Method for generating a parvovirus B19 virus-like particle
The invention provides a process for generating parvovirus VP1/VP2 virus like particles (VLPs). The invention further provides methods for purification of the parvovirus VLPs and immunogenic compositions that contain the VLPs. The invention also includes recombinant nucleic acid molecules that encode parvovirus VP1 and VP2, and host cells that contain the recombinant nucleic acids.
1. A recombinant nucleic acid comprising a nucleotide sequence encoding parvoviris VP1 protein (VP1) operably linked to a first control element and a nucleotide sequence encoding parvovirus VP2 protein (VP2) operably linked to a second control element, wherein the recombinant nucleic acid comprises equivalent amounts of the nucleotide sequence encoding VP1 and the nucleotide sequence encoding VP2, the first control element is a weak promoter suitable for expression in a yeast cell and the second control element is a strong promoter suitable for expression in a yeast cell, wherein the second control element is an ADH/GAPDH promoter comprising SEQ ID NO: 19 and the first control element is a variant of said ADH/GAPDH promoter that results in decreased expression of VP1 relative VP2, wherein, when recombinant nucleic acid is expressed in a yeast cell, VP1 is produced in a lower abundance relative to VP2, and virus like particles (VLP) are formed that comprise VP1 and VP2.
2. The recombinant nucleic acid of claim 1 , wherein the variant comprises a modification selected from the group consisting of: a deletion of at least a portion of the TATA box upstream of the nucleic acid encoding VP1, a deletion of a junction site upstream of the nucleic acid encoding VP1, introduction of a transcription start upstream of the nucleic acid encoding VP1, introduction of a transcription stop site upstream of the nucleic acid encoding VP1, and a combination thereof.
3. The recombinant nucleic acid of claim 1 , wherein said nucleic acid is a nucleic acid selected from the group consisting of single stranded DNA, double stranded DNA, and RNA.
4. The recombinant nucleic acid of claim 3 , wherein the recombinant nucleic acid comprises a plasmid.
5. The recombinant nucleic acid of claim 4 , wherein the plasmid comprises a detectable marker.
6. A yeast cell comprising a recombinant nucleic acid according to claim 1 .
7. The yeast cell of claim 6 , wherein said recombinant nucleic acid is integrated into the genome of the yeast cell or is carried on an extra-chromosomal element.
8. The recombinant nucleic acid of claim 1 , wherein the first control element comprises a nucleotide sequence selected from the group consisting of nucleotide 1-1379 of SEQ ID NO:20, SEQ ID NO:21, and nucleotide 7-24 of SEQ ID NO:22; and the second control element comprises SEQ ID NO:19.