Immunogenetic restriction on elicitation of antibodies

Abstract

The present invention provides structural determinants important for binding to the stem domain of the HA protein of influenza virus, and methods of use thereof for production of high affinity neutralizing influenza virus antibodies based upon these determinants. The present invention further provides tools for determining the efficacy of an influenza virus vaccine. The present invention further provides a molecular signature useful for determining the efficacy of an influenza virus vaccine in a subject, or for predicting prior immunologic exposure or antigen responsiveness to vaccine or influenza virus infection.

1 . An isolated monoclonal antibody that neutralizes an influenza virus comprising
 a. a heavy chain comprising a CDR1 comprising any one of the amino acid sequences SEQ ID NOs: 1-36 and 217-246; a CDR2 comprising any one of the amino acid sequences SEQ ID NOs: 37-72 and 247-276; and a CDR3 comprising any one of the amino acid sequences SEQ ID NOs: 73-108 and 277-306; and   b. a light chain comprising a CDR1 comprising any one of the amino acid sequences SEQ ID NOs: 109-144 and 307-336; a CDR2 comprising any one of the amino acid sequences SEQ ID NOs: 145-180 and 337-366; and a CDR3 comprising any one of the amino acid sequences SEQ ID NOs: 181-216 and 367-396.   
     
     
         2 . The antibody of  claim 1 , wherein said antibody comprises a V H  amino acid sequence selected from any one of SEQ ID NOS: 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, and 659; and a V L  amino acid sequence selected from any one of SEQ ID NOS: 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, and 660. 
     
     
         3 . The antibody according to any one of the preceding claims, wherein said antibody binds to the stem region of HA of the influenza virus. 
     
     
         4 . The antibody according to  claim 1 , wherein said influenza virus is an influenza A virus. 
     
     
         5 . The antibody according to  claim 1 , wherein said influenza virus is a Group I influenza virus. 
     
     
         6 . The antibody according to  claim 1 , wherein the antibody is a single chain Fv antibody, an F ab  fragment, an F ab′  fragment, or an F (ab′) 2  fragment. 
     
     
         7 . The antibody according to  claim 1  linked to a therapeutic agent. 
     
     
         8 . The antibody of  claim 7 , wherein said therapeutic agent is a toxin, a radiolabel, a siRNA, a small molecule, or a cytokine. 
     
     
         9 . A cell producing the antibody according to  claim 1 . 
     
     
         10 . A composition comprising the antibody of  claim 1  and a carrier. 
     
     
         11 . A nucleic acid sequence comprising a nucleic acid sequence selected from SEQ ID NOs: 397-468 and 541-600. 
     
     
         12 . A nucleic acid sequence encoding a polypeptide comprising an amino acid sequences selected from SEQ ID NOs: 469-540 and 601-660. 
     
     
         13 . A polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 469-540 and 601-660. 
     
     
         14 . A vector comprising the nucleic acid  claim 11 . 
     
     
         15 . A cell comprising the vector of  claim 14 . 
     
     
         16 . A method of preventing or treating a disease or disorder caused by an influenza virus, the method comprising administering to a person at risk of suffering from said disease or disorder, a therapeutically effective amount of the monoclonal antibody of  claim 1 . 
     
     
         17 . A method of improving the neutralization capacity or affinity of antibodies that bind to the HA protein of an influenza virus by mutating at least one amino acid in the VH domain, wherein the at least one mutation is selected from the following: a serine at position 24, a valine at position 27, an isoleucine or proline at position 28, a serine at position 29, an arginine at position 30, a valine at position 34, a serine at position 52, glycine, or alanine at position 52a, a lysine at position 58, a glutamine at position 73, a phenylalanine at position 74, a methionine, isoleucine, valine, or leucine at position 53, a phenylalanine at position 54, a tyrosine at positions 97, 98, and a tyrosine at position 99, or any combination thereof. 
     
