Attenuated negative strand viruses with altered interferon antagonist activity for use as vaccines and pharmaceuticals

Abstract

The present invention relates, in general, to attenuated negative-strand RNA viruses having an impaired ability to antagonize the cellular interferon (IFN) response, and the use of such attenuated viruses in vaccine and pharmaceutical formulations. The invention also relates to the development and use of IFN-deficient systems for selection of such attenuated viruses. In particular, the invention relates to attenuated influenza viruses having modifications to the NS1 gene that diminish or eliminate the ability of the NS1 gene product to antagonize the cellular IFN response. The mutant viruses replicate in vivo but demonstrate reduced pathogenicity, and therefore are well suited for live virus vaccines, and pharmaceutical formulations.

What is claimed: 
     
         1 . A vaccine formulation, comprising: an attenuated negative strand RNA virus having an interferon antagonist phenotype that (a) is responsible for attenuation, and (b) permits the attenuated virus to grow to higher titers in interferon-deficient host systems as compared to interferon-competent host systems, when propagated under the same conditions; and a physiologically acceptable excipient. 
     
     
         2 . The vaccine formulation of  claim 1  in which the attenuated virus is selected from naturally occurring viruses, mutagenized viruses or reassortants. 
     
     
         3 . The vaccine formulation of  claim 1  in which the attenuated virus is selected from genetically engineered mutants. 
     
     
         4 . The vaccine formulation of  claim 3  in which the attenuated virus is a chimeric virus that expresses an epitope of a foreign pathogen. 
     
     
         5 . The vaccine formulation of  claim 1 ,  2 ,  3  or  4  in which the attenuated virus is an influenza virus. 
     
     
         6 . A vaccine formulation comprising an attenuated influenza virus that has a mutation in the NS1 gene responsible for the attenuated phenotype, and a physiologically acceptable excipient. 
     
     
         7 . The vaccine formulation of  claim 1 ,  2 ,  3  or  4  in which the attenuated virus is a respiratory syncytial virus. 
     
     
         8 . The vaccine formulation of  claim 1 ,  2 ,  3  or  4  in which the attenuated virus is a parainfluenza virus. 
     
     
         9 . The vaccine formulation of  claim 1 ,  2 ,  3  or  4  in which the attenuated virus is a vesicular stomatitis virus. 
     
     
         10 . The vaccine formulation of  claim 1 ,  2 ,  3  or  4  in which the attenuated virus is Newcastle disease virus. 
     
     
         11 . The vaccine formulation of  claim 1  in which the interferon-deficient host system is STAT1 negative and the interferon-competent host system is STAT1 positive. 
     
     
         12 . The vaccine formulation of  claim 5  in which the interferon-deficient host system is an embryonated chicken egg of about 6 to about 8 days old, and the interferon-competent host system is an embryonated chicken egg of about 10 to about 12 days old. 
     
     
         13 . The vaccine formulation of  claim 8  in which the interferon-deficient host system is an embryonated chicken egg of about 6 to about 8 days old, and the interferon-competent host system is an embryonated chicken egg of about to about 12 days old. 
     
     
         14 . The vaccine formulation of  claim 9  in which the interferon-deficient host system is an embryonated chicken egg of about 6 to about 8 days old, and the interferon-competent host system is an embryonated chicken egg of about 10 to about 12 days old. 
     
     
         15 . The vaccine formulation of  claim 10  in which the interferon-deficient host-system is an embryonated chicken egg of about 6 to about 8 days old, and the interferon-competent host system is an embryonated chicken egg of about 10 to about 12 days old. 
     
     
         16 . The vaccine formulation of  claim 1  or  11  in which the titer of attenuated virus propagated in the interferon-deficient host system is at least one log greater than the titer of attenuated virus propagated in the interferon-competent host system. 
     
     
         17 . The vaccine formulation of  claim 12  in which the titer of attenuated virus propagated in the interferon-deficient host system is at least one log greater than the titer of attenuated virus propagated in the interferon-competent host system. 
     
     
         18 . The vaccine formulation of  claim 13  in which the titer of attenuated virus propagated in the interferon-deficient host system is at least one log greater than the titer of attenuated virus propagated in the interferon-competent host system. 
     
     
         19 . The vaccine formulation of  claim 14  in which the titer of attenuated virus propagated in the interferon-deficient host system is at least one log greater than the titer of attenuated virus propagated in the interferon-competent host system. 
     
     
         20 . The vaccine formulation of  claim 15  in which the titer of attenuated virus propagated in the interferon-deficient host system is at least one log greater than the titer of attenuated virus propagated in the interferon-competent host system. 
     
     
         21 . The vaccine formulation of  claim 5  in which the attenuated influenza virus concentration is about 10 4  to about 5×10 6  pfu per dose. 
     
     
         22 . The vaccine formulation of  claim 6  in which the attenuated influenza virus concentration is about 10 4  to about 5×10 6  pfu per dose. 
     
     
         23 . A pharmaceutical formulation, comprising: an attenuated negative strand RNA virus having an interferon antagonist phenotype that (a) is responsible for attenuation, and (b) permits the attenuated virus to grow to higher titers in interferon-deficient host systems as compared to interferon-competent host systems, when propagated under the same conditions; and a physiologically acceptable excipient. 
     
