Genetically modified recombinant vaccinia ankara (RMVA) vaccines of improved stability and methods of preparation thereof
A vaccine composition comprising an immunologically effective amount of recombinant modified vaccinia Ankara (rMVA) virus comprising IE1, IE2 and pp65 or antigenic fragments thereof, which is genetically stable after at least 10 passages. A method of improving the stability of such rMVA upon passage by including one or more of the modifications: (1) inserting one or more nucleic acid sequences encoding the CMV antigens or antigenic fragments thereof into one or more insertion sites including but not limited to 044L/045L, IGR3, G1L/18R, and Del3 but not including Del2; (2) codon optimizing the nucleic acid sequences encoding the CMV antigens by removing consecutive cytosines or guanines; and (3) introducing one or more mutations in the amino acid sequences of the CMV antigens.
1. An expression system for co-expressing cytomegalovirus (CMV) antigens comprising a genetically modified recombinant Vaccinia Ankara (rMVA) vector inserted with three nucleic acid sequences encoding three CMV antigens or antigenic portions thereof, wherein the three CMV antigens or antigenic fragments thereof include (a) an IE1 exon 4 (IE1/e4) antigen or antigenic portion thereof inserted in rMVA insertion site IGR3, (b) an IE2 exon 5 (IE2/e5) antigen or antigenic portion thereof inserted in rMVA insertion site 044L/045L, and (c) pp65 antigen or antigenic portion thereof inserted in rMVA insertion site Del3.
2. The expression system of claim 1 , wherein each of the three nucleic acid sequences are operably linked to and under the control of a single promoter.
3. The expression system of claim 2 , wherein the promoter is an mH5 promoter.
4. The expression system of claim 1 , wherein the nucleic acid sequence of the IE1/e4 or IE2/e5 antigen or antigenic portion thereof is codon optimized.
5. The expression system of claim 1 , wherein one or more of the three the amino acid sequences of the CMV antigens comprise one or more mutations to improve the genetic stability of the rMVA upon viral passaging.
6. The expression system of claim 1 , wherein the rMVA expressing the CMV antigens is genetically stable for at least 10 passages.
7. An immunogenic composition comprising an rMVA vector inserted with three nucleic acid sequences encoding three CMV antigens or antigenic portions thereof, wherein the three CMV antigens or antigenic fragments thereof include (a) an IE1 exon 4 (IE1/e4) antigen or antigenic portion thereof inserted in rMVA insertion site IGR3, (b) an IE2 exon 5 (IE2/e5) antigen or antigenic portion thereof inserted in rMVA insertion site 044L/045L, and (c) pp65 antigen or antigenic portion thereof inserted in rMVA insertion site Del3.
8. The vaccine composition of claim 7 , wherein each of the three nucleic acid sequences are operably linked to and under the control of a single promoter.
9. The vaccine composition of claim 8 , wherein the promoter is mH5 promoter.
10. The vaccine composition of claim 7 , wherein the nucleic acid sequence of the IE1/e4 or IE2/e5 antigen or antigenic portion thereof is codon optimized.
11. The vaccine composition of claim 7 , wherein one or more of the amino acid sequences of the CMV antigens comprise one or more mutations to improve the genetic stability of the rMVA upon viral passaging.
12. The vaccine composition of claim 7 , wherein the rMVA expressing the CMV antigens is genetically stable for at least 10 passages.
13. A method of eliciting or modifying an immune response in a subject by administering the vaccine of claim 7 to a subject in need thereof.
14. The method of claim 13 , wherein the subject is a mammal.
15. The method of claim 13 , wherein the subject is human.
16. The method of claim 4 , wherein the nucleic acid sequence of the IE1/e4 or IE2/e5 antigen or antigenic portion thereof is a four nucleotide optimized (4nt) nucleic acid sequence of the IE2/e5 antigen or antigenic portion thereof.
17. The method of claim 5 , wherein the one or more mutations is a mutation in the IE2/e5 antigen or antigenic portion thereof comprising (i) H363A, (ii) H369A, or (iii) both H363A and H369A.
18. The method of claim 10 , wherein the nucleic acid sequence of the IE1/e4 or IE2/e5 antigen or antigenic portion thereof is a four nucleotide optimized (4nt) nucleic acid sequence of the IE2/e5 antigen or antigenic portion thereof.
19. The method of claim 11 , wherein the one or more mutations is a mutation in the IE2/e5 antigen or antigenic portion thereof comprising (i) H363A, (ii) H369A, or (iii) both H363A and H369A.