using the general scheme set forth above with modifications known to or easily devisable by those skilled in the art without undue experimentation. In addition, the cold-adopted influenza vaccines that have proven efficacious are set forth in Table 10. The following Table sets forth the Type A and Type B reassortants:

TABLE 8
CAIV

	TYPE A	TYPE B
	REASSORTANT	REASSORTANT
	A/Victoria/75 (H3N2)	B/Tecumseh/63/80
	A/Victoria/75 (H3N2)	B/Texas/1/84
	A/Swine/New Jersey/8/76/ (H1N1)	B/Ann Arbor/1/86
	A/Alaska/6/77 (H3N2)	B/Yamagata/16/88
	A/Alaska/6/77 (H3N2)	B/Bangkok/163/90
	A/USSR/90/77 (H1N1)	B/Panama/45/90
	A/Hong Kong/77 (H1N1)	B/Panama/45/90
	A/California/10/78 (H1N1)
	A/Alaska/6/77 (H3N2)
	A/Peking/2/79 (H3N2)
	A/Washington D.C./897/80 (H3N2)
	A/Shanghai/31/80 (H3N2)
	A/Korea/1/82 (H3N2)
	A/Dunedin/6/83 (H1N1)
	A/Bethesda/1/85 (H3N2)
	A/Texas/1/85 (H1N1)
	A/Kawasaki/9/86 (H1N1)
	A/Wyoming/1/87 (H3N2)
	A/Los Angeles/2/87 (H3N2)
	A/Shanghai/11/87 (H3N2)
	A/Shanghai/16/89 (H3N2)
	A/Guizhou/54/89 (H3N2)
	A/Chick/Germany/N/49 (H10N7)
	A/Equine/Miami/1/63 (H3N8)
	A/Beijing/352/89 (H3N2)
	A/Yamagata/32/89 (H1N1)
	A/Texas/36/91 (H1N1)
	A/Beijing/352/89 (H3N2)
	A/Los Angeles/2/87 (H3N2)

SPECIFIC EXAMPLE 4

CA Influenza Virus Reassortant

Vaccine Pools

Facilities. The inoculation, harvesting, pooling, and filling operations were performed in a Biohazard Laminar Flow Hood (Type A/B3). All containers and equipment utilized were sterilized within 72 hr prior to use.

Production Substrate. Ten-day old incubated, specific pathogen free—complement fixation avian leukosis (SPF-COFAL) negative embryonated hens' eggs from SPAFAS, Inc. (Norwich, Conn.) were used. Quality Control Sheets for the flocks were obtained and retained to maintain traceability of eggs.

Cold-adapted Reassortant Vaccine Donor Strain. The cold-adapted reassortant vaccine donor strain passage will vary between SPF egg passage 1 (SE1) and SE4. These passages (SE1-SE4) of the donor virus were produced as follows: The virus was thawed and diluted 1:100 to 1:10,000 (strain-dependent) in HBSS. Ten-day old embryonated eggs were inoculated via the allantoic route with 0.1 ml of the indicated diluent. All eggs were incubated at 33° C. for 40-72 hr (strain-dependent) at which time they were chilled at 4° C. for 1-2 hr prior to the harvesting of the allantoic fluid. This material was passed once to prepare the seed lot.

A. Virus Seed Production

The virus seed lot was used as the seed for the production of all vaccine pools. All work was done in production facilities.

Inoculation. The seed virus was thawed and diluted 1:100 to 1:10,000 (strain-dependent) in HBSS containing 1% of 10×SPG (sucrose, 2.18M; KH₂PO₄, 0.038M; K₂HPO₄, 0.072M; potassium glutamate, 0.049M). The ten-day old embryonated eggs were inoculated via the allantoic route with 0.1 ml of the indicated diluent. All eggs were incubated at 33° C. for 40-72 hr (strain-dependent) at which time they were candled and any dead embryo was discarded. All live eggs were chilled overnight at 4° C. prior to the harvesting of the allantoic fluid.

