WO1996010631A1

WO1996010631A1 - Nucleic acid encoding mutant matrix proteins useful for attenuation or enhancement of influenza a virus

Info

Publication number: WO1996010631A1
Application number: PCT/US1995/012357
Authority: WO
Inventors: Yoshihiro Kawaoka; Maria R. Castrucci
Original assignee: St. Jude Children's Research Hospital
Priority date: 1994-09-30
Filing date: 1995-10-02
Publication date: 1996-04-11
Also published as: AU3727895A

Abstract

Attenuated or enhanced influenza viruses (IAV's) are provided using RNA or DNA encoding mutated matrix proteins (MMPs). Replacing MP nsRNA with nsRNA encoding attenuating MP (AMP) or enhancing MP (EMP) gives attenuated or enhanced IAVs, respectively. Also provided are methods of making and using AMP or EMP DNA or RNA for attenuating or enhancing IAV's.

Description

Nucleic Acid Encoding Mutant Matrix Proteins Useful for Attenuation or Enhancement of Influenza A Virus

Background of the Invention

Related Applications

This application is a continuation-in-part of U.S. Application No. 08/316,419, filed September 30, 1994, which disclosure is entirely incorporated herein by reference.

Statement as to Rights to Inventions Made Under Federally-Sponsored Research and Development

Part of the work performed during development of this invention utilized U.S. Government funds under Grant No. NTH ROI AI29 599 and Grant No. CA 21765 from the National Institutes of Health. The U.S. Government has certain rights in this invention.

Field of the Invention

The present invention, in the fields of virology, molecular biology and vaccines, relates to mutated nucleic acid and encoded mutated matrix proteins, useful for providing attenuated or enhanced influenza A virus (IAV) by the replacement or addition, in an LAV, of a matrix protein encoding negative strand RNA (nsRNA), with either (1) an attenuating nsRNA, encoding an attenuating matrix protein (AMP); or (2) an enhancing nsRNA, encoding an enhanced matrix protein (EMP), to provide, respectively, attenuated IAVs or enhanced IAVs useful for the production of attenuated or enhanced viral cultures. Related Art

Vaccines

Vaccines are preparations administered to animals, including humans, to effect prophylactic or therapeutic treatment of disease states through induction of specific immunity. Prophylactic vaccines are given to healthy individuals with the intention of preparing or priming the immune system for more effective defenses against particular infections in the future. In the event of an infection or infestation by a pathogen, the immune system of a vaccinated individual can usually mount an effective secondary immune response and can more rapidly recognize and eliminate the respective pathogens. Therapeutic vaccines are also given to diseased individuals with the intent of stimulating or modulating the immune system which of itself has either failed to mount an immune response or has mounted an ineffective immune response. In many diseases, the causative pathogens or toxins (e.g. , influenza, polio, and rabies viruses; pneumococcus bacteria; diphtheria and tetanus toxins) can be effectively targeted and neutralized in the extracellular fluid by the mechanisms of humoral immunity through antibodies that bind to the pathogens or toxins and thereby lead to their inactivation or destruction (see, e.g. , Plotkin et al. , Vaccines, Saunders, Philadelphia, 1988). In these cases, prophylactic or therapeutic vaccination with preparations that elicit a humoral immune response is generally sufficient for protection or treatment.

For many years, live, attenuated vaccines have been used to induce immunity against viral infections such as polio. These preparations contain live virions which cause mild, subclinical infections of the vaccinated individuals. In the course of such infections, viral vectors will enter certain host cells and code for the synthesis of virus-specific proteins (Zweerink et al. , Eur. J. Immunol. 7:630, 1977). These endogenously produced antigenic proteins will be processed into smaller 8-9 amino acid peptides and presented as MHC Class I antigens on the cell's surface, thereby eliciting cell-mediated immune responses.

Attenuation of viruses involves mutation or modification of one or more of the genes encoding the non-essential viral proteins. The mutation or modification results in sufficiently reduced infectivity and/or replication ability of the virus to induce an immune response, but not to cause pathologies related to viral infection.

Influenza A Viruses (IAVs) and Current Attenuated live Vaccines

Influenza infection in a host consists of five interrelated steps: entry into the host, primary replication, spread within the host, secondary replication and termination of replication owing to effective host defense mechanisms, including immunity. Successful completion of the first four steps results in disease, whereas the failure of a step results in either limited or nonproductive infection, or even total failure of infection. Influenza A virus (LAV) has eight negative-sense RNA (nsRNA) segments which encode at least 10 polypeptides, including RNA-directed RNA polymerase proteins (PB2, PB1 and PA), nucleoprotein (NP), neuraminidase (NA), hemagglutinin (HA), the matrix proteins (M, and M₂) and non- structural proteins (NS1 and NS2) (Krug et al., "Expression and Replication of the Influenza Virus Genome, " In The Influenza Viruses, R. M. Krug (ed.),

Plenum Press, New York (1989), p. 89-152).

Influenza A viruses are enclosed by lipid envelopes, derived from the plasma membrane of the host cell. The HA and NA molecules are embedded in the envelope by sequences of hydrophobic amino acids (Air et al., Structure, Function, and Genetics (5:341-356 (1989); Wharton et al ,

"Structure, Function, and Antigenicity of the Hemagglutinin of Influenza Virus" in The Influenza Viruses, R.M. Krug (ed.), Plenum Press, New York, (1989), p. 153-174). Within the lipid envelope exists the Ml protein, a major structural protein (Lamb, R. A., Genes and proteins of the influenza viruses In The Influenza Viruses, R. M. Krug (ed.), Plenum Press, New York (1989) p. 1-87).

The viral proteins from different viruses can be recombined or

"reassorted" by co-infection in host cells and selection of particular reassortant viruses by rescue from the infected cells. Accordingly, reassortment of new strains of pathogenic viruses with non-pathogenic viruses can provide attenuated new strains which can be used for vaccination.

Cold-adapted live influenza vaccines have been extensively investigated and potentially hold promise for use in the general population (Sears, S.D. et al. , J. Infect. Dis. 158: 1209-1219 (1988); Steinhoff, M.C. et al. , J. Infect.

Dis. 765:1023-1028 (1991); Steinhoff, M.C. et al. , J. Infect. Dis. 162:394- 401). The major concern with these vaccines is that the limited number of attenuating mutations (Cox, N.J. et al. , Virology 167:554-567 (1988); Herlocher, M.L. et al. , Proc. Natl. Acad. Sci. USA 90:6032-6036 (1993)) could permit the generation of a revertant virus in the field.

However, much of the cost associated with influenza epidemics in the U.S. originates from suboptimal immune responses to vaccination using such attenuated vaccines. Although inactivated vaccines reduce the frequency and severity of influenza, they do not provide the same high level of protection as seen in population with natural immunity. This fact is well-illustrated by the

Russian influenza epidemic of 1977, in which an H1N1 virus (genetically almost identical to that circulating in humans in 1950 (Nakajima, K. et al. , Nature (London) 274:334-339 (1978); Scholtissek, C. et al. , Virology 89:613- 617 (1978)) produced disease largely in persons who were born after the original H1N1 virus had disappeared from humans in 1957 (Kung, H.C. et al. , Bull. W.H.O. 56:913-918 (1978)). This episode also demonstrates the persistence of immunity conferred by natural influenza infection (up to 20 years), providing a strong rationale for the development of live influenza vaccines. Additionally, the production of attenuated viruses has been plagued by the problems associated with attenuated viral replication in culture which is necessary to provide sufficient quantities of attenuated virus for use in vaccines, and the lack of specificity of vaccines for currently active strains. The direct medical costs for treating influenza in the U.S. exceed $4.6 billion each year. Lost productivity because of days lost from work costs well over $11.6 billion each year. These bleak statistics generally reflect our inadequate understanding of influenza virus structure and how its subtle molecular changes can promote resistance to vaccines and antiviral drugs. Although the NA and M gene products are known to contribute to the virulence of influenza A viruses, the molecular mechanisms of these effects are largely unknown. Accordingly, it would be useful to provide methods and/or modified or enhanced helper viruses which would provide enhanced growth of attenuated viruses in culture. In addition, enhanced growth would provide superior methods and results for the production of attenuated influenza A viruses, useful as vaccines.

Replication of Influenza Virus

From attachment to uncoating. An influenza virus particle binds to cells via interaction between the receptor binding site of the HA and the terminal sialic acid of the cell surface receptor. After binding, the attached virion undergoes endocytosis. The low pH of the endocytotic vesicle triggers a conformational change in the cleavage-activated HA, initiating fusion of the viral and vesicular membranes. Fusion releases the contents of the virion (ribonucleoprotein complex; RNP) into the cytoplasm of the cell (uncoating). Prior to fusion, M2 proteins, by ion channeling, introduce protons into the inside of the virion, exposing the core to low pH. Such an event is thought to allow the Ml protein to dissociate from the RNP, disrupting their low pH- sensitive interaction (Zhirnov, O.P., Virology 176:274-279 (1990)), and allowing the migration of RNP to the nucleus (Helenius, A., Cell 69:577-57% (1992)). Transcription and translation. Once the RNP migrates into the host cell nucleus, the associated polymerase complexes begin primary transcription of mRNA. The primary transcripts are then used for translation of early viral proteins (e.g. , NP, NSl, PA, PBl, PB2). Later in infection, the principal translation products are Ml, HA, and NA proteins. HA and NA proteins are post-translationally processed and transported to the cell surface, where they integrate into the cell membrane.

Virion morphogenesis and budding. Ml protein transported into the nucleus is associated with the migration of RNP out of the nucleus for assembly into progeny viral particles in the cytoplasm. Few details of the assembly process are known. Presumably, RNP in association with the Ml protein buds outward through the cell membrane. Interactions between Ml and the cytoplasmic domains of HA, NA, or M2 have been proposed as signals for budding, but direct evidence for this relationship is lacking. Because influenza virus particles are formed at the cell surface, morphogenesis is regarded as part of the budding process. A virus could initiate morphogenesis but not complete budding. Thus, morphogenesis is defined as "the generation of virus particles", and budding as "the generation and release of virus particles".

Reverse genetics

Several research groups have established a system (reverse genetics) for incorporating cloned genes into negative-strand RNA viruses. Using this technique, Palese and colleagues generated influenza viruses (designated as transfectant viruses) with genes derived from cloned cDNA. This method involves (i) preparation of RNA, containing exactly the same 5' and 3' sequences as viral RNA, from cloned influenza virus genes with RNA polymerase, (ii) encapsulation of the RNA with influenza virus NP and polymerase proteins, (iii) transfection of the encapsulated RNA, and (iv) infection with a helper influenza virus to rescue the transfected RNA Luytjes et al., Cell 59:1107-1113 (1989); Enami et al., J. Virol. 65:2711- 2713 (1991).

The Matrix Gene

The matrix (M) gene of IAVs encodes two matrix proteins (MPs), Ml and M2 (Lamb, R. A. "Genes and Proteins of the Influenza Viruses, " p. 1-87.

In R.M. Krug (ed.), The Influenza Viruses. Plenum Press, New York (1989)).

Ml protein. Dogma holds that the Ml protein underlies and adds rigidity to the lipid bilayer, but direct evidence for this role is lacking. Immunogold labeling with anti-Ml monoclonal antibodies detectably labeled

Ml in virions when first treated with a protease or a detergent (Murti et al., Virology 186:294-299 (1992)). Recent cryoelectron microscopy studies suggested that the Ml can modify the lipid bilayer, causing thickening of the viral envelope (Fujiyoshi et al., EMBO J. 5:318-326 (1994)). Ml appears to escort RNP from the nucleus to the cytoplasm (Helenius, A., Cell 69:577-

578 (1992)). The Ml proteins of influenza A and B viruses have a "zinc- finger" motif (Wakefield et al, Nucl. Acids Res. 77:8569-8580 (1989)), and purified virus contains zinc; however, the amount of zinc is not correlated with its RNA binding activity, implying that zinc binding is not important for this activity. Although serine residues between amino acids 108 and 126 of

WSNMl arephosρhorylated (Gregoriadese<^* α/., Virus Res. 76:27-42 (1990)), the role of this property in viral replication is unknown. The influenza B Ml protein is also phosphorylated, suggesting its role in viral replication.

M2 protein. This integral membrane protein (Zebedee et al., J. Virol. 62:2762-2772 (1988)) is a homotetramer (Holsinger et al, Virology 755:32-43

(1991); Panayotov et al, Virology 186:352-355 (1992); Sugrue et al, Virology 180:617-624 (1991)) that is abundantly expressed at the surface of virus-infected cells but is a relatively minor component of virions (Zebedee et al, J. Virol 62:2762-2772 (1988)). Sharing eight ammo-terminal residues with Ml, the M2 protein comprises 97 amino acids, 24 as the ecto-, 19 as the transmembrane, and 54 as the cytoplasmic domain. With the exception of those in H3N8 equine viruses, M2 proteins are palmitoylated at Cys-50 (Sugrue et al, Virology 179:51-56 (1990)). The M2 protein is also phosphorylated at Thr-65 (Hay et al, EMBO J. 4:3021-3024 (1985)). The

M2 proteins have been proposed to function as an ion channel that permits protons to enter the virion during uncoating and that modulates the pH of intracellular compartments, an essential function for prevention of, the acid- induced conformational change of the intracellularly cleaved HA in the trans¬ Golgi network (Hay et al. , EMBO J. 4:3021-3024 (1985)). The activity of the M2 ion channel is blocked by the anti-influenza drug amantadine hydrochloride. The functional role of the M2 cytoplasmic region, the longest among the influenza viral membrane proteins, is unknown.

Citation of documents herein is not intended as an admission that any of the documents cited herein is pertinent prior art, or an admission that the cited documents is considered material to the patentability of any of the claims of the present application. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.

Summary of the Invention

The present invention is directed to overcoming one or more deficiencies of the related arts.

The present invention provides attenuated or enhanced influenza viruses (IAV's) using RNA or DNA encoding mutated matrix proteins (MMPs).

Replacing MP nsRNA with nsRNA encoding attenuating MP (AMP) or enhancing MP (EMP) gives attenuated or enhanced IAVs, respectively. Also provided are methods of making and using AMP or EMP DNA or RNA for attenuating or enhancing IAV's. The present invention provides mutant nucleic acid, derived from, or corresponding to, influenza A virus (LAV) matrix genes, encoding mutant matrix protein (MMP). The mutant nucleic acid is provided as either (i) attenuating nucleic acid encoding an attenuating matrix protein (AMP); or (ii) enhancing nucleic acid encoding an enhanced matrix protein (EMP), which

MMP is capable of either attenuating or enhancing, respectively, an influenza A virus (LAV) having at least one AMP or EMP encoding nucleic acid. The nucleic acid can be DNA or RNA, such as negative strand RNA (nsRNA). An AMP of the present invention has at least one attenuating mutation which confers attenuating activity, as described herein and/or as known in the art.

An EMP of the present invention has at least one enhancing mutation which confers growth enhancing activity, as described herein and/or as known in the art.

The present invention also provides attenuated IAVs, and isolated forms thereof, which comprise an pathogenic nsRNA encoding at least one neuraminidase (NA) and hemagglutinin (HA) from at least one pathogenic IAV; and at least one attenuating nsRNA encoding an AMP having at least one attenuating mutation.

Attenuated IAVs of the present invention are suitable for use as live, attenuated flu vaccines. An attenuated IAV of the present invention is preferably capable of inducing an immune response in an animal to at least one pathogenic IAV strain, which response causes a subclinical LAV infection in the animal, as compared to a clinical LAV infection when a native or non- attenuated pathogenic IAV infection occurs. The at least one pathogenic NA and HA, encoded by the pathogenic nsRNA in an attenuated IAV of the present invention, is derived from at least one pathogenic AV strain.

An AMP, used in an attenuated virus of the invention, can comprise at least one selected from an Ml AMP and an M2 AMP, wherein the at least one attenuating mutation can inhibit the level of at least one of transcription, replication, translation or virion incorporation of an attenuated IAV of the present invention, when the LAV infects a host cell. The attenuating mutation can encode at least one amino acid modification selected from the group consisting of a substitution, a deletion and an insertion.

An attenuated virus according to the present invention can further comprise a selection marker selected from the group consisting of a drug resistance marker, a temperature sensitive marker, and an antigenic marker. The present invention also provides a vaccine composition comprising an attenuated IAV of the present invention, and a pharmaceutically acceptable carrier or diluent. The vaccine composition can further comprise an adjuvant which enhances an IAV immune response to the pathogenic virus in an animal administered the vaccine composition.

The present invention also provides a method for obtaining an attenuated IAV capable of being used as a vaccine for at least one pathogenic

LAV strain, the method comprising isolating an attenuated IAV which comprises (1) a pathogenic nsRNA encoding at least one NA and HA from at least one pathogenic IAV; and (2) at least one attenuating nsRNA encoding an

AMP having at least one attenuating mutation.

Such a method according to the present invention can further comprise, prior to the isolating step, a further step of reassorting, in a host, (i) a helper virus having (1) the attenuating nsRNA and (2) sensitivity to at least one selection marker; with (ii) at least one pathogenic nsRNA encoding (1) at least one neuraminidase (NA) and hemagglutinin (HA) from the at least one pathogenic IAV strain; and (2) resistance to the selection marker, to provide the attenuated influenza A virus. The host may be selected from a prokaryotic and a eukaryotic cell, with mammalian cells preferred. The selection marker can be selected from a group consisting of a drug resistance marker, a temperature sensitive marker and an antigenic marker.

