Attenuation of human respiratory syncytial virus by genome scale codon-pair deoptimization
Inventors
Collins, Peter L. • Le Nouën, Cyril • Brock, Linda G. • Buchholz, Ursula J. • DiNapoli, Joshua Marc • Mueller, Steffen • Wimmer, Eckard
Assignees
Research Foundation of the State University of New York • US Department of Health and Human Services
Publication Number
US-11371024-B2
Publication Date
2022-06-28
Expiration Date
2034-02-07
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Abstract
Described herein are RSV polynucleotide sequences that make use of multiple codons that are containing silent nucleotide substitutions engineered in multiple locations in the genome, wherein the substitutions introduce a numerous synonymous codons into the genome. Due to the large number of defects involved, the attenuated viruses disclosed herein provide a means of producing attenuated, live vaccines against RSV.
Core Innovation
Human respiratory syncytial virus (RSV) is a major cause of pediatric infection, hospitalizations, and deaths worldwide, with no commercially available vaccines. Severe infection can lead to long-term airway dysfunction and exacerbate conditions such as asthma. RSV is a single-stranded negative-sense RNA virus encoding 11 proteins, complicating vaccine development.
The invention addresses the need for effective and stable live attenuated RSV vaccines by employing genome-scale codon pair deoptimization (CPD), a synthetic attenuated virus engineering technique. CPD introduces numerous synonymous codon substitutions throughout multiple RSV genes without altering the amino acid sequences, thereby suboptimally arranging codon pairs to attenuate viral replication while preserving antigenic properties.
By extensively recoding RSV open reading frames with silent nucleotide substitutions, the invention alters parameters such as codon bias, codon pair bias, RNA secondary structure, and others. This causes defects that yield stably attenuated viruses, enabling production of live vaccines that are genetically stable, immunogenic, and have reduced pathogenicity in vitro and in vivo.
Claims Coverage
The claims disclose a recombinant polynucleotide and related recombinant RSV genome or antigenome featuring codon pair deoptimized nucleotide sequences encoding RSV proteins. They also cover recombinant viruses with specified amino acid changes and methods of production and use.
Recombinant polynucleotide with codon pair deoptimized sequences encoding RSV proteins
A recombinant nucleotide sequence encoding RSV proteins NS1, NS2, G, F, or L or combinations thereof, wherein the codon pair bias is reduced by at least 0.05 relative to corresponding wild type RSV subgroup A sequences, the nucleotide identities range from 65% to 95% depending on the protein, and the amino acid sequences are identical or differ by up to four substitutions, additions, or deletions compared to wild type.
Recombinant RSV genome or antigenome comprising codon pair deoptimized sequences
An RSV genome or antigenome comprising the recombinant polynucleotide codon pair deoptimized sequences as encoded by specific plasmids pRSV_Min A, pRSV_Min B, pRSV_Min L, pRSV_Min FLC, or variants thereof.
Inclusion of specific nucleotide changes causing amino acid substitutions
The recombinant RSV genome or antigenome further includes nucleotide changes in codons encoding amino acid 136 of N protein, 114 of P protein, and/or 88 of M2-1 protein, resulting in amino acids other than lysine, glutamic acid, or asparagine at those respective positions.
Methods of producing recombinant RSV and inducing an immune response
Methods comprising expressing the recombinant codon pair deoptimized RSV genomes in a cell to produce recombinant RSV, and administering the produced virus via various routes to induce an immune response in animals, including humans.
Vaccine composition containing codon pair deoptimized RSV
A vaccine composition comprising the recombinant RSV produced by expression of the codon pair deoptimized genome or antigenome, where the virus is attenuated and may further include previously characterized RSV mutations or deletions.
Extended codon pair bias reduction and calculation relative to human genome
Variations where the codon pair bias reduction is at least 0.1 or 0.2 relative to wild type, and codon pair bias is calculated with reference to the human genome.
The claims define recombinant nucleotide sequences and RSV genome constructs that employ extensive codon pair deoptimization in key RSV proteins, optionally combined with specific amino acid mutations, to produce genetically stable, attenuated RSV suitable for vaccine development, along with methods for producing and administering such recombinant RSV viruses.
Stated Advantages
The invention provides stably attenuated live RSV vaccines with reduced replication compared to wild-type while maintaining antigenic properties.
Recombinant viruses exhibit enhanced genetic and phenotypic stability in vitro and in vivo.
Codon pair deoptimization allows for titration of attenuation by adjusting the extent of synonymous codon substitutions.
Preservation of wild type amino acid sequences ensures potential for robust cellular and humoral immunity against wild type virus epitopes.
Documented Applications
Use as live attenuated vaccine against human respiratory syncytial virus infection in humans and animals.
Production of recombinant RSV vaccines administered via injection, aerosol delivery, nasal spray, nasal droplets, oral inoculation, or topical application.
Use in eliciting immune responses to RSV proteins in susceptible hosts, including infants, children, adults, and immunocompromised individuals.
Combination with known attenuating mutations or other RSV strains to enhance vaccine efficacy and cross-protection.
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