Harvest and Clarification. Allantoic fluids were harvested and pooled in approximately 180 ml amounts. The harvested allantoic fluid was incubated at 37° C. (water bath) for 60 min to elute any virus adsorbed to red blood cells. Each bottle was then clarified by centrifugation at 1400 g for 15 min. 10×SPG was added to each harvest to achieve a 10% v/v suspension for virus stabilization. The harvest bottles were pooled. Sterility assays were carried out on the pool (dual sterility tests in both fluid thioglycollate and tryptone soya broth at 33° C. and 22° C.). The seed pool was assayed for hemagglutinin activity and aliquotted in the appropriate volumes needed for vaccine production.

B. Virus Pool Production

Inoculation. The seed virus was thawed and diluted 1:100 to 1:10,000 (strain-dependent) in HBSS containing 1% of 10×SPG. The ten-day old embryonated eggs were inoculated via the allantoic route with 0.1 ml of the indicated diluent. For negative controls, approximately 30 eggs were inoculated via the allantoic route with 0.1 ml of the indicated diluent. All eggs were incubated at 33° C. for 40-72 hr (strain-dependent) at which time they were candled and dead embryos were discarded. All live eggs were chilled overnight at 4° C. prior to the harvesting of the allantoic fluid.

Harvest and Clarification. Allantoic fluids were harvested and pooled in approximately 180 ml amounts. The harvested allantoic fluid was incubated at 37° C. (water bath) for 60 min to elute any virus adsorbed to red blood cells. Each bottle (control and infected) was then clarified by centrifugation at 1400 g for 15 min. 10×SPG was added to each harvest to achieve a 10% v/v suspension for virus stabilization. Aliquots were removed from each harvest bottle to form a sample master pool. Sterility assays were carried out on each individual bottle and on the sample master pool; dual sterility tests in both fluid thioglycollate and tryptone soya broth at 33° C. and 22° C. were conducted. The master pool was assayed for hemagglutinin activity and virus characterization (phenotype and genotype assays).

Pooling, Treatment and Dispensation. When the preliminary tests (sterility and virus characterization) proved satisfactory, the sterile harvests were thawed and pooled. Fluids were passed through sterile gauze pads to remove any membranous material that may be present. Antibiotics were added to the final pools to achieve the following concentrations: neomycin 100 mcg/ml, amphotericin B (I.V.) 5 mcg/ml.

Control Fluids: This pool was distributed into the appropriate aliquots needed for subsequent testing for adventitious agents. During dispensation the fluid was kept chilled in an ice-water bath. The fluids were stored at <−75° C. in a mechanical freezer.

Virus-infected Fluids: This pool was distributed into the appropriate aliquots needed for subsequent safety testing. The remainder of the fluid was distributed into aliquots for use as a live cold-adapted influenza virus vaccine. During dispensation the fluid was kept chilled in an ice-water bath. The fluids were stored at <−75° C. in a mechanical freezer.

Tests for Adventitious Agents. The following are microbial sterility tests: 1) pre-antibiotic testing for bacteria with fluid thioglycollate at 22° C. and 33° C., and tryptone soya broth media at 22° C. and 33° C.; and 2) post-antibiotic testing for bacteria in Lowenstein-Jensen egg medium, and for mycoplasma and brucella.

Identity in tissue culture is tested using serum-neutralization in Primary African Green Monkey Kidney (AGMK) cells.

Tissue culture tests for adventitious agents are performed using: 1) Primary African Green Monkey Kidney (AGMK) cells; 2) Primary Bovine Embryonic Kidney (BEK) cells; 3) Primary Human Amnion (PHA) cells; 4) Primary Rabbit Kidney (PRK) cells; 5) Human Diploid Fibroblast (MRC-5) cells; and 6) Human Carcinoma of the Cervix (HeLa) cells.

