Modified H5 promoter improves stability of insert genes while maintaining immunogenicity during extended passage of genetically engineered MVA vaccines

Abstract

We have engineered recombinant (r) Modified Vaccinia Ankara (MVA) to express multiple antigens under the control of either of two related vaccinia synthetic promoters (pSyn) with early and late transcriptional activity or the modified H5 (mH5) promoter which has predominant early activity. We sequentially passaged these constructs and analyzed their genetic stability by qPCR, and concluded that rMVA expressing multiple antigens using the mH5 promoter exhibit remarkable genetic stability and maintain potent immunogenicity after serial passage. In contrast, rMVA expressing antigens using engineered vaccinia synthetic E/L (pSyn I or II) promoters are genetically unstable. Progressive accumulation of antigen loss variants resulted in a viral preparation with lower immunogenicity after serial passage. Metabolic labeling, followed by cold chase revealed little difference in stability of proteins expressed from mH5 or pSyn promoter constructs. We conclude that maintenance of genetic stability which is achieved using mH5, though not with pSyn promoters, is linked to timing, not the magnitude of expression levels of foreign antigen, which is more closely associated with immunogenicity of the vaccine.

Fig. 1

Schematic map of pp65 and IE1/e4 gene expression cassette of pSyn-pp65-IE1/e4-MVA and WB detection of pp65 and IE1 e4 expression levels of pSyn-pp65-IE1/e4-MVA. (A) Schematic map of viral DNA genome of pSyn-pp65-IE1/e4-MVA generated via homologous recombination as described previously . (B) Western blot detection of pp65 and IE1/e4 expression level of pp65-IE1/e4-MVA infected CEF cells of serial passages 1–10. Top panel shows a membrane blotted with mAb 28–103 specific for pp65; middle panel shows a membrane blotted with p63-27 specific for IE1/e4; and the bottom panel shows a membrane blotted with mAb 19C2 that detects VV-BR5. (C) WB detection of pp65 expression of 18 pSyn-pp65-IE1/e4-MVA individual isolates. 18 individual pSyn-pp65-IE1/e4-MVA viruses were isolated from passage 10 by virus plaque purification and expanded on CEF cells to prepare cell lysates for WB. Each lane represented single individual isolate from passage 10. Samples #4, #6, #7 and #13 marked with a star were selected for viral genomic DNA extraction and Southern blot analysis as described in Section 2.

Fig. 2

Western blot detection of pp65 and IE1 e4 protein expression and Southern blot detection of pp65 and IE1 e4 gene insertion in pSyn-pp65-IE1/e4-MVA individual isolates. (A) Western blot detection of pp65 and IE1 e4 protein expression of selected individual isolates of pSyn-pp65-IE1 e4-MVA. The membrane shown in Panel A (i) was blotted with mAb 28–103 specific for pp65, the membrane shown in Panel A (ii) was blotted with p63-27 specific for IE1/e4 and the membrane shown in Panel A (iii) was blotted with mAb 19C2 specific for the VV-BR5 viral protein. (B) Southern blot detection of pp65 and IE1 e4 gene insertion of selected individual isolates of pSyn-pp65-IE1/e4-MVA. MVA viral genomic DNA was digested with restriction enzymes to excise 3.9 kb fragments of pp65-IE1 gene expression cassettes, separated by 1% agarose gel and transferred to nylon membrane filter. This filter was hybridized with the ³²P-radiolabled DNA probe specific for both pp65 and IE1 e4 gene and exposed to x-ray film. Notes: Lanes 1 and 2 (Panels A and B) are two individual isolates selected randomly from passage 1 of pSyn-pp65-IE1/e4-MVA. Lanes 3 and 4 (Panel A and B) are the two individually isolates of #4 and #6 marked with asterisk from Fig. 2 with no expression of pp65 and IE1 e4. Lanes 5 and 6 (Panels A and B) are the two individual isolates #7 and #13 marked with asterisk in Fig. 2 with elevated pp65 and IE1/e4 expression levels.

Fig. 3

Immunogenicity of pp65-IE1/e4-MVA passage 1 and passage 10 immunized HHD II mice (HLA A2.1). Splenocytes from HHD II mice immunized with pSyn-pp65-IE1/e4-MVA from passage 1 (P1) or passage 10 (p10) were subjected to IVS separately with either pp65 A2 or IE1 A2 peptides loaded blast cells for one week. After IVS, the splenocytes were incubated with mock A2, pp65A2 or IEA2 peptides overnight and harvested for ICC described in Section 2. Average levels of IFN-γ producing specific for the CMV pp65- or IE1-A2 epitope (x-axis) for all immunized mice is shown in Y-axis. IFN-γ producing CD8⁺ T-cells to mock during the ICS procedure were subtracted. Error bars represent the SEM for all immunized mice.

