Optimization of gene sequences of chimeric virus-like particles for expression in insect cells

Abstract

Chimeric virus-like particles that exhibit conformational antigenic epitopes capable of eliciting neutralizing antibodies are disclosed herein. The chimeric virus-like particles of the invention comprise a recombinant viral capsid protein that encapsulates a recombinant viral protein during self-assembly into a chimeric virus-like particle, wherein the chimeric virus-like particle exhibits confirmational antigenic epitopes capable of eliciting neutralizing antibodies. Pharmaceutical compositions, vaccines, and diagnostic test kits containing the chimeric virus-particles are also provided.

1 . A chimeric virus-like particle comprising a recombinant viral capsid protein that encapsulates a recombinant viral protein during self assembly into a chimeric virus-like particle, wherein the chimeric virus-like particle exhibits conformational antigenic epitopes capable of eliciting neutralizing antibodies.  
     
     
         2 . The chimeric virus-like particle of  claim 1 , wherein the recombinant viral capsid protein and the recombinant viral protein are from the same or a different virus.  
     
     
         3 . The chimeric virus-like particle of  claim 1 , wherein the recombinant viral capsid protein or the recombinant viral protein is from an enveloped virus, a non-enveloped virus, or both.  
     
     
         4 . The chimeric virus-like particle of  claim 3 , wherein the enveloped virus comprises influenza virus, hepatitis C virus, and human immunodeficiency virus.  
     
     
         5 . The chimeric virus-like particle of  claim 3 , wherein the non-enveloped virus comprises rotavirus, calicivirus, hepatitis E virus, papillomavirus, influenza virus, and hepatitis C viris.  
     
     
         6 . The chimeric virus-like particle of  claim 5 , wherein the papillomavirus comprises human papillomavirus.  
     
     
         6 . The chimeric virus-like particle of  claim 5 , wherein the viral capsid protein and the viral protein are from the same or a different human papillomavirus genotype.  
     
     
         7 . The chimeric virus-like particle of  claim 6 , wherein the viral capsid protein comprises a HPV L1 protein and the viral protein comprises a HPV L2, or a HPV L2 fusion protein.  
     
     
         8 . The chimeric virus-like particle of  claim 7 , wherein the HPV L2 fusion protein comprises HPV L2, fused with HPV E2, HPV E6, and/or HPV E7.  
     
     
         9 . The chimeric virus-like particle of  claim 5  wherein the human papillomavirus genotypes comprise HPV-16, HPV-18, HPV-45, HPV-31, HPV-33, HP]V-35, HPV-51, HPV-52, HPV-6, HPV-11, HPV-42, HPV-43, HPV-44, or a combination thereof.  
     
     
         10 . The chimeric virus-like particle of  claim 1 , wherein the viral capsid protein is encoded by a codon optimized polynucleotide comprising SEQ ID No. 1, or a polynucleotide having a sequence that is substantially homologous to SEQ ID No. 1.  
     
     
         11 . The chimeric virus-like particles of  claim 1 , wherein the codon optimized polynucleotide encoding the viral protein comprises SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, or a polynucleotide having a sequence that is substantially homologous to SEQ ID No.2, SEQ ID No. 3, SEQ ID No. 4, or SEQ ID No. 5.  
     
     
         12 . The chimeric virus-like particle of  claim 8 , wherein the fusion protein comprises a heterologous protein.  
     
     
         13 . A method for producing chimeric virus-like particles comprising: 
 (a) infecting an insect cell with a first recombinant baculovirus carrying a first codon-optimized polynucleotide that encodes a first viral protein;    (b) infecting the insect cell of the step (a) with a second recombinant baculovirus carrying a second codon-optimized polynucleotide that encodes a second viral protein,    wherein the first viral protein comprises a recombinant viral capsid protein that encapsulates the second viral protein during assembly of virus-like particles.    
     
     
         14 . The method of  claim 13 , wherein the first recombinant baculovirus and the second recombinant baculovirus are co-infected into the insect cell.  
     
     
         15 . The method of  claim 13 , wherein the insect cell is from the cell line designated ATCC PTA4047.  
     
     
         16 . The method of  claim 13 , wherein the recombinant viral capsid protein comprises a HPV L1 protein and the second viral protein is a recombinant viral protein comprising a HPV L2 or a HPV L2 fusion protein.  
     
     
         17 . The method of  claim 14 , wherein co-infection of the first and second recombinant baculoviruses results in an expression ratio of at least about 3:1 for the second viral protein versus the recombinant viral capsid protein.  
     
     
         18 . A codon optimized polynucleotide comprising at least one of the following characteristics: (a) an increased number of nucleotide sequences that are utilized at high levels in insect cells, (b) a ratio of GC nucleotide pairs to AT nucleotide pairs of approximately 1: 1, (c) a minimum number of palindromic and stem-loop DNA structures, and (d) a minimum number of transcription and post-transcription repressor elements.  
     