     
         18 . A method of screening an immunogen or vaccine composition to induce broadly neutralizing influenza antibodies:
 a. contacting a population of B-cells having at least one copy of the 51p1 allele with the immunogen or vaccine composition under conditions capable of eliciting antibodies from the B-cells;   b. collecting the antibodies elicited from said B-cells in step (a); and   c. determining the presence or absence of the antibodies from step (b) that are encoded by the VH1-69 germline gene or the 51p1 allele;   wherein the presence of antibodies encoded by the VH1-69 germline gene or the 51p1 allele indicates that the immunogen or vaccine composition is capable of inducing broadly neutralizing influenza antibodies.   
     
     
         19 . The method of  claim 18 , wherein step (c) is performed by measuring the reactivity of the antibodies with a reagent that specifically detects antibodies encoded by the VH1-69 germline gene or the 51p1 allele. 
     
     
         20 . The method of  claim 19 , wherein said reagent is anti-51p1 monoclonal G6 antibody or an antigen-binding fragment thereof. 
     
     
         21 . A method of predicting the efficacy of an influenza vaccine in a subject comprising
 a. obtaining a blood or serum sample from the subject;   b. isolating the genomic DNA from the sample; and   c. determining the copy number of the 51p1 and hv1263 genes;   wherein one or more copies of the 51p1 gene indicates that the influenza vaccine will elicit broadly neutralizing influenza antibodies in said subject, and wherein one or more copies of the hv1263 gene without at least one copy of the 51p1 gene indicates that the influenza vaccine will not be efficacious in eliciting broadly neutralizing influenza antibodies in said subject.   
     
     
         22 . A method of predicting the efficacy of an influenza vaccine in a subject comprising
 a. obtaining a blood or serum sample from the subject;   b. isolating the serum-derived immunoglobulins from the sample;   c. analyzing the reactivity of the serum-derived immunoglobulins to an antibody that specifically recognizes antibodies encoded by the IGVH1-69 germline gene;   wherein reactivity to said antibody indicates that the influenza vaccine will elicit broadly neutralizing influenza antibodies in said subject.   
     
     
         23 . The method of  claim 22 , wherein said antibody that specifically recognizes antibodies encoded by the IGVH1-69 germline gene comprises anti-51p1 monoclonal G6 antibody or an antigen-binding fragment thereof. 
     
     
         24 . The method of  claim 22 , wherein said analyzing the reactivity of the serum-derived immunoglobulins of step (c) is performed by immunoblotting or ELISA. 
     
     
         25 . A method of predicting the efficacy of an influenza vaccine in a subject comprising
 a. obtaining a blood sample from the subject;   b. isolating a nucleic acid from the sample; and   c. determining the presence or absence of a broadly-neutralizing antibody molecular signature by nucleic acid analysis;
 wherein the presence of said broadly-neutralizing antibody molecular signature indicates that the influenza vaccine will be or has been efficacious in eliciting broadly neutralizing antibodies in said subject. 
   
     
     
         26 . The method of  claim 25 , wherein said nucleic acid is genomic DNA or RNA. 
     
     
         27 . The method of  claim 25 , wherein said nucleic acid analysis is next generation sequencing. 
     
