Influenza viruses and uses thereof
Described herein are chimeric influenza virus gene segments and nucleic acid sequences encoding such chimeric influenza virus gene segments. A chimeric influenza virus gene segment described herein comprises packaging signals found in the non-coding and coding regions of one type of influenza virus gene segment and an open reading frame of a different type of influenza virus gene segment or fragment thereof. Also described herein are recombinant influenza viruses comprising two or more chimeric influenza virus gene segments and the use of such viruses in the prevention and/or treatment of influenza virus disease.
1. A recombinant influenza virus comprising
a) a first chimeric influenza virus gene segment comprising, in 3′ to 5′ order:
(i) a 3′ non-coding region of a first type of influenza virus gene segment;
(ii) a 3′ proximal coding region of the first type of influenza virus gene segment, wherein any start codon in the 3′ proximal coding region of the first influenza virus gene segment is mutated;
(iii) an open reading frame of a second type of influenza virus gene segment, wherein at least one packaging signal in the 3′ or 5′ proximal nucleotides in the open reading frame is mutated;
(iv) a 5′ proximal coding region of the first type of influenza virus gene segment; and
(v) a 5′ non-coding region of the first type of influenza virus gene segment; and
b) a second chimeric influenza virus gene segment comprising, in 3′ to 5′ order:
(i) a 3′ non-coding region of the second type of influenza virus gene segment;
(ii) a 3′ proximal coding region of the second type of influenza virus gene segment, wherein any start codon in the 3′ proximal coding region of the second influenza virus gene segment is mutated;
(iii) an open reading frame of the first type of influenza virus gene segment, wherein at least one packaging signal in the 3′ or 5′ proximal nucleotides in the open reading frame is mutated;
(iv) a 5′ proximal coding region of the second type of influenza virus gene segment; and
(v) a 5′ non-coding region of the second type of influenza virus influenza gene segment.
2. The recombinant influenza virus of claim 1 , wherein only progeny of the virus comprising the chimeric influenza virus gene segments is capable of forming plaques in a plaque assay.
3. A recombinant influenza virus comprising
a) a first chimeric influenza virus gene segment comprising, in 3′ to 5′ order:
(i) a 3′ non-coding region of a third type of influenza virus gene segment;
(ii) a 3′ proximal coding region of the third type of influenza virus gene segment, wherein any start codon in the 3′ proximal coding region of the third influenza virus gene segment is mutated;
(iii) an open reading frame of a first type of influenza virus gene segment, wherein at least one packaging signal in the 3′ or 5′ proximal nucleotides in the open reading frame is mutated;
(iv) a 5′ proximal coding region of the third type of influenza virus gene segment; and
(v) a 5′ non-coding region of the third type of influenza virus gene segment; and
b) a second chimeric influenza virus gene segment comprising, in 3′ to 5′ order:
(i) a 3′ non-coding region of the first type of influenza virus gene segment;
(ii) a 3′ proximal coding region of the first type of influenza virus gene segment, wherein any start codon in the 3′ proximal coding region of the first influenza virus gene segment is mutated;
(iii) an open reading frame of a second type of influenza virus gene segment, wherein at least one packaging signal in the 3′ or 5′ proximal nucleotides in the open reading frame is mutated;
(iv) a 5′ proximal coding region of the first type of influenza virus gene segment; and
(v) a 5′ non-coding region of the first type of influenza virus gene segment; and
c) a third chimeric influenza virus gene segment comprising, in 3′ to 5′ order:
(i) a 3′ non-coding region of the second type of influenza virus gene segment;
(ii) a 3′ proximal coding region of the second type of influenza virus gene segment, wherein any start codon in the 3′ proximal coding region of the second influenza virus gene segment is mutated;
(iii) an open reading frame of the third type of influenza virus gene segment, wherein at least one packaging signal in the 3′ or 5′ proximal nucleotides in the open reading frame is mutated;
(iv) a 5′ proximal coding region of the second type of influenza virus gene segment; and
(v) a 5′ non-coding region of the second type of influenza virus gene segment.
4. The recombinant influenza virus of claim 3 , wherein only progeny comprising the first, second and third chimeric influenza virus gene segments is capable of forming plaques in a plaque assay.
5. The virus of claim 3 , wherein the first, second, and third influenza virus gene segments encode the HA, NA, and NS proteins, respectively.
6. The recombinant influenza virus of claim 1 , which comprises nine gene segments.
7. The recombinant influenza virus of claim 1 , wherein: (a) the first influenza virus gene segment is NS, and wherein a mRNA 5′ splice site of the 3′ proximal coding region is mutated; or (b) the first influenza virus gene segment is M, and wherein a distal mRNA 5′ splice site of the 3′ proximal coding region is mutated.
8. The recombinant influenza virus of claim 1 , wherein the virus comprises an attenuating mutation.
9. A substrate comprising the recombinant influenza virus of claim 1 .
10. A pharmaceutical composition or immunogenic composition comprising the recombinant influenza virus of claim 1 .
11. A method for eliciting an immune response against an influenza virus in a subject, wherein the method comprises administering the recombinant influenza virus of claim 1 to the subject.
12. A method of preventing or treating an influenza virus disease, or treating an influenza virus infection, in a subject, wherein the method comprises administering the recombinant influenza virus of claim 1 to the subject.
13. A method for propagating a recombinant influenza virus, wherein the method comprises infecting a substrate with the recombinant influenza virus of claim 1 ; and purifying the virus subsequently from the substrate.
14. The recombinant influenza virus of claim 1 , wherein said mutation of at least one packaging signal in the 3′ or 5′ proximal nucleotides is a silent mutation.
15. The recombinant influenza virus of claim 1 , wherein at least one packaging signal in the 3′ and the 5′ proximal nucleotides in each open reading frame is mutated.