A Novel Monoclonal Antibody Against a Modified Vaccinia Ankara (MVA) Envelope Protein as a Tool for MVA Virus Titration by Flow Cytometry

Abstract

Modified vaccinia Ankara (MVA) virus is a widely used vaccine platform, making accurate titration essential for vaccination studies. However, the current plaque forming unit (PFU) assay, the standard for MVA titration, is prone to observer bias and other limitations that affect accuracy and precision. To address these challenges, we developed a new flow cytometry-based quantification method using a highly specific monoclonal antibody (mAb) for the detection of MVA-infected cells, as a more accurate titration assay. Through previous work, we serendipitously identified three MVA-specific hybridoma antibody clones, which we characterized through ELISA, immunoblot, and flow cytometry, confirming their specificity for MVA. Sequencing confirmed that each antibody was monoclonal, and mass spectrometry results revealed that all mAbs target the MVA cell surface binding protein (CSBP, MVA105L). We next optimized the titration protocol using the most effective mAb, 33C7 by refining culture conditions and staining protocols to enhance sensitivity and minimize background. Our optimized method demonstrated superior sensitivity, reliability, and reduced processing time when compared with the traditional PFU assay, establishing it as a more accurate and efficient approach for MVA titration.

Keywords: D8L; MVA105L; OPG105; TCID50; cell surface binding protein; flow cytometry; modified vaccinia Ankara; monoclonal antibody; plaque-forming unit assay; virus titration.

Figure 1

Generation and characterization of antibodies against modified vaccinia Ankara (MVA) virus. (A) Schematic diagram of the BALB/c mouse (n = 1) immunization schedule used to generate MVA/human herpesvirus protein (HHVp)-specific murine antibodies. Mouse was bled or immunized with either MVA expressing His-tagged MVA-HHVp (V) or purified His-tagged HHVp produced from cells infected with this virus (P) on the indicated days. Harvested spleen at the end of the study period was used to generate hybridomas via the myeloma cell fusion method. Created in BioRender. Cua, S. (2023) BioRender.com/o30l999 (B) Confirmatory ELISA screen of hybridoma supernatants to detect anti-HHVp antibodies. Culture supernatants from hybridomas generated in (A) were subjected to an initial HHVp-specific antibody ELISA screen. After expansion of the resulting ELISA-positive hybridoma clones, culture supernatants were further screened by a confirmatory ELISA (shown), using soluble semi-purified His-tagged HHVp produced in an MVA system as the target antigen. Each bar represents the mean OD_405nm of three replicates + the standard deviation; samples with a mean OD_405nm > 0.1 (dotted line) were considered positive. (C) Immunoblot assessment of hybridoma supernatants to detect anti-HHVp antibodies. Culture supernatants from hybridoma clones that were ELISA-positive in (B) were used as primary antibodies in immunoblot assay against lysates of MVA-eGFP-infected BHK-21 cells (MVA-eGFP) or semi-purified His-tagged HHVp (s-p HHVp) produced in an MVA system. Shown are the blots of hybridoma clones (3/9) that detected a protein band of the same size in both MVA-eGFP and s-p HHVp samples (indicated with red arrow), suspected to be an MVA-specific protein. Remaining blots (6/9), which did not detect this protein band, are shown in Figure A1B. (D) SDS-PAGE analysis of purified hybridoma supernatant antibodies. Antibodies from hybridoma supernatants shown in (C) were purified via protein A (9E8 and 33C7) or G (38D11) affinity chromatography and subjected to SDS-PAGE followed by Coomassie blue staining to assess antibody purity. Heavy and light chains are indicated with red arrows. See also Figure A1.

