Figure 1.
Schematic representation of the novel Pr4LS5E and PrS5E synthetic hybrid promoters.
The Pr4LS5E promoter consists of four late elements (dark grey boxes) which include from 5’ to 3’: the ATI late promoter (ATI), the short synthetic late promoter (PrSSL) and the MVA123L and MVA124L late promoters (PrMVA123L and PrMVA124L). The Pr4LS5E contains downstream of these four late elements one PrS promoter sequence (white box) followed by five modified early promoter elements derived from the p7.5k promoter (7.5e; light-grey boxes). The PrS5E promoter consists only of the PrS promoter sequence followed by the five 7.5e elements. The promoters were used to drive the expression of the ovalbumin (OVA) gene. The position of the promoter sequences relative to the start (ATG) of the OVA open reading frame (ORF) is shown. Arrows indicate the direction of transcription. The OVA expression cassettes were introduced into the MVA genome at the intergenic region (IGR) between the MVA genes MVA044L and MVA045L (IGR 44/45) shown as black arrows in the direction of gene transcription.
Figure 2.
Expression kinetics of novel synthetic hybrid promoters.
The kinetics and strength of the indicated promoters driving the expression of ovalbumin (OVA) were analysed in HeLa cells at the mRNA level (A) and at the protein level (B). (A) Samples were collected at 0.5, 1, 2, 4 and 8 hours post infection (p.i.). Total RNA was reverse transcribed and OVA cDNA was detected by qPCR. Fold OVA expression levels were calculated relative to wild-type MVA infected samples after normalization to actin levels using the delta-delta CT method. The average of three independent qPCR runs done in duplicates is shown. Error bars represent ± standard error of the mean (SEM) values. *, indicates the promoter was significantly different (p < 0.007) when compared to all other constructs (B) Infected cells were harvested at 2, 4, 6 and 8 hours p.i., stained intra-cellularly for OVA protein and analysed by flow cytometry. Samples staining positive for OVA (OVA+) were gated based on empty vector (MVA-EGFP) infected samples and the median fluorescence intensity (MFI) of OVA+ cells was calculated. For each virus group, three wells were infected using independent virus dilutions. The average MFIs above the background obtained from empty vector infected samples (MFI = 250) are shown. Error bars represent ± SEM values. Statistical differences between the indicated groups are shown by asterisks. * indicates p < 0.05; ** indicates p < 0.007.
Figure 3.
(A) The MVA sequence 250 nucleotides (nt) upstream of the MVA13.5L open reading frame (ORF) with a ‘left’ orientation is shown (GenBank accession number AY603355). The ATG encoding for the methionine (Met) within the MVA13.5L ORF is shown in bold. The arrow indicates the direction of transcription for the MVA13.5L gene. The two transcriptional start sites (TSS) for the MVA13.5L gene are indicated by black arrow heads. TSS1 correspond to nt 9 978 and TSS2 corresponds to nt 10 058 in the MVA genome. The two 44 nt repeats, denoted R1 and R2, are shown as grey sequences. The 36 nt spacer region between R1 and R2, denoted SP, is shown as sequences in italic. The sequences used to construct the PrMVA13.5-long promoter (9 965–10 088 nt) are underlined with a solid line. Sequences used to construct the PrMVA13.5-short promoter (9 965-10 019 nt) are underlined with a dashed line. (B) The features of the native PrMVA13.5-long and PrMVA13.5-short promoter are depicted graphically. The PrMVA13.5-long consists of the two repeated motifs (R1 and R2) shown as grey boxes separated by the spacer region (SP). The PrMVA13.5-short consists of only the R2 motif and 11 nt of the spacer region (SP). The TSS1 is shown as a black arrowhead. The promoters were used to generate an OVA expression cassette introduced into the MVA genome at IGR 44/45. The position of the promoter sequences relative to the start (ATG) of the OVA open reading frame (ORF) is shown. Arrows indicate the direction of transcription.
Figure 4.
Expression kinetics of the novel MVA13.5L promoter constructs.
