GENETICALLY STABLE RECOMBINANT MODIFIED VACCINIA ANKARA (RMVA) VACCINES AND METHODS OF PREPARATION THEREOF

Abstract

A vaccine comprising an immunologically effective amount of recombinant modified vaccinia Ankara (rMVA) virus which is genetically stable after serial passage and produced by a) constructing a transfer plasmid vector comprising a modified H5 (mH5) promoter operably linked to a DNA sequence encoding a heterologous foreign protein antigen, wherein the expression of said DNA sequence is under the control of the mH5 promoter; b) generating rMVA virus by transfecting one or more plasmid vectors obtained from step a) into wild type MVA virus; c) identifying rMVA virus expressing one or more heterologous foreign protein antigens using one or more selection methods for serial passage; d) conducting serial passage; e) expanding an rMVA virus strain identified by step d); and f) purifying the rMVA viruses from step e) to form the vaccine. One embodiment is directed to a fusion cytomegalovirus (CMV) protein antigen comprising a nucleotide sequence encoding two or more antigenic portions of Immediate-Early Gene-1 or Immediate-Early Gene-2 (IEfusion), wherein the antigenic portions elicit an immune response when expressed by a vaccine.

Description

Claims

1. A cytomegalovirus (CMV) vaccine comprising: a) an immunologically effective amount of a recombinant modified vaccinia Ankara (rMVA) virus, wherein the rMVA virus comprises a fusion nucleotide sequence which encodes an IEfusion CMV protein antigen, said fusion nucleotide sequence comprising a nucleotide sequence encoding an Immediate-Early Gene-1 (IE1) antigenic portion directly fused to a nucleotide sequence encoding an Immediate-Early Gene-1 (IE2) antigenic portion, wherein (i) the nucleotide sequence encoding the IE1 antigenic portion includes a nucleotide sequence encoding IE1 exon 4 (IE1/e4);(ii) the nucleotide sequence encoding the IE2 antigenic portion is a nucleotide sequence encoding IE2 exon 5 (IE2/e5); or(iii) both (i) and (ii), andb) a nucleotide sequence which encodes at least one CMV antigen or a combination of antigens selected from the group consisting of HCMV-pp65, glycoprotein B (gB), a UL128 complex and members thereof, wherein the UL128 complex comprises five members including UL128, UL130, UL131a, gH, and gL.

2. The CMV vaccine of claim 1, wherein the fusion nucleotide sequence comprises SEQ ID NO:11.

3. (canceled)

4. The CMV vaccine of claim 1, wherein the rMVA virus is genetically stable after serial passage.

5. The cytomegalovirus (CMV) vaccine of claim 4, wherein the vaccine is produced by: a) constructing a transfer plasmid vector comprising a modified H5 (mH5) promoter operably linked to a DNA sequence encoding the IEfusion CMV protein antigen, wherein the expression of said DNA sequence is under the control of the mH5 promoter;b) generating the rMVA virus by transfecting one or more plasmid vectors obtained from step a) into cells infected with wild type MVA; andc) identifying rMVA virus expressing one or more of the IEfusion CMV protein antigens using one or more selection methods for serial passage;d) conducting serial passage;e) expanding an rMVA virus strain identified by step d); andf) purifying the rMVA virus strain from step e) to form the vaccine.

6. The vaccine composition of claim 1, wherein the identification of rMVA virus carrying the MVA virus vector is accomplished by one or more gene-in selection methods, one or more gene-out selection methods, or a combination of gene-in and gene-out selection methods.

7. The vaccine composition of claim 1, wherein the serial passage is at least 10 passages.

8. The vaccine composition of claim 1, wherein the transfer plasmid vector comprises a nucleotide sequence selected from SEQ ID NO:9 or SEQ ID NO:10.

9. The vaccine composition of claim 1, wherein the transfer plasmids comprise nucleotide sequences SEQ ID NO:9 and SEQ ID NO:10.

10. A method of modifying an immune response in a mammalian subject by administering a vaccine composition of claim 1 to the subject.

11. The method of claim 10, wherein the subject is a human.

12. The method of claim 10, wherein the subject is a human stem cell donor or a human solid organ transplant donor.

13. The method of claim 10, wherein the subject is a human with an immunodeficiency disease such as HIV or a heritable immunodeficiency and subject is susceptible to infection by human cytomegalovirus.