A method for obtaining an attenuated LAV of the present invention may further comprise removing the resistance to the selection marker from the attenuated LAV to provide a sensitive attenuated IAV lacking resistance to the selection marker.

The method optionally further comprises selecting the attenuated LAV using an antibody binding an epitope specific for said atenuated AV. The antibody is selected from polyclonal, monoclonal or a fragment thereof.

The present invention further provides a method for vaccinating an animal against an IAV strain, comprising administering to the animal an LAV immune response effective amount of a vaccine composition comprising an attenuated LAV of the present invention. The present invention also provides a method for eliciting an immune response to an IAV in an animal which is prophylactic or therapeutic for an LAV infection, the method comprising administering to an animal a vaccine composition comprising an attenuated influenza A virus of the present invention, which is protective for the animal against a clinical LAV pathology caused by infection of at least one IAV strain.

An enhancing nucleic acid of the present invention, can comprise nucleic acid encoding at least one Ml EMP or M2 EMP, wherein at least one enhancing mutation in an enhancing nucleic acid can stimulate the level of at least one of transcription, replication, translation or virion incorporation of an enhanced IAV of the present invention, e.g. , when the IAV infects a host cell.

The enhancing mutation can encode at least one amino acid modification selected from the group consisting of a substitution, a deletion and an insertion.

An enhanced LAV of the present invention can further comprise a selection marker selected from the group consisting of a drug resistance marker, a temperature sensitive marker and an antigenic marker.

The present invention also provides a method for obtaining an enhanced IAV, the method comprising isolating an enhanced IAV which comprises at least one enhancing nsRNA encoding an EMP having at least one enhancing mutation which is capable of enhancing the growth of the enhanced

IAV. Such a method according to the present invention can further comprise, prior to the isolating step, a step of reassorting, in a host, (i) a helper virus having (1) the enhancing nsRNA and (2) sensitivity to at least one selection marker; with (ii) at least one nsRNA encoding (1) at least one neuraminidase (NA) and hemagglutinin (HA) from the at least one LAV strain; and (2) selection resistant nsRNA encoding at least one protein conferring resistance to the selection marker to provide the enhanced LAV.

The method optionally further comprises selecting the enhanced IAV using an antibody binding an epitope specific for said enhanced LAV. The antibody is selected from polyclonal, monoclonal or a fragment thereof.

The NA and HA in such a method can be derived from at least one IAV strain. The enhancing nsRNA can encode at least one of an Ml EMP and an M2 EMP. The enhancing mutation can encode at least one animo acid modification selected from the group consisting of a substitution, a deletion and an insertion. The enhanced IAV can be selected from any known or discovered IAV strain. The host may be selected from a prokaryotic and a eukaryotic cell. The selection marker can be selected from a group consisting of a drug resistance marker, a temperature sensitive marker and an antigenic marker. A method for obtaining an enhanced IAV of the present invention may further comprise removing the resistance to the selection marker from the enhanced LAV to provide a sensitive enhanced IAV lacking resistance to the selection marker.

Other objects, features, advantages, utilities and embodiments of the present invention will be apparent to skilled practitioners from the following detailed description and examples relating to the present invention. Brief Description of the Figures

Fig. 1. Amino acid sequence of Ml consensus sequence (SEQ ID NO:l).

Fig. 2. Amino acid sequence of M2 consensus sequence (SEQ ID NO:2).

Fig. 3. Amino acid and nucleotide sequences of M2 mutants (SEQ ID NOS.4-11). The C-terminal portion of the M2 protein is shown. Mutated nucleotides to introduce a stop codon are underlined. Plus (+) and minus (-) signs signify that viruses with the indicated M2 protein were not generated. Fig. 4. Nucleotide sequence of an M gene of one strain of IAV (SEQ

ID NO:3) and two exemplary variants thereof, as known in the art. See, e.g. , Ortin et al. Gene 25:233-239 (1983), which is entirely incorporated herein by reference.

Detailed Description of the Invention

It has now been discovered that the matrix gene of influenza A viruses

(IAVs) can be specifically mutated as mutant nucleic acids, which encode mutant matrix proteins (MMPs). An MMP can be either an attenuating matrix protein (AMP) or an enhancing matrix protein (EMP). An AMP or EMP is sufficient in itself to either attenuate or enhance, respectively, the growth of an LAV containing the mutant nucleic acid, as a negative strand RNA

(nsRNA).

Accordingly, the present invention relates to mutant nucleic acid (e.g. , cDNA or nsRNA) as attenuating nucleic acid, encoding AMPs, or enhancing nucleic acid, encoding EMPs, and to compositions, vaccines, mutant LAV and methods of making and using thereof.

The present invention also relates to attenuated IAVs comprising both (i) pathogenic nsRNA encoding at least one NA and HA and (ii) attenuating nsRNA encoding an AMP having at least one attenuating mutation, and to methods of making and using thereof. The now discovered attenuated IAVs and methods provide a utility for attenuating newly found pathogenic strains of LAV, e.g. , as attenuated IAVs, to be used as live, attenuated vaccines. In a preferred embodiment, an attenuated IAV of the present invention induces an immune response in an animal infected with the attenuated virus, but the infection is subclinical, such that the infection is suitable for vaccination purposes.

The present invention also relates to enhanced IAVs comprising enhancing nsRNA encoding at least one EMP having at least one enhancing mutation, and to methods of making and using thereof.

The present invention also provides high titers of IAV using specific matrix gene enhancing mutations, encoding EMPs, which mutations facilitate inactivated vaccine production by providing a high growth master strain, superior to those presently used.

Virulence of IAVs

In the context of the present invention, the term "virulence" is intended to mean the capacity of a virus, compared to other closely related viruses, to produce disease in a host (Tyler et al, "Pathogenesis of Viral Infections" In Virology, Fields et al., (eds.) Raven Press, Ltd., New York (1990), pp. 191-

239). Thus, more efficient virus replication should directly influence virulence by promoting spread of the pathogen within the host, as well as secondary replication, ultimately overwhelming host defense mechanisms. Accordingly, attenuated IAVs of the present invention have reduced virulence to the extent that they produce subclinical infections while still eliciting an

LAV specific immune response in a vaccinated animal.

High yield property. The term "high yielding" as used in the literature can be ambiguous, referring to either high HA or high infectivity titers assayed in either eggs or tissue culture. In the context of the present invention, "enhancing" viruses are defined as those capable of producing high infectivity titers in in vitro replication systems (e.g. , tissue culture). A non- limiting example of such a culture is one using mammalian cells, e.g. , Madin- Darby canine kidney (MDCK) cells. Different influenza viruses show different infectivity titers. For example, the WSN and A/Puerto Rico/8/34

(H1N1)(PR8) strains, both of which are lethal to mice, produce high infectivity titers (approximately 10⁹ plaque-forming units (PFUs)/ml) in MDCK cells in contrast to ordinary viruses (e.g. , A/Aichi/2/68 (H3N2)(Aichi); approximately 10⁷ PFUs/ml). A recent study showed a correlation between the increased virulence of A/FM 1/47 (HlNl) virus in mice and its high-yield property in vitro (Smeenk et al, J. Virol. 65:530-534 (1994)). The amino acid residues potentially responsible for the high growth of influenza viruses have been investigated using comparisons between high- and low-growth viruses (Klimov et al, Virus Res. 19: 105-114 (1991); Yasuda et al, Arch. Virol 755:283-294 (1993)).

Filamentous vs spherical particles. Influenza virus particles are known to be pleomorphic (Hoyle, L., "Morphology and Physical Structure" In The Influenza Viruses, Springer- Verlag, New York (1968), pp. 49-68); however, clinical isolates of early passages in eggs or tissue cultures contain more filamentous than spherical particles, whereas laboratory strains passaged extensively in such cultures contain spherical virions predominantly (Hoyle, L., "Morphology and Physical Structure" In The Influenza Viruses, Springer- Verlag, New York (1968), pp. 49-68). The filamentous virions possess many of the serologic, hemagglutinating and enzymatic characteristics of the spherical particles. The M gene is now discovered to be a major determinant of this morphologic difference. The morphologic change and general reduction of virulence in many viruses during passage in vitro suggest an association of the shape of influenza virus with virulence.

In general, viruses produce higher infectivity titers but become less virulent after repeated passaging in vitro, whereas the high-yield phenotype correlates with increased virulence (Smeenk et al, J. Virol. 65:530-534 (1994)) and a filamentous virion shape with high infectivity titers. This seeming contradiction originates from the polygenic nature of viral replicative efficiency. During adaptation to in vitro passage, multiple viral genes mutate, affecting the growth of the virus in ways that obscure the direct relationship between high infectivity titers and virulence.

It has now been discovered that mutations in the M gene, as at least one nsRNA encoding an Ml or an M2 matrix protein (MP), provide attenuation or enhanced growth of influenza A virus, such that live IAV reassortant and/or reverse genetics produced viruses and vaccines, having attenuation or growth enhancement can be provided according to the present invention.

Mutant Matrix .Protein (MMP) as Attenuating Matrix Protein (AMP) and Enhanced Matrix Protein (EMP)

An MMP, as an AMP or EMP, according to the present invention, can refer to any AMP or EMP, or a subset thereof, which is capable of replacing a matrix protein (MP), or nsRNA encoding therefor, in an IAV to provide an attenuated IAV (using an AMP or encoding nsRNA) or an enhanced IAV

(using an EMP or encoding nsRN A) according to the present invention, where the AMP or EMP has at least one amino acid substitution, deletion or insertion of a wild type MP form or variant which provides attenuating (AMP) or enhancing (EMP) activity. MMPs, as AMPs or EMPs are incorporated into a virus by use of mutant nucleic acid encoding therefore, e.g. , nsRNA.

Such nsRNA can be provided using genetic engineering to manipulate the corresponding cDNA. The cDNA can thus be mutated to provide mutant cDNA-encoding attenuating or enhancing mutations, as part of an attenuating or enhancing nucleic acid.

Any subset of an AMP or an EMP, e.g. , as a peptide fragment of an AMP, (according to the present invention) can be prepared by recombinant DNA methods discussed in more detail below, and/or by any other method capable of producing an attenuating or enhancing nucleic acid encoding, respectively, an AMP or EMP having a conformation similar to an attenuating (AMP) or enhancing (EMP) portion of an MP and having attenuating or enhancing activity, according to suitable screening assays, e.g. , as described herein and/or as known in the art. The minimum peptide or encoding nucleic acid sequence to provide an AMP or EMP having attenuating or enhancing activity is based on the smallest unit containing or comprising a particular domain, consensus sequence, or repeating unit thereof of an MP having attenuating or enhancing activity. Accordingly, an MMP of the present invention alternatively includes polypeptides having attenuating or enhancing activity as comprising a portion of an MP amino acid sequence which substantially corresponds to at least one 10-252 or 43 to 252 amino acid fragment and/or consensus sequence of a known wild type MP variant or group of MP variants. Such an MMP can have homology of at least 80% to a known MP, such as 80-99% homology, or any range or value therein, while providing attenuating or enhancing activity. An AMP or EMP, or encoding nucleic acid, of the present invention is not naturally occurring or is naturally occurring but is in a purified or in an isolated form which does not occur in nature. Preferably, an MMP of me present invention substantially corresponds to a MP domain of an MP or a group of MPs, as a consensus sequence, such as a consensus sequence of 2 or more naturally occurring wild type variants of M2 or Ml proteins.

Percent homology may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (J. Mol Biol 48:443 (1970), as revised by Smith and Waterman (Adv. Appl. Math. 2:482 (1981). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e. , nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences. The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745 (1986), as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.

In a preferred embodiment, the AMP of the present invention is a mutant form of at least one Ml or M2 MP.

The following Table 1, presents alternative variants of Ml matrix protein, which can be used or mutated to provide AMPs or EMPs, or nucleic acid encoding therefor, according to present invention. See, e.g. , Ito et al., J. Virol. 65:5491-5498 (1991), which is entirely incorporated herein by reference to alternative MP sequences.

TABLE 1: Ml MATRIX PROTEIN VARIANTS

(1-61 of SEQ ID NO: 1)

CONSENSUS MS LTEVETYVLSIVPSGPLKAEIAQRLEDVFAGKNTD EALMEWLKTRPILSPLTKGILG

M1_EP N

M1_FPVR

M1_FPV V V

M1_PR8 I V

M1_AA60 I

M1_S57

M1_K68

M1_M88 1_BK

M1_A68

M1_FW l_OD

M1_PC

M1_WS

M1_SI30-SJ

M1_SM54

M1_SW61

M1_SMS2

M1_STN

M1_SI88

M1_SW37

M1_WI

Ml_SO

M1_GM

M1_GM79

M1_TM833

Ml GM78 Ml TM81 1

Ml PINALB 1

Ml CV85-C0 1

Ml ETN 1

Ml SHK 1

Ml BH 1

Ml CKP-COP 1 V

Ml CKPAV 1 V

Ml SN 1

Ml DC 1

Ml MNY 1

Ml EK 1

Ml_FPVDOB 1

Ml WSN 1

M1_USSR 1

(62 - 122 OF SEQ ID NO : l )

CONSENSUS 62 FVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDRAVKLYRKLKREITFHGAKEVALSYSAG

Ml EP 62 K I K DV G T

Ml FPVR 62 K Y T

Ml FPVW 62 K Y T

Ml PR8 62 K

Ml AA60 62

Ml S57 62

Ml K68 62

Ml M88 62

Ml BK 62

Ml A68 62

Ml FW 62

Ml UD 62

Ml PC 62

Ml HS 62 K

Ml SI30-SJ 62 K

Ml SM54 62 c K

Ml SW61 62 c K

Ml SM52 62 c K

Ml STN 62 K

Ml SI88 62 K

Ml SW37 62 K

Ml WI 62 K

Ml SO 62 K T

Ml GM 62 T

Ml GM79 62 T

Ml TM833 62 T

Ml GM78 62 T

Ml TM81 62 T

Ml PINALB 62 T

Ml CV85-CO 62 T

Ml ETN 62 T

Ml SHK 62 T

Ml BH 62 T

Ml CKP-COP 62 T

Ml CKPAV 62 T

Ml SN 62 T

Ml DC 62 T

Ml MNY 62 T

Ml EK 62 T

Ml FPVDOB 62 T

Ml WSN 62

M1_USSR 62

CONSENSUS 1 12233 AIASCMGLIYNRMGTVTTEVAFGI.VCATCEQIADSQHRSHRQMVTTTNPLIRHENRMVLAS

(123 -181 OF SEQ ID NO : l )

Ml EP 123

Ml FPVR 123

Ml FPVW 123

Ml PR8 123 A

Ml AA60 123 A VL

Ml S57 123 A Ml K68 123 A

Ml M88 123 A A

Ml BK 123 A A

Ml A68 123 A

Ml FW 123 A

Ml UD 123 A A

Ml PC 123 A A

Ml WS 123 A

Ml SI30-SJ 123

Ml SM54 123

Ml SW61 123

Ml SM52 123

Ml STN 123

Ml SI88 123

Ml SW37 123

Ml WI 123

Ml SO 123

Ml GM 123

Ml GM79 123

Ml TM833 123

Ml GM78 123

Ml TM81 123

Ml PINALB 123

Ml CV85-CO 123

Ml ETN 123

Ml SHK 123

Ml BH 123

Ml CKP-COP 123

Ml CKPAV 123

Ml SN 123

Ml DC 123

Ml MNY 123

Ml EK 123

Ml FPVDOB 123

Ml WSN 123 A

M1_USSR 123 A

CONSENSUS 1 18844 ITTAKAMEQMAGSSEQAAEAMEVASQARQMVQAMRTGTHPSSSAGLKDDLLENLQAYQKRM

( 184-252 OF SEQ ID NO : l)

Ml EP 184 I N V N T

Ml FPVR 184

Ml FPVW 184

Ml PR8 184 N

Ml AA60 184 V N

Ml S57 184 A RA C

Ml K68 184 A P

Ml M88 184 A T

Ml BK 184 A T

Ml A68 184 A R

Ml FW 184 A R

Ml UD 184 A

Ml PC 184 A

Ml WS 184 DI

Ml SI30-SJ 184 I

Ml SM54 184

Ml SW61 184

Ml SM52 184

Ml STN 184

Ml SI88 184

Ml SW37 184

Ml WI 184

Ml SO 184

Ml GM 184

Ml GM79 184

Ml TM833 184

Ml GM78 184

Ml TM81 1Θ4

Ml PINALB 184

Ml CV85-CO 184

Ml ETN 184 Ml SHK 184 H R

Ml BH 184 H R G

Ml CKP-COP 184 H R

Ml CKPAV 184 H R

Ml SN 184 H

Ml DC 184 R

Ml MNY 184

Ml EK 184 T V K

Ml FPVDOB 184 R V

Ml WSN 184 DI V

M1_USSR 184 EV Al N

CONSENSUS 245 GVQMQRFK

Ml EP 245

Ml FPVR 245

Ml FPVW 245 L

Ml PR8 245

Ml AA60 245

Ml S57 245

M1^*~K68 245

Ml M88 245 V

Ml BK 245

Ml A 8 245

Ml FW 245

Ml UD 245

Ml PC 245

Ml WS 245

Ml SI30-SJ 245

Ml SM54 245

Ml SW61 245

Ml SM52 245

Ml STN 245 R

Ml SI88 245

Ml SW37 245

Ml WI 245

Ml SO 245

Ml GM 245

Ml GM79 245

Ml TM833 245

Ml GM78 245

Ml TM81 245

Ml PINALB 245

Ml CV85-CO 245

Ml ETN 245

Ml SHK 245

Ml BH 245

Ml CKP-COP 245

Ml CKPAV 245 I

Ml SN 245 I

M1~DC 245 I

Ml MNY 245

Ml EK 245

Ml FPVDOB 245 L

Ml WSN 245

Ml USSR 245

The following Table 2, presents alternative variants of M2 matrix protein, which can be used or mutated to provide AMPs or EMPs, or nucleic acid encoding therefor, according to present invention. See, e.g., Ito et al, J. Virol 65:5491-5498 (1991), which is entirely incorporated herein by reference to alternative MP sequences. TABLE 2: M2 MATRIX PROTEIN VARIANTS