The kinetics and strength of the PrMVA13.5 promoter constructs were compared to the two novel synthetic hybrid promoters (PrS5E and Pr4LS5E). (A) Cells were collected at 0.5, 1, 2, 4 and 8 hours post infection (p.i.). Total RNA was reverse transcribed and OVA cDNA was detected by qPCR. The fold OVA mRNA levels relative to wildtype MVA were determined using the delta-delta CT method. The average of two independent qPCR runs is shown. Error bars represent ± standard error of the mean (SEM) values. *, indicates the promoter was significantly different when compared to all other promoters (p < 0.0002). (B) Infected cells were harvested at 2, 4, 6 and 8 hours p.i., stained intra-cellularly for OVA protein and analysed by flow cytometry. OVA positive (OVA+) cells were gated and the median fluorescence intensity (MFI) of these cells calculated. For each virus group, three wells were infected using independent virus dilutions. The average MFIs of samples with values above the background obtained from empty vector infected samples (MFI = 150) are shown. Error bars represent ± SEM values. *, indicates the promoter was significantly different when compared to all other promoters (p < 0.02). (C) Infected MDBK cells were lysed at 2, 4, 6 and 8 hours p.i. All samples were standardized to a total protein concentration of 0.1µg/µL and then used for the determination of OVA protein by ELISA. Values shown for each group are the average of three samples infected with independent virus dilutions, processed independently and assayed in duplicate. Values are reported as nanograms (ng) of OVA per µg of total lysate. Error bars represent ± SEM values. *, denotes a significant difference (p < 0.006) between the indicated promoters.
Figure 5.
OVA specific CD8 T cell responses.
Mice were immunized with different recombinant MVAs expressing ovalbumin (OVA) under the control of the indicated promoters. CD8 T cells from blood were analysed for OVA- and vector- specific responses one week after each immunization by staining for CD8 and CD44 markers along with MHC class I dextramers complexed with the TSYKFESV peptide from the B8R protein or with the SIINFEKL peptide from the OVA protein. CD8 T cells were gated from the lymphocyte population and the frequencies of OVA (gray bars) and B8R (white bars) specific CD8 T cells were obtained as a percentage from the total CD8 T cell population. The frequency of both OVA and B8R specific CD8 T cells were obtained for each mouse and the average for each group calculated. The average frequency of OVA and B8R specific CD8 T cells for each group (+/- SEM) is shown after the first (A), second (B) and third (C) immunizations. *, indicates values with significant difference (p < 0.02) when compared to the PrMVA13.5-short, PrS and PrH5m promoters. (D) The frequencies of OVA and B8R specific CD8 T cells were used to calculate OVA- to B8R- specific (OVA/B8R) ratios for individual mice after each immunization (immun). Values reported are the average for each group. Error bars represent +/- SEM. *, indicates values with significant difference (p < 0.05) when compared to the PrS; **, indicates values with significant difference when compared to the PrMVA13.5-short; ***, indicates values with significant differences when compared to the PrH5m.
Figure 6.
OVA and B8R specific CD8 T cells at the early memory phase.
(A) The frequency of OVA- and B8R-specific CD8 T cells were detected for the indicated groups from spleens 10 weeks after the last vaccination using dextramer staining and including memory phenotype markers (CD127 and CD62L). The average for each group is shown +/- SEM values. (B) The average OVA-to-B8R ratios (OVA/B8R) 10 weeks after the third immunization are shown for each group +/- SEM values. Each promoter construct group consisted of 4-5 mice. The empty vector and PBS control groups consisted of 3 mice each. *, indicates the promoter was significantly different when compared to the PrMVA13.5-long promoter (p < 0.05).
Figure 7.
OVA and vector specific antibody titers.
Antibody titers specific for MVA (A) or OVA (B) were determined three weeks after the first (white bars), second (gray bars) and third (black bars) immunization (immun). Individual endpoint titers were obtained for each mouse and the respective log10 transformed values were calculated. The values reported are the average log10 transformed titers for each group +/- SEM values. *, indicates values with significant differences (p > 0.05) compared to PrS, PrH5m, pHyb, and PrMVA13.5-long. **, indicates values with significant differences (p < 0.05) compared to PrS, PrH5m, pHyb, PrS5E and PrMVA13.5-long.