Replication-competent recombinant viral vaccines and method of producing same

A replication-competent recombinant poliovirus which expresses an exogenous polypeptide as a component of a recombinant polyprotein precursor that is proteolytically processed, with the result that the exogenous polypeptide is released from the recombinant polyprotein precursor, in which the recombinant genome comprises:

• a) an exogenous nucleic acid sequence encoding an exogenous polypeptide to be expressed;
• b) an exogenous sequence which is a polioviral proteolytic cleavage site for the poliovirus 3C protease or for the poliovirus 2A protease;
• and
• c) the genome of a parent poliovirus,
• wherein the exogenous nucleic acid sequence of (a) and nucleic acid sequence encoding an artificial proteolytic cleavage site for the poliovirus 3C protease or the poliovirus 2A protease of (b) are present in the recombinant poliovirus genome in the following order:
• 5' untranslated region of the poliovirus genome--unique poliovirus start codon--exogenous nucleic acid sequence of (a)--nucleic acid sequence encoding the artificial proteolytic cleavage site of (b)--second codon of the parent poliovirus genome--remainder of the parent poliovirus genome and when expressed, the exogenous polypeptide is expressed as a component of the polyprotein precursor produced by the recombinant poliovirus, the polyprotein precursor is proteolytically processed by the recombinant poliovirus and the exogenous polypeptide is not included in a mature poliovirus particle produced by the replication-competent poliovirus.

A replication-competent recombinant poliovirus which expresses an exogenous polypeptide as a component of a recombinant polyprotein precursor that is proteolytically processed, with the result that the exogenous polypeptide is released from the recombinant polyprotein precursor, in which the recombinant genome comprises:

• a) an exogenous nucleic acid sequence encoding an exogenous polypeptide to be expressed;
• b) exogenous sequences which are polioviral proteolytic cleavage sites for the poliovirus 3C protease or for the poliovirus 2A protease;
• and
• c) the genome of a parent poliovirus,
• wherein the exogenous nucleic acid sequence of (a) and nucleic acid sequences encoding artificial proteolytic cleavage sites for the poliovirus 3C protease or the poliovirus 2A protease of (b) are present in the recombinant poliovirus genome in the following order:
• 5' untranslated region of the poliovirus genome--poliovirus unique start codon--polio protein encoding region(s)--artificial 3C or 2A protease recognition site--exogenous nucleic acid sequence--artificial 3C or 2A protease recognition site--remainder of the poliovirus, and when expressed, the exogenous polypeptide is expressed as a component of the polyprotein precursor produced by the recombinant poliovirus, the polyprotein precursor is proteolytically processed by the recombinant poliovirus and the exogenous polypeptide is not included in a mature poliovirus particle produced by the replication-competent poliovirus.

A method of producing a replication-competent recombinant poliovirus, which expresses an exogenous polypeptide as a component of a recombinant polyprotein precursor that is proteolytically processed with the result that the exogenous polypeptide is released from the recombinant polyprotein precursor, comprising the steps of:

• a) providing a poliovirus which in its natural life cycle produces a polyprotein precursor which is proteolytically processed;
• b) introducing into the genome of the poliovirus of (a)
• (1) an exogenous nucleic acid sequence encoding a polypeptide to be expressed;
• and
• (2) an exogenous sequence which is a polioviral proteolytic cleavage site and which is cleaved by a poliovirus protease, wherein the protease proteolytically processes a protein precursor produced by the poliovirus provided in (a),
• wherein (b)(1) and (b)(2) are inserted into the genome of the poliovirus of (a) at a location such that they do not disrupt a viral sequence necessary for viral replication and when expressed, the exogenous polypeptide is expressed as a component of the polyprotein precursor produced by the recombinant poliovirus, the polyprotein precursor is proteolytically processed by the recombinant poliovirus and the exogenous polypeptide is not included in a mature poliovirus particle produced by the replication-competent poliovirus.

A method of producing a replication-competent recombinant poliovirus which expresses an exogenous polypeptide as a component of a recombinant polyprotein precursor that is proteolytically processed with the result that the exogenous polypeptide is released from the polyprotein precursor, the method comprising the steps of:

• a) providing a parent poliovirus;
• b) introducing into the genome of the parent poliovirus of (a)(1) an exogenous nucleic acid sequence encoding a polypeptide to be expressed;
• and
• (2) a nucleic acid sequence encoding an artificial proteolytic cleavage site for a protease which proteolytically processes a polyprotein precursor produced by the parent poliovirus provided in (a),
• wherein the exogenous nucleic acid sequence of (b)(1) and a nucleic acid sequence encoding an artificial proteolytic cleavage site of (b)(2) which is the poliovirus 3C protease or the poliovirus 2A protease are present in the recombinant poliovirus genome in the following order:
• 5' untranslated region of the parent poliovirus genome--unique poliovirus start codon--exogenous nucleic acid sequence of (b)(1)--nucleic acid sequence encoding the artificial proteolytic cleavage site of (b)(2)--second codon of the parent poliovirus genome--remainder of the parent poliovirus genome, and when expressed, the exogenous polypeptide is expressed as a component of the polyprotein precursor produced by the recombinant poliovirus, the polyprotein precursor is proteolytically processed by the recombinant poliovirus and the exogenous polypeptide is not included in a mature poliovirus particle produced by the replication-completion poliovirus.

A method of producing a replication-competent recombinant poliovirus which expresses an exogenous polypeptide as a component of a recombinant polyprotein precursor that is proteolytically processed with the result that the exogenous polypeptide is released from the polyprotein precursor, the method comprising the steps of:

• a) providing a parent poliovirus;
• b) introducing into the genome of the parent poliovirus of (a)
• (1) an exogenous nucleic acid sequence encoding a polypeptide to be expressed;
• and
• (2) exogenous sequences which are polioviral proteolytic cleavage sites and which are cleaved by a poliovirus protease, wherein the protease proteolytically processes a polyprotein precursor produced by the parent poliovirus in (a),
• wherein the unit of the exogenous nucleic acid sequence of (b)(1) and nucleic acid sequences encoding artificial proteolytic cleavage sites of (b)(2) is located at a site within the poliovirus genome selected from the group consisting of:
• 1) the junction between Vp1 and 2A;
• 2) the junction between 2A and 2B;
• and 3) the junction between 2C and 3A, and when expressed, the exogenous polypeptide is expressed as a component of the polyprotein precursor produced by the recombinant poliovirus, the polyprotein precursor is proteolytically processed by the recombinant poliovirus and the exogenous polypeptide is not included in a mature poliovirus particle produced by the replication-competent poliovirus.