(1-61 of SEQ IDNO:2)

CONSENSUS 1 MMSS:LLTEVETPIRNEWGCRCNDSSDPLVIAASIIGILHLILWILDRLFFKCIYRRLKYGLKR

M2 PR8 1 N F

M2 A68 1 V FFEH

M2 PC 1 V FFEH

M2 UD 1 V FFEH

M2 S57 1 V FF H

M2 BK 1 V FF H

M2 K68 1 V FF H

M2 AA60 1 V FF H

M2 FW 1 V F H

M2 M88 1 V LF

M2 USSR 1 V LF

M2 WS 1 N F

M2 WSN 1 N F

M2 SO 1 A F

M2 SW37 1 A E

M2 SM52 1 A E

M2 SM54 1 A E

M2 SW61 1 A F

M2 SI30-SJ 1 A

M2 SI88 1 K AV F

M2 WI 1 K A F

M2 STN 1 S A F

M2 FPVR 1 G

M2 MNY 1 G S

M2 CV85-CO 1 G S

M2 SHK 1 G S L

M2 TM81 1 G S L

M2 SN 1 G FS L

M2 FPVDOB 1 G S S

M2 TM833 1 G K S

M2 ETN 1 G K SG F G

M2 EK 1 G K SG F L

M2 PINALB 1 G K S

M2 BH 1 TK GWE £ S RLFFKCIYRRLKYGLKR

M2 FPVW 1 T GWE 3 S RLFFKCIYRRLKYGLKR

M2 DC 1 T GWE YSG RLFFKCIYRCLKHGLKR

M2 GM 1 HT SGWE RLFFKCIYRRLKYGLKR

M2 GM79 1 HT SGWE RLFFKCIYRRLKYGLKR

M2 GM78 1 HT SGWE F RLFFKCIYRRLKYGLKR

M2 CKP-COP 1 LT GWE K S I FYRLFFKCIYRRLKYGLKR

M2 CKPAV 1 LT GWE KYS I RLFFKCIYRRLKYGLKR

M2_EP 1 KSGWE L Al F RLFFKCAYRRFRHGLKR

CONSENSUS 62 GPSTEGVPESMREEYRQEQQSAVDVDDGHFVNIELE (62-97 OF SEQ ID NO:2)

M2 PRβ 62 K

M2 A68 62 K A

M2 PC 62 K A

M2~ ^"UD 62 K A

M2 S57 62 K A

M2 BK 62 K A

M2 K68 62 K A

M2 AA60 62 K A

M2 FW 62 K A

M2 MB8 62 K N A

M2 USSR 62 K N A

M2 WS 62 K N

M2 WSN 62 K N

M2 SO 62 K

M2 SW37 62 K

M2 SM52 62 L K

M2 SM54 62 L K

M2 SW61 62 K

M2 SI30- ■SJ 62 K

M2 SI8B 62 K

M2 WI 62 K M2 STN 62 K

M2 FPVR 62

M2 MNY 62

M2 CV85-CO 62

M2 SHK 62

M2 TM81 62

M2 SN 62

M2 FPVDOB 62 N

M2 TM833 62 N

M2 ETN 62 N

M2 EK 62 N

M2 PINALB 62 S

M2 BH 62 V

M2 FPVW 62 K N

M2 DC 62 N

M2 GM 62 Q K

M2 GM79 62 Q K

M2 GM78 62 Q K

M2 CKP-COP 62 A V

M2 CKPAV 62 A V

M2 EP 62 G I D N N

Thus, one of ordinary skill in the art, given the teachings and guidance presented herein, will know how to substitute other amino acid residues in other positions of an MP to obtain an AMP or EMP, including substituted, deletional or insertional variants.

An MMP of the present invention also includes mutants of MP variants, wherein at least one amino acid residue in the polypeptide has been replaced, inserted or deleted by at least one different amino acid, which mutation either confers attenuation by the resulting AMP, or confers enhancement by the resulting EMP, on an IAV strain containing the AMP or

EMP, or nucleic acid encoding therefor.

An amino acid or nucleic acid sequence of an MMP of the present invention is said to "substantially correspond" to an MP amino acid or nucleic acid sequence respectively, if the sequence of amino acids or nucleic acid in both molecules provides polypeptides having biological activity that is substantially similar, qualitatively or quantitatively, to the corresponding fragment of at least one MP domain, but where the MMP sequence also has attenuating or enhancing activity. Such "substantially corresponding" MMP sequences include conservative amino acid or nucleotide substitutions, or degenerate nucleotide codon substitutions wherein individual amino acid or nucleotide substitutions are well known in the art. Accordingly, MMPs of the present invention, or nucleic acid encoding therefor, include a finite set of substantially corresponding sequences as substitution peptides or polynucleotides which can be routinely obtained by one of ordinary skill in the art, without undue experimentation, based on the teachings and guidance presented herein. For a detailed description of protein chemistry and structure, see Schulz, G.E. et al., Principles of Protein Structure, Springer- Verlag, New York, 1978, and Creighton, T.E., Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, 1983, which are hereby incorporated by reference. For a presentation of nucleotide sequence substitutions, such as codon preferences, see Ausubel et al. , eds, Current Protocols in Molecular Biology, Greene Publishing Assoc, New York, NY (1987, 1988, 1989, 1990, 1991, 1992, 1993, 1994, 1995) at §§ A.l.l-A.1.24, and Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), at Appendices C and D.

Amino Acid Substitutions of MMPs Alternative or in Addition to Attenuating or Enhancing Mutations. Substitutions of an AMP or EMP of the present invention include, alternatively or in addition to attenuating or enhancing mutations, substitutions of at least one amino acid residue which has been replaced, inserted or deleted by at least one different amino acid.

Such substitutions preferably are made in accordance with the following list as presented in Table 3, which substitutions can be determined by routine experimentation to provide modified structural and functional properties of a synthesized polypeptide molecule, while maintaining MP, attenuating and/or enhancing biological activity, as determined by suitable activity assays. In the context of the present invention, the term MMP, AMP, MMP or "substantially corresponding to" includes such substitutions. Table 3

Original Exemplary

Residue Substitution

AΪa GΪyjSeir

Arg Lys

Asn Gln;His

Asp Glu

Cys Ser

Gin Asn

Glu Asp

Gly Ala;Pro

His Asn;Gln

He Leu;Val

Leu Ile;Val

Lys Arg;Gln;Glu

Met Leu;Tyr;Ile

Phe Met;Leu;Tyr

Ser Thr

Thr Ser

Tip Tyr

Tyr Trp;Phe

Val Ile;Leu

Accordingly, based on the above examples of specific substitutions, alternative substitutions can be made by routine experimentation, to provide alternative MMPs of the present invention, e.g. , by making one or more conservative substitutions of MP fragments which provide MP activity.

Alternatively, another group of substitutions of MMPs of the present invention are those in which at least one amino acid residue in the protein molecule has been removed and a different residue inserted in its place according to the following Table 4. The types of substitutions which can be made in the protein or peptide molecule of the present invention can be based on analysis of the frequencies of amino acid changes between a homologous protein of different species, such as those presented in Table 1-2 of Schulz et al , infra. Based on such an analysis, alternative conservative substitutions are defined herein as exchanges within one of the following five groups: Table 4

1. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr (Pro, Gly);

2. Polar, negatively charged residues and their amides: Asp, Asn, Glu, Gin;

3. Polar, positively charged residues: His, Arg, Lys; 4. Large aliphatic, nonpolar residues: Met, Leu, He, Val (Cys); and

5. Large aromatic residues: Phe, Tyr, Tip.

The three amino acid residues in parentheses above have special roles in protein architecture. Gly is the only residue lacking any side chain and thus imparts flexibility to the chain. This however tends to promote the formation of secondary structure other than α-helical. Pro, because of its unusual geometry, tightly constrains the chain. Pro generally tends to promote /3-turn-like structures, although in some cases Cys can be capable of participating in disulfide bond formation which is important in protein folding. Note that Schulz et al. would merge Groups 1 and 2, above. Note also that Tyr, because of its hydrogen bonding potential, has significant kinship with

Ser, and Thr, etc.

Conservative amino acid substitutions, included in the term "substantially corresponding" or "corresponding", according to the present invention, e.g. , as presented herein, are well known in the art and would be expected to maintain biological and structural properties of the polypeptide after amino acid substitution. Most deletions and insertions, and substitutions according to the present invention are those which do not produce radical changes in the characteristics of the protein or peptide molecule. "Characteristics" is defined in a non-inclusive manner to define both changes in secondary structure, e.g. α-helix or /3-sheet, as well as changes in physiological activity, e.g. in receptor binding assays.

However, when the exact effect of the substitution, deletion, or insertion is to be confirmed, one skilled in the art will appreciate that the effect of the substitution or substitutions will be evaluated by routine MP activity screening assays, either immunoassays or bioassays, to confirm biological activity, such as, but not limited to, attenuation or enhancement of growth.

Amino acid sequence insertions as included in an MMP can also include amino and/or carboxyl-terminal fusions of from one residue to polypeptides of essentially unrestricted length, as well as intrasequence insertions of single or multiple amino acid residues. Intrasequence insertions can range generally from about 1 to 10 residues, more preferably 1 to 5. An example of a terminal insertion includes a fusion of a signal sequence, whether heterologous or homologous to the host cell, to an MMP to facilitate secretion from recombinant bacterial hosts.

One additional group of variants according to the present invention is that in which at least one amino acid residue in the peptide molecule, and preferably, only one, has been removed and a different residue inserted in its place. Most deletions, insertions and substitutions of MMPs according to the present invention are those which maintain or improve the attenuating or growth enhancing characteristics of the MMP. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. For example, an MMP made by site-specific mutagenesis of an MP-encoding nucleic acid and expression of an MMP in cell culture or, alternatively, by chemical synthesis, can be tested for attenuating or enhancing activity (e.g. , as is known or as described herein). The activity of the cell lysate or purified MMP can be screened in a suitable screening assay for the desired characteristic, e.g. , attenuating or enhancing activity in any of the several assays.

Modifications of protein or peptide properties, such as redox or thermal stability, hydrophobicity, susceptibility to proteolytic degradation or the tendency to aggregate with carriers or into multimers, can also be assayed by methods well known to the ordinarily skilled artisan. Also included in the scope of the invention are salts of the MMPs of the present invention. As used herein, the term "salts" refers to both salts of carboxyl groups and to acid addition salts of amino groups of the protein or peptide molecule. Amino acid sequence variations in an MMP of the present invention can be prepared by mutations in the DNA. Such MMPs include, for example, deletions, insertions or substitutions of nucleotides coding for different amino acid residues within the amino acid sequence. Any combination of deletion, insertion, and substitution can also be made to arrive at the final construct, provided that the final construct encodes an MMP having some attenuating or enhancing activity. Preferably improved attenuating or enhancing activity is found over that of the corresponding MP. Obviously, mutations that will be made in nucleic acid encoding an MMP must not place the sequence out of reading frame and preferably will not create complementary domains that could produce secondary mRNA structures (see, e.g. , EP Patent Application

Publication No. 75,444; Ausubel, infra; Sambrook, infra).

Mutant nucleic acid, as attenuating or enhancing nucleic acid of the present invention, can be prepared by site-directed mutagenesis of nucleotides in the DNA or nsRNA encoding an MP, thereby producing mutant nucleic acid encoding an AMP or EMP, and thereafter reverse transcribing the EMP or AMP encoding DNA to produce nsRNA. MMPs typically exhibit the same qualitative biological activity as the naturally occurring MP (see, e.g. , Ausubel, infra; Sambrook, infra), except for the additional attenuating or enhancing activity produced by the at least one mutation. Knowledge of the three-dimensional structures of proteins is crucial in understanding how they function. The three-dimensional structures of hundreds of proteins are currently available in the protein structure databases (in contrast to several hundred thousand known protein and peptide sequences in sequence databases, e.g. , Genbank, Chemical Abstracts REGISTRY, etc.). Analysis of these structures shows that they fall into recognizable classes or motifs. It is possible to model the three-dimensional structure of protein based on homology to a related protein of known structure. Examples are known where two proteins that have relatively low sequence homology, but are found to have almost identical three dimensional structure. Such homologous variants of MPs or MMPs are also included in AMPs or EMPs of the present invention.

Once an MP structure or characteristics have been determined using the above analysis, attenuating or enhancing nucleic acid can be recombinantly or synthetically produced, or optionally purified, to provide commercially useful amounts of attenuating or enhancing nucleic acid for use in diagnostic or research applications, according to known method steps (see, e.g.,

Ausubel, infra, and Sambrook, infra, which references are herein entirely incorporated by reference), e.g. , in LAV vaccine or master IAV strain production.

Screening Assays

For screening activity of MMP containing IAVs for attenuation or enhancement, any known and/or suitable screening assay can be used, as is known in the art. For example, virus replication can be used to screen both attenuation or enhancement. Other activities suitable, alone or in any combination, for screening include, but are not limited to, quantitative and/or qualitative measurement of transcription, replication, translation, virion incorporation, virulence, viral yield, and/or morphogenesis, using such methods as reverse genetics, reassortment, complementation, and infection. See, e.g., The Influenza Viruses, R.M. Krug (ed.), Plenum Press, New York, (1989).

Attenuated nsRNA, AMPs and Attenuated IAVs

Attenuated IAVs of the present invention comprise at least one pathogenic nsRNA and at least one attenuated nsRNA. The pathogenic nsRNA encodes at least one neuraminidase (NA) and at least one hemagglutinin (HA) from at least one pathogenic IAV strain. The inclusion of at least one NA and HA encoding negative strand RNA provides a host immune response specificity of the resulting attenuated virus for the at least one pathogenic IAV strain from which the NA an HA encoding RNA are derived. The additional inclusion of at least one attenuating nsRNA, which codes for an AMP having at least one mutation which attenuates the resulting IAV, provides a attenuated IAV having utility as a vaccine against at least one pathogenic IAV strain. According to the present invention, one or more sources of NA or HA encoding nsRNA, or cDNA encoding therefore, can be used to provide attenuated IAVs which can be used to vaccinate against one or more pathogenic strains of LAV. For example, at least one, such as 1-20 NA and/or HA encoding nsRNA can be included such that the attenuated live virus can be used as a vaccine against at least one or more pathogenic IAV strains, such as 1-2, 2-4, 5-8, 9-20 or 1-20 strains. An attenuated LAV according to the present invention is attenuated to the degree such that, while the attenuated virus is capable of inducing a pathogenic IAV strain specific immune response in an animal, the immune response involves a subclinical IAV infection in the animal. Such an attenuated virus or vaccine composition of the present invention can be used for prophylactic or therapeutic treatment, as described herein.

An attenuated IAV of the present invention can contain an attenuating nsRNA encoding for an Ml AMP. When the attenuated virus contains an attenuating nsRNA encoding an attenuating Ml matrix protein, it is preferred that the attenuating mutation comprise at least one mutation in an Ml domain selected from the group consisting of a 5' non-coding domain, a 3' non-coding domain, a lipid binding domain, a zinc finger domain, a phosphorylation site, a morphology domain, a transcription inhibiting domain and an RNA binding domain, which domains are known in the art. Such domains include 1-252 amino acids, or any range or value therein, corresponding to positions 1-252 of an Ml amino acid sequence. Attenuating and Enhancing Mutations

An attenuated or enhanced virus according to the present invention can contain at least one mutation in at least one matrix protein wherein the mutation encodes at least one amino acid modification selected from the group consisting of a substitution, a deletion or an insertion. The attenuating or enhancing mutation causes at least part of the attenuation or enhancement of the virus, and the amino acid modification affects the functioning of the virus in an animal host, such that an IAV, as either inhibiting an IAV with an attenuating mutation, or stimulating the growth of an IAV with an enhancing mutation. In an attenuated LAV of the present invention, it is preferred that the attenuated IAV induce a suitable immune response in the infected animal, which immunizes the animal while only producing a subclinical influenza A virus infection in the animal. In an enhanced IAV of the present invention, the growth of the virus is enhanced in a statistically significant manner, over an LAV not having the enhancing nucleic acid and/or EMP.

Ml and M2 Attenuating and Enhancing Mutations

According to the present invention, an Ml or M2 AMP having one or more mutations in an Ml or M2 matrix protein can provide attenuating or enhancing activity on an influenza A virus. The mutation can be in any coding or non-coding region which is sufficient to encode an effect in the transcription, replication, translation or virion incorporation of a mutated IAV of the present invention, when the LAV infects a host cell, or other mutation in the matrix amino acid sequence which confers the attenuating or enhancing activity of a IAV containing the attenuating or enhancing nucleic acid. As a non-limiting example, an Ml attenuating mutation can comprise at least one mutation in an Ml MP domain selected from the group consisting of a 5' non-coding domain, a 3' non-coding domain, a zinc finger domain, a phosphorylation domain, a transcription inhibiting domain, a morphology changing domain and an RNA binding domain.