Fig. 4

Genetic stability of pSyn-pp65-IE1/e4-MVA serial passages and pSyn-pp65-IEfusion-MVA determined by qPCR: (A) pSyn-pp65-IE1/e4-MVA genomic DNA was extracted as described in Section 2. pSC11 plasmid containing CMV genes (pp65, IE1/e4 and IE2/e5) was used to prepare absolute standards. The qPCRs were performed using primers specific for pp65, IE1/e4 and TK gene. The copy numbers for pp65 gene, IE1 gene and MVA backbone copies were calculated using ABI software (SDS3.2) and the genetic stability of the mH5-pp65-IEfusion-MVA was determined by computing the ratio of the pp65 gene insert and the MVA backbone or the ratio of the IE1/e4 gene insert and the MVA backbone as indicated in Y-axis. The ratio at passage 1 is normalized to 1 and each consecutive passage was normalized based on passage 1. The qPCR for each DNA sample were performed for three times independently in duplicates and average ratio and error bar shown in the figure represented three independent determinants. (B) pSyn-pp65-IEfusion-MVA viral genomic DNA was extracted and qPCR was performed using pp65, IEfuson and TK specific primers as described in Section 2. The copy numbers for pp65 gene, IEfusion gene and MVA backbone were analyzed using ABI software (SDS3.2) and the genetic stability of the mH5-pp65-IEfusion-MVA was determined by computing the ratio of the pp65 gene insert and the MVA backbone or the ratio of the IEfusion gene insert and the MVA backbone. The ratios at passage 1 for pp65 and IE1/e4 gene were normalized to 1. The qPCR for each DNA sample were performed for three times independently in duplicates and average ratio and error bar shown in the figure represented three independent determinants.

Fig. 5

Schematic representation of mH5-pp65-IEfusion-MVA and qPCR to determine genetic stability of 10 serial passages of mH5-pp65-MVA and mH5-pp65-IEfusion-MVA. (A) Schematic representation of mH5-pp65-IEfusion-MVA. (B) qPCR to determine genetic stability of 10 serial passages of mH5-pp65-MVA. mH5-pp65-MVA viral genomic DNA was extracted and qPCR was performed using pp65, and TK specific primers as described in Section 2. The copy numbers for pp65 gene and MVA backbone were analyzed using ABI software (SDS3.2) and the genetic stability of the mH5-pp65-IEfusion-MVA was determined by computing the ratio of the pp65 gene insert and the MVA backbone. The ratios at passage 1 were normalized to 1. The qPCR for each DNA sample were performed for three times independently in duplicates and average ratio and error bar shown in the figure represented three independent determinants. (C) qPCR to determine genetic stability of 10 serial passages of mH5-pp65-IEfusion-MVA. mH5-pp65-IEfusion-MVA genomic DNA was extracted and qPCR was performed using pp65, IEfusion and TK specific primers as described in Section 2. The copy numbers for pp65 gene, IEfusion gene and MVA backbone were analyzed using ABI software (SDS3.2) and the genetic stability of the mH5-pp65-IEfusion-MVA was determined by computing the ratio of the pp65 gene insert and the MVA backbone or the ratio of the IEfusion gene insert and the MVA backbone. The ratios at passage 1 for pp65 and IE1/e4 gene were normalized to 1. (D) Similar to (C) except 10 serial passages were conducted on CEF and shown are results computed using pp65 and TK-specific primers. The qPCR for each DNA sample were performed for three times independently in duplicates and the ratios and error bar shown in the figure presented average of three independent determinants.

Fig. 6

Immunogenicity of mH5-pp65-IEfusion-MVA of passage 1 and 7 using human PBMC and in HHD II mice (HLA A2.1). (A) Immunogenicity of mH5-pp65-IEfusion-MVA of passages 1 and 7 using human PBMC. PBMCs from healthy donors who were ex vivo positive responders to CMV antigens were incubated with antigen presenting cells infected with either passage 1 or passage 7 of mH5-pp65-IEfusion-MVA for 7 days followed by overnight incubation with diluent (mock), pp65, IE1 or IE2 peptide libraries in the presence of brefeldin A. Cells were then harvested and stained with anti-human CD8 or CD4 mAb, permeabilized and stained with anti-human IFN-γ mAb and evaluated by flow cytometry. Average percentages of IFN-γ producing CD8 or CD4 lymphocytes are shown (N = 4). Error bars represent standard deviation. (B) Immunogenicity of mH5-pp65-mH5-IEfusion-MVA of passages 1 and 7 in HHD II mice (HLA A2.1). Splenocytes from HHD II mice immunized with mH5-pp65-IEfusion-MVA from passage 1 (P1) or passage 7 (P7) were subjected to IVS separately with either pp65A2, IE1A2 peptides or IE2 peptide library loaded HLA-A*0201 EBV-lymphoblastoid cells (LCL) derived from a healthy CMV positive volunteer for 8 days. After IVS, the splenocytes were incubated with mock A2, pp65A2, IE1A2 peptides or IE2 peptide library overnight and harvested for ICC described in Section 2. Average levels of CD8+ T-cell IFN-γ production specific for the CMV pp65A2, IE1A2 epitopes or IE2 peptide library shown (x-axis) for all immunized mice. IFN-γ production to mock stimulated cells during the ICS procedure was subtracted. Error bars represent the SEM for all immunized mice.

Fig. 7

Metabolic radio-labeling of CMV-pp65 detected by immunoprecipitation after viral infection of CEF. mH5-pp65-MVA (lanes 2–5) and pSyn-pp65-MVA (lanes 6–9) viruses were used to infect primary CEF plated on 60 mm TC dishes at an MOI of 10 for 1 h, followed by depletion of intracellular stores of Met + Cys for 1 h, and labeled with ³⁵S [Met + Cys] for an additional 30 min. Excess unlabeled Met + Cys was diluted into fresh medium, and further incubation times are indicated in hours (O, 1, 4 and 10) above the gel profile. At the conclusion of the “chase” period, cell lysates were made and immunoprecipitation was conducted as described in Section 2. The CMV-pp65 antigen detected by the mAb 28–103 is indicated by an arrow to the right and adjacent to the gel profile. The 1st lane at the far left (Con) represents a control CEF culture that was radio-labeled after infection with a Gus-MVA virus which expresses the α-glucoronidase bacterial marker without CMV-pp65 .