Attenuated human parainfluenza virus, methods and uses thereof
Inventors
SKIADOPOULOS, Mario H. • Murphy, Brian R. • Collins, Peter L. • Nolan, Sheila
Assignees
US Department of Health and Human Services
Publication Number
US-9034343-B2
Publication Date
2015-05-19
Expiration Date
2026-01-10
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Abstract
The invention provides self replicating infectious recombinant paramyxoviruses. The recombinant paramyxovirus preferably have one or more attenuating mutations. In some embodiments, the recombinant paramyxovirus has a separate variant polynucleotide encoding a P protein and a separate monocistronic polynucleotide encoding a V protein. In some embodiments, recombinant paramyxovirus have at least one temperature sensitive mutation and one non-temperature sensitive mutation. Also provided are compositions and methods for using the recombinant paramyxoviruses as described herein.
Core Innovation
The invention provides self-replicating infectious recombinant paramyxoviruses, particularly human parainfluenza virus type 2 (HPIV2), with one or more attenuating mutations. These recombinant viruses feature a genome or antigenome that complies with the “rule of six,” ensuring efficient replication, and may include variant polynucleotides encoding separate P and V proteins. Separation of the P and V genes into monocistronic polynucleotides allows for mutations in the V protein without affecting the P protein, enabling targeted attenuation.
The problem addressed is the lack of currently available vaccines for human parainfluenza viruses, which cause significant respiratory diseases in infants, children, and adults, with no effective immunogenic compositions for protection. Existing vaccine efforts face issues including limited cross-protection and the stability of attenuating mutations. Paramyxoviruses have overlapping P/V genes limiting mutation options. The invention seeks to create stable, safely attenuated recombinant HPIV2 variants with improved immunogenicity and genetic stability, suitable for use as vaccines or vectors to express heterologous antigens.
Claims Coverage
The patent claims focus on recombinant infectious HPIV2 encoding separate monocistronic polynucleotides for P and V proteins with a separating spacer, covering genetic structure, mutations, locations, and phenotypes.
Encoding P and V proteins on separate monocistronic polynucleotides in HPIV2 genome
A recombinant, infectious HPIV2 comprising a partial or complete polyhexameric genome or antigenome with separate monocistronic polynucleotides encoding a P protein that does not encode V protein and a V protein monocistronic polynucleotide, separated by a non-coding spacer sequence comprising gene end, intergenic, and gene start transcription signals.
Inclusion of major nucleocapsid, phosphoprotein, and large polymerase proteins
The recombinant HPIV2 further comprises a major nucleocapsid (N) protein, a nucleocapsid phosphoprotein (P), and a large polymerase (L) protein.
Attenuated phenotype of recombinant virus
The recombinant HPIV2 exhibits an attenuated phenotype.
Gene order with P ORF and spacer upstream of V ORF
The P open reading frame (ORF) and spacer sequence are located upstream of the V ORF.
Cis-acting gene start transcription signal with specific phasing
The gene start transcription signal is cis-acting and includes a first adenosine at position 6n+1.
Variant V protein encoded by monocistronic polynucleotide
The monocistronic polynucleotide encoding the V protein includes a nucleotide sequence encoding a variant V protein having at least 80% amino acid sequence identity with SEQ ID NO:45.
Location options for monocistronic V gene
The monocistronic polynucleotide encoding the V protein is located either at the 3' terminus immediately adjacent to the N ORF or as the first ORF after the N ORF.
Attenuating amino acid mutations with codon stability
The genome or antigenome further comprises at least one amino acid mutation that is an attenuating mutation, stabilized by at least two nucleotide changes in the codon encoding the mutation.
Nucleotide substitutions introducing stop codons in overlapping V ORF
The variant polynucleotide encoding P protein has one or more nucleotide substitutions that introduce stop codons in the overlapping V ORF without altering the P protein amino acid sequence.
Variant V protein with substitutions or deletions at specific residues
The variant V protein comprises amino acid substitutions or deletions at or between residues corresponding to positions 67-72, 105-108, 121-127, 130-140, 167-172, or any amino acid of 174-225 of SEQ ID NO:45.
The claims cover recombinant HPIV2 viruses with separated monocistronic P and V genes separated by a spacer, encoding variant proteins with specific attenuating mutations, their genome organization, locations of gene units, and resulting attenuated phenotypes, thereby providing genetically stable attenuated vaccine candidates and vectors.
Stated Advantages
Allows introduction of mutations in the V protein without affecting P protein, overcoming limitations due to overlapping reading frames.
Recombinant viruses exhibit attenuation phenotypes appropriate for vaccine development, including temperature sensitive and host-range restricted mutations.
Containment of stable mutations via codon substitutions requiring multiple nucleotide changes reduces risk of reversion to wild type.
Provide safe and stable live virus vaccines capable of eliciting protective immune responses against HPIV infection and for delivering heterologous antigens.
Documented Applications
Use of recombinant HPIV2 viruses as live attenuated vaccines to protect against HPIV infections and disease.
Use as vectors to express heterologous antigens from various pathogens, including measles virus, respiratory syncytial virus subgroups, mumps virus, human papilloma virus, HIV types 1 and 2, herpes simplex virus, cytomegalovirus, rabies virus, Epstein Barr virus, filoviruses, bunyaviruses, flaviviruses, alphaviruses, human metapneumoviruses, and influenza virus.
Administering immunogenic compositions comprising recombinant HPIV2 to elicit protective immune responses such as production of IgA antibodies in animals.
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