Recombinant respiratory syncytial virus stains with mutations in the M2-2 ORF providing a range of attenuation phenotypes
Inventors
Collins, Peter L. • Buchholz, Ursula J. • Luongo, Cindy
Assignees
US Department of Health and Human Services
Publication Number
US-10655109-B2
Publication Date
2020-05-19
Expiration Date
2036-12-12
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Abstract
Provided herein are novel recombinant respiratory syncytial viruses (RSV) having an attenuated phenotype that contain mutations in the M2-2 open reading frame that interfere with the expression of the M2-2 protein. The M2-2 mutations may be present in combination with mutations at other loci. Using methods described herein, combinations of mutations are provided to achieve desired levels of attenuation. The recombinant RSV strains described here are suitable for use as live-attenuated RSV vaccines. Also provided are polynucleotide sequences of the described viruses, as well as methods for producing and using the viruses.
Core Innovation
Provided herein are novel recombinant respiratory syncytial viruses (RSV) having an attenuated phenotype that contain mutations in the M2-2 open reading frame that interfere with the expression of the M2-2 protein. The M2-2 mutations may be present in combination with mutations at other loci to achieve desired levels of attenuation. These recombinant RSV strains are suitable for use as live-attenuated RSV vaccines.
Human respiratory syncytial virus (RSV) causes significant morbidity and mortality worldwide, especially in pediatric populations. Development of RSV vaccines has been challenging due to factors like immune enhancement upon inactivated RSV vaccination, incomplete immune protection due to RSV replication at superficial respiratory epithelial cells, moderate RSV titers in cell culture, difficulty identifying attenuating mutations that balance attenuation with immunogenicity, and genetic instability leading to mutation reversion.
The disclosed mutations that silence the M2-2 ORF, mainly large deletions removing most of the M2-2 protein coding sequence combined with point mutations disabling start codons, provide genetically stable attenuation. Combining these M2-2 mutations with other mutations allows creation of recombinant RSV strains with graded attenuation phenotypes. These strains are infectious, attenuated, self-replicating, and elicit a high titer of neutralizing antibodies in humans.
Claims Coverage
The patent claims cover recombinant RSV viruses with specific deletions and mutations in the M2-2 ORF, alone or in combination with additional mutations to modulate attenuation and other properties.
M2-2 open reading frame deletion mutations
A recombinant RSV with a deletion in the M2-2 ORF comprising either a 241-nucleotide deletion combined with nucleotide mutations at specific positions (ΔM2-2), or a 234-nucleotide deletion combined with nucleotide mutations creating AclI or HindIII restriction sites (ΔM2-2-AclI, ΔM2-2-HindIII).
Deletion of non-coding region mutation (6120)
Inclusion of a deletion of 112 nucleotides and five silent mutations in the downstream nontranslated region of the SH gene, termed the 6120 mutation, to adjust viral attenuation and facilitate molecular manipulation.
Additional protein-coding mutations for attenuation and phenotype modification
Incorporation of nucleotide mutations encoding amino acid substitutions such as V267I (N protein), E218A and T523I (F protein), C319Y and H1690Y (L protein) (cp mutations); K51R (NS2), T24A (N); deletion of the SH gene (ΔSH); replacements of G and F protein genes with codon optimized or clinical isolate sequences; amino acid substitutions K66E and Q101P in the F protein (HEK); and reversing the gene order of G and F proteins.
Mutation to stabilize polymerase protein and provide temperature sensitivity
Nucleotide mutations introducing a Y1321K substitution along with an S1313 residue in the L protein (1030s mutation) to increase genetic stability and provide a temperature-sensitive phenotype.
The claims define recombinant RSVs attenuated by deletions and mutations in the M2-2 ORF, optionally combined with a deletion in the SH gene noncoding region and various amino acid substitutions or gene replacements. These modifications provide recombinant RSV strains with a spectrum of attenuation phenotypes suitable for viably replicating, immunogenic live-attenuated RSV vaccines.
Stated Advantages
The M2-2 deletion mutations provide genetic stability and reduce risk of de-attenuation, a major concern in RSV vaccine development.
The recombinant RSV strains elicit substantially greater immunogenicity in human subjects compared to previous vaccine candidates.
The 6120 mutation facilitates manipulation of the antigenomic cDNA in bacteria and can increase replication efficiency in vitro and in vivo.
Combinations of mutations provide an array of attenuated strains with graded attenuation phenotypes allowing tailored balancing of attenuation and immunogenicity.
Documented Applications
Use of the recombinant RSV strains as live-attenuated vaccines to elicit protective immune responses against RSV infections in human subjects, including infants between 1 and 18 months of age.
Production of recombinant RSV by reverse genetics using isolated polynucleotide molecules encoding modified RSV genomes or antigenomes.
Combining recombinant RSV vaccines with adjuvants and physiologically acceptable carriers for administration via nasal spray, aerosol or injection, to stimulate immunity against RSV.
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