     
         19 . The codon optimized polynucleotide of  claim 18  encoding a viral capsid protein, or encoding a viral protein that becomes encapsulated into a virus like particle by the viral capsid protein in an insect cell.  
     
     
         20 . The codon optimized polynucleotide of  claim 19  comprising a polynucleotide encoding a protein from an enveloped virus, a non-enveloped virus, or both.  
     
     
         21 . The codon optimized polynucleotide of  claim 20 , wherein the enveloped virus comprises influenza virus, hepatitis C virus, and human immunodeficiency virus.  
     
     
         22 . The codon optimized polynucleotide of  claim 20 , wherein the non-enveloped virus comprises rotavirus, calicivirus, hepatitis E virus, papillomavirus, influenza virus, and hepatitis C virus.  
     
     
         23 . The codon optimized polynucleotide of  claim 20 , wherein the protein is HPV L2 protein or a peptide fragment thereof.  
     
     
         24 . The codon optimized polynucleotide of  claim 20 , wherein the protein is a HPV L2 fusion protein comprising heterologous proteins.  
     
     
         25 . The codon optimized polynucleotide of  claim 18  represented by SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, or a sequence that is substantially homologous to SEQ ID No.2, SEQ ID No. 3, SEQ ID No. 4, or SEQ ID No. 5.  
     
     
         26 . A vector comprising the codon optimized polynucleotide of  claim 18  operatively linked to a baculovirus regulatory control element, which vector is capable of replication in an insect host cell.  
     
     
         27 . An insect host cell comprising the vector of  claim 26 .  
     
     
         28 . A pharmaceutical composition for treating or preventing a papillomavirus related disease or disorder comprising administering to a subject in need thereof a composition containing chimeric virus-like particles that exhibit conformational antigenic epitopes capable of eliciting neutralizing antibodies in the subject, wherein the chimeric virus-like particles comprise a recombinant HPV L1 that encapsulates a recombinant TPV L2 and/or HPV L2 fusion protein, and an acceptable carrier or diluent.  
     
     
         29 . The pharmaceutical composition of  claim 28 , where the HPV L1, HPV L2, and/or HPV L2 fusion proteins are from the same or a different HPV genotypes.  
     
     
         30 . A vaccine composition for inducing immunity against a papillomavirus infection in a human comprising a composition containing chimeric virus-like particles that exhibit conformational antigenic epitopes capable of eliciting neutralizing antibodies in the human, wherein the chimeric virus-like particles comprise a recombinant HPV L1 that encapsulates a recombinant HPV L2 and/or HPV L2 fusion protein, and an adjuvant.  
     
     
         31 . The vaccine composition of  claim 30 , wherein the immunity is humoral immunity, cell mediated immunity, or both.  
     
     
         32 . The vaccine composition of  claim 30 , wherein the infecting papillomavirus comprises a human papillomavirus genotype selected from the group consisting of BPV-16, HPV-18, BPV-45, HPV-31, HPV-33, HPV-35, HPV-51, HPV-52, HPV-6, HPV-11, HPV42, HPV-43, HPV-44, and combinations thereof.  
     
     
         32 . The vaccine composition of  claim 30  comprising a monovalent formulation.  
     
     
         33 . The vaccine composition of  claim 30  comprising a multivalent formulation.  
     
     
         34 . The vaccine composition of  claim 30 , comprising 
 a) a polypeptide which is encoded by a polynucleotide molecule represented by SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, or a polynucleotide molecule that is substantially homologous to SEQ ID No.2, SEQ ID No. 3, SEQ ID No. 4, or SEQ ID No. 5;    b) a polynucleotide molecule represented by SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, or a polynucleotide molecule that is substantially homologous to SEQ ID No.2, SEQ ID No. 3, SEQ ID No. 4, or SEQ ID No. 5; or    c) a vector carrying a polynucloetide molecule represented by SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, or a polynucleotide molecule that is substantially homologous to SEQ ID No.2, SEQ ID No. 3, SEQ ID No. 4, or SEQ ID No. 5; and    an adjuvant.    
     
     
         35 . A diagnostic test kit for detection of papillomavirus infection, comprising a composition containing chimeric virus-like particles that exhibit conformational antigenic epitopes capable of eliciting neutralizing antibodies in a subject, wherein the chimeric virus-like particles comprise a recombinant HPV L1 that encapsulates a recombinant HPV L2 and/or HPV L2 fusion protein, and a detection agent.  
     
     
         36 . The diagnostic test kit of  claim 34 , wherein the papillomavirus infection is caused by one or more human papillomavirus genotypes.  
     
     
         37 . A method of treating or preventing a papillomavirus related disease or disorder comprising administering to an individual in need thereof an effective amount of the pharmaceutical composition of  claim 28 .  
     
     
         38 . A method of protecting an individual against a papillomavirns infection, comprising administering to the individual an effective amount of the vaccine of  claim 29.