Genetically stable recombinant modified vaccinia Ankara (RMVA) vaccines and methods of preparation thereof
Inventors
Diamond, Don J. • Wang, Zhongde
Assignees
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Publication Number
US-12337032-B2
Publication Date
2025-06-24
Expiration Date
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.
Core Innovation
The invention provides genetically stable recombinant modified vaccinia Ankara (rMVA) vaccines. The rMVA virus comprises a fusion nucleotide sequence that encodes an IEfusion CMV protein antigen, where the fusion nucleotide sequence includes SEQ ID NO: 11. The fusion nucleotide sequence is engineered for expression under a modified H5 (mH5) promoter to support genetic stability and to maintain antigen expression and immunogenicity after serial passage.
The document contrasts genetic instability observed with pSyn-driven constructs, where serial passage can lead to loss of insert cassettes and declining expression, resulting in reduced immunogenicity after serial passage. By replacing promoter control with the modified H5 promoter (mH5), the described rMVA strains maintain improved genetic stability while preserving immunogenicity over serial passage.
The CMV antigen design is centered on an IEfusion construct comprising Immediate-Early Gene-1 (IE1) exon4 fused to Immediate-Early Gene-2 (IE2) exon5 (SEQ ID NO: 11). The IEfusion CMV protein antigen may be optionally co-expressed with CMV pp65. Genetic stability is assessed using qPCR-based insert-to-backbone ratios, and immunogenicity is evaluated using T-cell IFN-γ responses in HLA-transgenic mice and human PBMC stimulation/assays including HCT recipient assays.
Claims Coverage
The independent claims cover three inventive features: an rMVA immunogenic composition with an IEfusion CMV antigen sequence defined by SEQ ID NO: 11; a method for producing a genetically stable rMVA immunogenic composition using an mH5 promoter-driven foreign antigen expression cassette followed by selection and serial passage; and an immunogenic composition produced by the same workflow, including an IE1 antigenic portion directly fused to SEQ ID NO: 11.
IEfusion CMV protein antigen fusion nucleotide sequence including SEQ ID NO: 11
An immunogenic composition comprising an immunologically effective amount of a recombinant modified vaccinia Ankara (rMVA) virus wherein the rMVA virus comprises a fusion nucleotide sequence encoding an IEfusion CMV protein antigen, the fusion nucleotide sequence comprising a nucleotide sequence comprising SEQ ID NO: 11.
mH5 promoter operably linked to a foreign protein antigen and serial-passage stability selection workflow
A method for producing a genetically stable rMVA immunogenic composition comprising constructing a transfer plasmid vector with a modified H5 (mH5) promoter operably linked to a DNA sequence encoding a heterologous foreign protein antigen under control of the mH5 promoter; generating rMVA virus by transfecting the plasmid vectors into cells infected with wild type MVA; identifying rMVA virus expressing the heterologous foreign protein antigens using one or more selection methods for serial passage; conducting serial passage; expanding an rMVA virus strain identified by serial passage; and purifying the rMVA virus strain to form the immunogenic composition, wherein expression and immunogenicity of the foreign protein antigens are stable after serial passage, and wherein at least one foreign protein antigen is an IEfusion CMV protein antigen comprising a nucleotide sequence comprising SEQ ID NO: 11.
Genetically stable rMVA immunogenic composition including IE1 antigenic portion directly fused to SEQ ID NO: 11
An immunogenic composition comprising an immunologically effective amount of an rMVA virus genetically stable after serial passage and produced by constructing a transfer plasmid vector with an mH5 promoter operably linked to a DNA sequence encoding an IEfusion CMV protein antigen under control of the mH5 promoter; generating rMVA by transfecting one or more plasmid vectors into cells infected with wild type MVA; identifying rMVA virus expressing the IEfusion CMV protein antigens using one or more selection methods for serial passage; conducting serial passage; expanding an rMVA virus strain identified after serial passage; and purifying the rMVA virus strain to form the immunogenic composition, wherein expression and immunogenicity of the foreign protein antigens are stable after serial passage, and wherein at least one foreign protein antigen is an IEfusion CMV protein antigen comprising an IE1 antigenic portion directly fused to a nucleotide sequence comprising SEQ ID NO: 11.
Across the independent claims, the central inventive coverage is directed to an immunogenic composition with an rMVA virus expressing an IEfusion CMV antigen encoded by a fusion sequence comprising SEQ ID NO: 11, and to a production method using an mH5 promoter operably linked to a foreign antigen expression cassette, followed by selection and serial passage to yield rMVA with expression and immunogenicity stable after serial passage, including embodiments where the IEfusion antigen includes an IE1 antigenic portion directly fused to SEQ ID NO: 11.
Stated Advantages
Improved genetic stability of the rMVA strains compared with pSyn-driven constructs after serial passage.
Maintained antigen expression and immunogenicity after serial passage.
Genetically stable rMVA strains are suitable for forming a genetically stable virus seed for large-scale GMP manufacturing.
Documented Applications
An immunogenic composition that modulates an immune response in a mammalian subject by administering the composition.
Use of immunogenicity testing involving HLA-transgenic mice to measure T-cell IFN-γ responses.
Use of immunogenicity testing involving human PBMC stimulation and assays using HCT recipients.
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