Figure A1

Modified vaccinia Ankara (MVA) infection of BHK-21 cells for lysate generation and human herpesvirus 4 protein (HHVp) production, and HHVp-specific antibody immunoblot assessment. (A) Microscopy analysis of BHK-21 cells infected with MVA-eGFP or MVA expressing His-tagged HHVp. BHK-21 cells were infected with MVA-eGFP or MVA expressing His-tagged HHVp (MVA-HHVp). Shown are representative phase (transmitted light) and GFP (green fluorescence) channel micrographs for each virus 16 h after infection; uninfected cells were used as a mock control. For generation of reagents for antibody generation and characterization, infected cells were incubated until achieving 90–100% infection based on eGFP expression as assessed by microscopy, and were harvested and processed for either lysing (MVA-eGFP-infected cells) or for protein purification (MVA-HHVp-infected cells). (B) Immunoblot assessment of hybridoma supernatants to detect anti-HHVp antibodies. As described in Figure 1C, culture supernatants from hybridoma clones that were ELISA-positive in Figure 1B were used as primary antibodies in immunoblot assay against lysates of MVA-eGFP-infected BHK-21 cells or semi-purified His-tagged HHVp (s-p HHVp). Shown are the blots of hybridoma clones (6/9) that did not detect a suspected MVA-specific protein band. Remaining blots (3/9), positive for suspected MVA protein, are shown in Figure 1C.

Figure A2

Modified vaccinia Ankara (MVA) infection of BHK-21 and CEF cells for lysate and flow cytometry sample generation. BHK-21 or CEF cells were infected with eGFP-expressing wildtype MVA (MVA-eGFP). Shown are representative phase (transmitted light) and GFP (green fluorescence) channel micrographs for each cell type 22 h after infection. For generation of reagents for antibody characterization, infected cells were incubated until achieving 90–100% infection based on eGFP expression as assessed by microscopy, and were harvested and processed for either lysing or for flow cytometry.

Figure A3

Characterization of recombinant 9E8, 33C7, and 38D11 antibodies. (A) SDS-PAGE analysis of purified recombinant 9E8, 33C7, and 38D11 antibodies. After 9E8, 33C7, and 38D11 hybridoma sequencing (Table 1), the CDR1, CDR2, and CDR3 sequences of each antibody were cloned into antibody expression plasmids, which were used to produce recombinant 9E8, 33C7, and 38D11 (r9E8, r33C7, and r38D11) in ExpiCHO-S cells. Recombinant antibodies were purified from the supernatants of transfected ExpiCHO-S cells via protein A (r9E8 and r33C7) or G (r38D11) affinity chromatography and subjected to SDS-PAGE followed by Coomassie Brilliant Blue staining to assess antibody purity. Heavy and light chains are indicated with red arrows. (B) Immunoblot assessment of purified r9E8, r33C7, and r38D11 antibodies against MVA-infected cells. Purified r9E8, r33C7, and r38D11 were used as primary antibodies in immunoblot assay against lysates of MVA-eGFP-infected BHK-21 or CEF cells. Uninfected and BHK-21 and CEF cell lysates were used as negative controls. As a loading control, all samples were additionally stained with β-actin primary antibody. Expected protein sizes are indicated with red arrows.

Figure A4

Titration of nocodazole as a viral morphogenesis inhibitor in MVA-eGFP-infected BHK-21 cells. (A) Flow cytometry assessment of MVA-eGFP-infected BHK-21 cells cultured in varying concentrations of nocodazole. BHK-21 cells were infected with MVA-eGFP and subsequently cultured with varying concentrations of nocodazole to inhibit viral morphogenesis and secondary infection, followed by harvesting and processing for eGFP-based flow cytometry assay. Shown is the resulting infection curve, with each dot representing the mean percentage (%) ± standard deviation of cells from triplicate infections that were positive for eGFP signal at each corresponding nocodazole culture condition. A range of 0.2–0.5 µg/mL nocodazole concentration was chosen as the optimal culture condition for modified vaccinia Ankara-infected cells for subsequent flow cytometry viral titrations. (B) Microscopy analysis of MVA-eGFP-infected BHK-21 cells cultured in varying concentrations of nocodazole. Shown are representative phase (transmitted light) and GFP (green fluorescence) channel micrographs of cells from (A) 18–20 h after infection for 0, 0.2, and 2 µg/mL nocodazole culture conditions, before harvesting; uninfected cells were used as a mock control.

Figure A5

Gating strategy for 33C7-based flow cytometry modified vaccinia Ankara (MVA) titrations. Shown is the gating strategy used for 33C7-based flow cytometry for viral titration of MVA in MVA-eGFP-infected BHK-21 cells, using uninfected cells as a negative control. On the top row, representative flow cytometry plots are shown illustrating the gating of BHK-21 cells, followed by gating of single cells and live cells via a viability dye. On the middle and bottom rows, shown are the subsequent gating of infected cells based on the detected eGFP (green arrow) and secondary antibody AF-555 (red arrow) signals, respectively. For eGFP and AF-555 gates, the percentage (%) of cells that were positive for each corresponding signal is shown within each gate.

