Barcoded influenza viruses and deep mutational scanning libraries including the same

Abstract

Methods to create barcoded influenza viruses without disrupting the function of the viral proteins and the proper packaging of the viral genome segments are described. The barcoded influenza viruses can be used within deep mutational scanning libraries to map influenza resistance mutations to therapeutic treatments. The libraries can also be used to predict influenza strains that may become resistant to therapeutic treatments and/or more easily evolve to infect new species. The libraries include features that allow efficient collection and assessment of informative data, obviating bottlenecks of previous approaches.

What is claimed is: 
     
         1 . A method for barcoding an influenza virus genome segment with minimal to no effects on viral fitness comprising:
 inserting a nucleic acid barcode and a copy of a 5′ viral RNA genome packaging signal between the end of the corresponding genome segment open reading frame and the naturally occurring non-coding portion of the 5′ viral RNA genome packaging signal.   
     
     
         2 . A method of  claim 1 , further comprising inserting a copy of the 3′ viral genome packaging signal between the non-coding portion of the naturally occurring 3′ viral RNA genome packaging signal and the beginning of the genome segment open reading frame. 
     
     
         3 . A method of  claim 1  or  2 , wherein the copy of the 3′ viral RNA genome packaging signal lacks a start codon. 
     
     
         4 . A method of  claim 2 , wherein the copy of the 5′ viral RNA genome packaging signal and the copy of the 3′ viral RNA genome packaging signal lack a start codon. 
     
     
         5 . A method of  claim 1  or  2 , wherein the copy of the 5′ viral RNA genome packaging signal has at least 80% sequence identity with the naturally occurring 5′ viral RNA genome packaging signal and/or wherein the copy of the 3′ viral RNA genome packaging signal has at least 80% sequence identity with the naturally occurring 3′ viral RNA genome packaging signal. 
     
     
         6 . A method of  claim 2 , wherein the copy of the 5′ viral RNA genome packaging signal has at least 80% sequence identity with the naturally occurring 5′ viral RNA genome packaging signal and the copy of the 3′ viral RNA genome packaging signal has at least 80% sequence identity with the naturally occurring 3′ viral RNA genome packaging signal. 
     
     
         7 . A method of  claim 1  or  2 , wherein the copy of the 5′ viral RNA genome packaging signal has at least 90% sequence identity with the naturally occurring 5′ viral RNA genome packaging signal and/or wherein the copy of the 3′ viral RNA genome packaging signal has at least 90% sequence identity with the naturally occurring 3′ viral RNA genome packaging signal. 
     
     
         8 . A method of  claim 2 , wherein the copy of the 5′ viral RNA genome packaging signal has at least 90% sequence identity with the naturally occurring 5′ viral RNA genome packaging signal and the copy of the 3′ viral RNA genome packaging signal has at least 90% sequence identity with the naturally occurring 3′ viral RNA genome packaging signal. 
     
     
         9 . A method of  claim 1  or  2 , wherein the copy of the 5′ viral RNA genome packaging signal has at least 95% sequence identity with the naturally occurring 5′ viral RNA genome packaging signal and/or wherein the copy of the 3′ viral RNA genome packaging signal has at least 95% sequence identity with the naturally occurring 3′ viral RNA genome packaging signal. 
     
     
         10 . A method of  claim 2 , wherein the copy of the 5′ viral RNA genome packaging signal has at least 95% sequence identity with the naturally occurring 5′ viral RNA genome packaging signal and the copy of the 3′ viral RNA genome packaging signal has at least 95% sequence identity with the naturally occurring 3′ viral RNA genome packaging signal. 
     
     
         11 . A method of  claim 1  or  2 , wherein the copy of the 5′ viral RNA genome packaging signal has at least 99% sequence identity with the naturally occurring 5′ viral RNA genome packaging signal and/or wherein the copy of the 3′ viral RNA genome packaging signal has at least 99% sequence identity with the naturally occurring 3′ viral RNA genome packaging signal. 
     
     
         12 . A method of  claim 2 , wherein the copy of the 5′ viral RNA genome packaging signal has at least 99% sequence identity with the naturally occurring 5′ viral RNA genome packaging signal and the copy of the 3′ viral RNA genome packaging signal has at least 99% sequence identity with the naturally occurring 3′ viral RNA genome packaging signal. 
     