As another non-limiting example, an Ml attenuating mutation can comprise at least one mutation in an M2 MP domain selected from the group consisting of a 5' non-coding domain, a 3' non-coding domain, a domain having at least one cysteine involved in oligomerization, a phosphorylation domain, a transmembrane domain, and a C-terminus domain.

As a further non-limiting example, an Ml enhancing mutation can comprise at least one mutation in an Ml MP domain selected from the group consisting of a 5 ' non-coding domain, a 3 ' non-coding domain, a high yielding domain, a phosphorylation domain, a transcription stimulating domain, a morphology changing domain and an RNA binding domain.

As an additional non-limiting example, an M2 enhancing mutation can comprise at least one mutation in an M2 MP domain selected from the group consisting of a 5' non-coding domain, a 3' non-coding domain, a morphology domain, a phosphorylation domain, and a transcription stimulating domain.

Phosphorylation Domains

Phosphorylation and dephosphorylation are key events in cellular functions, including signal transduction (Fantl et al, Annu. Rev. Biochem. 62:453-481 (1993)). Recently, Liu and Brown (Liu et al, Virology 196:703-

711 (1993)) demonstrated the importance of phosphorylation and dephosphorylation of the membrane glycoprotein tail of Sindbis virus for interaction with the viral core.

Ml Phosphorylation Domains

Attenuating Ml Phosphorylation Mutations. AMPs generated according to the present invention can contain at least one change from Ser or Thr to Ala, Gly or Pro. Phosphorylation of these mutant Ml proteins can be performed as known or described in Gregoriades et al, J. Virol -^9:229-235 (1984) and Gregoriades et al, Virus Res. 16:27-42 (1990). Substitution mutations of Ml where at least one Ser and Thr replaced with Ala, Gly or Pro does not contain phosphorylated residues, showing which residues are phosphorylated. Replacement of phosphorylated residues with other amino acids, preferably Ala, can generate AMPs according to the present invention. A non-limiting example of a group of attenuating mutations which can provide an Ml AMP of the present invention can include at least one substitution of a Ser or Thr in the phosphorylation domain of amino acids 94-129 of a IAV Ml MP with an animo acid selected from the group consisting of Ala, Gly or Pro, with Ala preferred. Further non-limiting examples of such substitutions include at least one of Alal lό, Alallδ, Alal20 and Ala 128, corresponding to substitutions of serine residues (or AlalOδ or Alal26, corresponding to substitutions at Thr residues, or substitutions are made for one or more negative variance or consensus sequence sera.

Enhancing Ml Phosphorylation Mutations. EMPs are generated according to the present invention that contain at least one change from Ala, Gly or Pro to Ser or Thr. Phosphorylation of these mutant Ml proteins is performed. Replacement of Ala, Gly or Pro with phosphorylated residues, such as Ser or Thr, can generate EMPs according to the present invention.

Such phosphorylation domains can comprise at least two amino acids corresponding to positions 94-129 of said naturally occurring Ml MP. An enhancing nucleic acid can include a substitution of a Ser or Thr for at least one selected from the group consisting of Alall6, Glyllό, Alallδ, Gly 118, Alal20, Glyl20, Alal2δ or Glyl2δ.

M2 Phosphorylation Domains

Attenuating M2 Phosphorylation Mutations. AMPs generated according to the present invention can contain at least one change from Ser or Thr to Ala, Gly or Pro. Phosphorylation of these mutant M2 proteins can be -34-

performed as known or described. Substution mutations of M2 where at least one Ser and Thr replaced with Ala, Gly or Pro does not contain phosphorylated residues, showing which residues are phosphorylated.

Replacement of phosphorylated residues with other amino acids, preferably Ala, can generate AMPs according to the present invention.

A non-limiting example of a group of attenuating mutations which can provide an M2 AMP of the present invention can include at least one substitution of a Ser or Thr in the phosphorylation domain of amino acids

64-65 of a LAV M2 MP with an animo acid selected from the group consisting of Ala, Gly or Pro, with Ala preferred. Further non-limiting examples of such substitutions include an Ala for at least one selected from the group consisting of Ser64 and Thr65.

Enhanced M2 Phosphorylation Mutations. EMPs can be generated according to the present invention that contain at least one change from Ser or Thr to Ala, Gly or Ser. Replacement of Ser or Thr with phosphorylated residues with other amino acids, such as Ala, Gly or Pro, can generate EMPs according to the present invention.

A non-limiting example of such enhancing mutations can include mutations of at least one amino acid corresponding to positions 64-65 of an M2 MP. An enhancing nucleic acid can include, e.g. , a substitution of at least one of Ser or Thr for at least one selected from the group consisting of

Ala64, Gly64, Pro64, Ala65, Gly65 and Pro65.

Λf7 Zinc Finger Domains

Although the zinc-finger motif has been identified in the Ml of influenza- A and B viruses, and the Ml in the purified virus contains zinc

(Elster et al., J. Gen. Virol. 75:37-42 (1994)), the function of this common structural feature was up to now unknown.

Attenuated Ml Zinc Finger Mutations. Attenuating mutations which can provide Ml AMPs of the present invention include zinc-finger substitutions of a least one His or Cys residue encompassing positions corresponding to one or more of 148 to 162 of a naturally occurring Ml matrix protein.

Such attenuating substitutions can include, but are not limited to, substituting a Cys by an amino acid selected from the group consisting Ser,

Gly and Ala with Ser preferred; or substitution of a His residue by an amino acid selected from the group consisting of Tyr, Trp and Phe, with Tyr preferred. Further non-limiting examples of such substitutions, e.g., of a zinc-finger domain of an Ml MP, include at least one of Serl4δ, Serl51, Tyrl59 and Tyrl62, e.g. , for at least one of Cysl4δ, Cysl51, Hisl59 and

Hisl62. Two transfectant viruses (one containing mutations at Cysl51 and Hisl59, the other at Cysl4δ, Cysl51, Hisl59, and Hisl62 in the Ml) have been generated. Both viruses produce smaller plaques than the wild type virus, as attenuated viruses. One or more of such zinc-finger substitutions can attenuate an Ml matrix protein to provide an AMP according to the present invention.

Enhanced Ml Zinc Finger Mutations. Enhanced mutations which can provide Ml EMPs of the present invention include zinc-finger substitutions of a least one His or Cys residue encompassing positions corresponding to one or more of 148 to 162 of a naturally occurring Ml matrix protein.

Such enhancing substitutions can include, but are not limited to, substituting a Cys for an amino acid selected from the group consisting Ser, Gly and Ala with Ser preferred; or substitution of a His residue for an amino acid selected from the group consisting of Tyr, Trp and Phe, with Tyr preferred. Further non-limiting examples of such substitutions, e.g. , of a zinc-finger domain, of an Ml MP include at least one of Alal49, Alal55, Serl57, Serl61, Serl4S, Serl51, Tyrl59 and Tyrl62, e.g. , by at least one of Cys or His. One or more of such zinc-finger substitutions can enhance an Ml matrix protein to provide an EMP according to the present invention. In many zinc-binding proteins, a change of one of the four Cys or His residues abolishes their activities (Coleman, J. E., Annu. Rev. Biochem. 67:897-946 (1992); Medina et al, Proc. Natl. Acad. Sci. USA 55:7620-7624 (1991); Webster et al, Proc. Natl. Acad. Sci. USA 55:9989-9993 (1991)).

Zinc blot assays (Schiff et al, Proc. Natl. Acad. Sci. USA 55:4195- 4199 (1988)) are used according to the present invention to confirm that the mutant Ml proteins have lost the zinc-binding activity, as preliminary screening before determination of attenuation or enhancement. This assay involves fractionation of the viral proteins by SDS-PAGE, transfer of the proteins to nitrocellulose, and blotting with "Zn.

Ml and M2 Morphology Domains

Clinical isolates of early passages in vitro contain more filamentous than spherical particles, whereas laboratory strains passaged extensively in such cultures contain spherical virions predominantly (Hoyle, L., "Morphology and Physical Structure" In The Influenza Viruses, Springer- Verlag, New York (1968), p. 49-68). This observation and general knowledge of the reduction of virulence in many viruses during passage in vitro suggest an association of the shape of influenza virus with virulence.

Ml Attenuating Morphology Domain Mutations. Non-limiting examples of an Ml protein attenuating substitution which can provide an Ml AMP of the present invention include, but are not limited to, morphology changing domain substitution of at least one of Asn207, Ser207, His222 and

Pro222, by at least one selected from the group consisting of Asn, Ser, His or Pro. Alternatively or additionally, an attenuating mutation can comprise at least one substitution of at least one of Asn207 and His222 by at least one of Gln207, Ser207 or His207 and one of His222, Asn222 and Gln222. Non-limiting examples of an Ml protein enhancing substitutions which can provide an Ml EMP of the present invention include, but are not limited to, morphology changing domain substitutions of at least one of an Ser207, His222 and/or Pro222 for a corresponding amino acid selected from the group consisting of Asn207, Gly207, Arg222 and His222. M2 Attenuating Morphology Domain Mutations. Non-limiting examples of an M2 protein attenuating substitution which can provide an M2 AMP of the present invention include, but are not limited to, morphology changing domain substitution of at least one of Asn82 by Ser82. Alternatively or additionally, an attenuating mutation can comprise at least one substitution of at least one of Asn207 and His222 by at least one of Glnδ2 by Glnδ2 or Serδ2.

Ml Enhancing Morphology Domain Mutations. Non-limiting examples of an Ml protein enhancing substitutions which can provide an Ml EMP of the present invention include, but are not limited to, morphology changing domain substitutions of at least one of an Asn207 and/or His222 for a corresponding amino acid selected from at least one of the group consisting of Ser207, Gln207, Arg222 and Pro222. Alternatively or additionally, an attenuating mutation can comprise at least one substimtion of at least one of Asn207 and His222 by at least one of Gln207 or His207 and one of Pro222,

Arg222 and Gln222.

M2 Enhancing Morphology Domain Mutations. Non-limiting examples of an M2 protein enhancing substitutions which can provide an M2 EMP of the present invention include, but are not limited to, morphology changing domain substitution of an Asn for SerS2. Alternatively or additionally, an attenuating mutation can comprise at least one substitution of Ser82 by Asn, Gin or Pro.

Ml Lipid Binding Domains

Further non-limiting example of attenuating substitutions to provide an Ml AMP include substitutions in a lipid bonding domain, as amino acids

62-68 and/or 114-133 of an Ml. Further non-limiting examples for specific attenuating substitutions which can provide an Ml AMP include substitution of Arg for at least one of Phe62, Val63, Phe64, Leu66, Val68, Vail 15, Leull7, Tyrll9, Leul30 and Ilel31. Ml Transcription Inhibiting Domains

Additional examples of Ml AMPs which contain attenuating mutations include substimtions of at least one amino acid in a transcription inhibiting domain. Non-limiting examples of such substitutions include at least one of a Gly for Pro69 and a Pro for at least one of Glul41 and Thrl40.

Ml Transcription Stimulating Domains

Additional examples of Ml EMPs which contain attenuating mutations include substimtions of at least one amino acid in a transcription stimulating domain. Non-limiting examples of such substitutions include Gly for at least one of Pro69, Glnl41 and Thrl40.

Ml RNA Binding Domains

A further example of a Ml domain which can be mutated to provide an Ml AMP of the present invention include substimtions in an RNA binding domain which can include, as a non-limiting example, amino acids 90-108 and/or 128-164 of a native Ml amino acid sequence. Non-limiting examples of such substitutions include Ala for at least one of Arg95, Lys98, ArglOl, Lysl02, Lysl04, Argl05, Argl34, Argl60, Hisl62 and Argl63.

M2 Attenuating Oligomerization Domains

Additionally, a domain having a cysteine involved in oligomerization of an M2 MP can be modified by an amino acid substimtion in an M2 MP sequence to provide an M2 AMP of the present invention. Such domains include, as non-limiting examples, sequences corresponding to amino acids Cysl7, Cysl9 and Cys50 of an M2 amino sequence. Such a attenuating mutations can include, but are not limited to, a replacement of Cys with a Ser, Gly or Ala, such a replacement of at least one of Cysl7, Cysl9 and Cys50 by an Ala, Gly or Ser, with Ser preferred.

M2 Attenuating C-Terminal Domains

Additional, M2 AMPs of the present invention can be provided by deleting 1-54 amino acids from the C-terminus. Such deletions include, but are not limited to, a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55 amino acid deletions. Such deletions correspond to positions 44-97 of any M2 matrix protein.

Ml or M2 (M-Gene) 3' or 5' Non-Coding Domains

When an attenuated IAV of the present invention contains a mutation in either the Ml or M2, 3' or 5', non-coding domain, as an M-gene domain it is preferred that the mutation inhibit the level of at least one of transcription, replication, translation or virion incorporation of said attenuated

IAV in a host cell capable of being infected by said attenuated influenza A virus, such that the resulting virus is attenuated.

Attenuating or enhancing mutations in a 3' non-coding region or domain of an M-gene nsRNA can also provide an Ml or M2 AMP or EMP of the present invention. Such modifications, include, but are not limited to at least one 3' mutation selected from the group consisting of 1U by G, A or C; 2C by G, A or U; 3G by U, C or A; 4U by G, A or C; 5U by G, A or C; 6U by G, A or C; 7U by G, A or C; 8C by G, U or A; 9G by A, U or C; 10U by G, A or C; 11C by G, A or U; 12C by G, A or C; 13C by G, A or U; 14A by U, G or A and 15C by G, U or A; according to SEQ ID NO:3.

Preferred attenuating 3' non-coding region substimtion are at least one selected from the group consisting of 1A, 2G, 2A, 2U, 3A, 5G, 5C, 10A and 11G. Preferred enhancing substimtions are at least one selected from the group 1G, 1C, 4G, 4A, 6G, 6C, 8A and 14U. .See e.g. , Piccone et al. , Virus Res. 25:99-112 (1993), which is entirely incorporated herein by reference. Additional mutations in the 5 ' non-coding regions or domains of an Ml or M2 nsRNA can also provide an Ml or M2 AMP or EMP of the present invention. Such modifications, include, but are not limited to 5' mutations at least one selected from the group consisting of 1015G by A, U or C; 1016G by A, U or C; 1017A by G, U or C; 1018A by G, C or U; 1019C by A, G or U; 1020A by G, U or C; 1021A by G, C or U; 1022A by G, C or U; 1023G by A, C or U; 1024A by G, C or U; 1025U by A, G or C; 1026G by

A, U or C; 1027G by A, U or C; and 1027A by U, G or C; according to SEQ ID NO:3.

Preferred attenuating 5' non-coding region substimtions include, but are not limited to, at least one selected from the group consisting of 1017C, 1017U, 1017A, 1019U, 1020U, 1021U, 1023C, 1023U, 1023A, 1024C,

1024U, 1027C, 1027U, 1027G. Preferred enhancing substimtions include, but are not limited to, at least one selected from the group consisting of 1015A, 1016C, 1018C, 1022C, 1022G, 1024G, 1025A, 1025G, 1026U and 1026A. See e.g. , Piccone et al , J. Virol 66:443 l-433δ (1992).

M2 Transmembrane Domains

Alternatively or additionally, modifications in the sequence of the transmembrane domain corresponding to amino acids 25-43 of an M2 MP. Non-limiting examples of such modifications can include, but are not limited to, substimtion of a Leu, He or Val for at least one selected from Ala, Gin, Glu, Asn, Asp, Lys, Leu, Pre and Arg. Preferred substimtions include, but are not limited to at least one selected from the group consisting of Leu26, Val27, Val28, Ile32, Ile33, Ile35, Leu36, Leu38, Leu40, Ile42 and Leu43. Enhanced nsRNA, EMPs and Enhanced IAVs.

Enhanced IAVs of the present invention comprise at least one enhancing nsRNA. The at least one enhanced nsRNA, which codes for an

EMP having at least one mutation which attenuates the resulting virus, can provide an enhanced LAV having utility, e.g. , as a master strain for culturing

IAVs.

An enhanced LAV of the present invention can contain an enhanced nsRNA encoding for an EMP selected from an Ml EMP or an M2 EMP. When the enhanced virus contains an enhancing nsRNA encoding an Ml EMP, it is preferred that the enhancing mutation comprises at least one mutation in a domain an Ml matrix protein or encoding nsRNA selected from the group consisting of a 5' non-coding domain, a 3' non-coding domain, a zinc finger, a lipid binding domain, a phosphorylation site, a transcription inhibiting domain, a morphology domain, and an RNA binding domain, which domains are known in the art. Such domains include 1-100 amino acids of

1-252 of an M2 MP, or any range or value therein.

Alternatively, when an enhancing nsRNA of the present invention encodes an M2 EMP, the enhancing mutation is preferably selected from at least one mutation in an M2 domain selected from the group consisting of a 5' non-coding domain, a 3' non-coding domain, a domain containing at least one cystine residue involved in oligomerization; a transmembrane domain and a phosphorylation domain. Such domains can include 1-60 amino acids corresponding to positions 1-97 of an M2 amino acid sequence.

Recombinant Cloning and/or Production of MMPs.

