Recombinant respiratory syncytial virus strains 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-11332721-B2
Publication Date
2022-05-17
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 (ORF) that interfere with the expression of the M2-2 protein. These mutations may be present individually or in combination with mutations at other loci, enabling the achievement of desired levels of viral attenuation. The resulting recombinant RSV strains are suitable for use as live-attenuated RSV vaccines. Additionally, polynucleotide sequences of these viruses, methods for their production, and their use are described.
The problem addressed is the need for live attenuated RSV strains that replicate efficiently in vitro, are maximally immunogenic, satisfactorily attenuated, and genetically stable. Historically, RSV vaccine development has been complicated by issues such as immune enhancement from inactivated vaccines, inefficient immune protection at the site of viral replication, attenuation-immunogenicity balance, low virus yields, difficulties identifying and developing attenuating mutations, and genetic instability leading to reversion to non-attenuated phenotypes.
The disclosed innovation provides mutations that interfere with M2-2 protein expression, resulting in novel RSV strains with a range of attenuation phenotypes. These mutations, alone or combined with other mutations, enable the tuning of attenuation magnitude. Notably, large deletions ablating most of the M2-2 ORF offer genetic stability, addressing concerns about de-attenuation. The mutant strains show infection, attenuation, self-replication, and notably elicit higher titers of neutralizing antibodies in human subjects. The RSV genome backbones used include D46 and LID, with various accompanying mutations such as “6120,” “cp,” “HEK,” and “1030s” to modulate attenuation and replication.
Claims Coverage
The patent includes one independent claim that covers recombinant RSV with specific genomic modifications related to the M2-2 open reading frame and optionally additional mutations.
M2-2 ORF large deletion with start codon mutations
A recombinant RSV genome comprising a deletion of 241 nucleotides at positions 8189-8429 of SEQ ID NO: 1 in the M2-2 ORF, combined with nucleotide mutations disrupting translation start codons at positions T8161, T8167, and T8179 of SEQ ID NO: 1 (referred to as "ΔM2-2").
Additional deletion in the SH gene region (6120 mutation)
A deletion of 112 nucleotides corresponding to positions 4499-4610 of SEQ ID NO: 1 combined with nucleotide mutations C4489T, C4492T, A4495T, A4497G, and G4498A, referred to as the “6120” mutation.
Introduction of Y1321K substitution and stabilizing S1313 residue in the L protein (1030s mutation)
Nucleotide mutations encoding a Y1321K substitution and a stabilizing S1313 residue in the L protein, with codons AAA and TCA respectively, known as the “1030s” mutation.
Combinations of ΔM2-2 with 6120 and 1030s mutations
Genomes containing the ΔM2-2 deletion combined with the 6120 deletion and the 1030s substitution, and nucleotide sequences at least 90% identical to the LID/ΔM2-2/1030s sequence (SEQ ID NO: 16).
Use of additional amino acid substitutions and gene modifications
Incorporation of amino acid substitutions V267I (N protein), E218A and T523I (F protein), C319Y and H1690Y (L protein) collectively called “cp,” K51R in NS2 (“NS2”), T24A in N (“N”), deletions of SH gene (“ΔSH”), codon optimization and replacement of G and F gene sequences from clinical isolates, F protein mutations K66E and Q101P (“HEK”), and reversal of order of G and F genes.
The claims cover recombinant RSV variants attenuated through a specific large deletion in the M2-2 ORF disrupting M2-2 expression, optionally combined with one or more additional mutations affecting other proteins or genomic regions, including deletions, point mutations, and gene rearrangements. These inventive features enable a range of attenuation phenotypes suitable for live-attenuated RSV vaccines.
Stated Advantages
Recombinant RSV strains with genetic stability due to large deletions in the M2-2 ORF, reducing risk of reversion to a non-attenuated form.
RSV strains exhibit a range of attenuation phenotypes, allowing tuning of vaccine replication and immunogenicity.
Increased immunogenicity demonstrated by surprisingly high titers of neutralizing antibodies in human subjects.
Mutations that allow efficient replication in vitro, supporting effective vaccine manufacturing.
Flexibility in combining mutations for desired levels of attenuation and immunogenicity.
Possibility to incorporate codon optimization and antigen gene modifications for improved antigen expression and immunogenicity.
Documented Applications
Use of the recombinant RSV strains as live attenuated vaccines for immunization against RSV infection, including administration to human subjects such as infants and children.
Methods of eliciting an immune response in a subject by administering an immunogenically effective amount of the recombinant RSV vaccine.
Use of recombinant RSV as vectors to express protective antigens of other respiratory pathogens, such as parainfluenza virus.
Production of purified RSV proteins by infection of permissive host cells with recombinant RSV strains for vaccine or diagnostic uses.
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