Genetically stable live attenuated respiratory syncytial virus vaccine and its production
Inventors
Collins, Peter L. • Luongo, Cindy L. • Buchholz, Ursula J. • Murphy, Brian R.
Assignees
US Department of Health and Human Services
Publication Number
US-10980872-B2
Publication Date
2021-04-20
Expiration Date
2033-03-13
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Abstract
Provided herein are recombinant respiratory syncytial viruses that contain mutations that make the disclosed viruses attractive vaccine candidates. The viruses disclosed contain attenuating mutations designed to have increased genetic and phenotypic stability. Desired combinations of these mutations can be made to achieve desired levels of attenuation. Exemplary vaccine candidates are described. Also provided are polynucleotides capable of encoding the described viruses, as wells as methods for producing the viruses and methods of use.
Core Innovation
The invention provides recombinant respiratory syncytial viruses (RSVs) that contain specific mutations to create attenuated strains with increased genetic and phenotypic stability, making them suitable as live vaccine candidates. These recombinant viruses include mutations at positions such as codons 1321 and 1313 of the RSV large polymerase protein (L), wherein desired combinations of amino acid substitutions or deletions at these sites provide improved stability and desired levels of attenuation. The viruses may also contain other point mutations and gene deletions, and are engineered to optimize immunogenicity and stability for vaccine use.
The problem addressed by the invention arises from the challenges in developing live-attenuated RSV vaccines due to factors including the difficulty in producing consistently attenuated strains, the genetic instability characteristic of RNA viruses leading to reversion to more virulent forms, and challenges in virus growth and purification. Previous biologically-derived vaccine candidates were often either under-attenuated or over-attenuated, with some displaying genetic instability during use. Additionally, RNA viruses such as RSV have a high propensity for mutation, making it essential to identify mutations that confer stable attenuation without loss of immunogenicity or viability.
Claims Coverage
The claims include multiple inventive features focusing on nucleic acid molecules encoding recombinant infectious RSVs with specific deletions and mutations and methods of producing such viruses.
Deletion of the codon encoding serine at position 1313 of the L protein
A nucleic acid molecule comprising a genome or antigenome of a recombinant RSV with a deletion of the codon encoding serine at position 1313, optionally including other RSV proteins such as NS1, NS2, G, F, M, M2-2, and SH.
Combination with deletion of NS2 gene
The recombinant RSV genome or antigenome may further comprise a deletion of a gene encoding the NS2 protein in combination with the deletion at codon 1313.
Mutation at codon encoding tyrosine at position 1321 of the L protein
The genome or antigenome may further comprise a mutation at the codon encoding the tyrosine at position 1321 of the L protein, including specific mutations such as an AAA codon encoding lysine (1321K).
Specific mutation sets combining mutations at positions 1321, 1313, 831, and deletions
The genome or antigenome may comprise mutations including L protein mutations 1321K, 1321E, 1321P, 1321G, 1321K(AAA), 1321E(GAA), 1321P(CCT), 1321G(GGA), or 1321G(GGT); mutation Q831L in L protein; a set of cold passage mutations in N, F, and L proteins; a gene-start signal mutation at the M2 gene; and deletion of the SH gene.
Mutation of amino acid residue 1314 of the L protein to leucine combined with deletion at 1313
A nucleic acid molecule encoding a recombinant RSV with deletion of the codon encoding serine at position 1313 and a mutation of amino acid residue 1314 of the L protein from isoleucine to leucine, optionally encoded by the codon CTG, and optionally comprising other RSV proteins.
Methods and vectors for production of recombinant RSV
Claims include vectors comprising the nucleic acid molecules encoding the described recombinant RSVs, isolated cells comprising these vectors, and methods of producing recombinant RSVs by expressing the nucleic acid molecules in cells.
The claims primarily cover recombinant RSV nucleic acid molecules encoding viruses harboring a deletion of the serine codon at position 1313 of the L protein, optionally combined with mutations at position 1321 of the L protein including specific amino acid substitutions, mutations at 1314, and deletion of NS2, as well as methods and vectors related to producing these recombinant RSVs. These inventive features focus on enhanced genetic and phenotypic stability, attenuation, and provisions for vaccine use.
Stated Advantages
The disclosed recombinant RSVs have improved genetic and phenotypic stability compared to previous vaccine candidates.
The deletions and mutations result in temperature sensitive phenotypes and attenuation suitable for live vaccine development.
Certain deletion mutations, such as Δ1313, maintain replication efficiency at permissive temperatures important for vaccine manufacture.
Combining mutations enables calibration of attenuation levels and resistance to phenotypic reversion.
Alternative codon usage reduces likelihood of reversion to wild-type virus, enhancing vaccine safety and efficacy.
Documented Applications
Use of the recombinant RSVs as live attenuated vaccines for the prevention of RSV infection, administered via intranasal, aerosol, droplet, oral, or topical routes.
Use of recombinant RSVs to induce an immune response in mammals including humans, non-human primates, rodents, and other susceptible animals.
Use in producing immune responses against related viruses such as human parainfluenza virus (PIV) and metapneumovirus (MPV).
Use as vectors to deliver heterologous genes or immunomodulatory proteins, including cytokines, to mammals.
Methods of producing recombinant RSVs in cells using polynucleotides encoding genomes or antigenomes containing the specified mutations and deletions.
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