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-9624475-B2
Publication Date
2017-04-18
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 attenation. 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 relates to recombinant respiratory syncytial virus (RSV) vaccines containing specific mutations that confer attenuated phenotypes with improved genetic and phenotypic stability. These mutations, present individually or in combination, particularly in the RSV large polymerase protein (L), are designed to reduce the likelihood of reversion to wild-type or non-attenuated forms, thereby enhancing vaccine safety and efficacy. Exemplary mutations occur at amino acid residues 1321, 1313, 1314, 1316, 649, 874, and regions 1744-1764 in the L protein, including codon deletions and substitutions making the viruses viable and temperature sensitive.
The problem addressed is the historical difficulty in developing RSV live attenuated vaccines due to challenges such as moderate virus titers, loss of infectivity during handling, difficulty balancing attenuation and immunogenicity, limited suitable animal models, and, critically, the genetic instability inherent to RNA viruses leading to reversal of attenuating mutations. Previous biologically-derived vaccine candidates suffered from under- or over-attenuation and genetic instability, undermining their utility.
The invention solves these problems by engineering recombinant RSV strains with mutations that stabilize attenuating positions in the L protein, specifically developing alternative codons and amino acid substitutions to prevent reversion. A notable example is stabilizing mutations at position 1321 combined with codon changes or deletion of codon 1313 to eliminate compensatory mutations that negate attenuation. The invention further provides methods to produce these recombinant viruses from polynucleotides, including cDNA, allows combining these mutations with other known attenuating mutations (e.g., cp mutations, gene deletions), and demonstrates vaccine candidate phenotypes with appropriate temperature sensitivity, in vitro growth titers, and attenuation in animal models.
Claims Coverage
The patent claims cover recombinant infectious respiratory syncytial virus particles incorporating mutations primarily in the L protein codon encoding amino acid 1321 and optionally position 1313, alongside other mutations or gene deletions. The claims focus on stabilizing attenuating mutations with increased genetic stability for vaccine candidates.
Mutation of codon encoding amino acid 1321 of RSV L protein
The virus has a mutation of at least two nucleotides in the codon encoding tyrosine at position 1321 of the L protein, causing replacement by glutamic acid, lysine, glycine, proline, threonine, cysteine, glutamine, valine, alanine, or isoleucine, optionally with presence of other structural and nonstructural proteins.
Attenuated RSV with specified amino acid replacements at position 1321
The L protein codon mutation causes substitution specifically of glutamic acid, lysine, glycine, or proline at position 1321, with codon choices including GAA/GAG for glutamic acid, AAA/AAG for lysine, GGA/GGC/GGT/GGG for glycine, or CCT/CAA/CCG for proline.
Mutation of codon encoding serine at L protein position 1313
The virus further comprises mutation in the codon encoding serine at position 1313 of L protein to a different codon encoding serine, particularly the codon TCA, to enhance genetic stability and prevent compensatory mutations.
Combination of mutations at positions 1321 and 1313 with other known mutations
The RSV genome comprises combinations of L protein mutations at 1321 and 1313 (e.g., RSV 1321K/S1313(TCA)) combined with other attenuating mutations such as Q831L in L, N V267I, F E218A and T523I, L C319Y and H1690Y (cp mutations), a gene-start mutation in M2, deletion of SH gene, and deletion of NS2 gene.
Specific recombinant RSV strains
Examples of recombinant RSV strains claimed include RSV ΔNS2/1321K(AAA)/1313(TCA) and RSV ΔNS2/1321K(AAA)/1313(TCA)/cp/ΔSH, which incorporate the above mutations and deletions in defined combinations for use as live attenuated vaccine candidates.
The claims cover recombinant RSVs with engineered mutations in the L protein codons for amino acids 1321 and 1313, alone or combined with other known vaccine attenuating mutations or gene deletions, resulting in attenuated viruses with increased genetic and phenotypic stability suitable for vaccine use.
Stated Advantages
Increased genetic stability of attenuating mutations reduces reversion to wild-type virus phenotype, enhancing vaccine safety.
The mutations provide phenotypic stability, including temperature-sensitive phenotypes favorable for live attenuated vaccines.
Some deletion mutations, notably deletion of codon 1313, unexpectedly yield viable viruses with temperature sensitivity and effective attenuation without compromising replication at permissive temperature, facilitating vaccine manufacture.
Combination of mutations allows for tuning of virus attenuation levels to balance safety and immunogenicity.
Stable attenuated RSV candidates identified show promising replication and attenuation profiles in experimental animal models and are suitable for clinical vaccine development.
Documented Applications
Use as live attenuated RSV vaccines for protection against RSV infection and related lower respiratory tract diseases in humans, including infants and high-risk adults.
Method of expressing recombinant RSV viruses from isolated polynucleotides such as cDNA, and production of vaccine compositions comprising these attenuated viruses.
Use of recombinant RSV viruses as vectors for heterologous genes, including other respiratory viruses (e.g., parainfluenza virus) or immunomodulatory proteins such as cytokines.
Use of modified RSV viruses with enhanced genetic stability and attenuation for immunization protocols administered intranasally or via other routes to induce protective immune responses.
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