Genetically stable recombinant modified vaccinia Ankara (RMVA) vaccines and methods of preparation thereof

A method for producing a genetically stable rMVA immunogenic composition, 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;
• and
• 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 virus strain from step e) to form the immunogenic composition;
• wherein the expression and immunogenicity of said foreign protein antigens are stable after serial passage in the rMVA immunogenic composition obtained from step f),
• wherein at least one of the foreign protein antigens is an IEfusion CMV protein antigen comprising a nucleotide sequence comprising SEQ ID NO:
• 11.

An immunogenic composition 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;
• and
• c) 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);
• and
• f) purifying the rMVA virus strain from step e) to form the immunogenic composition;
• wherein the expression and immunogenicity of said foreign protein antigens are stable after serial passage in the rMVA immunogenic composition 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 comprising SEQ ID NO:
• 11.