Live attenuated influenza virus vaccines comprising microRNA response elements
AssigneeIcahn School of Medicine at Mount Sinai
InventorBenjamin tenOever
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Abstract
The invention is directed to novel live attenuated influenza virus (LAIV) vaccines comprising one or more microRNA (miRNA) Response Element(s) (MRE) within an influenza virus genome. The MREs useful for the present invention can be derived from any miRNA which is highly expressed in influenza-targeted cells of an animal in need of vaccination but are not expressed or are expressed at very low levels in species (e.g., embryonated chicken eggs) or cell lines used for a large-scale vaccine production. This allows efficient vaccine production but renders the vaccine virus susceptible to attenuation in the influenza-targeted cells of vaccinated animals expressing a cognate miRNA.
Claims65
A composition comprising a recombinant influenza virus wherein said influenza virus comprises a microRNA Response Element (MRE) sequence, wherein the MRE sequence is selected such that the mean free energy (MFE) of MRE interaction with its corresponding microRNA is less than −35 kcal/mol.
An isolated nucleic acid molecule comprising an influenza virus coding sequence and a MRE sequence inserted within said coding sequence, wherein the MRE sequence is selected such that the MFE of MRE interaction with its corresponding microRNA is less than −35 kcal/mol.
An isolated nucleic acid molecule comprising an influenza virus coding sequence and an artificial 3′ untranslated region (3′ UTR) comprising a MRE sequence inserted between the stop codon of the transcript and the polyadenylation site, wherein the MRE sequence is selected such that the MFE of MRE interaction with its corresponding microRNA is less than −35 kcal/mol.
An isolated nucleic acid molecule comprising an influenza virus coding sequence and a first MRE sequence inserted within said coding sequence, wherein the MRE sequence is selected such that the MFE of MRE interaction with its corresponding microRNA is less than −25 kcal/mol;
- wherein the isolated nucleic acid molecule further comprises a second MRE sequence;
- and
- wherein both of the MRE sequences correspond to miR-93 and are inserted into the coding sequence of influenza virus protein NP, wherein the first MRE sequence is at the nucleotide sequence encoding NP amino acids 62-69 and the second MRE sequence is at the nucleotide sequence encoding NP amino acids 258-265.
An isolated nucleic acid molecule comprising an influenza virus coding sequence and a first MRE sequence inserted within said coding sequence, wherein the MRE sequence is selected such that the MFE of MRE interaction with its corresponding microRNA is less than −25 kcal/mol;
- wherein the isolated nucleic acid molecule further comprises a second MRE sequence;
- and
- wherein both of the MRE sequences correspond to miR-92 and are inserted into the coding sequence of influenza virus protein NS1, wherein the first MRE sequence is at the nucleotide sequence encoding NS1 amino acids 131-137 and the second MRE sequence is at the nucleotide sequence encoding NS1 amino acids 150-156.
An isolated nucleic acid molecule comprising an influenza virus coding sequence and a first MRE sequence inserted within said coding sequence, wherein the MRE sequence is selected such that the MFE of MRE interaction with its corresponding microRNA is less than −25 kcal/mol;
- wherein the isolated nucleic acid molecule further comprises a second and a third MRE sequence;
- and
- wherein all three of the MRE sequences which correspond to miR-92 and are inserted into the coding sequence of influenza virus protein HA, wherein the first MRE sequence is at the nucleotide sequence encoding HA amino acids 68-74, the second MRE sequence is at the nucleotide sequence encoding HA amino acids 195-201, and the third MRE sequence is at the nucleotide sequence encoding HA amino acids 526-532.
An isolated nucleic acid molecule comprising an influenza virus coding sequence and a first MRE sequence inserted within said coding sequence, wherein the MRE sequence is selected such that the MFE of MRE interaction with its corresponding microRNA is less than −25 kcal/mol;
- wherein the isolated nucleic acid molecule further comprises a second and a third MRE sequence;
- and
- wherein all three of the MRE sequences correspond to miR-19 and are inserted into the coding sequence of influenza virus protein HA, wherein the first MRE sequence is at the nucleotide sequence encoding HA amino acids 15-22, the second MRE sequence is at the nucleotide sequence encoding HA amino acids 561-568, and the third MRE sequence is at the nucleotide sequence encoding HA amino acids 327-334.