Known method steps for synthesizing oligonucleotides probes useful for cloning and expressing DNA encoding an MMP, as an AMP or EMP, of the present invention, based on the teaching and guidance presented herein, are disclosed by, for example, Ausubel, infra; Sambrook, infra; and Wu et al , Prog. Nucl. Acid. Res. Molec. Biol. 27:101-141 (197δ), which references are entirely incorporated herein by reference.

A suitable oligonucleotide, or set of oligonucleotides, which is capable of encoding (or which is complementary to a sequence encoding) an MP fragment of a matrix gene is identified as above, isolated or synthesized, and hybridized by means well known in the art, against a DNA or, more preferably, a cDNA preparation derived from cells having matrix genes and which are capable of expressing an MP. Single stranded oligonucleotide probes complementary to an attenuating or enhancing activity encoding sequence can be synthesized using method steps (see, e.g. , Ausubel, infra;

Sambrook, infra).

Such a labeled, detectable probe can be used by known procedures or as a basis for synthesizing PCR probes for amplifying a cDNA generated from an isolated RNA encoding a target nucleic acid or amino acid sequence. As a further non-limiting example host cell or IAV, transformants can be selected by use of selection media appropriate to the vector or, virus or MP used, RNA analysis or by the use of antibodies specific for a target protein as an MP or MMP used in a method according to the present invention.

A target, detectably labeled probe of this sort can be oligonucleotide that is complementary to a polynucleotide encoding a target protein, as an

MP. Alternatively, a synthetic oligonucleotide can be used as a target probe which is preferably at least about 10 nucleotides in length (such as 10, 15, 16, 17, lδ, 19, 20, 21, 22, 23, 24, 25, 26, 27, 2δ, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more, or any combination or range therein, in increments of 1 nucleotide), in order to be specific for a target a nucleic acid to be detected, amplified or expressed. The probe can correspond to such lengths of a DNA or RNA encoding an MP, such as a sequence encoding a peptide corresponding to a portion of SEQ ID NO:l or SEQ ID NO:2, wherein the probe sequence is selected according to the host cell containing the DNA, e.g. , as presented in Table A1.4 of Ausubel, infra. MMP encoding mutant nucleic acids of the present invention can include 30-756, such as 30-300, 40- 200, 30-100, 101-200, and 201-300 nucleotides, or any range or value therein, substantially complementary to a portion of nucleotides encoding SEQ ID NO:l or SEQ ID NO:2, or complementary to SEQ NO:3 or 4, wherein the codons can be substimted by codons encoding the same or conservatively substimted amino acids, as well known in the art.

Culturing of the host or rescue virus and introduction of complementary nsRNA into an IAV can be induced by methods known per se. A nucleic acid sequence encoding an MMP of the present invention can be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed, e.g., by Ausubel, infra, and are well known in the art. The cultured and/or amplified cDNA is then incorporated into IAVs, according to known techniques, e.g. , using reverse genetics. This method involves (i) preparation of RNA, containing exactly the same 5' and 3' sequences as viral RNA, from cloned influenza virus genes (e.g. , cDNA) with RNA polymerase, (ii) encapsulation of the RNA with influenza virus NP and polymerase proteins, (iii) transfection of the encapsidated RNA, and (iv) infection with a helper influenza virus to rescue the transfected RNA (Luytjes et al., Cell 59:1107-1113 (1989); Enami et al, Proc. Natl Acad. Sci. USA 57:3δ02-3δ05 (1990); Enami et al, J. Virol. 65:2711-2713 (1991), which references are entirely incorporated herein by reference). Host cells comprising a nucleic acid which encodes an MMP of the present invention may be grown under conditions that provide expression of a desired MMP or mutant nsRNA in recoverable or commercially useful amounts. See, e.g., Ausubel, infra, at §§ 1 and 13; Palese, U.S. Patent No. 5,166,057, which are entirely incorporated herein by reference. A mutant nucleic acid can also be recombinantly expressed in a host cell after the nucleic acid is incorporated into a plasmid or viral vector capable -44-

of autonomous replication in the recipient host. Any of a wide variety of vectors can be employed for this purpose. See, e.g. , Ausubel, infra, §§ 1.5, 1.10, 7.1, 7.3, δ.l, 9.6, 9.7, 13.4, 16.2, 16.6, and 16.8-16.11. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector can be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.

Temperature Sensitive Mutants

In a preferred embodiment, a substimtion in a matrix protein to induce attenuation, involves a replacement of an alanine for a charged amino acid in an attenuating matrix protein, which mutation results in a cold sensitive mutant. In another alternative or additional embodiment, the mutation in a matrix protein involves a substimtion of a conserved amino acid for a similar amino acid, as described herein.

In a preferred embodiment, the deletion is selected from the group consisting of 1-54 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 51, 52, 53, or 54, or any range or value therein, from the C-terminal or N-terminal ends of at least one of an Ml or M2 MP.

In another additional or alternative preferred embodiment, the insertion is selected from the group consisting of 1-54 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 51, 52, 53, or 54, or any range or value therein, from the C-terminal or

N-terminal ends of at least one of an Ml or M2 MP. IAVs Used in Accordance with the Present Invention

According to the present invention, any influenza A virus strain can be used to obtain an attenuated influenza A virus which is capable of inducing an immune response in an animal, but which responds involves a subclinical infection.

The present invention also provides attenuated viruses wherein the mutant negative strand RNA and pathogenic negative strand RNA are derived from influenza A virus strains which have the same or similar host strains, such as a host strains including the same order genus or species. In a preferred embodiment, the attenuated virus contains mutant negative strand

RNA and pathogenic negative strand RNA which are derived from influenza A viruses both capable of infecting human hosts or primate hosts.

An attenuated virus according to the present invention can further comprise a selection marker used to select for a reassortant or recombinant influenza A virus containing a mutant negative strand RNA according to the present invention. Such selection marker can be selected from the group consisting of a drug resistance marker, a temperature sensitive marker, and an antigenic marker. When the selection marker is a drug resistance marker, the drug resistance can be used in any form which provides the ability to select an attenuated influenza A virus containing both the pathogenic negative strand RNA and the mutant negative strand RNA. As known in the examples, a drug resistance marker useful in the present invention is a drug selected from the group consisting of an amantadine-like compound, amantadine, rimantadine or other tricyclic symmetric amine. When the drug resistance invoke resistance to an amantadine-like compound, such a compound can be selected from the group consisting of amantadine, rimantidine, or other tricyclic symmetric amine analog. See, e.g. , Katzung, infra, at 674-681; Hay, EMBO J. 4:3021 (1985) In a preferred embodiment, the drug is amantadine. As an additional selection step, rescued IAVs can be further selected using an MMP antibody, e.g. , as described herein. According to a different aspect of the present invention, a method is provided for obtaining an attenuated influenza A virus which is capable of being used as a vaccine for at least one pathogenic influenza A virus strain. The method can comprise isolating an attenuated influenza A virus of the present invention, as described herein. The method may further comprise, prior to the isolating step, reassorting in a host a helper virus having said at least one mutant negative strand RNA and sensitivity to at least one selection marker, with at least one of said pathogenic negative strand RNA encoding (1) at least one NA and HA from at least one pathogenic influenza virus strain and selection RNA encoding at least one selection protecting conferring resistance to the selection marker. The use of helper virus containing the mutant negative strand RNA which helper virus also has sensitivity to a selection marker, the selected reassortant having resistance to the selection marker, are also discovered to be obtainable as the reassortant attenuated virus containing both the pathogenic negative strand RNA and the mutant negative strand RNA. Such attenuated influenza A viruses are found to be useful as vaccines for inducing a suitable immune response against the at least one pathogenic influenza A virus strain from which the at least one NA and HA encoding pathogenic negative strand RNA is derived. Additionally, such attenuated viruses induce a suitable immune response while at the same time inducing a subclinical influenza A viral infection. Currently, such attenuated vaccines of the present invention are useful for vaccine compositions and methods for prophylactic and therapeutic vaccine treatment of animals, preferably primates and humans. In methods for generating attenuated influenza A viruses of the present invention, the pathogenic negative strand

RNA can be derived from at least one pathogenic influenza A virus strain which is not limited to any particular strains. Currently, any known or discovered influenza A virus strain can be used as a source of the at least one NA and HA encoding pathogenic negative strand RNA. According for methods for generating attenuated viruses of the present invention, any host cell which is suitable for replicating and/or reassorting attenuated viruses of the present invention may be used. Such host cells may be selected from prokaryotic or eukaryotic cells. It is preferred that the eukaryotic cells be selected from the group consisting of a mammalian cell, an insect cell, a yeast cell and a bird cell, with mammalian cells preferred. Non-limiting examples of cell lines suitable for methods of the present invention, include MDCK, MDBK, VERO and CV-1 cells, readily available from commercial stores (e.g., ATCC, Rockville, MD).

The present invention also provides methods for generating reassortant viruses wherein the resistance to the selection marker is removed from the attenuated virus to provide an attenuated virus having sensitivity to the selection marker or attenuated virus which lacks resistance to the selection marker. For example, if amantadine resistance is used as a selection marker for obtaining reassortant attenuated viruses of the present invention, which are to be used as vaccines for human treatment, then it is preferred that such resistance to amantadine be removed.

Such removal can be used by either removing the selection marker by using culture systems which, while allowing rescue of mutant M gene, do not support replication of amantadine resistant viruses. From our experience with reverse genetics of the NA gene, it is expected that mutant viruses with changes in the M gene generated by reverse genetics exert host range alteration; that is, the mutants do not grow in certain culture systems (e.g. , MDBK cells or eggs) which support the replication of the parent virus. Thus, to rescue the M gene of an amantadine-sensitive virus with attenuating mutations, the host range mutants can be used as helper viruses in such nonpermissive culture. Unlike the first strategy, this system allows the generation of viruses with temperature-sensitive attenuating mutations. As an alternative to amantadine resistance, for example, in use in generating human influenza A attenuated vaccines, a temperature sensitive helper virus can be used as a selection system. Helper viruses can be eliminated during the reassortant process by use of the appropriate temperature selection. A non- limiting example of such a helper virus is tS51 virus containing a temperature sensitive mutation in the Ml protein.

MP or MMP Antibodies Having Binding Affinity to an AMP or EMP Peptide and a Hybridoma Containing the Antibody

In another embodiment, the invention relates to the making and using of an anti-MP antibody (e.g., an M2 antibody) or an anti-MMP antibody (e.g. , an AMP or EMP antibody). An AMP or EMP antibody binds specifically to an epitope of either an AMP or an EMP, respectively. Preferably, the AMP or EMP antibody binds an epitope that distinguishes an AMP or EMP from an non-attenuating or non-enhancing MP, respectively.

For example, an AMP or EMP antibody binds an epitope within or resulting from the attenuating or enhancing amino acid mutation in the AMP or EMP, respectively. An MP antibody binds to an epitope specific for an MP. Anti-MP or anti-MMP antibodies are useful for the analysis, isolation or purification of: (a) MPs; (b) MMPs; or (c) MP- or MMP-containing IAVs.

An MP or MMP of the present invention can be used as an antigen to provide antibodies or hybridomas.

MP or MMP antibodies of the present invention include monoclonal and polyclonal antibodies, fragments, and single chain antibodies. Such antibodies are provided by known method steps, such as hybridoma or recombinant technology. Antibody fragments which contain the idiotype of an MP or MMP antibody molecule can be generated by known techniques. See, e.g., Kohler and Milstein, Nature 256:495-497 (1975); U.S. Patent No. 4,376,110; Ausubel et al, eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Assoc. and Wiley Interscience, N.Y., (19δ7,

1992); Harlow and Lane ANTIBODIES: A LABORATORY MANUAL Cold Spring Harbor Laboratory (19δδ); Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1992, 1993), the contents of which references are incorporated entirely herein by reference. Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, GILD, or any subclass thereof.

A monoclonal antibody (MAb) contains a substantially homogeneous population of antibodies specific to antigens, which population contains substantially similar epitope binding sites. A hybridoma producing a MAb of the present invention can be cultivated in vitro, in situ or in vivo. Preferred methods of antibody production include hybridoma or recombinant techniques, which provide high titers of antibody.

An antibody is said to be "capable of binding" a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody. The term "epitope" is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody. Epitopes or "antigenic determinants" usually consist of surface groupings of molecules such as amino acids or sugar side chains and can have specific three dimensional structural characteristics, as well as specific charge characteristics.

Immunoassays

Antibodies directed against an MP or MMP can be used to detect an MP, MMP or an enhanced or attenuated IAV, using known techniques, based on the teaching and guidance presented herein.

When such antibodies are used in screening methods to select reassortants (as attenuated or enhanced IAVs), the antibodies distinguish either: (a) human IAV proteins from non-human LAV proteins; or (b) MMPs from MPs. In (a), these antibodies distinguish a host cell LAV MP from a helper cell IAV MP, when helper cells are used that express non-human LAV proteins. Host cell-specific anti-LAV antibodies therefore select reassortants having proteins of the host cell-specific virus. Thus, antibodies specific for a host cell MP can be used to select attenuated or enhanced IAVs from other IAVs having MPs derived from the helper virus. Alternatively , in (b) above, antibodies which distinguish an MMP from an MP can be used to select an attenuated or enhanced IAV of the present invention. The antibodies bind an epitope found in an MMP (e. g. , in an AMP or EMP), but not found in an MP. Such antibodies bind an MMP in an attenuated or enhanced LAV to select rescued assortants (alternatively or in addition to amantidine selection).

One screening method of the invention is an immunoassay employing an enzyme immunoassay (El A). These assays are based on the use of specific antibodies (monoclonal or polyclonal) to an MP or an MMP, including those present in an LAV. For these assays, samples containing attenuated or enhanced IAVs can be used in addition to those containing an MP or an MMP. An MP- or MMP-specific antibody can be detectably labeled by linking it to an enzyme for use in an EIA. This enzyme, when later exposed to an appropriate substrate, will produce a product detectable by spectrophotometric, fluorometric or by visual means. Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuc lease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Calorimetric methods also can be used which employ a chromogenic substrate for the enzyme. Detection can also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

Detection can also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibodies, it is possible to detect or measure an MP, MMP, or attenuated or enhanced LAV, through the use of a radioi munoassay (RIA). RIAs are described in Laboratory Techniques and Biochemistry in Molecular Biology, Work et al ,

North Holland Publishing Company, NY (1978). The radioactive isotope can be detected by such means such as the use of a gamma coimter, a scintillation counter or by autoradiography. See, e.g., Harlow and Lane, infra, Colligan, infra.

The binding activity of a given sample of anti-MP or anti-MMP antibody can be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.

Other steps, such as washing, stirring, shaking, filtering and the like, can be added to the assays as is customary or necessary for the particular situation.

It is also possible to label an anti-MP or anti-MMP antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can be then be detected by its fluorescence. Among the most commonly used fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. The antibody can also be detectably labeled using fluorescence emitting metals such as ^1S2EU or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as ethylene-di-amine tetraacetic acid (EDTA).

The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound can be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting luminescence qualitatively or quantitatively. Non-limiting examples of bioluminescent compounds for purposes of labeling include luciferin, luciferase and aequorin.

An antibody molecule of the present invention can be adapted for utilization in a immunometric assay, also known as a "two-site", "forward" or "sandwich" assay. In a typical immunometric assay, a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support or carrier and a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid- phase antibody, antigen, and labeled antibody. See, e.g., Harlow and Lane, infra, Colligan, infra.

An antigenic MP or MMP peptide can be modified or administered with an adjuvant in order to increase the peptide antigenicity. Methods of increasing the antigenicity of a peptide are well-known in the art. Such procedures include coupling the antigen with a heterologous protein (such as globulin or β-galactosidase) or through the inclusion of an adjuvant during immunization.

The specific concentrations of detectably labeled antibody, MP, MMP, or IAV, the temperature and time of incubation, as well as other assay conditions, can be varied depending on various factors including the concentration of an MP or MMP in the sample, the nature of the sample, and the like. The binding activity of a given lot of anti-MP or anti-MMP antibody can be determined according to well-known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation. The assay of the present invention is also ideally suited for the preparation of a kit. Such a kit can comprise a carrier being compartmentalized to receive in close confinement therewith one or more containers, such as vials, tubes and the like, each container comprising the separate elements of the immunoassay. For example, there can be a container or recepticle containing a first antibody immobilized on a solid phase support, and a further container means containing a second detectably labeled antibody in solution. Further containers can contain standard solutions comprising serial dilutions of the MP or MMP to be detected. The standard solutions of an MP or MMP can be used to prepare a standard curve with the concentration of MP or MMP plotted on the abscissa and the detection signal on the ordinate. The results obtained from a sample containing an MP or an MMP can be interpolated from such a plot to give me concentration of the MP or the MMP. See, e.g. , Harlow and Lane, infra; Colligan, infra.

Pharmaceutical Compositions

Pharmaceutical preparations of the present invention, suitable for inoculation or for parenteral administration, include attenuated virus containing sterile aqueous or non-aqueous solutions, suspensions, and emulsions, which can also contain auxiliary agents or excipients which are known in the art. See, e.g. , Berkow et al , eds., The Merck Manual, 15th edition, Merck and Co., Rah way, N.J., 1987; Goodman et al , eds.,

Goodman and Gilman's The Pharmacological Basis of Therapeutics, 8th edition, Pergamon Press, Inc., Elmsford, N.Y., (1990); Avery 's Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3rd edition, ADIS Press, LTD., Williams and Wilkins, Baltimore, MD. (1987), Katzung, ed. Basic and Clinical Pharmacology, Fifth

Edition, Appleton and Lange, Norwalk, Conn. (1992), which references and references cited therein, are entirely incorporated herein by reference as they show the state of the art. A composition may also include other immunomodulators, such as cytokines which accentuate an immune response to a viral infection.

