Compositions, preparations and uses of human papillomavirus L1 protein

Abstract

Large quantities of soluble multimers of human papillomavirus L1 proteins can be produced in bacterial expression systems and used as therapeutic and diagnostic tools. L1 multimers can be used in immunogenic vaccine compositions, as diagnostic reagents, and as tools for mapping cell surface receptor interactions. The invention was made using U.S. Government funds, and therefore the U.S. Government has rights in the invention.

1 . A method of immunizing a human against a human papillomavirus, comprising the step of: 
 administering to a human a composition which comprises a multimer of a human papillomavirus L1 protein, wherein the L1 protein is truncated at its amino terminus, at a dose effective to induce an immune response against the L1 protein in the human.    
     
     
         2 . The method of  claim 1  wherein the multimer of the human papillomavirus L1 protein is a T=1 icosahedral particle comprising at least one pentamer of L1 protein.  
     
     
         3 . The method of  claim 2  wherein the at least one pentamer of the human papillomavirus L1 protein comprises a human papillomavirus L2 protein.  
     
     
         4 . The method of  claim 1  wherein the human papillomavirus is selected from the group consisting of HPV 1, HPV2, HPV3, HPV4, HPV5, HPV6, HPV7, HPV8, HPV9, HPV11, HPV12, HPV14, HPV15, HPV16, HPV17, HPV18, HPV19, HPV20, HPV21, HPV22, HPV23, HPV24, HPV25, HPV26, HPV27, HPV28, HPV29, HPV31, HPV33, HPV34, HPV35, HPV36, HPV37, HPV38, HPV42, HPV43, HPV44, HPV45, HPV51, HPV52, HPV56, HPV58, and HPV66.  
     
     
         5 . The method of  claim 1  wherein the human papillomavirus is a human papillomavirus 16.  
     
     
         6 . A method of detecting the presence of antibodies against a human papillomavirus in a biological sample, comprising the steps of: 
 contacting a biological sample with a multimer of a human papillomavirus L1 protein, wherein the L1 protein is truncated at its amino terminus; and    detecting antibodies which bind to the multimer, wherein detection of antibodies which are bound to the multimer identifies the presence of antibodies against the human papillomavirus in the biological sample.    
     
     
         7 . The method of  claim 6  wherein the multimer is a T=1 icosahedral particle.  
     
     
         8 . The method of  claim 6  wherein the human papillomavirus is selected from the group consisting of HPV1, HPV2, HPV3, HPV4, HPV5, HPV6, HPV7, HPV8, HPV9, HPV11, HPV12, HPV14, HPV15, HPV16, HPV17, HPV18, HPV19, HPV20, HPV21, HPV22, HPV23, HPV24, HPV25, HPV26, HPV27, HPV28, HPV29, HPV31, HPV33, HPV34, HPV35, HPV36, HPV37, HPV38, HPV42, HPV43, HPV44, HPV45, HPV51, HPV52, HPV56, HPV58, and HPV66.  
     
     
         9 . The method of  claim 6  wherein the multimer is bound to a solid support.  
     
     
         10 . The method of  claim 6  wherein the multimer is in solution.  
     
     
         11 . The method of  claim 6  wherein the multimer comprises a detectable label.  
     
     
         12 . A method of detecting a specific subtype of human papillomavirus in a biological sample, comprising the steps of: 
 contacting a biological sample with an antibody which specifically binds to an L1 protein of a specific subtype of human papillomavirus, wherein the L1 protein is truncated at its amino terminus; and    detecting L1 protein which is bound to the antibody, wherein detection of L1 protein which is bound to the antibody identifies the presence of the specific subtype of human papillomavirus in the biological sample.    
     
     
         13 . The method of  claim 12  wherein the human papillomavirus is selected from the group consisting of HPV1, HPV2, HPV3, HPV4, HPV5, HPV6, HPV7, HPV8, HPV9, HPV11, HPV12, HPV14, HPV15, HPV16, HPV17, HPV18, HPV19, HPV20, HPV21, HPV22, HPV23, HPV24, HPV25, HPV26, HPV27, HPV28, HPV29, HPV31, HPV33, HPV34, HPV35, HPV36, HPV37, HPV38, HPV42, HPV43, HPV44, HPV45, HPV51, HPV52, HPV56, HPV58, and HPV66.  
     
     
         14 . The method of  claim 12  wherein the human papillomavirus is HPV16.  
     
     
         15 . The method of  claim 12  wherein the antibody is bound to a solid support.  
     
     
         16 . The method of  claim 12  wherein the antibody is in solution.  
     
     
         17 . The method of  claim 12  wherein the antibody comprises a detectable label.  
     
     
         18 . A solid support comprising a multimer of a human papillomavirus L1 protein, wherein the L1 protein is truncated at its amino terminus.  
     
     
         19 . The solid support of  claim 18  wherein the multimer is a T=1 icosahedral particle.  
     
     
         20 . The solid support of  claim 18  wherein the human papillomavirus is selected from the group consisting of HPV1, HPV2, HPV3, HPV4, HPV5, HPV6, HPV7, HPV8, HPV9, HPV11, HPV12, HPV14, HPV15, HPV16, HPV17, HPV18, HPV19, HPV20, HPV21, HPV22, HPV23, HPV24, HPV25, HPV26, HPV27, HPV28, HPV29, HPV31, HPV33, HPV34, HPV35, HPV36, HPV37, HPV38, HPV42, HPV43, HPV44, HPV45, HPV51, HPV52, HPV56, HPV58, and HPV66.  
     
