the present invention will now be described with reference to the following examples which are provided for the purpose of illustration and are not intended to be construed as being limiting on the present invention , and further , with reference to the figures , wherein : fig1 shows the effect of the multiple subtype natural human alpha interferon product multiferon ™ on the cytopathogenicity of human encephalomyocarditis virus ( emcv ) on a549 cells , wherein the multiferon ™ concentration required to obtain 50 % cytopathic effect ( cpe ) for human a549 cells challenged with emc virus is shown for different concentrations of emc virus ; fig2 shows the effect of increasing concentrations of multiferon ™ on survival of cells infected with emcv ; fig3 shows cytotoxicity ( dashed line ) and antiviral ( unbroken line ) profiles for multiferon ( 10 - 10000 iu / ml ) treatment of mdbk cells infected with 100 tcid 50 h5n1 avian influenza virus ( a / vn / 1203 / 04 ); fig4 shows cytotoxicity ( dashed line ) and antiviral ( unbroken line ) profiles for ribavirin ( 0 . 1 - 100 μg / ml ) treatment of mdbk cells infected with 100 tcid 50 h5n1 avian influenza virus ( a / vn / 1203 / 04 ); fig5 shows cytotoxicity ( dashed line ) and antiviral ( unbroken line ) profiles for repeat experiment with multiferon ( 0 . 1 - 100 iu / ml ) treatment of mdbk cells infected with 100 tcid 50 h5n1 avian influenza virus ( a / vn / 1203 / 04 ); and fig6 shows a comparison of ic 50 concentrations ( in pg / ml ) for multiferon , interferon alpha 2a , and interferon beta 1a protection of mdbk cells from h5n1 avian influenza virus . interferons are widely known to be species specific as the target for the interferon is the infected cell rather than the virus itself . the effect of each anti - viral treatment will be tested in quadruplicate . briefly , 100 microlitres of serial 10 - fold dilutions of each treatment was incubated with 100 microlitres of cells to give a final cell count of 20 , 000 cells per well in a 96 - well plate . incubation at 37 ° c . in 5 % co 2 was carried out overnight for the interferon preparations and for one hour for ribavirin ™. 10 microlitres of virus at a concentration of 10 , 000 pfu / well was then added to each test well . the plates were then incubated at 37 ° c . in 5 % co 2 for three days , with the plates being observed daily for cytopathic effects . the end point is the diluted concentration that inhibited the cytopathic effect in all four set - ups by 50 %. to determine cytotoxicity , 100 microlitres of serial 10 - fold dilutions of each treatment was incubated with 100 microlitres of cells giving a final cell count of 20 , 000 cells per well in a 96 - well plate , without viral challenge . the plates were then incubated at 37 ° c . in 5 % co 2 for three days and toxicity effects observed for using an inverted microscope . multiferon ™ was added to human lung epithelial cells ( cell line a549 ) prior to addition of virus . the human encephalomyocarditis virus ( emcv ) was then used to infect a549 cells and the effect of multiferon ™ on the cytopathogenicity of emcv was determined by assessing the interferon concentration required to obtain 50 % cytopathic effect ( cpe ) for the human a549 cells . the results shown in fig1 show the concentration of multiferon ™ needed to obtain 50 % cytopathic effect in the human cells at varying viral titres . as would be expected , a higher viral concentration requires a higher effective multiferon ™ concentration . in fig2 , multiferon ™ can be observed to inhibit the cytopathic effect caused by emcv infection in a titration dependent manner . these results show that multiferon ™ successfully inhibited cytopathic effect in emcv - infected cells , in a titration - dependent manner . madin darby bovine kidney ( mdbk ) cells were used to test the efficacy of compounds to h5n1 avian influenza virus ( h5n1 ; strain a / vn / 1203 / 04 ). the antiviral evaluation assay examined the effects of compounds at seven half - log concentrations each . recombinant human interferon alpha 2a and recombinant human interferon beta 1a ( pbl biomedical laboratories , piscataway , n . j .) as well as ribavirin ™ ( mp biomedicals , irvine , calif .) were included in each run as positive control compounds . multiferon and controls were run in duplicate assays in triplicate for h5n1 as well as duplicate toxicity wells . subconfluent cultures of mdbk cells were plated out into 96 - well plates for the analysis of cell numbers ( cytotoxicity ) or antiviral activity ( cpe ) and the next day drugs were added to the appropriate wells . one hundred 50 % tissue culture infectious doses ( tcid 50 ) of h5n1 or media were added to appropriate wells and cells were processed 72 hours later when the virus induced peak cpe . the effective drug concentration which reduced h5n1 cpe levels by 25 % ( ic 25 ), 50 % ( ic 50 ) and 90 % ( ic 90 ) were calculated by regression analysis with semi log curve fitting . h5n1 levels were assessed as relative luminescence units ( rlu ) using celltiter - glo ®. the toxic concentration of drug that reduced cell numbers by 50 % ( tc 50 ) and 90 % ( tc 90 ) were calculated in the same manner . selectivity ( therapeutic ) indices ( si tc / ic ) at 50 % ( si 50 ) and 90 % ( si 90 ) were calculated . at assay termination , cell viability and drug cytotoxicity were assessed using the celltiter - glo ® luminescent cell viability assay reagent ( promega , madison , wis .) per the manufacturer &# 39 ; s instructions . this reagent is a homogeneous method for determining the number of viable cells in culture based on