Heterogeneity in the vaccine may be provided by mixing specific species for at least one influenza A virus strain. For example, the vaccine preparation may contain one or more specific attenuated viruses of the present invention, which viruses contain at least one NA or HA encoding RNA and at least one mutant matrix encoding RNA. Using HA and NAs or attenuated viruses according to the present invention, vaccines can be provided for variations in a single strain of influenza A virus or for more than one strain of influenza A virus, using techniques known in the art. A pharmaceutical composition according to the present invention may further or additionally comprise at least one viral chemotherapeutic compound selected from gamma globulin, amantadine, guanidine, hydroxybenzimidazole, interferon-α, interferon-/3, interferon-γ, thiosemicarbarzones, methisazone, rifampin, ribvirin, a pyrimidine analog, a purine analog, foscamet, phosphonoacetic acid, acyclovir, dideoxynucleosides, or ganciclovir. See, e.g. , Katzung, infra, and the references cited therein on pages 798-800 and 680-6δl, respectively, which references are herein entirely incorporated by reference.

Pharmaceutical Purposes

The administration of the vaccine (or the antisera which it elicits) may be for either a "prophylactic" or "therapeutic" purpose. When provided prophylactically, the compound (s) are provided in advance of any symptom of influenza A viral infection. The prophylactic administration of the compound(s) serves to prevent or attenuate any subsequent infection. When provided therapeutically, the attenuated viral vaccine is provided upon the detection of a symptom of actual infection. The therapeutic administration of the compound(s) serves to attenuate any actual infection. See, e.g., Berker, infra, Goodman, infra, Avery, infra and Katzung, infra, which are entirely incorporated herein by reference, including all references cited therein. An attenuated vaccine or vaccine composition of the present invention may, thus, be provided either prior to the onset of infection (so as to prevent or attenuate an anticipated infection) or after the initiation of an actual infection. A composition is said to be "pharmacologically acceptable" if its administration can be tolerated by a recipient patient. Such an agent is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. A vaccine or composition of the present invention is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient.

The "protection" provided need not be absolute, i.e. , the disease need not be totally prevented or eradicated, provided that there is a statistically significant improvement relative to a control population. Protection may be limited to mitigating the severity or rapidity of onset of symptoms of the disease.

As would be understood by one of ordinary skill in the art, when the vaccine of the present invention is provided to an individual, it may be in a composition which may contain salts, buffers, adjuvants, or other substances which are desirable for improving the efficacy of the composition. Adjuvants are substances that can be used to specifically augment a specific immune response. Normally, the adjuvant and the composition are mixed prior to presentation to the immune system, or presented separately, but into the same site of the animal being immunized. Adjuvants can be loosely divided into several groups based upon their composition. These groups include oil adjuvants, mineral salts (for example, AlK(SO₄)₂, AlNa(SO₄)₂, AlNH₄(SO₄), silica, kaolin, and carbon), polynucleotides (for example, poly IC and poly AU acids), and certain natural substances (for example, wax D from Mycobacterium tuberculosis, as well as substances found in Corynebacterium parvum, or Bordetella pertussis, and members of the genus Brucella. Among those substances particularly useful as adjuvants are the saponins such as, for example, Quil A. (Superfos A S, Denmark). Examples of materials suitable for use in vaccine compositions are provided in Remington 's Pharmaceutical Sciences (Osol, A, Ed, Mack Publishing Co, Easton, PA, pp. 1324-1341 (19δ0), which reference is incorporated herein by reference). Pharmaceutical Administration

A vaccine of the present invention may confer resistance to one or more strains of an IAV by either passive immunization or active immunization. In active immunization, a live attenuated vaccine or composition is administered prophylactically, according to a method of the present invention. In another embodiment of passive immunization, the vaccine is provided to a host (i.e. a human or mammal) volunteer, and the elicited antisera is recovered and directly provided to a recipient suspected of having an infection caused by an IAV strain. In a second embodiment, the vaccine is provided to a female (at or prior to pregnancy or parturition), under conditions of time and amount sufficient to cause the production of antisera which serve to protect both the female and the fetus or newborn (via passive incorporation of the antibodies across the placenta). The present invention thus concerns and provides a means for preventing or attenuating infection by an IAV strain, or by organisms which have antigens that can be recognized and bound by antisera to the polysaccharide and/or protein of the conjugated vaccine. As used herein, a vaccine is said to prevent or attenuate a disease if its administration to an individual results either in the total or partial attenuation (i.e. suppression) of a symptom or condition of the disease, or in the total or partial immunity of the individual to the disease.

At least one attenuated IAV of the present invention may be administered by any means that achieve the intended purpose, using a pharmaceutical composition as previously described.

For example, administration of such a composition may be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, or buccal routes. Parenteral administration can be by bolus injection or by gradual perfusion over time. A preferred mode of using a pharmaceutical composition of the present invention is by subcutaneous, intramuscular or intravenous application. See, e.g. , Berker, infra, Goodman, infra, Aveiy, infra and Katzung, infra, which are entirely incorporated herein by reference, including all references cited therein. A typical regimen for preventing, suppressing, or treating a disease or condition which can be alleviated by a cellular immune response by active specific cellular immunotherapy, comprises administration of an effective amount of a vaccine composition as described above, administered as a single treatment, or repeated as enhancing or booster dosages, over a period up to and including between one week and about 24 months.

According to the present invention, an "effective amount" of a vaccine composition is one which is sufficient to achieve a desired biological effect. It is understood that the effective dosage will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. The ranges of effective doses provided below are not intended to limit the invention and represent preferred dose ranges. However, the most preferred dosage will be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation. See, e.g., Berkow et al , eds., The Merck Manual, 16th edition, Merck and Co., Rahway, N.J., 1992;

Goodman et al. , eds. , Goodman and Gilman 's The Pharmacological Basis of Therapeutics, δth edition, Pergamon Press, Inc., Elmsford, N.Y., (1990); Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3rd edition, ADIS Press, LTD., Williams and Wilkins, Baltimore, MD. (19δ7), Ebadi, Pharmacology, Little, Brown and Co.,

Boston, (1985); and Katsung, infra, which references and references cited therein, are entirely incorporated herein by reference.

Generally speaking, the dosage for a human adult will be from about lO O⁷ plaque forming units (PFU)/kg or colony forming units (CFU)/kg. However, the dosage should be a safe and effective amount as determined by conventional methods, using existing vaccines as a starting point. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions, which may contain auxiliary agents or excipients which are known in the art. Examples of non- aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and iηjectable organic esters such as ethyl oleate. Carriers or occlusive dressings can be used to increase skin permeability and enhance antigen absorption. Liquid dosage forms for oral administration may generally comprise a liposome solution containing the liquid dosage form. Suitable forms for suspending liposomes include emulsions, suspensions, solutions, syrups, and elixirs containing inert diluents commonly used in the art, such as purified water. Besides the inert diluents, such compositions can also include adjuvants, wetting agents, emulsifying and suspending agents, or sweetening, flavoring, or perfuming agents. See, e.g. , Berker, infra, Goodman, infra, Avery, infra and Katzung, infra, which are entirely incorporated herein by reference, included all references cited therein.

Subjects

The recipients of the vaccines of the present invention may be any vertebrate animal which can acquire specific immunity via a cellular or humoral immune response, where said response is mediated by an MHC class I (cellular response) or a class LI (humoral response) protein. MHC proteins have been identified in mammals, birds, bony fish, frogs and toads. Among mammals, the preferred recipients are mammals of the Orders Primata (including humans, apes and monkeys), Arteriodactyla (including horses, goats, cows, sheep, pigs), Rodenta (including mice, rats, rabbits, and hamsters), and Carnivora (including cats, and dogs). Among birds, the preferred recipients are turkeys, chickens and other members of the same order. The most preferred recipients are humans. Having now generally described the invention, the same will be more readily understood through reference to the following example which is provided by way of illustration, and is not intended to be limiting of the present invention.

Example 1: Production and Efficacy of Live Attenuated

Influenza A Vaccines Having Mutated Matrix Genes

Materials and Methods

Viruses and cells. Influenza A/equin 63 (H3Nδ) (Eq/MLA), A/Puerto Rico/δ/34 (HlNl) (PRδ), and A/duck/Oklahoma/4/77 (H1N4) viruses were obtained from a repository at St. Jude Children's Research Hospital. The

Madin-Darby bovine kidney (MDBK) cell line was cultured in Eagle's minimal essential medium (MEM) containing 10% fetal calf serum. Madin- Darby canine kidney (MDCK) cells were cultured in the same conditions as MDBK cells, except that 5% calf serum was used. Reverse genetics. A plasmid (pPRδM-10) containing the PRδ M gene was constructed as described by Huddleston and Brownlee (Huddleston, J.A. et al , Nucl. Acids Res. 70:1029-1037 (1982)). A second plasmid (pUCT3PRM), containing the PR8 M gene flanked by the Ksp632I site and T3 RNA polymerase promoter sequence, was made by cloning the polymerase chain reaction (Saiki, R.K. et al. , Science 25P:4δ7-491 (19δδ)) product made with pPRδM-10 as a template and with primers 5 '- ATCGATGAATTCTCTTCGAGCGAAAGCAGGTAGATATTG-3' (SEQ ID NO: 12) and 5'-GAGGACAAGCTTATTAACCCTCACTAAAAG- TAGAAACAACτGAGTTTTTTACT-3' (SEQ ID NO: 13). pUCT3PRM contains a T3 RNA polymerase promoter upstream and a Ksp632I site downstream of the M gene, so that viral sense RNA transcripts are generated when digested with Ksp632I, filled-in with Klenow fragment, and transcribed with T3 RNA polymerase (Enami, M. et al. , Proc. Natl Acad. Sci. USA 57:3802-3805 (1990)). pUCT3COOH- 1, pUCT3COOH-5, and pUCT3COOH-10 were constructed by replacing M gene nucleotides, which convert the M2 carboxyl-terminal Glu, amino acid residue 93, and 89 codon to a stop codon, respectively, using oligonucleotide- directed mutagenesis (Kunkel, T.A. et al. , Methods in Enzymology 154:367-

3δ2 (19δ7)) (Fig. 1).

Nucleoprotein (NP) and polymerase (P) proteins were purified from A/duck/Oklahoma/4/77 by glycerol and glycerol-cesium chloride (CsCl) gradients as previously described (Parvin, J.D. et al , J. Virol. 65:5142-5152 (19δ9)). An artificial M ribonucleoprotein (RNP) complex was prepared by transcribing puCT3PRM, pUCT3COOH-l, pUCT3COOH-5, or pUCT3COOH-10 with T3 RNA polymerase in the presence of the NP and P, after these plasmids were digested with Ksp632I and filled-in with Klenow fragment, as described (Enami, M. et al , Proc. Natl. Acad. Sci. USA 57:3δ02-3δ05 (1990); Enami, M. et al. , J. Virol. 65:2711-2713 (1991)). The

M RNP complex was then transfected into 70-90% confluent MDBK cells infected 1 hr before transfection with Eq/MIA at a multiplicity of infection of 1.

Alternative Selection 1: Amantidine selection was carried out as follows. Eighteen hours after transfection, MDCK cells were infected with transfectants in supernatant fluid in the presence of amantadine (lμ/ml). Three days later, viruses in the supernatant were plaque-purified in MDCK cells three times in the presence of amantadine (lμ/ml), and then inoculated into embryonated eggs. Alternative Selection 2: Alternatively, amantidine and antibody selection of transfectant viruses (containing the M gene encoding the amantadine resistant M2 protein) was carried out as follows. Eighteen hours after transfection, MDCK cells were infected with transfectants in supernatant fluid in the presence of amantadine (1 μg/ml). Three days later, viruses were further identified with the use of a monoclonal antibody specific for human

IAV M2. This antibody reacts with the M2 protein encoded by the transfected M gene, but not with the M2 protein produced by the helper virus. Appropriate numbers of plaques were picked, according to the ratio of transfectant and helper viruses identified with the monoclonal antibody specific for the M2 protein. Pure populations of the transfectant viruses were obtained by plaque purifying three times in the presence of amantadine (1 μg/ml).

The M gene of the viruses was sequenced as disclosed (Katz, J.M. et al , J. Gen. Virol 73:1159-1165 (1992)), to confirm the origin of the gene and the intended mutations and to ensure no unwanted mutations as described.

Immunologic methods. HA titration and hemagglutination inhibition (HI) tests were performed with receptor-destroying enzyme (RDE)-treated antisera in microliter plates (Palmer, D.F. et al , Immunol Ser. 6:51-52 (1975)).

Studies in ferrets. To determine viral replication in vivo, we anesthetized two 5-month-old ferrets (Marshall Farms, North Rose, NY) with ketamine-HCl (50 mg) intramuscularly and infected them intranasally with 1 ml of virus (approximately 10⁷ plaque-forming units (PFU)). The animals' nostrils were washed out with 1 ml of phosphate-buffered saline (PBS), pH 7.2, and the virus in nasal wash samples was titrated with MDCK cells.

For protection assays, ferrets were infected with the wild type virus (10⁷ PFU) 3 weeks after initial infection, and virus titers in nasal wash samples were examined as described above.

Results

Generation of an influenza virus containing the Mgene derived from cloned cDNA. To generate a transfectant virus with the M gene derived from cloned cDNA, we followed the basic reverse genetics procedure of Palese and colleagues (Enami, M. et al , Proc. Natl Acad. Sci. USA 57:3δ02-3δ05 (1990); Enami, M. et al., J. Virol 65:2711-2713 (1991)), which require a strong selection system for the virus with the rescued gene, because the majority of virus in the transfection supernatant are helper virus. We therefore chose amantadine resistance conferred by the M2 protein of PRδ strain, a naturally amantadine-resistant virus, as a selection method to rescue its M gene, using amantadine-sensitive Eq/MIA as a helper virus. PCR amplification and partial sequencing of the M gene of viruses derived from individual plaques grown in the presence of amantadine showed that approximately 50% of the plaques represented viruses with the PRδ M gene (designated Eq/MLA-PRδM). All remaining plaques were considered amantadine-resistant mutants of the helper Eq/MIA virus, and were not examined further. We, however, did not obtain viruses with the PRδ M gene by direct plaque formation of the transfection supernatant in the presence of amantadine; more than 20 plaques examined were all Eq/MIA. Thus, our selection system permits generation of influenza A viruses containing the M gene with mutations introduced in vitro.

The carboxyl-terminal Glu in the M2 protein is not essential for viral replication. Using the selection system described above, we attempted to attenuate influenza A virus by introducing mutations in an M gene product (M2 protein). The total number of M2 amino acid residues is identical among all influenza A viruses examined (Hay, A.J. et al , EMBO J. 4:3021-3024 (19δ5)), suggesting that the entire M2 protein is important for normal functioning. We therefore attempted to generate influenza A viruses with carboxyl-terminal deletions in their M2 proteins, assuming that any rescued M2 deletion mutants would likely be attenuated. Attempts to generate viruses with either five or ten deleted residues (COOH-5 and COOH-10) were unsuccessful; however, a mutant with a carboxyl terminal Glu deletion (COOH-1) was rescued with comparable efficiency to that of viruses with the parental (PRδ M gene).

The COOH-1 mutant produced slightly smaller plaques on MDCK cells than did the transfectant virus with the PRδ M gene (2-mm vs. 3-mm diameter on day 3), suggesting that the former was attenuated. When examined in ferrets, the COOH-1 mutant had a 10-fold lower titer than did the parental

Eq/NHA-PRδM virus three days after infection and recovered up to four days instead of five days as did the parental virus. These findings show that the COOH-1 virus is attenuated in ferrets.

To determine if the COOH-1 virus confers protective immunity against the wild type virus, we inoculated ferrets with Eq/MLA-PRδM virus (10⁷ PFU) three weeks after infection with COOH-1. Both the duration of virus shedding (4 vs. 5 days) and titer (10⁷ vs. 10^s PFU) were reduced in ferrets previously infected with COOH-1 as compared to results in non-immunized controls (Table X). These findings indicate that the COOH-1 virus confers protective immunity against the wild type virus. We next examined the stability of the mutation during replication in ferrets. Sequencing of the M gene of the COOH-1 mutant, recovered from two ferrets on the last day of shedding (4 days after infection), failed to reveal additional mutations in five plaque-purified clones (two from ferret no. 1 and three from ferret no. 2).

Discussion

In this study, we established a rescue system for the M gene of influenza A viruses, whose efficiency is sufficient for generating transfectants that contain a mutation in the M2 or Ml protein. The establishment of an M gene rescue system provides an elegant means to produce live, attenuated influenza vaccines. In a preferred embodiment a master strain used in vaccine production can contain attenuating mutations in genes for internal proteins in addition to HA and NA gene from a currently circulating strain. The present invention provides such attenuating mutations, and screening methods for determining an optimal balance between attenuation and viral replication. Amantadine resistance, the selection pressure used to rescue the M gene from cloned cDNA, is not a desirable feature for vaccine strains, because amantadine and its derivative, rimantadine, are the only licensed antiviral drugs against influenza A. Thus, an alternative system that would generate viruses with attenuating mutations in the M gene, without introducing amantadine resistance, is also provided by use of alternative selection systems, or by the removal of the amantadine resistance after selection of attenuated viruses of the present invention. In one alternative, this objective is met by production of attenuated viruses with a specific M-gene-determined host range. Several influenza viruses with mutations in the NA gene have been generated by reverse genetics, and their host range differs from that of the parent virus (Luo, G. et al , Virus Res. 29:141-153 (1993); Castrucci, M.R. et al , J. Virol 67:759-764 (1993)). Thus, according to the present invention, viruses have similarly different host ranges, useful for selection of attenuated or enhanced viruses are used as helper viruses in the generation of amantadine- sensitive, or other selection sensitive transfectant viruses.

