Attenuated recombinant alphaviruses incapable of replicating in mosquitoes and uses thereof
The present invention discloses an attenuated recombinant alphavirus that is incapable of replicating in mosquito cells and of transmission by mosquito vectors. These attenuated alphavirus may include but is not limited to Western Equine Encephalitis virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus or Chikungunya virus. The present invention also discloses the method of generating such alphaviruses and their use as immunogenic compositions.
1. A live, attenuated Chikungunya (CHIK) virus comprising: a CHIK virus polynucleotide having (i) an inactivated subgenomic promoter, and (ii) an insertion of an encephalomyelocarditis virus internal ribosomal entry site (EMCV IRES) functional only in cells of vertebrate origin, between one end of nonstructural protein 4 (nsP4) coding sequence and initiating AUG of a subgenomic RNA coding sequence of the CHIK virus, wherein the subgenomic promoter is inactivated by clustered point mutations and wherein the CHIK virus is attenuated.
2. The live, attenuated CHIK virus of claim 1 , wherein the subgenomic promoter is inactivated by clustered point mutations and the clustered point mutations are located in the 5′ UTR of the subgenomic RNA.
3. The live, attenuated CHIK virus of claim 1 , wherein the mutation of the subgenomic promoter does not modify the amino acid sequence of the carboxy terminus of non-structural protein 4.
4. A pharmaceutical composition comprising the live, attenuated, Chikungunya (CHIK) virus of claim 1 and a pharmaceutically acceptable carrier.
5. A method for inducing an anti-Chikungunya (CHIK) virus immune response in a subject, comprising administering to the subject the composition of claim 4 .
6. The method of claim 5 , wherein the subject is a human, a horse or other domestic animal.
7. An expression vector comprising a polynucleotide encoding the live, attenuated Chikungunya (CHIK) virus of claim 1 .
8. An isolated host cell comprising the expression vector of claim 7 .
9. The live, attenuated CHIK virus of claim 2 , wherein the clustered point mutations comprise synonymous mutations at position 11, 12, 14, 17, 20, 22, 24, 25, 26, 27, 28, 29 and 30 of SEQ ID NO:1.