Attenuated virus mutated at sites of evolutionarily conserved RNA structure
Inventors
Tuller, Tamir • Goz, Eli • ATAR, Shimshi • Zur, Hadas
Assignees
Synvaccine Ltd • Ramot at Tel Aviv University Ltd • Yeda Research and Development Co Ltd
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Publication Number
US-11111481-B2
Publication Date
2021-09-07
Expiration Date
Abstract
Attenuated viruses and methods of designing them are disclosed. In one embodiment, there is disclosed an attenuated form of a virulent virus comprising an RNA encoding a viral protein or a nucleic acid sequence transcribable to said RNA, wherein the folding energy or structure of the RNA is changed at positions of evolutionarily conserved RNA structures with respect to that of said RNA encoding said viral protein in the virulent virus so as to bring about attenuation of the virus.
Core Innovation
The invention provides an attenuated form of a virulent virus in which an RNA encoding a viral protein, or a nucleic acid sequence transcribable to said RNA, is modified by at least one synonymous nucleotide substitution. The substitution is made in a region of evolutionarily conserved local RNA folding energy, where the region is characterized by folding energy below a predetermined threshold and the substitution increases folding energy, or folding energy above a predetermined threshold and the substitution decreases folding energy.
The predetermined threshold is derived from the average local folding energy of a randomized sequence of the virulent virus, where the randomized sequence encodes an amino acid sequence identical to the amino acid sequence of the virulent virus. The attenuated virus preserves protein identity by requiring that the viral protein of the attenuated virus comprises an amino acid sequence identical to the amino acid sequence of the viral protein of the virulent virus, and the substitution decreases replicative fitness of the attenuated form of a virus as compared to the virulent virus.
The disclosed scope includes additional refinements in conserved RNA structure and folding-energy change, and discusses computational and analytical approaches for identifying evolutionarily conserved local folding features and for optimizing synonymous substitutions to achieve attenuation. The concept is described as applicable to multiple virus types, including flaviviruses such as dengue virus, and is framed around using local RNA secondary structure signals and folding-energy thresholds to guide synonymous nucleotide substitutions while preserving encoded proteins and optionally nucleic-acid composition features.
Claims Coverage
The independent claim covers an attenuated virulent virus defined by synonymous nucleotide substitutions in evolutionarily conserved local RNA folding-energy regions with thresholding based on a randomized-sequence average, while keeping the encoded viral protein amino-acid sequence identical and reducing replicative fitness. Dependent claims refine the folding-energy change magnitude, preserve codon/GC/dinucleotide-related biases, narrow to specific dengue virus sequence identifiers, and relate the attenuated virus to pharmaceutical composition and immune-response elicitation for prophylactic or therapeutic use.
Synonymous substitution in evolutionarily conserved local RNA folding energy region
An attenuated form of a virulent virus comprising an RNA encoding a viral protein, or a nucleic acid sequence transcribable to said RNA, comprising at least one nucleotide in a region of evolutionarily conserved local RNA folding energy synonymously substituted to another nucleotide.
Folding-energy threshold derived from randomized sequence average
The region of evolutionarily conserved local RNA folding energy comprises folding energy below a predetermined threshold and said substitution increases said folding energy, or wherein said region of evolutionarily conserved RNA folding energy comprises folding energy above a predetermined threshold and said substitution decreases said folding energy, wherein said predetermined threshold is derived from the average local folding energy of a randomized sequence of the virulent virus, wherein said randomized sequence encodes an amino acid sequence which is identical to the amino acid sequence of said virulent virus.
Protein amino-acid sequence preserved with reduced replicative fitness
The viral protein of said attenuated virus comprises an amino acid sequence which is identical to the amino acid sequence of said viral protein of the virulent virus and said at least one substitution decreases replicative fitness of said attenuated form of a virus as compared to said virulent virus.
Quantified total change relative to maximum folding-energy change
The attenuated virus includes a total change in local RNA folding energy that is at least 20% of a maximum achievable change under a defined set of synonymous substitutions relative to a predetermined folding-energy threshold.
Quantified folding-energy magnitude thresholds for the substitution
The attenuated virus is modified by a substitution that increases folding energy by more than 3 kcal/mol or decreases folding energy by more than 9 kcal/mol.
Preservation of codon usage bias and/or GC content
Any nucleotide substitutions preserve the virulent virus overall codon usage bias, GC content, or both.
Specific dengue virus genomes identified by SEQ ID NOs
The attenuated dengue virus has a dengue virus genome encoded by a sequence selected from SEQ ID NOs: 1671-1734.
Pharmaceutical composition enabling immune response elicitation and immunization
A method of immunizing or eliciting an immune response in a subject against a virus-associated disease by administering a prophylactically or therapeutically effective dose of the pharmaceutical composition, optionally after exposure to a pathogenic virus.
Overall, the claim set centers on attenuation via synonymous nucleotide substitutions located in regions of evolutionarily conserved local RNA folding energy, where directionality of folding-energy change is determined relative to a predetermined threshold derived from randomized-sequence average folding energy, while maintaining an identical encoded viral protein amino-acid sequence and decreasing replicative fitness. Dependent refinements further constrain quantitative folding-energy change, preserve codon/GC/dinucleotide-related biases, narrow to enumerated dengue sequences, and connect the attenuated virus to pharmaceutical composition for prophylactic or therapeutic immune-response elicitation.
Stated Advantages
Decreases replicative fitness of the attenuated form of a virus as compared to the virulent virus.
Preserves an identical amino-acid sequence for the encoded viral protein while attenuating replicative fitness.
Imparts attenuation based on evolutionarily conserved local RNA folding energy using predetermined thresholds derived from randomized-sequence average local folding energy.
Documented Applications
Vaccine composition concepts are tied to attenuated viruses, and the document relates the attenuated virus to pharmaceutical/immunogenic compositions intended to elicit protective immune responses.
Immunizing or eliciting an immune response in a subject against a virus-associated disease by administering a prophylactically or therapeutically effective dose of a pharmaceutical composition, optionally after exposure to a pathogenic virus.
Documented example support includes dengue virus (DENV) and flaviviruses (including dengue) as virus types for which attenuation design is discussed.
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