     
         21 . The solid support of  claim 18  wherein the human papillomavirus is HPV16.  
     
     
         22 . The solid support of  claim 18  wherein the multimer comprises a detectable label.  
     
     
         23 . A solid support comprising an antibody which specifically binds to an L1 protein of a specific human papillomavirus.  
     
     
         24 . The solid support of  claim 23  wherein the human papillomavirus is selected from the group consisting of HPV1, HPV2, HPV3, HPV4, HPV5, HPV6, HPV7, HPV8, HPV9, HPV11, HPV12, HPV14, HPV15, HPV16, HPV17, HPV18, HPV19, HPV20, HPV21, HPV22, HPV23, HPV24, HPV25, HPV26, HPV27, HPV28, HPV29, HPV31, HPV33, HPV34, HPV35, HPV36, HPV37, HPV38, HPV42, HPV43, HPV44, HPV45, HPV51, HPV52, HPV56, HPV58, and HPV66.  
     
     
         25 . The solid support of  claim 23  wherein the human papillomavirus is HPV18.  
     
     
         26 . The solid support of  claim 23  wherein the human papillomavirus is HPV16.  
     
     
         27 . The solid support of  claim 23  wherein the antibody comprises a detectable label.  
     
     
         28 . A method of producing a soluble multimer of a human papillomavirus L1 protein, comprising the steps of: 
 expressing a recombinant human papillomavirus L1 protein in a bacterial host cell, wherein the recombinant human papillomavirus L1 protein comprises amino acids 13-505 as shown in SEQ ID NO:1; and    treating the recombinant human papillomavirus L1 protein to remove bacterial host cell proteins, whereby a soluble multimer of the human papillomavirus L1 protein is formed.    
     
     
         29 . The method of  claim 28  wherein the human papillomavirus is selected from the group consisting of HPV1, HPV2, HPV3, HPV4, HPV5, HPV6, HPV7, HPV8, HPV9, HPV11, HPV12, HPV14, HPV15, HPV16, HPV17, HPV18, HPV19, HPV20, HPV21, HPV22, HPV23, HPV24, HPV25, HPV26, HPV27, HPV28, HPV29, HPV31, HPV33, HPV34, HPV35, HPV36, HPV37, HPV38, HPV42, HPV43, HPV44, HPV45, HPV51, HPV52, HPV56, HPV58, and HPV66.  
     
     
         30 . The method of  claim 28  wherein the human papillomavirus is a human papillomavirus type 16.  
     
     
         31 . The method of  claim 28  wherein the human papillomavirus 16 L1 protein is truncated by 12 consecutive amino acids at its amino terminus.  
     
     
         32 . The method of  claim 30  wherein the human papillomavirus 16 L1 protein is truncated by 11 consecutive amino acids at its amino terminus.  
     
     
         33 . The method of  claim 30  wherein the human papillomavirus 16 L1 protein is truncated by 10 consecutive amino acids at its amino terminus.  
     
     
         34 . The method of  claim 30  wherein the human papillomavirus 16 L1 protein is truncated by 9 consecutive amino acids at its amino terminus.  
     
     
         35 . The method of  claim 28  wherein the bacterial host cell is an  E. coli  cell.  
     
     
         36 . The method of  claim 28  wherein the step of treating removes GroEL protein from the expressed human papillomavirus L1 protein.  
     
     
         37 . The method of  claim 28  wherein the soluble multimer is a pentamer.  
     
     
         38 . The method of  claim 37  wherein twelve soluble pentamers of the human papillomavirus L1 protein are capable of associating to form a T=1 icosahedral particle.  
     
     
         39 . The method of  claim 38  wherein the T=1 icosahedral particle is stable at a pH above 4.5.  
     
     
         40 . The method of  claim 39  wherein the T=1 icosahedral particle is stable at neutral pH.  
     
     
         41 . The method of  claim 39  wherein the T=1 icosahedral particle is held together by means of crosslinking between soluble L1 pentamers.  
     
     
         42 . The method of  claim 41  wherein the T=1 icosahedral particle is held together by means of at least one disulfide bond.  
     
     
         43 . The method of  claim 28  wherein the soluble multimer of the human papillomavirus L1 protein comprises a human papillomavirus L2 protein.  
     
     
         44 . The method of  claim 28  wherein the recombinant human papillomavirus L1 protein comprises at least one cysteine residue.  
     
     
         45 . A method of producing an T=1 icosahedral particle which comprises twelve pentamers of a human papillomavirus 16 L1 protein, comprising the steps of: 
 expressing a recombinant human papillomavirus 16 L1 protein in an  E. coli  cell, wherein the recombinant L1 protein is truncated by 9, 10, 11, or 12 consecutive amino acids at its amino terminus;    treating the recombinant L1 protein to remove GroEL protein, whereby soluble pentamers of L1 protein are formed; and    placing the soluble pentamers at pH 4-5 at a concentration of at least 0.2 mg/ml, whereby T=1 icosahedral particles comprising twelve L1 pentamers are formed.