An isolated nucleic acid molecule comprising an influenza virus coding sequence and a first MRE sequence inserted within said coding sequence, wherein the MRE sequence is selected such that the MFE of MRE interaction with its corresponding microRNA is less than −25 kcal/mol;
- wherein the isolated nucleic acid molecule further comprises a second MRE sequence;
- and
- wherein both of the MRE sequences which correspond to miR-16 and are inserted into the coding sequence of influenza virus protein HA, wherein the first MRE sequence is at the nucleotide sequence encoding HA amino acids 2-9 and the second MRE sequence is at the nucleotide sequence encoding HA amino acids 439-445.
An isolated nucleic acid molecule comprising an influenza virus coding sequence and a first MRE sequence inserted within said coding sequence, wherein the MRE sequence is selected such that the MFE of MRE interaction with its corresponding microRNA is less than −25 kcal/mol;
- wherein the isolated nucleic acid molecule further comprises a second and a third MRE sequence;
- and
- wherein all three of the MRE sequences which correspond to miR-34 and are inserted into the coding sequence of influenza virus protein PA, wherein the first MRE sequence is at the nucleotide sequence encoding PA amino acids 426-433, the second MRE sequence is at the nucleotide sequence encoding PA amino acids 634-641, and the third MRE sequence is at the nucleotide sequence encoding PA amino acids 709-716.
An isolated nucleic acid molecule comprising an influenza virus coding sequence and a first MRE sequence inserted within said coding sequence, wherein the MRE sequence is selected such that the MFE of MRE interaction with its corresponding microRNA is less than −25 kcal/mol;
- wherein the isolated nucleic acid molecule further comprises a second MRE sequence;
- and
- wherein both of the MRE sequences which correspond to miR-25 and are inserted into the coding sequence of influenza virus protein M1, wherein the first MRE sequence is at the nucleotide sequence encoding M1 amino acids 111-118 and the second MRE sequence is at the nucleotide sequence encoding M1 amino acids 127-134.
An isolated nucleic acid molecule comprising an influenza virus coding sequence and a first MRE sequence inserted within said coding sequence, wherein the MRE sequence is selected such that the MFE of MRE interaction with its corresponding microRNA is less than −25 kcal/mol;
- wherein the isolated nucleic acid molecule further comprises a second and a third MRE sequence;
- and
- wherein all three of the MRE sequences correspond to miR-17 and are inserted into the coding sequence of influenza virus protein PB1, wherein the first MRE sequence is at the nucleotide sequence encoding PB1 amino acids 374-381, the second MRE sequence is at the nucleotide sequence encoding PB1 amino acids 418-424, and the third MRE sequence is at the nucleotide sequence encoding PB1 amino acids 677-683.
An isolated nucleic acid molecule comprising an influenza virus coding sequence and an artificial 3′ untranslated region (3′ UTR) comprising a first MRE sequence inserted between the stop codon of the transcript and the polyadenylation site, wherein the first MRE sequence is selected such that the MFE of MRE interaction with its corresponding microRNA is less than −25 kcal/mol;
- wherein the isolated nucleic acid molecule further comprises second, third, and fourth MRE sequences;
- and
- wherein all four of the MRE sequences correspond to miR-142 and are inserted into an artificial 3′ UTR of influenza virus protein NP, wherein each of the MRE sequences are found between the viral stop codon and the polyA tail sequence.
An isolated nucleic acid molecule comprising an influenza virus coding sequence and an artificial 3′ untranslated region (3′ UTR) comprising a first MRE sequence inserted between the stop codon of the transcript and the polyadenylation site, wherein the first MRE sequence is selected such that the MFE of MRE interaction with its corresponding microRNA is less than −25 kcal/mol;
- wherein the isolated nucleic acid molecule further comprises second, third, and fourth MRE sequences;
- and
- wherein all four of the MRE sequences which correspond to miR-142 and are inserted into an artificial 3′ UTR of influenza virus protein NS1, wherein each of the MRE sequences is found between the viral stop codon and the polyA tail sequence but before a duplicated NS2/NEP ORF.
Dependencies are inferred from the claim text.