14. The method of claim 10, wherein the subject is a human subject that has received a stem cell transplant (HCT) or a solid organ transplant from a healthy donor.

15. A method for producing a genetically stable rMVA vaccine, comprising: a) constructing a transfer plasmid vector comprising a modified H5 (mH5) promoter operably linked to a DNA sequence encoding a heterologous foreign protein antigen, wherein the expression of said DNA sequence is under the control of the mH5 promoter;b) generating rMVA virus by transfecting one or more plasmid vectors obtained from step a) into cells infected with wild type MVA-; andc) identifying rMVA virus expressing one or more heterologous foreign protein antigens using one or more selection methods for serial passage;d) conducting serial passage;e) expanding an rMVA virus strain identified by step d); andf) purifying the rMVA virus strain from step e) to form the vaccine;wherein the expression and immunogenicity of said foreign protein antigens are stable after serial passage in the rMVA vaccine obtained from step f),wherein at least one of the foreign protein antigens is an IEfusion CMV protein antigen comprising a nucleotide sequence encoding an Immediate-Early Gene-1 (IE1) antigenic portion directly fused to a nucleotide sequence encoding an Immediate-Early Gene-1 (IE2) antigenic portion, wherein (i) the nucleotide sequence encoding the IE1 antigenic portion includes a nucleotide sequence encoding IE1 exon 4 (IE1/e4);(ii) the nucleotide sequence encoding the IE2 antigenic portion is a nucleotide sequence encoding IE2 exon 5 (IE2/e5); or(iii) both (i) and (ii), andwherein the at least one of the foreign protein antigens further comprise a CMV antigen selected from the group consisting of pp65, CMV pp150, glycoprotein B (gB) and antigenic fragments thereof, the UL128 complex or members thereof, wherein UL128 members comprise UL128, UL130, UL131a, gH, and gL.

16. (canceled)

17. The method of claim 15, wherein the IEfusion CMV protein antigen comprises a nucleotide sequence of SEQ ID NO:11.

18. (canceled)

19. The method of claim 15, wherein the wherein the identification of rMVA virus carrying the MVA virus vector is accomplished by one or more gene-in selection methods, one or more gene-out selection methods, or a combination of gene-in and gene-out selection methods.

20. The method of claim 15, wherein the serial passage is at least 10 passages.

21. A CMV vaccine comprising an immunologically effective amount of an rMVA virus which is genetically stable after serial passage and produced by: a) constructing a transfer plasmid vector comprising a modified H5 (mH5) promoter operably linked to a DNA sequence encoding an IEfusion CMV protein antigen, wherein the expression of said DNA sequence is under the control of the mH5 promoter;b) generating rMVA virus by transfecting one or more plasmid vectors obtained from step a) into cells infected with wild type MVA; andc) identifying rMVA virus expressing the IEfusion CMV protein antigens using one or more selection methods for serial passage;d) conducting serial passage;e) expanding an rMVA virus strain identified by step d); andf) purifying the rMVA virus strain from step e) to form the vaccine;wherein the expression and immunogenicity of said foreign protein antigens are stable after serial passage in the rMVA vaccine obtained from step f),wherein at least one of the foreign protein antigens is an IEfusion CMV protein antigen comprising a nucleotide sequence encoding an Immediate-Early Gene-1 (IE1) antigenic portion directly fused to a nucleotide sequence encoding an Immediate-Early Gene-1 (IE2) antigenic portion, wherein (i) the nucleotide sequence encoding the IE1 antigenic portion includes a nucleotide sequence encoding IE1 exon 4 (IE1/e4);(ii) the nucleotide sequence encoding the IE2 antigenic portion is a nucleotide sequence encoding IE2 exon 5 (IE2/e5); or(iii) both (i) and (ii), andwherein the at least one foreign protein antigens further comprise a CMV antigen selected from the group consisting of pp65, CMV pp150, glycoprotein B (gB) and antigenic fragments thereof, the UL128 complex or members thereof, wherein UL128 members comprise UL128, UL130, UL131a, gH, and gL.

22. (canceled)