quantitation of the atp present , an indicator of metabolically active cells . the homogeneous assay procedure involves adding the single reagent ( celltiter - glo ® reagent ) directly to cells cultured in assay medium . the homogeneous “ add - mix - measure ” format results in cell lysis and generation of a “ glow - type ” luminescent signal ( half - life generally greater than five hours ) that is proportional to the amount of atp present . the amount of atp is directly proportional to the number of cells present in culture and readout is determined by rlu . the toxic concentration of drug that reduced cell numbers by 50 % ( tc 50 ) and 90 % ( tc 90 ) were calculated in spreadsheets by regression analysis with semi log curve fitting . selectivity ( therapeutic ) indices ( si = tc / ic ) at 50 % ( si 50 ) and 90 % ( si 90 ) were calculated . multiferon ™ was tested for anti - h5n1 activity using a concentration range from 10000 iu / ml down to 0 . 1 iu / ml . fig3 details results following treatment with multiferon at a concentration range of 10000 iu / ml down to 10 iu / ml . in this experiment , the lowest concentration of multiferon ™ used , 10 iu / ml , protected 100 % of cells from h5n1 infection ( unbroken line in fig3 ) indicating that multiferon is highly efficient at protecting cells in vitro from h5n1 infection . in contrast , 12 . 11 iu / ml of interferon beta 1a only protected 50 % of cells from h5n1 infection ( graph not shown ; a summary of the results is presented in table 1 below ). fig3 ( dashed line ) shows that multiferon ™ was not toxic at any concentrations tested up to 10 , 000 iu / ml . in contrast , fig4 ( dashed line ) shows that ribavirin ™ was toxic at concentrations above that which protected 80 % of cells from h5n1 infection , i . e . it was not possible to reach full protection of cells using ribavirin ™. fig5 details results from a repeat study where the concentration range for multiferon ™ was reduced to 100 iu / ml down to 0 . 1 iu / ml to determine an ic 50 concentration for multiferon ™. ribavirin ™, interferon beta 1a and interferon alpha 2a were included in this study . multiferon ™ was demonstrated to be & gt ; 17 - fold stronger than interferon beta 1a and & gt ; 51 - fold stronger than interferon alpha 2a in protecting cells from h5n1 infection ( graphs not shown , results also summarized in table 3 ). table 3 details the comparison of ic 50 concentrations for v to interferon alpha 2a and interferon beta 1a , in iu / ml and in pg / ml . this takes into account differences in specific activity of the products tested , demonstrating multiferon ™ is & gt ; 20 - fold stronger than either interferon alpha 2a or interferon beta 1a when ic 50 concentrations in pg / ml are compared . data taken from column 4 of table 3 is graphed in fig6 , which shows a comparison of ic 50 concentrations ( in pg / ml ) for multiferon ™, interferon alpha 2a , and interferon beta 1a protection of mdbk cells from h5n1 avian influenza virus . it is surprising that multi - subtype forms of interferon alpha provide a robust treatment or preventative therapy against avian influenza . the present invention provides an important broad spectrum , first line of defense therapeutic product which can protect against infection with avian influenza h5n1 and likely any reassortment or variant derived therefrom . leukocyte derived natural multi - subtype forms of interferon alpha have no or very little tendency to give rise to neutralising antibodies , and accordingly provide a higher response rate than recombinant interferon alpha 2 products when used therapeutically in humans . for patients who develop anti - interferon antibodies to recombinant interferon alpha 2 products , it has proven useful to follow up the treatment with a natural form of alpha interferon ( milella et al ., 1995 ). all documents referred to in this specification are herein incorporated by reference . various modifications and variations to the described embodiments of the inventions will be apparent to those skilled in the art without departing from the scope of the invention . although the invention has been described in connection with specific preferred embodiments , it should be understood that the invention as claimed should not be unduly limited to such specific embodiments . indeed , various modifications of the described modes of carrying out the invention which are obvious to those skilled in the art are intended to be covered by the present invention . antonelli g . ( 1997 ) j interferon cytokine res jul ; 17 suppl 1 : s39 - 46 . easterday , b . c ., et al . ( 1997 ) influenza , p . 583 - 605 . in b . w . calnek , h . j . barnes , c . w . beard , l . r . mcdougald , and y . m . saif ( ed .). diseases in poultry . iowa state university press , ames . foster g r , et al . 1996 . j . interferon res . 16 : 1027 - 1033 . goodbourn , s . e . y ., et al . ( 2000 ) j . gen . virol . 81 : 2341 - 2364 . weck , p . k . et al . ( 1981 ) j . gen . virol . 57 233 - 237
the present invention provides methods and uses for an interferon composition in the treatment of avian influenza . transmission of avian influenza virus h5n1 to humans has been shown to be highly pathogenic . the present invention provides for methods of treatment that provide a broad spectrum , first line defense against avian influenza infection in humans . the methods of the present invention can be further extended to treat strains of avian influenza viruses that have resulted from antigenic drift , this potentially resulting in avian influenza based virus which is highly pathogenic and transmissible between humans .