     
         28 . The method of  claim 25 , wherein said broadly-neutralizing antibody molecular signature comprises any one of the following:
 a. at least one copy of the 51p1 allele;   b. a nucleic acid encoding an immunoglobulin variable heavy chain comprising any one of the following: a serine at position 24, a valine at position 27, an isoleucine or proline at position 28, a serine at position 29, an arginine at position 30, a valine at position 34, a serine at position 52, a glycine or an alanine at position 52a, a lysine at position 58, a glutamine at position 73, a phenylalanine at position 74, a methionine, isoleucine or leucine at position 53, a phenylalanine at position 54, a tyrosine at position 97, a tyrosine at position 98, and a tyrosine at position 99, or any combination thereof;   c. a nucleic acid encoding an immunoglobulin variable heavy chain comprising a phenylalanine at position 54, a hydrophobic amino acid at position 53, and a tyrosine at amino acid positions 97, 98, and/or 99;   d. a nucleic acid encoding an immunoglobulin comprising a heavy chain comprising:
 i. any one of the following: a serine at position 24, a valine at position 27, an isoleucine or proline at position 28, a serine at position 29, an arginine at position 30, a valine at position 34, a serine at position 52, a glycine or an alanine at position 52a, a lysine at position 58, a glutamine at position 73, a phenylalanine at position 74, a methionine, isoleucine, valine or leucine at position 53, or a phenylalanine at position 54; and 
 ii. tyrosine at least at two amino acid positions selected from 97, 98, and/or 99; 
   e. a nucleic acid encoding an immunoglobulin comprising a heavy chain comprising a glycine at position 52a and a tyrosine at positions 97, 98 or 99, or any combination thereof;   f. a nucleic acid encoding an immunoglobulin comprising a heavy chain comprising a glycine at position 52a and either a methionine at position 53 or a valine and position 52;   g. a nucleic acid encoding an immunoglobulin comprising a heavy chain comprising a valine at position 27, a serine at position 52, and a glutamine at position 73;   h. a nucleic acid encoding an immunoglobulin comprising a heavy chain comprising a serine at position 52 and a glutamine at position 73;   i. a nucleic acid encoding an immunoglobulin comprising a heavy chain comprising a proline at position 28 and an arginine at position 30;   j. a nucleic acid encoding an immunoglobulin comprising a heavy chain comprising a proline at position 28, an arginine at position 30, and an alanine at position 52a;   k. a nucleic acid encoding an immunoglobulin comprising a heavy chain comprising an arginine at position 30 and an alanine at position 52a;   l. a nucleic acid encoding an immunoglobulin comprising a heavy chain comprising an isoleucine at position 28, an arginine at position 30, and an alanine at position 52a; and   m. a nucleic acid encoding an immunoglobulin comprising a heavy chain comprising an isoleucine at position 28 and an arginine at position 30.   
     
     
         29 . A method of predicting prior immunologic exposure or memory to an influenza virus or antigen responsiveness to vaccine or an influenza virus infection comprising
 a. obtaining a blood sample from the subject;   b. isolating a nucleic acid from the sample; and   c. determining the presence or absence of a broadly-neutralizing antibody molecular signature by nucleic acid analysis.   
     
     
         30 . A method of predicting prior immunologic exposure or memory to an influenza virus or antigen responsiveness to vaccine or influenza virus infection comprising
 a. obtaining a blood sample from the subject;   b. isolating at least one B cell population via surface markers;   c. isolating RNA from said at least one B cell population;   d. determining the presence or absence of a broadly-neutralizing antibody molecular signature by nucleic acid analysis of the isolated RNA from step (c); and   e. calculating the ratios or absolute frequency of B cell receptor precursors in the at least one B cell population comprising the broadly-neutralizing antibody molecular signature;   wherein said ratio is used to predict prior exposure or memory to an influenza virus or antigen responsiveness to vaccine or influenza virus infection.   
     