Animal tests for adventitious agents are performed using: 1) adult mice (ICR); 2) suckling mice (CD-1); and 3) adult guinea pigs. Guinea pig tests are conducted for M. tuberculosis, Q-fever and B. abortus antibodies.

A test for reverse transcriptase by assaying for the detection of RNA-dependent DNA-polymerase activity is also performed.

Final container/pool testing is performed by the following tests: microbial sterility is tested with fluid thioglycollate at 22° C. and 33° C. and fluid soybean-casein digest; COFAL testing is performed to test for avian leukosis virus; general safety testing using mice and guinea pigs; virus characterization including infectivity with TCID₅₀ in Madin-Darby Canine Kidney (MDCK) cells, plaquing efficiency with Madin-Darby Canine Kidney (MDCK) cells with Plaque Forming Unit (PFU) determination at 34, 36, 37, 38 and 39° C., and SPF derived Primary Chick Kidney (SPCK) cells with Plaque Forming Unit (pfu) determination at 25, 33 and 39° C. for confirmation of phenotypic markers; antigenic analyses using hemagglutinin inhibition assay and neuraminidase inhibition assay; reactogenicity in ferrets; hemagglutinin activity; and passage level, wherein the final passage of the vaccine will vary between SPF Egg Passage 3 (SE3) and SE6.

SPECIFIC EXAMPLE 5

Characterization of CA Vaccines

A. CA Vaccine Evaluation

Production lots of cold-adapted influenza vaccines were evaluated prior to distribution to certify that they were identical to the seed strains from which they were produced. The production lots underwent three different tests to certify that they were identical to the seed strains: phenotypic evaluation, genotypic evaluation and ferret reactogenicity studies.

1. A vaccine comprising a reassortant influenza A virion, the virion comprising: a polynucleotide coding for the surface protein HA and a polynucleotide coding for the surface protein NA, each of HA and NA of a selected wild type influenza virus; a polynucleotide coding for PB1; a polynucleotide coding for PA and a polynucleotide coding for M, each PB1, PA and M of a selected cold-adapted influenza virus; and a polynucleotide coding for PB2 which comprises the sequence of SEQ ID NO 15; the polynucleotides being operatively linked to allow packaging of the reassorted polynucleotides into the virion.

2. The vaccine of claim 1, wherein the polynucleotide coding for M is either of SEQ ID NOS 5 or 7; the polynucleotide coding for PB1 is SEQ ID NO 13; and the polynucleotide coding for PA is SEQ ID NO 11.

3. The vaccine of claim 1 wherein the polynucleotide coding for PB2 consists essentially of the sequence of SEQ ID NO 15 and further characterized as consisting of cytosine at nucleotide 1933.

4. The vaccine of claim 1, wherein the polynucleotide coding for PB2 consists essentially of the sequence of SEQ ID NO 15 and further characterized as consisting of guanine at nucleotides 141 and 821 and cytosine at nucleotide 1933.

5. The vaccine of claim 1, wherein the polynucleotide for PB2 consists of the sequence of SEQ ID NO 15.

6. A vaccine comprising a reassortant influenza A virion, the virion comprising: a polynucleotide coding for the surface protein HA and a polynucleotide coding for the surface protein NA, each of HA and NA of a selected wild type influenza virus; a polynucleotide coding for PB1; a polynucleotide coding for PA and a polynucleotide coding for M, each PB1, PA and M of a selected cold-adapted influenza virus; and a polynucleotide coding for PB2 wherein the polynucleotide has a cytosine at a nucleotide which corresponds to nucleotide 1933 of SEQ ID 15, the polynucleotides being operatively linked to allow packaging of the reasserted polynucleotides into the virion.

7. A vaccine of any of claims 1, 2, 3, 4 or 5, further comprising a reassortant influenza B virion.

8. A composition comprising the vaccine of any of claims 3, 4, 5, or 7 and a pharmaceutically acceptable carrier.

9. The composition of claim 8, wherein the composition is formulated for intranasal administration.