Figure A6

Schematic diagrams of plaque-forming unit (PFU) and median tissue culture infectious dose (TCID₅₀) assays for modified vaccinia Ankara (MVA) viral titration. (A) Schematic diagrams of MVA titration PFU and TCID₅₀ assay experimental setups. On Day-1, cells are seeded in either 6-well plates (×4) or a 96-well plate (×1) for PFU and TCID₅₀ assays, respectively. On Day 0, the viral stock to be tested is serially diluted 10-fold from 10¹ to 10¹¹ and then added to the seeded cells in duplicate for PFU assay, or to each column (8 wells) of the 96-well plate for TCID₅₀ assay, leaving one set of duplicates and one column uninfected, respectively, as controls. Cells are incubated for 1 h under standard cell culture conditions, then the virus is removed, and the cells are further incubated for an additional 24 h. On Day 1, the cells are fixed, and staining and data acquisition can proceed immediately or at a later time. Created in BioRender. Reidel, I. (2024) BioRender.com/h70v781 (B) Schematic diagram of MVA titration PFU assay data acquisition and calculation. To calculate viral titer via PFU assay, the stained plates are analyzed by microscopy. Viral plaques, which appear as dark cell clumps (brown if using DAB substrate), are counted in duplicate wells at the dilution that yields 10-100 plaques. These counts are then used in the shown formula to calculate the viral stock IU/mL. A calculation example is provided for a theoretical scenario in which 1 mL of 10⁷-diluted virus yielded 35 and 42 viral plaques in each duplicate well, respectively. Created in BioRender. Reidel, I. (2024) BioRender.com/v20y722 (C) Schematic diagram of MVA titration TCID₅₀ assay data acquisition and calculation. To calculate viral titer via TCID₅₀ assay, the fixed and stained plate is analyzed by microscopy. The last dilution that resulted in all 8 wells displaying viral plaques (i.e., 100% infectivity) and the number of wells that displayed viral plaques in all subsequent dilutions, are recorded, and then used in the shown formula to calculate the viral stock infectious units per mL (IU/mL). A calculation example is provided for a theoretical scenario in which using an infection volume of 0.1 mL, the last dilution with 100% infectivity in 8 wells was 10⁶, and the number of infected wells for the subsequent dilutions (10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹) were 4, 1, 0, 0, and 0, respectively. Created in BioRender. Reidel, I. (2024) BioRender.com/j18g391.

Figure A7

Comparison of plaque-forming unit (PFU) modified vaccinia Ankara (MVA) titration outcomes after 24 h versus 48 h virus incubations. Commercial polyclonal anti-MVA antibody was used as primary antibody for PFU assay immunostaining to titrate MVA in MVA-eGFP-infected BHK-21 cells, after either a 24 h or 48 h incubation following infection. On the top, shown are representative GFP (eGFP signal) and phase (transmitted light, antibodies) channel micrographs for selected virus dilutions under each condition after immunostaining; uninfected cells were used as a mock control. On the bottom, a table is shown with the resulting virus titers (infectious units per mL, IU/mL) as calculated under each condition.

Figure 2

Characterization of 9E8, 33C7, and 38D11 purified antibodies against modified vaccinia Ankara (MVA) virus. (A) Immunoblot assessment of purified 9E8, 33C7, and 38D11 antibodies against MVA-infected cells. Purified hybridoma supernatant antibodies 9E8, 33C7, and 38D11 were used as primary antibodies in immunoblot assay against lysates of MVA-eGFP-infected BHK-21 or CEF cells. Uninfected BHK-21 and CEF cell lysates were used as negative controls. As a loading control, all samples were additionally stained with β-actin primary antibody. Expected protein sizes are indicated with red arrows. (B) Flow cytometry assessment of purified 9E8, 33C7, and 38D11 antibodies against MVA-infected cells. Purified hybridoma supernatant antibodies 9E8, 33C7, and 38D11 were used as primary antibodies in flow cytometry assay against BHK-21 or CEF cells infected with MVA-eGFP, using Alexa Fluor 647-conjugated anti-mouse IgG secondary antibody. Uninfected BHK-21 and CEF cells were used as negative controls. For each antibody and condition (infected versus uninfected), shown is the mean percentage (%) + standard deviation of cells from quadruplicate measurements that were positive for the antibody signal but negative for eGFP (Ab+ GFP−), negative for the antibody signal but positive for eGFP (Ab− GFP+), or positive for both the antibody signal and eGFP (Ab+ eGFP+). See also Figure A2.