     
         13 . A method of  claim 1  or  2 , wherein the copy of the 5′ viral RNA genome packaging signal has 100% sequence identity with the naturally occurring 5′ viral RNA genome packaging signal and/or wherein the copy of the 3′ viral RNA genome packaging signal has 100% sequence identity with the naturally occurring 3′ viral RNA genome packaging signal. 
     
     
         14 . A method of  claim 2 , wherein the copy of the 5′ viral RNA genome packaging signal has 100% sequence identity with the naturally occurring 5′ viral RNA genome packaging signal and the copy of the 3′ viral RNA genome packaging signal has 100% sequence identity with the naturally occurring 3′ viral RNA genome packaging signal. 
     
     
         15 . A method of  claim 1 , wherein the nucleic acid barcode comprises 4-100 nucleotides in length. 
     
     
         16 . A method of  claim 1 , wherein the nucleic acid barcode comprises 10-30 nucleotides in length. 
     
     
         17 . A method of  claim 1 , wherein the nucleic acid barcode is 18 nucleotides in length. 
     
     
         18 . A method of  claim 1 , wherein the open reading frame encodes hemagglutinin (HA), neuraminidase (NA), M1 matrix protein (M1), M2 ion channel protein (M2), nuclear protein (NP), nonstructural protein 1 (NS1), nonstructural protein 1 (NS2), or a subunit of an RNA-dependent RNA polymerase complex selected from PB1, PB2, and PA. 
     
     
         19 . A barcoded influenza virus comprising one or more barcoded influenza virus genome segments formed according to a method of  claim 1  or  2 . 
     
     
         20 . The barcoded influenza virus of  claim 19 , wherein the influenza virus is an influenza A virus, an influenza B virus, an influenza C virus, or an influenza D virus. 
     
     
         21 . A deep mutational scanning library comprising barcoded influenza virus genome segments formed according to a method of  claim 1  or  2 . 
     
     
         22 . The deep mutational scanning library of  claim 21 , wherein the set of barcoded variant nucleotide sequences collectively encode viral protein variants comprising at least 17 amino acid substitutions at at least 95% of amino acid positions of the viral protein. 
     
     
         23 . The deep mutational scanning library of  claim 21 , wherein the set of barcoded variant nucleotide sequences collectively encode (i) viral protein variants comprising at least 19 amino acid substitutions at all amino acid positions of the viral protein or (ii) a random or selected number of substitutions at a pre-determined subset of sites within a protein of interest. 
     
     
         24 . A method of identifying mutations in a viral protein that affect the sensitivity of the virus to a selection pressure using a barcoded deep mutational scanning library wherein the method comprises:
 Obtaining the library of  claim 21 ;   Culturing the virions;   Exposing the virions to the selection pressure;   Sequencing barcodes of variant nucleotide sequences from surviving virions; and   Linking sequenced barcodes to encoded viral protein variants to identify mutations in each surviving variant relative to a reference under the selection pressure, thereby identifying mutations in a viral protein that affect the sensitivity of a virus to the selection pressure.   
     
     
         25 . The method of  claim 24 , wherein the reference comprises a counterpart viral protein of a wild-type virus, of a parental virus, or of a baseline clinical isolate. 
     
     
         26 . The method of  claim 24 , wherein the reference comprises an absolute standard obtained from a glycoprotein of an influenza strain that is not recognized by the sera or antibodies of the species under consideration. 
     
     
         27 . The method of  claim 24 , wherein the reference comprises an absolute standard obtained from a glycoprotein of an influenza strain that is not recognized by the sera or antibodies of humans. 
     
     
         28 . The method of  claim 24 , wherein the selection pressure comprises a therapeutic compound. 
     
     
         29 . The method of  claim 24 , further comprising calculating a percentage of viral protein variants that the therapeutic compound is effective against, thereby identifying the percentage of viral entry protein variants of a virus that the therapeutic compound is effective against. 
     
