The present invention relates to nucleic acid molecules, polypeptides, antibodies and pharmaceutical composition containing same, which can be utilized for treating and detecting influenza virus infection in vertebrates such as avian, swines and humans.
Influenza viruses have been a major cause of mortality and morbidity in man throughout recorded history. Influenza epidemics occur at regular intervals, which vary widely in severity but which always cause significant mortality and morbidity, most frequently in the elderly population. An influenza infection produces an acute set of symptoms including headache, cough, fever and general malaise. In severe cases or situations involving pre-existing pulmonary or cardiovascular disease, hospitalization is required. Pneumonia due to direct viral infection or due to secondary bacterial or viral invasion is the most frequent complication. For a review on the clinical aspects of influenza virus infection see Douglas (1990) New England Journal of Medicine, 322:443-450.
Influenza viruses are currently divided into three types: A, B, and C, based upon differences in internal antigenic proteins; while the A and B types are closely related and account for most infections, the type C influenza virus represents a distant third in disease-causing potential and is probably of little public health concern. Although overall gene homology is less than 30%, between the A and B types, these viruses share a common ancestor and include eight RNAs of negative sense polarity. Hemagglutinin (HA) and neuraminidase (NA) are expressed on the surface of the lipid containing virus particles and are primarily responsible for the antigenic changes observed in influenza viruses.
New strains of influenza caused by antigenic drift appear at regular frequency, usually annually, and begin a cycle of infection, which travels around the globe. Little is known about how individual epidemics are initiated. Major new subtypes of influenza appear less frequently but can result in major pandemics.
It will be appreciated that up to 20% of the population may develop influenza infection in any given year and influenza epidemics are responsible for 20,000 deaths per year in the U.S. [Palese (2002) J. Clin. Invest. 110:9-13]. By far the most catastrophic impact of influenza during the past 100 years was the pandemic of 1918, which cost more than 500,000 lives in the U.S. and lowered life expectancy by almost 10 years [Heilman (1990) Clin. North Am. 37:669-688].
Given the impact of influenza on individuals and on society the challenge at present is to generate highly potent prophylactic tools which can be used to prevent influenza infection in subjects which are at considerable risk of infection such as young children and the elderly population.
Several approaches have been undertaken to uncover novel anti influenza agents.
Inactivated influenza virus vaccines—The most effective way to deal with the influenza virus for a population at risk of severe complications is by prevention. To be effective, current vaccines must contain an A, B and preferably C virus components. To prepare vaccines, the viral strains are grown in embryonated eggs, and the virus is then purified and made noninfectious by chemical inactivation. Use of the available influenza vaccine is an effective way to lower the mortality in a population, however due to the ever-changing nature of the virus, the development of a vaccine with the appropriate composition to protect against the currently circulating virus strains is complex and expensive. Moreover, patient compliance in receiving the vaccine is generally very low. Thus, large numbers of patients at risk of serious complications from influenza virus go unprotected.
Cold adapted influenza virus vaccines—The generation of temperature sensitive influenza viruses as live vaccines has been attempted because the pathogenicity in animals and mammals is significantly attenuated [Wareing (2001) Vaccine 19:3320-3330; Maasab (1990) Adv. Biotechnol. Processes 14:203-242]. Typically, to generate cold adapted viruses the influenza viruses are passaged in chicken kidney cells and in embryonated eggs to adapt growth thereof at 25° C. Thus, the annually adapted vaccine formulations can be genetically engineered to include the two genes which encode major viral surface antigens (i.e., HA and NA) reflecting the antigens found in current strains, whereas the remaining six genes derived from the cold-adapted master strains. Such live-virus vaccines can induce local neutralizing immunity and cell-mediated immune responses, which may be associated with a longer lasting and cross-reactive immunity than is elicited by chemically inactivated virus preparations. However, the use of live vaccines requires extensive monitoring against unexpected complications, which might arise from the spread of virulent revertants essentially explaining the nonexistence of licensure for such therapy in the U.S.
Genetically engineered live influenza virus vaccines—The advent of techniques for engineering site-specific changes in the genomes of RNA viruses rendered it possible to develop new vaccine approaches [Enami (1990) Proc. Natl. Acad. Sci. USA 87:3802-3805; Garcia-Sastre (1998) Trends. Biotechnol. 16:230-235]. Thus, generation of virus particles which undergo only a single cycle of replication has been demonstrated by Watanbe and co-workers [(2002), J. Virol. 76:767-773]. Infection of cells with a preparation of virus particles lacking the NEP expressing gene (NS2) produces viral proteins but does not result in the formation of infectious particles. Thus, these preparations induce a protective antibody response and stimulate a strong cell-mediated immune response without allowing the replication of infectious virus. Another approach for virus attenuation is the generation of a replication defective strain which M2 gene is eliminated. Such a deletion mutant grows efficiently in tissue culture but only poorly in mice and thus represents a potential live virus vaccine candidate [Watanbe (2001) J. Virol. 75:5656-5662]. However, frequently the infectious titers of such engineered viruses are too low to be useful in a clinical setting.
DNA vaccination—This approach involves the topical administration or administration via injection of plasmid DNA encoding one or more influenza proteins. However, to date reports on DNA vaccination against influenza have been limited to studies in animal models and no therapeutic efficacy has been demonstrated in human subjects [Donnelly (1995) Nat. Med. 1:583-587; Ljungberg (2000) 268:244-25-; Kodihalli (2000) Vaccine 18:2592-2599].
Antiviral agents—Four antiviral agents are approved at present in the U.S.; amantidine and rimantidine are chemically related inhibitors of the ion channel M2 protein which is involved in viral uncoating [Hay (1985) EMBO J 4:3021-3024], and zanamivir and oseltamivir are NA inhibitors [Palese (1976) J. Gen. Virol. 33:159-63], preventing the proper release of influenza virus particles from the cytoplasmic membrane. These antiviral drugs are important adjuncts for any medical intervention against influenza, and may be used in prophylaxis against the virus (excluding zanamivir which has not yet been approved). Furthermore, these agents can be of significant value in case a new pandemic strain emerge against which a vaccine has not been developed.
Despite overall advantages, the widespread use of currently available antiviral agents is limited by concerns over side effects, patients compliance and the possible emergence of drug-resistant variants.
Antisense—Attempts at the inhibition of influenza virus using antisense oligonucleotides have been reported. Leiter and co-workers have targeted phosphodiester and phosphorothioate oligonucleotides to influenza A and influenza C viruses. Leiter, J., Agrawal, S., Palese, P. & Zamecnik, P. C., Proc. Natl. Acad. Sci. USA, 87:3430-3434 (1990). In this study polymerase PB1 gene and mRNA were targeted in the vRNA 3′ region and mRNA 5′ region, respectively. Sequence-specific inhibition of influenza A was not observed although some specific inhibition of influenza C was noted. No other influenza virus segments or mRNAs were targeted.
There is thus a widely recognized need for, and it would be highly advantageous to have compositions, which can be used to diagnose and treat influenza virus infection devoid of the above limitations.