Figure 3

Identification of 9E8, 33C7, and 38D11 antibody targets. (A) Immunoblot assessment of purified antibodies 9E8, 33C7, and 38D11 against HEK-293T cells expressing modified vaccinia Ankara (MVA) proteins. Purified hybridoma supernatant antibodies 9E8, 33C7, and 38D11 were used as primary antibodies in immunoblot assay against lysates of HEK-293T cells transfected with expression plasmids coding for His-tagged cell surface-binding protein (CSBP-His) and His-tagged IMV heparin binding surface protein (IMV HBP-His), MVA proteins identified as possible antibody targets in Table 2. Un-transfected cells were used as negative controls. As a positive antibody control, samples were also processed with anti-His primary antibody. As a loading control, all samples were additionally stained with β-actin primary antibody. Expected protein sizes are indicated with red arrows. (B) Flow cytometry assessment of purified antibodies 9E8, 33C7, and 38D11 against HEK-293T cells expressing MVA proteins. Purified hybridoma supernatant antibodies 9E8, 33C7, and 38D11 were used as primary antibodies in flow cytometry assay against HEK-293T cells transfected with CSBP-His and IMV HBP-His expression plasmids. Un-transfected cells were used as a negative control. To control for secondary antibody background, all samples were also processed in the absence of primary antibody (2° Ab only). For each antibody and condition, shown is the mean percentage (%) + standard deviation of cells from quadruplicate measurements that were positive for the antibody signal.

Figure 4

Optimization of experimental conditions for monoclonal antibody (mAb)-based flow cytometry modified vaccinia ankara (MVA) detection in MVA-eGFP-infected cells. (A) Comparison of 33C7-based flow cytometry assessment in non-permeabilized versus permeabilized cells infected with MVA-eGFP in the absence or presence of nocodazole. Purified 33C7 was used as primary antibody in flow cytometry assay against BHK-21 cells that were infected with MVA-eGFP under different conditions. Infected cells were either cultured in the absence or presence of nocodazole to inhibit viral morphogenesis. Following harvest, cells were processed for 33C7 staining, either without permeabilization (extracellular staining), or following permeabilization (extra- and intracellular staining). Uninfected cells were used as a negative control. To control for secondary antibody background, all samples were also processed in the absence of primary antibody (2° Ab only). Shown are representative flow cytometry plots of triplicate infections for each condition, with the Y-axis representing the eGFP signal and the X-axis representing the secondary antibody AF-555 signal. For each plot, the percentage of cells that were either positive or negative for each fluorescent signal are shown in each corresponding quadrant. (B) Comparison of 9E8, 33C7, and 38D11 as primary antibodies in flow cytometry-based detection of MVA in MVA-eGFP-infected cells. Purified hybridoma supernatant antibodies 9E8, 33C7, and 38D11 were used in flow cytometry assay as primary antibodies against permeabilized BHK-21 cells that were infected with MVA-eGFP in the presence of nocodazole. Uninfected cells were used as negative controls. For each antibody and condition (infected versus uninfected), shown is the mean percentage (%) + standard deviation of cells from triplicate infections that were positive for the antibody signal but negative for eGFP (mAb+ GFP−), negative for the antibody signal but positive for eGFP (mAb− GFP+), or positive for both the antibody signal and eGFP (mAb+ eGFP+). (C) Titration of 33C7 as primary antibody for flow cytometry detection of MVA in MVA-eGFP-infected cells. MVA-eGFP-infected BHK-21 cells cultured in nocodazole were permeabilized and processed for flow cytometry with varying concentrations of primary antibody 33C7 to identify an optimal 33C7 concentration. Unstained cells were used as a negative control. Shown are two resulting antibody titration curves, one corresponding to the population of cells that were mAb+ GFP+ and the other corresponding to the cells that mAb+ GFP−, with each dot representing the mean percentage (%) ± standard deviation of cells from triplicate infections that were positive for the antibody signal at each corresponding 33C7 concentration. The green line depicts the mean % eGFP+ cells in the unstained control set of cells. 3 µg/mL was chosen as the optimal 33C7 primary antibody concentration for staining. See also Figure A4.