     
         30 . The method of  claim 24 , further comprising selecting a therapeutic compound with the highest efficacy against the virus by repeating the exposing, sequencing, linking, and calculating steps for a multitude of therapeutic compounds, and selecting the therapeutic compound effective with the highest efficacy against the virus. 
     
     
         31 . The method of  claim 30 , wherein the therapeutic compound is undergoing pre-clinical development. 
     
     
         32 . The method of  claim 30 , wherein the therapeutic compound is undergoing clinical development. 
     
     
         33 . The method of  claim 30 , wherein the therapeutic compound comprises viral entry and/or fusion inhibitors. 
     
     
         34 . The method of  claim 30 , wherein the therapeutic compound comprises an antibody, or sera from humans or animals following infection or vaccination. 
     
     
         35 . The method of  claim 34 , wherein the antibody is TNX-355 (ibalizumab), PGT121, or 3BNC117. 
     
     
         36 . The method of  claim 30 , wherein the therapeutic compound comprises a small molecule, a protein, a peptide, a polynucleotide, a polysaccharide, an oil, a solution, or a plant extract. 
     
     
         37 . The method of  claim 24 , wherein the selection pressure is selected from heat, cold, low pH, high pH, and a toxic agent. 
     
     
         38 . The method of  claim 24 , further comprising: calculating the fraction of each surviving virion associated with a particular variant relative to the reference at each antibody concentration; and generating an antibody neutralization curve for each variant nucleotide sequence associated with a surviving virion. 
     
     
         39 . The method of  claim 38 , wherein the antibody neutralization curve is visualized as sequence logo plots. 
     
     
         40 . The method of  claim 38 , wherein barcode counts for a given variant nucleotide sequence greater than barcode counts for the reference at each antibody concentration indicate that a virus comprising the viral protein encoded by the variant nucleotide sequence is resistant to the neutralization antibody. 
     
     
         41 . The method of  claim 40 , further comprising scoring a phenotype as a function of the concentration of the therapeutic compound to obtain an EC 50  value for each surviving virion associated with a variant viral protein. 
     
     
         42 . The method of  claim 41 , further comprising calculating a ratio of the EC 50  value for each surviving virion to an EC 50  value of the reference, wherein the ratio indicates a fold resistance change for each surviving virion associated with a variant viral protein. 
     
     
         43 . The method of  claim 41 , further comprising calculating the fold resistance change for each variant protein to other therapeutic compounds in the same class. 
     
     
         44 . The method of  claim 41 , wherein the phenotype comprises virus titer or target cell survival. 
     
     
         45 . The method of  claim 44 , wherein the virus titer is calculated from an assay selected from plaque assay and focus-forming assay. 
     
     
         46 . The method of  claim 44 , wherein target cell survival is calculated from a colorimetric MTT cytotoxicity assay. 
     
     
         47 . The method of  claim 24 , wherein the selection pressure comprises the ability of the virus to enter (i) a host cell of a target host species or (ii) a cell expressing a receptor protein of a species that is different from the species from which the cell was derived, wherein the ability is not dependent on presence of a functional unrelated viral entry protein. 
     
     
         48 . The method of  claim 47 , wherein adaptation to a host h of a variant amino acid sequence s is scored as 
       
         
           
             
               
                 
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         where s r  is the amino acid at site r of sequence s. 
       
     
     
         49 . The method of  claim 47 , wherein the target host is selected from human, bat, camel, rat, and bird. 
     
     
         50 . The method of  claim 47 , wherein the cells of a target host species are from human cell lines. 
     
     
         51 . The method of  claim 50 , wherein the human cell lines are derived from human liver, human lung, or human lung epithelia. 
     
     
         52 . The method of  claim 51 , wherein the human cell line derived from human liver comprises HuH7, the human cell line derived from human lung comprises Calu-3 or MRC-5, and/or the human cell line derived from human lung epithelia is A549 or BEAS-2B. 
     
     
         53 . The method of  claim 47 , wherein the cells of a target host species are from bat cell lines. 
     
     
         54 . The method of  claim 53 , wherein the bat cell lines are derived from fruit bat lung, fruit bat kidney, Egyptian fruit bat, or pipestrelle bat. 
     
     
         55 . The method of  claim 47 , wherein the target host species is human.