     
         31 . The method of  claim 29 , wherein said broadly-neutralizing antibody molecular signature comprises any one of the following:
 a. at least one copy of the 51p1-like allele;   b. a nucleic acid encoding an immunoglobulin variable heavy chain comprising any one of the following: a serine at position 24, a valine at position 27, an isoleucine or proline at position 28, a serine at position 29, an arginine at position 30, a valine at position 34, a serine at position 52, a glycine or an alanine at position 52a, a lysine at position 58, a glutamine at position 73, a phenylalanine at position 74, a methionine, isoleucine, valine or leucine at position 53, a phenylalanine at position 54, a tyrosine at position 98, and a typrosine at position 99, or any combination thereof;   c. a nucleic acid encoding an immunoglobulin variable heavy chain comprising a phenylalanine at position 54, a hydrophobic amino acid at position 53, and a tyrosine at amino acid positions 97, 98, and/or 99;   d. a nucleic acid encoding an immunoglobulin comprising a heavy chain comprising:
 i. any one of the following: a serine at position 24, a valine at position 27, an isoleucine or proline at position 28, a serine at position 29, an arginine at position 30, a valine at position 34, a serine at position 52, a glycine or an alanine at position 52a, a lysine at position 58, a glutamine at position 73, a phenylalanine at position 74, a methionine, isoleucine, valine or leucine at position 53, or a phenylalanine at position 54; and 
 ii. tyrosine at least at two amino acid positions selected from 97, 98, and/or 99; 
   e. a nucleic acid encoding an immunoglobulin comprising a heavy chain comprising a glycine at position 52a and a tyrosine at positions 97, 98 or 99, or any combination thereof;   f. a nucleic acid encoding an immunoglobulin comprising a heavy chain comprising a glycine at position 52a and either a methionine at position 53 or a valine and position 52;   g. a nucleic acid encoding an immunoglobulin comprising a heavy chain comprising a valine at position 27, a serine at position 52, and a glutamine at position 73;   h. a nucleic acid encoding an immunoglobulin comprising a heavy chain comprising a serine at position 52 and a glutamine at position 73;   i. a nucleic acid encoding an immunoglobulin comprising a heavy chain comprising a proline at position 28 and an arginine at position 30;   j. a nucleic acid encoding an immunoglobulin comprising a heavy chain comprising a proline at position 28, an arginine at position 30, and an alanine at position 52a;   k. a nucleic acid encoding an immunoglobulin comprising a heavy chain comprising an arginine at position 30 and an alanine at position 52a;   l. a nucleic acid encoding an immunoglobulin comprising a heavy chain comprising an isoleucine at position 28, an arginine at position 30, and an alanine at position 52a; and   m. a nucleic acid encoding an immunoglobulin comprising a heavy chain comprising an isoleucine at position 28 and an arginine at position 30.   
     
     
         32 . The method of  claim 30 , wherein said at least one B cell population is a CD27− and/or a CD 27+ population. 
     
     
         33 . The method of  claim 30 , wherein said determining step of step (e) comprises detecting the RNA encoding IgG, IgM, IgA, IgD, or IgE immunoglobulins, or any combination thereof. 
     
     
         34 . A kit for predicting the efficacy of an influenza vaccine in a subject comprising
 a. a reagent for detecting the 51p1-like and/or the hv1263 allele;   b. and instructions for their use.   
     
     
         35 . The kit of  claim 23 , wherein said reagent for detecting the 51p1-like allele is an anti-51p1 monoclonal G6 antibody. 
     
     
         36 . The kit of  claim 23 , wherein said reagent for detecting the 51p1-like allele is a primer pair that hybridizes to the 51p1-like allele. 
     
     
         37 . A method of identifying a subject that will be responsive to an influenza vaccine comprising:
 a. obtaining a blood or serum sample from the subject;   b. isolating the genomic DNA from the sample; and   c. determining the copy number of the 51p1 and hv1263 genes;   wherein the subject will be responsive to the influenza vaccine if said subject contains one or more copies of the 51p1 gene, and wherein the subject will not be responsive to the influenza vaccine if said subject has one or more copies of the hv1263 gene without at least one copy of the 51p1 gene; and   d. administering the influenza vaccine to the subject that is determined to be responsive to the influenza vaccine.   
     
     
         38 . A method of identifying a subject that will be or has been responsive to an influenza vaccine comprising:
 a. obtaining a blood sample from the subject;   b. isolating a nucleic acid from the sample; and   c. determining the presence or absence of a broadly-neutralizing antibody molecular signature by nucleic acid analysis;
 wherein the presence of said broadly-neutralizing antibody molecular signature indicates that the subject will be or has been responsive to the influenza vaccine; and 
   d. administering the influenza vaccine to the subject that is determined to be responsive to the influenza vaccine.