Figure 5

Validation of 33C7 binding to cells exposed to live versus inactivated modified vaccinia Ankara (MVA). (A) Microscopy analysis of BHK-21 cells infected with live or inactivated MVA-eGFP. BHK-21 cells were incubated with either MVA-eGFP (live) or UV-treated MVA-eGFP (UV MVA-eGFP; inactivated). Shown are representative phase (transmitted light) and GFP (green fluorescence) channel micrographs of cells 18 h after infection. (B) Confirmation of 33C7 binding specificity to live-MVA-infected cells via flow cytometry. Purified 33C7 was used in flow cytometry assay as a primary antibody against permeabilized BHK-21 cells that were incubated with either MVA-eGFP or UV MVA-eGFP in the presence of nocodazole. For each condition (MVA−eGFP− versus UV MVA-eGFP-infected), shown is the mean percentage (%) + standard deviation of cells from triplicate infections that were positive for the antibody signal but negative for eGFP (mAb+ GFP−), negative for the antibody signal but positive for eGFP (mAb− GFP+), or positive for both the antibody signal and eGFP (mAb+ eGFP+).

Figure 6

Validation of 33C7 and r33C7 as primary antibodies in flow cytometry modified vaccinia Ankara (MVA) titrations. (A) Comparison of eGFP- versus 33C7-based flow cytometry MVA-eGFP titer quantification in non-permeabilized versus permeabilized MVA-eGFP-infected cells. Purified 33C7 was used as primary antibody in flow cytometry assay against BHK-21 cells that were infected with varying dilutions of MVA-eGFP and cultured in the presence of nocodazole, to calculate viral titer. Following harvest, cells were processed for 33C7 staining, either without permeabilization (extracellular staining), or following permeabilization (extra- and intracellular staining). On the top row, shown are infection curves for each condition (extracellular versus extra- and intracellular staining), with each dot representing the mean percentage (%) ± standard deviation of cells from triplicate infections that were positive for eGFP or secondary antibody AF-555 signals at each corresponding virus dilution. On the bottom row, shown is a table comparing the infectious units per volume (IU/mL) calculated from each infection curve using eGFP versus AF-555 signals for each condition; each value represents the calculated mean infectious units per volume (IU/mL) ± standard deviation for each condition and signal, and the p-value after one-tailed Mann-Whitney test comparison of IU/mL for each signal within each condition is shown; * = significant, NS = non-significant. Permeabilization was chosen as the optimal staining condition. (B) Comparison of eGFP- versus 33C7- and r33C7-based flow cytometry MVA titer quantification in MVA-eGFP-infected cells under optimized experimental conditions. Purified 33C7 and r33C7 were used as primary antibody in flow cytometry assay against permeabilized MVA-eGFP-infected BHK-21 cells as in (A) to calculate infectious titer. On the top, shown are the resulting infection curves for each condition, where each dot represents the mean % ± standard deviation of cells from quadruplicate infections that were positive for the fluorescent signal at each corresponding virus dilution. On the bottom, a table is shown with the resulting virus titers (infectious units per mL, IU/mL) as calculated under each condition. See also Figure A5.

Figure 7

Validation of 33C7 and r33C7 as primary antibodies in plaque-forming unit (PFU) modified vaccinia Ankara (MVA) titrations. Purified 33C7 and r33C7 were used as primary antibody for PFU assay immunostaining to titrate MVA in MVA-eGFP-infected BHK-21 cells as described in Figure A6. As positive control, commercial rabbit polyclonal anti-MVA antibody was used as primary antibody in an additional set of infected cells. As an additional control, eGFP signal was also used to identify viral plaques for IU/mL calculations. On the top, shown are representative GFP (eGFP signal) and phase (transmitted light, antibodies) channel micrographs for select virus dilutions under each condition after immunostaining; uninfected cells were used as a mock control. On the bottom, a table is shown with the resulting virus titers (infectious units per mL, IU/mL) as calculated under each condition. See also Figure A6.