Multi plasmid system for the production of influenza virus
Vectors and methods for the production of influenza viruses suitable as recombinant influenza vaccines in cell culture are provided. Bi-directional expression vectors for use in a multi-plasmid influenza virus expression system are provided. Additionally, the invention provides methods of producing influenza viruses with enhanced ability to replicate in embryonated chicken eggs and/or cells (e.g., Vero and/or MDCK) and further provides influenza viruses with enhanced replication characteristics. A method of producing a cold adapted (ca) influenza virus that replicates efficiently at, e.g., 25° C. (and immunogenic compositions comprising the same) is also provided.
1. A reassortant influenza virus comprising:
i) an HA protein comprising a leucine at position 183 and an alanine at position 226; or
ii) an HA protein comprising a valine at position 186 and an isoleucine at position 226; or
iii) an HA protein comprising a valine at position 186 and an isoleucine at position 226 and an NA protein comprising a glutamate at position 119 and a glutamine at position 136;
wherein the HA protein is of the H3 type.
2. An immunogenic composition comprising the reassortant influenza virus of claim 1 .
3. A method of making the reassortant influenza virus of claim 1 , comprising:
(a) introducing at least one mutation in an influenza HA and/or NA gene, wherein the at least one mutation results in:
(i) an HA polypeptide comprising a leucine at position 183 and an alanine at position 226; or
(ii) an HA polypeptide comprising a valine at position 186 and an isoleucine at position 226; or
(iii) an HA polypeptide comprising a valine at position 186 and an isoleucine at position 226 and an NA comprising a glutamate at position 119 and a glutamine at position 136; wherein the HA protein is of the H3 type;
(b) introducing a plurality of vectors into a population of host cells, wherein the plurality of vectors corresponds to an influenza virus genome, wherein at least one of the plurality of vectors comprises an influenza HA and/or NA gene comprising the at least one mutation recited in (a), and wherein the population of host cells is capable of supporting replication of influenza virus;
(c) culturing the population of host cells; and
(d) recovering the reassortant influenza virus.
4. A method of making the reassortant influenza virus of claim 1 , comprising:
(a) introducing a plurality of vectors into a population of host cells, wherein the plurality of vectors corresponds to an influenza virus genome, wherein at least one of the plurality of vectors encodes:
(i) an HA polypeptide comprising a leucine at position 183 and an alanine at position 226; or
(ii) an HA polypeptide comprising a valine at position 186 and an isoleucine at position 226; or
(iii) an HA polypeptide comprising a valine at position 186 and an isoleucine at position 226 and an NA comprising a glutamate at position 119 and a glutamine at position 136; wherein the HA protein is of the H3 type;
and wherein the population of host cells is capable of supporting replication of influenza virus;
(b) culturing the population of host cells; and
(c) recovering the reassortant influenza virus.
5. A method of stimulating an immune response, comprising:
administering the immunogenic composition of claim 2 .
6. The method of claim 5 , wherein the immunogenic composition is administered intranasally.
7. The method of claim 5 , wherein the immunogenic composition is administered by subcutaneous injection.
8. The method of claim 5 wherein the immunogenic composition is administered by intramuscular injection.
9. The reassortant influenza virus of claim 1 , wherein the virus is a 6:2 reassortant.
10. The reassortant influenza virus of claim 1 , wherein the virus is a 7:1 reassortant.