Example 2: Elucidation of the Molecular Mechanisms of Virulence Attributable to the M Gene

Molecular Basis for High-Yield Growth Characteristics

Because of a correlation of increased virulence and high-yield property, high efficiency or enhancement of viral replication is provided by mutated M gene encoding an EMP of the present invention. Amino acid changes responsible for the high-yield property are provided according to the present invention, as demonstrated by increased IAV virulence in mice and ferrets having enhance Ml or M2 EMP encoding nucleic acid.

Identification of Critical Amino Acid Changes

Enhancing mutations are provided by the present invention, e.g. , by comparing the M genes of high-yield (WSN) and ordinary (Aichi) viruses

(Smeenk et al., J. Virol. 65:530-534 (1994), to identify amino acid residues that are responsible for supporting the high growth of an IAV and to provide virus (δ2). We intend to pinpoint such residues by using reverse genetics to generate viruses containing substimtions in the M gene products.

Suitable Amino Acid Mutations. Mutations are introduced in the M gene of the PRδ virus, a high-yielding strain (10⁹ PFUs/ml in MDCK cells) for which we already have a reverse genetics system. Among 11 positions (41, 204, 205 and 21δ in the Ml and 2δ, 31, 54, 56, 57, δ9 and 93 in the M2) where the Aichi and WSN M gene products differ, only two (41 in the Ml (Table 5) and 31 in the M2 are common to enhancing WSN and PRδ viruses. A change found at residue 31 of the M2 protein is responsible for amantadine resistance, which does not correlate with the enhancing phenotype. The Ala-to-Val change at residue 41 of the Ml is also found in a mouse- adapted A/Port Chalmers/73 (H3N2) virus. In addition, a change at residue 139 from Thr to Ala in the Ml has been identified in the mouse-adapted A FM/ 1/47 strain and shown to be responsible for enhancing characteristics (Smeenk et al, J. Virol 65:530-534 (1994)). This change has also been detected in another mouse-adapted A/WS/33 strain, NWS. Thus, an amino acid change at either residue 41 or 139 could result in enhancing properties.

Table 5. Amino acid residues at positions 41 and 139 of the Ml protein

Amino Natural isolates and their variants Transfectants to be made acid by reverse genetics resi¬

Aichi WSN PR8 PC PC-M FM FM-M 41A 139A 41A139A

41 Ala Val Val Ala Val Ala Ala Ala Val Ala

139 Thr Thr Thr Thr Thr Thr Ala Thr Ala Ala

Note: PC, A/Port Chalmers/73 (H3N2); FM, A/FM/1/47 (HlNl). "-M" indicates mouse- adapted. Changes specifically found in mouse-adapted strains are shown in bold-face type. Residues to be changed in the transfectant PR8 Ml are shown in italics. Generation of transfectants

Using the reverse genetics system described herein, enhanced viruses are generated containing at least one amino acid change at residue 41 from Val to Ala (41A), at residue 139 from Thr to Ala (139A), or double mutations at residues 41 from Val to Ala and 139 from Thr to Ala (41A139A) in the

PRδ Ml protein (Table 5). As a helper virus, a reassortant is used containing only the M gene from an amantadine-sensitive A/equine/Miami/ 1/63 (H3N8) and all the other genes from PRδ. Thus, upon rescue of the M gene, viruses are obtained containing mutations in that gene only. Virus replication in MDCK cells (plaque formation as well as 50% tissue culture infectious dose

(TCIDso) determination) is also tested.

It is expected that superior growth is provided by one or more of these mutants.

Additionally, the entire M gene is optionally sequenced before the rescue experiments are begun, to determine whether undesired or unspecified mutations are provided. The entire M genes of transfectant viruses are also optionally sequenced to ensure that they contain only desired mutations. Mutations may also be introduced into other genes upon rescue of the mutant M, thus affecting the viral phenotype. At least two viruses are generated for each mutant.

Correlation of the enhancing phenotype with virulence

When amino acid changes responsible for the enhancing phenotype in vitro are determined, their effects on virulence are next tested. Because the

PRδ strain is mouse-adapted, the effects of mutations in the M gene are determined using this animal model. Ferrets are also optionally used, which are routinely employed in virulence evaluations of human viruses.

Determination of virulence. Three animals per mutant virus are infected with 10⁴ PFU virus under anesthesia, and the virus titer (in nasal washes for ferrets and trachea and lung for mice) is examined in MDCK cells three days after infection (highest virus titers were found on day 3 in previous experiments). In mice, virus titers in trachea and lung are determined to examine the spread from upper respiratory tract to the lung. The virus titers in these organs of mice correlates with virulence

(P6).

A dose required to kill 50% of mice (MLD*,) and to infect animals (IDso) is also determined. A one log difference in the MLD_J0 and IDjo and more than a two log difference in virus titers in organs and nasal washes, based on previous findings (P3.P6), serves as criteria for a difference in virulence. A transfectant virus with the wild-type PRδ M gene is the control.

If viruses yielding higher titers in vitro are more virulent, then the enhancing phenotype does in fact confer virulence. This results allows prediction of increased virulence on the basis of the amino acid sequence in the Ml or M2 EMP. One or more amino acid changes correlate with virulence. Changes resulting in the enhancing phenotype in vitro but not virulence are useful to introduce into live vaccine strains for culturing. If neither substimtion correlates with virulence, replication in vitro may not relate to virulence, although the likelihood of this result is remote according, to recent findings (Smeenk et al, J. Virol. 65:530-534 (1994)).

Mechanism of enhancing growth induced by gene products

It is expected that the Ml protein is involved in the uncoating and transport of RNP from the nucleus to cytoplasm. It is also expected to alternatively or additionally contribute to budding. To determine if enhancing viruses have a short replication cycle (thus leading to high titers in a given period of time) or produce more virus particles in a single replication cycle than do low-yield viruses, one-step growth characteristics are tested using the transfectant viruses described herein. The differences in Ml functions -6δ-

between high- and low-yield viruses are then optionally determined, as described herein.

One step growth. Known method steps are followed (Burleson et al, Viral replication: one-step growth curve, In Virology: A laboratory manual, Academic Press, San Diego (1992), p. 100-106) to construct one-step growth curves for high- and low-yield viruses. If the replication cycle is faster in the enhancing viruses, it is expected that Ml promotes rapid uncoating or influences the efficiency of RNP transport from the nucleus. If, however, they generate more infectious virus particles than the low-yielding viruses but the time required for a single replication cycle is the same, then budding is the expected step affecting virus yield. An increased efficiency of RNP transport from the nucleus could also result in increased virus formation. The following experiments provide data to pinpoint the differences between high- and low- yield virus replication. Because one mutation may have multiple effects, it is optionally preferred to examine the mutants for each of the three aspects described below.

Uncoating

Kinetics of RNP transport

In the current view of uncoating, upon endocytosis, the core of influenza A virus is exposed to low pH by the function of the M2 ion channel.

Because association of the Ml with RNP interferes with transport of RNP to the nucleus (Martin et al, Cell 67:117-130 (1991)), the rapidity of dissociation of the Ml protein from RNP upon exposure to low pH may be responsible for a difference in uncoating. Thus, enhancing viruses may be uncoated faster than others, so that their replication cycles are faster than those of ordinary viruses. To test this expectation, the kinetics of migration of RNP into the nucleus by immunofluorescence assays are compared after infection with monoclonal antibodies (MAbs) to the NP and Ml proteins (Martin et al, J. Virol 65:232-244 (1991)), and by in situ hybridization using an influenza virus specific probe (e.g. , NP gene) Enami et al, Virology 194:822-827 (1993)). Kinetics of the NP and Ml transport of incoming virus particles has been quantitated by confocal microscopy (Martin et al, J. Virol 65:232-244 (1991)) after synchronizing infection by letting virus bind to cells at 4°C and penetrate at 37°C. Quantitative confocal microscopy is optionally preformed using a suitable imaging system, as is known in the art. MAbs to these proteins and the NP gene for the probe preparation are readily available from research or commercial sources. If the enhancing virus' NP and NP gene appear in the nucleus after infection more rapidly than those of the low-yield virus, it is expected that the uncoating step is responsible for the difference in growth rate and that the enhancing virus' Ml dissociates faster from RNP. Although the molecules in RNP to which the Ml binds (RNA, NP, polymerase proteins or NS2) are unknown, a change at residue 139 of the Ml protein, potentially important in generation of the enhancing phenotype, is located in the RNA binding region (Ye et al, J. Virol. 63:3586-3594 (1989)). Thus, the Ml of enhancing viruses may dissociate more rapidly than the Ml of low-yield viruses because of an altered Ml -RNA association.

pH-dependent dissociation of the Ml from RNP

If a difference in the kinetics of RNP transport between high- and low- yield viruses is determined, the molecular basis for this distinction is optionally determined. Because the Ml dissociates from the RNP upon exposure to low pH (Zhirnov, O.P., Virology 176:274-279 (1990)), a difference in pH threshold may influence the speed of uncoating. To determine the pH threshold of Ml dissociation from RNP, an assay is employed as method steps used by Zirnov (Zhirnov, O.P., Virology 176:274- 279 (1990)). Virus is ultracentrifuged through 15% glycerol in water/25% glycerol in Tris-Mes (morpholinoehanesulfonic acid) buffer, containing 1% Nonidet P-40, protease inhibitors, and 150 mM NaCl, adjusted to a pH ranging from 5 to 8. Viral envelope is disrupted under this condition and the core RNP is then precipitated. Since the dissociation of the Ml from RNP depends upon pH values of buffer, determination of the pH threshold for Ml dissociation is provided by comparing the ratio of Ml to NP in the pellet.

If the Ml of the enhancing virus dissociates from the RNP at higher pH values than does the low yield virus' Ml, a higher pH threshold is responsible for rapid uncoating, leading to rapid initiation of primary transcription. This shows that the limited introduction of protons into the viral core via the M2 ion channel can initiate dissociation of the Ml from RNP in enhancing viruses.

Transport of RNP from the nucleus to the cytoplasm

It is expected that altered transport of RNP from the nucleus to the cytoplasm contributes to the enhancing phenotype, and is confirmed by quantitating the movement of RNP with anti-NP and Ml Mabs using immunofluorescence assays (Martin et al, Cell 67:117-130 (1991); Martin et al., J. Virol 65:232-244 (1991); Rey et al., J. Virol. 66:5815-5δ24 (1992)) and in situ hybridization (Enami et al, Virology 194:822-827 (1993)) with a probe specific for NP vRNA as described earlier in this application. If the RNP of the enhancing virus is transported more efficiently

(appears more rapidly and/or in higher quantities), active participation of RNP transport in enhancing growth is shown. As described herein, one of the changes (residue 139) that is expected to affect the in vitro yield of virus is located in the RNA binding region (Ye et al, J. Virol. 63:3566-3594 (19δ9)). Thus, the mode of Ml binding to vRNA could differ between the high- and low-yield viruses. Alternatively, the differences are expected to lie in Ml binding to other viral proteins in RNP, such as the NP. Other expected mechanisms include, but are not limited to interaction of the Ml -RNP complex with cellular proteins for its transport. Budding

It is not presently known whether all of the Ml molecules act in concert to escort RNP from the nucleus and always associate with RNP or whether a function of the Ml is also associated with the lipid bilayer at the plasma membrane and awaits the Ml -RNP complex from the nucleus, as hypothesized for vesicular stomatitis virus assembly (Chong et al, J. Virol 67:407-414 (1993)). However, because the purified Ml associates with liposome (Bucher et al, J. Virol 36:5δ6-590 (19δ0); Gregoriades et al, J. Virol. 40:323-32δ (1981)), the Ml is presumed to interact with cellular membranes. A recent electron microscopic study (Fujiyoshi et al, EMBOJ.

73:318-326 (1994)) suggesting modification of the viral envelope by Ml proteins would support the latter possibility. Moreover, although no direct evidence is available, the Ml is now expected to associate with other viral proteins (i.e. , HA, NA, or M2), in ways that could be essential for budding. It is therefore expected that a difference in budding efficiency results in the enhancing phenotype by examining mutation of an Ml MP to form an Ml EMP of the present invention as an Ml or Ml -RNP complex with cellular membranes.

Interaction of the Ml with cellular membranes

To examine the level of Ml incorporation into cellular membranes, the

Ml protein from the cDNA is expressed in MDCK cells in the presence of (³⁵S)methionine, using either the vaccinia or Semliki forest virus (SFV) (Liljestrom et al, Biotechnology 9: 1356-1361 (1991)) expression system, both of which are currently in use in my laboratory (P3,P29). The vaccinia system expresses sufficient wild-type Ml proteins to correct the RNP-escorting defect of the ts51 Ml at a nonpermissive temperature (Dr. J. Ye, personal communication). Equivalent or greater amounts of protein are expressed by the SFV system (Liljestrom et al, Biotechnology 9:1356-1361 (1991)). The vaccinia system has proved useful in a variety of smdies, including virus assembly (Chong et al, J. Virol. 67:407-414 (1993); Chong et al., J. Virol. 65:441-447 (1994); Simpson et al, J. Virol 66:790-δ03 (1992)). The SFV system is also used (only four nonstructural SFV proteins are made). Cell membranes are fractionated according to published methods (Chong et al, J.

Virol 65:441-447 (1994)) known to give good separation of membranes derived from ER, Golgi apparatus, and the plasma membrane. The fractions containing these membranes are identified by measuring the sucrose density of individual fractions and the presence of the HA (expressed from cDNA) for the plasma membrane, Bip for the ER, or -mannosidase II for Golgi membranes.

These marker proteins are identified by radioimmunoprecipitation (HA and Bip) or enzyme activity (α-mannosidase II (Storrie et al, Methods Enzymol. 752:203-225 (1990))). The distribution of the Ml proteins is then compared between the high- and low-yield viruses expressed from the cDNA among membrane fractions. Because the entire M gene encodes the M2 protein as well, a construct containing only the Ml coding region is used, so that the effect of the M2 protein is eliminated. Experiments expressing other viral proteins are also performed (HA, NA, and M2) in addition to the Ml to examine their effect on the Ml association with membrane. The cDNA clones are provided as described herein and the protein in each fraction will be identified by either radioimmunoprecipitation or Western blotting. MAbs to these proteins are also available from research or commercial sources.

At least two major outcomes are expected. Finding more Ml in the plasma membrane of the enhancing virus suggests that the protein contributes to more efficient budding, thus leading to increased virion formation. Alternatively, differences can be found in the distribution of the Ml among other membrane fractions, such as the Golgi membrane, where a higher concentration of the protein could increase interactions with HA, NA, and M2 in this compartment. A difference in interaction of the Ml with membranes between high- and low-yield viruses only when expressing other influenza viral proteins, shows that differences in the Ml-viral protein interaction can affect virus yield. If so, critical interactions are then identified between the Ml and other viral proteins by expressing these proteins in different combinations, according to known method steps.

Interaction of the Ml-RNP complex with the plasma membrane

Because the Ml escorts RNP from the nucleus to cytoplasm and presumably to the budding site (i.e., plasma membrane), the level of the interaction of the Ml-RNP complex with the plasma membrane is expected to affect the budding efficiency. The Ml-RNP complex is prepared, e.g. , in pH 7.2 buffer by ultracentrifugation of lysate of cells infected with either high- or low-yield virus labeled with (³⁵S)methionine as described (Deshpande et al, Virology 139:32-42 (19δ4); Martin et al, J. Gen. Virol 73: Iδ55-lδ59 (1992); Zhirnov, O.P., Virology 776:274-279 (1990)). Under this condition, the Ml is still associated with RNP. By mixing the Ml-RNP complex with the membrane fractions, followed by sucrose density gradient centrifugation and analysis of each fraction on SDS-PAGE, changes in association of the Ml- RNP complex with the plasma membrane are detected. Experiments using cellular membranes expressing other viral proteins (HA, NA, M2, and corresponding Ml) are also performed. If the Ml-RNP complex associates with the plasma membrane without expression of other viral proteins, differences in plasma membrane Ml-RNP quantities are compared between the high- and low-yield viruses. If not, results with the membranes expressing other viral proteins show specific interactions of the Ml-RNP complex with these proteins. The findings of a greater quantity of the Ml-RNP complex in association with the plasma membrane (with or without other viral proteins) of enhancing viruses would show that association of the complex is a major factor in development of the enhancing phenotype. If a difference only with the expression of other viral proteins is shown, then the responsible proteins are identified by expressing them individually.

Role of filamentous versus spherical virulence

Recent evidence indicates that filamentous particles are more infectious than sphencalones (Shaw, C.-K. , Comparision of the M Gene in Filamentous and Spherical Influenza Virus, Ph.D. thesis. University of Michigan, Ann

Arbor (1993)). Clinical isolates of early passages in vitro contain more filamentous than spherical particles, whereas laboratory strains passaged extensively in such cultures contain spherical virions predominantly (Hoyle, L., "Morphology and Physical Structure" In The Influenza Viruses, Springer-

Verlag, New York (196δ), p. 49-6δ). This observation and general knowledge of the reduction of virulence in many viruses during passage in vitro suggest an association of the shape of influenza virus with virulence.