     
         24 . The pharmaceutical formulation of  claim 23  in which the attenuated virus is selected from naturally occurring viruses, mutagenized viruses or reassortants. 
     
     
         25 . The pharmaceutical formulation of  claim 23  in which the attenuated virus is selected from genetically engineered mutants. 
     
     
         26 . The pharmaceutical formulation of  claim 25  in which the attenuated virus is a chimeric virus that expresses an epitope of a foreign pathogen. 
     
     
         27 . The pharmaceutical formulation of  claim 23 ,  24 ,  25 , or  26  in which the attenuated virus is an influenza virus. 
     
     
         28 . A pharmaceutical formulation comprising an attenuated influenza virus that has a mutation in the NS1 gene responsible for the attenuated phenotype, and a physiologically acceptable excipient. 
     
     
         29 . The pharmaceutical formulation of  claim 23 ,  24 ,  25  or  26  in which the attenuated virus is a respiratory syncytial virus. 
     
     
         30 . The pharmaceutical formulation of  claim 23 ,  24 ,  25  or  26  in which the attenuated virus is a parainfluenza virus. 
     
     
         31 . The pharmaceutical formulation of  claim 23 ,  24 ,  25  or  26  in which the attenuated virus is a vesicular stomatitis virus. 
     
     
         32 . The pharmaceutical formulation of  claim 23 ,  24 ,  25  or  26  in which the attenuated virus is Newcastle disease virus. 
     
     
         33 . The pharmaceutical formulation of  claim 23  in which the interferon-deficient host system is STAT1 negative and the interferon-competent host system is STAT1 positive. 
     
     
         34 . The pharmaceutical formulation of  claim 27  in which the interferon-deficient host system is an embryonated chicken egg of about 6 to about 8 days old, and the interferon-competent host system is an embryonated chicken egg of about 10 to 12 days old. 
     
     
         35 . The pharmaceutical formulation of  claim 30  in which the interferon-deficient host system is an embryonated chicken egg of about 6 to about 8 days old, and the interferon-competent host system is an embryonated chicken egg of about 10 to 12 days old. 
     
     
         36 . The pharmaceutical formulation of  claim 31  in which the interferon-deficient host system is an embryonated chicken egg of about 6 to about 8 days old, and the interferon-competent host system is an embryonated chicken egg of about 10 to 12 days old. 
     
     
         37 . The pharmaceutical formulation of  claim 32  in which the interferon-deficient host system is an embryonated chicken egg of about 6 to about 8 days old, and the interferon-competent host system is an embryonated chicken egg of about 10 to 12 days old. 
     
     
         38 . The pharmaceutical formulation of  claim 23  or  33  in which the titer of attenuated virus propagated in the interferon-deficient host system is at least one log greater than the titer of attenuated virus propagated in the interferon-competent host system. 
     
     
         39 . The pharmaceutical formulation of  claim 34  in which the titer of attenuated virus propagated in the interferon-deficient host system is at least one log greater than the titer of attenuated virus propagated in the interferon-competent host system. 
     
     
         40 . The pharmaceutical formulation of  claim 35  in which the titer of attenuated virus propagated in the interferon-deficient host system is at least one log greater than the titer of attenuated virus propagated in the interferon-competent host system. 
     
     
         41 . The pharmaceutical formulation of  claim 36  in which the titer of attenuated virus propagated in the interferon-deficient host system is at least one log greater than the titer of attenuated virus propagated in the interferon-competent host system. 
     
     
         42 . The pharmaceutical formulation of  claim 37  in which the titer of attenuated virus propagated in the interferon-deficient host system is at least one log greater than the titer of attenuated virus propagated in the interferon-competent host system. 
     
     
         43 . The pharmaceutical formulation of  claim 27  in which the attenuated influenza virus concentration is about 10 4  to about 5×10 6  pfu per dose. 
     
     
         44 . The pharmaceutical formulation of  claim 28  in which the attenuated influenza virus concentration is about 10 4  to about 5×10 6  pfu per dose. 
     
     
         45 . An attenuated influenza virus containing a modified NS1 gene and an altered interferon antagonist phenotype. 
     
     
         46 . The attenuated influenza virus of  claim 45 , in which the NS1 gene is modified or truncated at the carboxy terminus. 
     
     
         47 . The attenuated influenza virus of  claim 45 , in which the NS1 gene is modified at the amino terminus. 
     
     
         48 . The attenuated influenza virus of  claim 45  which is NS1/99. 
     
     
         49 . A method for vaccinating a subject, comprising administering the vaccine formulation of  claim 1  or  6  to the subject at a dose effective to elicit an immune response. 
     
     
         50 . A method for the prevention of infectious disease in a subject, comprising administering the pharmaceutical formulation of  claim 23  or  28  to the subject at a dose effective to induce a cellular interferon response. 
     
     
         51 . A method for the treatment or prevention of tumors in a subject, comprising administering the pharmaceutical formulation of  claim 23  or  28  to the subject at a dose effective to induce a cellular interferon response or oncolysis.