Virion shape is expected to influence influenza pathogenesis, amino acid substimtions in the M gene products are provided according to the present invention by determining the morphology of influenza virions, testing the influence of virion shape on virulence, and then showing how the mutations influence virion morphology.

Determination of amino acid changes involved in morphology

Morphological amino acid residues. Shaw (Shaw, C.-K. , Comparision of the M Gene in Filamentous and Spherical Influenza Virus, Ph.D. thesis. University of Michigan, Ann Arbor (1993)) compared amino acid sequences of the Ml and M2 proteins of spherical WSN, filamentous NWS-F and a reassortant virus containing only the M gene from the NWS-F and all the other genes from WSN (WSN/NWS(M)). The WSN/NWS(M) virus used by

Shaw was originally filamentous, but over time, it had become spherical. There are two amino acid differences in the Ml and one in the M2 between NWS-F and WSN/NWS(M) viruses. The amino acid change at residue 222 relates to shifts in morphology, because the WSN strain differs from the NWS-F strain only at that position among the three residues (Table 6).

Table 6. Amino acid sequence divergence among the Ml and M2 proteins and its relationship to viral morphology

In addition, a natural isolate, A/Korea/426/68 Ml contains Pro, whereas the PRδ Ml contains His at position 222 (P14). Electron microscopic analysis showed that A/Korea/426/68 was filamentous but a transfectant virus containing the PRδ M gene was spherical, demonstrating a strong correlation between the Pro-222 in the Ml and the filamentous phenotype.

Amino acid residues responsible for virion shape are determined, and transfectant viruses are generated, each containing a change from Asn-207 to Ser, His-222 to Pro in the Ml, or Ser-δ2 to Asn in the M2 of PRδ strain by reverse genetics as described earlier. The transfectant viruses evaluated under the electron microscope. The filamentous A/Korea/426/68 containing Pro-222 in the Ml and a spherical transfectant containing the wild type PRδ Ml serve as controls.

Amino acid changes associated with a shift in virion shape from spherical to filamentous are expected to show that a single amino acid change in the Ml determines the ultimate morphology of influenza virus. If the transfectant viruses are spherical, a virus is generated containing all three changes. If this triple mutant is filamentous, viruses are generated containing different combination of changes to pinpoint those with the major influence.

Virion shape influences infectivity and virulence

In vitro infectivity

Changes in the shape of influenza A viruses are expected to affect infectivity, as shown by examining the ratio of PFUs per virion. Electron microscopy is used to count the number of virions as compared to an internal standard (latex beads). PFUs are determined with MDCK cells. If the filamentous virions are more infectious than the spherical ones on the basis of virion number, virion shape promotes the infectivity of influenza viruses in vitro.

Virulence

The MLD_JO, ID^ and virus titers are examined in organs in mice and in nasal washes in ferrets as described earlier. If the filamentous viruses are more infectious in animals, evidence of increased virulence (i.e. , reduced

MLDso, ID₅₀ and/or increased virus titers in organs and nasal wash) is shown. If replication levels differ between filamentous and spherical viruses, virion shape is directly related to virulence.

Molecular mechanisms by which the Ml protein affects virion shape

Because the change at residue 222 in the Ml is expected to be responsible for the alteration in virion shape, Ml functions are involved. Since virion shape is most likely to be determined during the budding process this step is expected to be involved in enhancement of virulence. lnteraction of the M1(-RNP) with the plasma membrane

Because virion shape can be influenced by interactions of the Ml with itself or other viral proteins, differences are distinguished between filamentous and spherical viruses in interactions of the Ml protein or Ml-RNP complex with the plasma membrane, in the presence or absence of the HA, NA and/or

M2, using previously described methods.

If more Ml protein is associated with the plasma membrane for filamentous than for spherical viruses, the degree of Ml association with the plasma membrane influences virus morphology. If the difference occurs only when the plasma membrane contains other proteins (HA, NA, or M2), this relationship depends on interaction of the Ml with other viral proteins. Using data obtained by methods described herein, proteins are identified which interact with the Ml and therefore have important roles in determining virion shape.

Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the inventions following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.

All references cited herein, including journal articles or abstracts, published or corresponding U.S. or foreign patent applications, issued U.S. or foreign patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by reference. Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: St. Jude Children's Research Hospital

(ii) INVENTORS: Castrucci, Maria R. Kawaoka, Yoshihiro

(iii) TITLE OF INVENTION: Nucleic Acid Encoding Mutant Matrix Proteins Useful for Attenuation or Enhancement of Influenza A Virus, Vaccines and Methods of making and Using Thereof

(iv) NUMBER OF SEQUENCES: 13

(v) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: STERNE, KESSLER, GOLDSTEIN & FOX

(B) STREET: 1100 New York Avenue, N. .

(C) CITY: Washington

(D) STATE: DC

(E) COUNTRY: USA

(F) ZIP: 20005

(vi) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30

( ii) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: TBA

(B) FILING DATE: 29-SEPT-1995

(C) CLASSIFICATION:

(Viii) PRIORITY APPLICATION DATA:

(A) APPLICATION NUMBER: 08/316,419

(B) FILING DATE: 30-SEPT-1994

(C) CLASSIFICATION: and

(A) APPLICATION NUMBER: 08/471,100

(B) FILING DATE: 6-JUNE-1995

(C) CLASSIFICATION:

(ix) ATTORNEY/AGENT INFORMATION:

(A) NAME: Fox, Samuel L.

(B) REGISTRATION NUMBER: 30,353

(C) REFERENCE/DOCKET NUMBER: 0656.048PC01

(x) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 202-371-2600

(B) TELEFAX: 202-371-2540

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 251 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: not relevant

(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser lie Val Pro 1 5 10 15

Ser Gly Pro Leu Lys Ala Glu lie Ala Gin Arg Leu Glu Asp Val Phe 20 25 30

Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr 35 40 45

Arg Pro lie Leu Ser Pro Leu Thr Lys Gly lie Leu Gly Phe Val Phe 50 55 60

Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gin Arg Arg Arg Phe Val 65 70 75 80

Gin Asn Ala Leu Asn Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala 85 90 95

Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu lie Thr Phe His Gly Ala 100 105 110

Lys Glu Val Ala Leu Ser Tyr Ser Gly Ala Leu Ala Ser Cys Met Gly 115 120 125

Leu lie Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Val Ala Phe Gly 130 135 140

Leu Val Cys Ala Thr Cys Glu Gin lie Ala Asp Ser Gin His Arg Ser 145 150 155 160

His Arg Gin Met Val Thr Thr Thr Asn Pro Leu lie Arg His Glu Asn 165 170 175

Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gin Met Ala 180 185 190

Gly Ser Ser Glu Gin Ala Ala Glu Ala Met Glu Val Ala Ser Gin Ala 195 200 205

Arg Gin Met Val Gin Ala Met Arg Thr lie Gly Thr His Pro Ser Ser 210 215 220

Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gin Ala Tyr Gin 225 230 235 240

Lys Arg Met Gly Val Gin Met Gin Arg Phe Lys 245 250

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 97 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: not relevant

(D) TOPOLOGY: not relevant

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: -81-

Met Ser Leu Leu Thr Glu Val Glu Thr Pro Xaa Arg Asn Glu Trp Gly 1 5 10 15

Cys Arg Cys Asn Asp Ser Ser Asp Pro Leu Val lie Ala Ala Ser lie 20 25 30 lie Gly lie Leu His Leu lie Leu Trp lie Leu Asp Arg Leu Phe Phe 35 40 45

Lys Cys lie Tyr Arg Arg Leu Lys Tyr Gly Leu Lys Arg Gly Pro Ser 50 55 60

Thr Glu Gly Val Pro Glu Ser Met Arg Glu Glu Tyr Arg Gin Glu Gin 65 70 75 80

Gin Ser Ala Val Asp Val Asp Asp Gly His Phe Val Asn lie Glu Leu 85 90 95

Glu

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1027 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Influenza virus

(B) STRAIN: A/Bangkok/1/79

(viii) POSITION IN GENOME:

(A) CHROMOSOME/SEGMENT: 7 (MBANGK)

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

AGCAAAAGCA GGTAGATATT GAAAGATGAG CCTTCTAACC GAGGTCGAAA CGTATGTTCT 60

CTCTATCGTT CCGTCAGGCC CCCTCAAAGC CGAAATCGCG CAGAGACTTG AAGATGTCTT 120

TGCTGGAAAG AACACAGATC TTGAGGCTCT CATGGAATGG CTAAAGACAA GACCAATCCT 180

GTCACCTCTG ACTAAGGGGA TTTTGGGATT TGTGTTCACG CTCACCGTGC CCAGTGAGCG 240

AGGACTGCAG CGTAGACGCT TTGTCCAAAA TGCCCTCAAT GGGAATGGGG ATCCAAATAA 300

CATGGACAGA GCAGTTAAAC TATACAGAAA ACTTAAGAGG GAGATAACAT TCCATGGGGC 360

CAAAGAAATA GCACTCAGTT ATTCTGCTGG TGCACTTGCC AGTTGCATGG GCCTCATATA 420

CAACAGGATG GGGGCTGTAA CCACTGAAGT GGCCTTTGGC CTGGTATGTG CAACCTGTGA 480

ACAGATTGCT GACTCCCAGC ACAGGTCTCT TAGGCAAATG GTGGCAACAA CCAATCCACT 540

AATAAGACAT GAGAACAGAA TGGTTCTGGC CAGCACTACA GCTAAGGCTA TGGAGCAAAT 600

GGCTGGATCA AGTGAGCAGG CAGCAGAGGC CATGGAGGTT GCTAGTCAGG CCAGGCAAAT 660

GGTGCAGGCA ATGAGAGCCA TTGGGACCCA TCCTAGCTCC AGTGCTGGTC TAAAAGATGA 720

TCTTCTTGAA AATTTGCAGA CCTATCAGAA ACGAATGGGG GTGCAGATGC AACGATTCAA 780 GTGACCCTCT TGTTGTTGCT GCGAGTATCA TTGGGATCTT GCACTTGATA TTGTGGATTC 840

TTGATCGTCT TTTTTTCAAA TGCATCTATC GATTCTTCAA ACACGGCCTG AAAAGAGGGC 900

CTTCTACGGA AGGAGTACCT GAGTCTATGA GGGAAGAATA TCGAAAGGAA CAGCAGAATG 960

CTGTGGATGC TGACGACAGT CATTTTGTCA GCATAGAGCT GGAGTAAAAA ACTACCTTGT 1020

TTCTACT 1027 (2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 48 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..45

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

GCT GTG GAT GCT GAC GAT GGT CAT TTT GTC AGC ATA GAG CTG GAG 45

Ala Val Asp Ala Asp Asp Gly His Phe Val Ser lie Glu Leu Glu 1 5 10 15

TAA 48

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Ala Val Asp Ala Asp Asp Gly His Phe Val Ser lie Glu Leu Glu 1 5 10 15

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 48 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..42

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GCT GTG GAT GCT GAC GAT GGT CAT TTT GTC AGC ATA GAG CTG 42

Ala Val Asp Ala Asp Asp Gly His Phe Val Ser lie Glu Leu 20 25

TAGTAA 48

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Ala Val Asp Ala Asp Asp Gly His Phe Val Ser lie Glu Leu 1 5 10

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 48 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..30

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

GCT GTG GAT GCT GAC GAT GGT CAT TTT GTC TGAATAGAGC TGGAGTAA 48

Ala Val Asp Ala Asp Asp Gly His Phe Val 15 20

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Ala Val Asp Ala Asp Asp Gly His Phe Val 1 5 10

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 48 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..15

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

GCT GTG GAT GCT GAC TAAGGTCATT TTGTCAGCAT AGAGCTGGAG TAA 48

Ala Val Asp Ala Asp 15

(2) INFORMATION FOR SEQ ID NO:ll:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

Ala Val Asp Ala Asp 1 5

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: not relevant

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: ATCGATGAAT TCTCTTCGAG CGAAAGCAGG TAGATATTG 39

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 52 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: not relevant

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: GAGGACAAGC TTATTAACCC TCACTAAAAG TAGAAACAAG GAGTTTTTTA CT 52

Claims

What Is Claimed Is:

1. An attenuating nucleic acid, in isolated form, comprising a polynucleotide in isolated form encoding at least one attenuating matrix protein (AMP) corresponding to at least one 10 amino acid portion of a naturally occurring influenza A virus (LAV) matrix protein (MP), wherein said

AMP comprises at least one attenuating mutation capable of attenuating an LAV comprising said attenuating nucleic acid.

2. An attenuating nucleic acid according to claim 1, wherein said MP is Ml MP or M2 MP.

3. A vector, comprising an attenuating nucleic acid according to claim 1.

4. A host cell, comprising an attenuating nucleic acid according to claim 1.

5. An attenuated influenza A virus (attenuated LAV), comprising (A) a pathogenic negative strand RNA (nsRNA) encoding at least one neuraminidase (NA) and hemagglutinin (HA) from at least one pathogenic influenza A virus (LAV) strain; and

(B) at least one attenuating nsRNA derived from an attenuating nucleic acid according to claim 1, wherein said attenuating nsRNA attenuates said attenuated LAV.

6. A vaccine composition, comprising an attenuated LAV according to claim 5, and a pharmaceutically acceptable carrier or diluent.

7. A method for obtaining an attenuated influenza A virus (attenuated LAV), comprising (A) isolating an attenuated LAV comprising (1) a pathogenetic negative strand RNA (nsRNA) encoding at least one neuraminidase (NA) and hemagglutinin (HA) from at least one pathogenic strain of an LAV; and (2) at least one attenuating nsRNA derived from an attenuating nucleic acid according to claim 1.

δ. A method according to claim 7, further comprising, prior to step (A),

(B) reassorting, in a reassorting host,

(i) a helper virus having ( 1 ) said attenuating nsRN A and (2) sensitivity to at least one selection marker; with

(ii) said pathogenic nsRNA encoding (1) at least one neuraminidase (NA) and hemagglutinin (HA) from said at least one pathogenic strain and (2) selection nsRNA encoding at least one selection protein conferring resistance to said selection marker, to provide said attenuated LAV.

9. A method according to claim δ, further comprising

(C) selecting said attenuated LAV using said selection marker.

10. A method according to claim δ, further comprising

(C) selecting said attenuated LAV using an antibody binding an epitope specific for said attenuated LAV.

11. An attenuated influenza A virus, obtained by a method according to claim 13.

12. An attenuated influenza A virus, obtained by a method according to claim 14. 13. A method of eliciting an immune response to at least one strain of an influenza A virus (LAV) in an animal, which immune response is prophylactic or therapeutic for an LAV infection, comprising

(a) administering to said animal an effective amount of a vaccine composition according to claim 12, which vaccine composition is effective to protect said animal against an IAV clinical pathology.

14. An attenuating composition, comprising an attenuating nucleic acid according to claim 1, and a carrier or diluent.

15. An enhancing nucleic acid, in isolated form, comprising a polynucleotide encoding at least one enhancing matrix protein (EMP) corresponding to at least one 10 amino acid portion of a naturally occurring influenza A virus (LAV) matrix protein (MP), wherein said EMP comprises at least one enhancing mutation capable of enhancing the growth of an IAV comprising said enhancing nucleic acid.

16. An enhancing nucleic acid according to claim 15, wherein said MP is Ml or M2.

17. A vector, comprising an enhancing nucleic acid according to claim 15.

lδ. A host cell, comprising an enhancing nucleic acid according to claim 15.

19. An enhanced influenza A virus (enhanced LAV), comprising at least one enhancing nsRNA derived from an enhancing nucleic acid according to claim 15, which enhances the growth of said enhanced influenza virus in culture. 20. A method for obtaining an enhanced influenza A virus (enhanced LAV), comprising

(A) isolating an enhanced IAV comprising at least one enhancing negative strand RNA (nsRNA) derived from an enhancing nucleic. acid according to claim 15, wherein said enhancing nsRNA enhances the growth of said enhanced IAV.

21. A method according to claim 20, wherein said method further comprises, prior to step (A), reassorting, in a reassorting host, (i) a helper virus having (1) said enhancing nsRNA and

(2) sensitivity to at least one selection marker; with

(ii) at least one LAV nsRNA encoding (1) at least one neuraminidase (NA) and hemagglutinin (HA) from at least one IAV strain and (2) selection nsRNA encoding at least one selection protein conferring resistance to said selection marker, to provide said enhanced IAV.

22. A method according to claim 20, further comprising

(C) selecting said enhanced IAV using said selection marker.

23. A method according to claim 20, further comprising

(C) selecting said enhanced LAV using an antibody binding an epitope specific for said attenuated LAV.

24. An enhanced influenza A virus, obtained by a method according to claim 20. 25. A method for enhancing the growth of at least one strain of influenza A virus (LAV) in culture, comprising introducing a growth enhancing effective amount of an enhancing nucleic acid according to claim 15 into at least one strain of an IAV.

26. An enhancing composition, comprising an enhancing nucleic acid according to claim 15, and a carrier or diluent.

27. An attenuated IAV vaccine, comprising an attenuated LAV according to claim 5 and a pharmaceutically acceptable carrier or diluent.