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monoclonal reafinity recombinant human antibodies targeting pdc surface antigens bdca2  (Miltenyi Biotec)


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    Miltenyi Biotec monoclonal reafinity recombinant human antibodies targeting pdc surface antigens bdca2
    The interaction of Vidu-AF647 with pDCs, as determined by flow cytometry, is dependent on anti-Qβ and is mediated through both CD32 and <t>BDCA2.</t> (A-C) PBMCs from healthy donors were cultured for 2 hours in medium (No Tx), Vidu-AF647 alone or Vidu-AF647 and anti-Qβ. (D-M) PBMCs from healthy donors were cultured for 2 hours or 20 hours with Vidu-AF647 and anti-Qβ with or without receptor blocking. IC or receptor-specific antibodies (anti-CD32 or anti-BDCA2) were added to PBMC cultures prior to Vidu-AF647 and anti-Qβ. Vidu-AF647 signal associated with CD45 + cells or BDCA4 + pDCs was determined by flow cytometry. (A) Gating on Vidu-AF647 + pDCs in PBMCs from one representative donor. (B) Frequency and (C) MdFI of Vidu-AF647 + pDCs (n=9 donors). (D) Frequency and (E) MdFI of Vidu-AF647 + CD45 + or pDCs after PBMC from healthy donors (n=6) were treated with IC (–) or anti-CD32 (+) followed by a 2-hour culture with anti-Qβ and Vidu-AF647. (F) Frequency and (G) MdFI of Vidu-AF647 + CD45 + cells or pDCs after PBMC from healthy donors (n=5) were treated with IC (–) or anti-CD32 (+) followed by a 20-hour culture with anti-Qβ and Vidu-AF647. (H) Frequency and (I) MdFI of Vidu-AF647 + CD45 + cells or pDCs after PBMC from healthy donors (n=6) were treated with IC (–) or anti-BDCA2 (+) followed by a 2-hour culture with anti-Qβ and Vidu-AF647. (J) Frequency and (K) MdFI of Vidu-AF647 + CD45 + cells or pDCs after PBMC from healthy donors (n=12) were treated with IC (–) or anti-BDCA2 (+) followed by a 2-hour culture with anti-Qβ and Vidu-AF647. (L) Contour plots from one representative donor showing Vidu-AF647 + pDCs and (M) frequency of Vidu-AF647 + CD45 + or pDCs (n= 3 donors) after IC, anti-CD32, anti-BDCA2 or anti-CD32/BDCA2 treatment of PBMCs followed by a 20-hour culture with Vidu-AF647 and anti-Qβ. Antibody pre-treatment was done at a final concentration of 1 μg/ml for 15–30 minutes; anti-Qβ and Vidu-AF647 were each used at a final concentration of 5 μg/ml. Statistical significance was determined using a paired t-test (B, C) or a two-way ANOVA with Sidak’s multiple comparisons test (D-K, M) : *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant.
    Monoclonal Reafinity Recombinant Human Antibodies Targeting Pdc Surface Antigens Bdca2, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 93/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/target+antigen/CD303+(BDCA-2)+Antibody%2C+anti-human%2C+REAfinity/pmc12951047-44-0-11
    Average 93 stars, based on 17 article reviews
    monoclonal reafinity recombinant human antibodies targeting pdc surface antigens bdca2 - by Bioz Stars, 2026-09
    93/100 stars

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    1) Product Images from "BDCA2 plays a central role in the binding, internalization and response of plasmacytoid dendritic cells to vidutolimod"

    Article Title: BDCA2 plays a central role in the binding, internalization and response of plasmacytoid dendritic cells to vidutolimod

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2026.1769287

    The interaction of Vidu-AF647 with pDCs, as determined by flow cytometry, is dependent on anti-Qβ and is mediated through both CD32 and BDCA2. (A-C) PBMCs from healthy donors were cultured for 2 hours in medium (No Tx), Vidu-AF647 alone or Vidu-AF647 and anti-Qβ. (D-M) PBMCs from healthy donors were cultured for 2 hours or 20 hours with Vidu-AF647 and anti-Qβ with or without receptor blocking. IC or receptor-specific antibodies (anti-CD32 or anti-BDCA2) were added to PBMC cultures prior to Vidu-AF647 and anti-Qβ. Vidu-AF647 signal associated with CD45 + cells or BDCA4 + pDCs was determined by flow cytometry. (A) Gating on Vidu-AF647 + pDCs in PBMCs from one representative donor. (B) Frequency and (C) MdFI of Vidu-AF647 + pDCs (n=9 donors). (D) Frequency and (E) MdFI of Vidu-AF647 + CD45 + or pDCs after PBMC from healthy donors (n=6) were treated with IC (–) or anti-CD32 (+) followed by a 2-hour culture with anti-Qβ and Vidu-AF647. (F) Frequency and (G) MdFI of Vidu-AF647 + CD45 + cells or pDCs after PBMC from healthy donors (n=5) were treated with IC (–) or anti-CD32 (+) followed by a 20-hour culture with anti-Qβ and Vidu-AF647. (H) Frequency and (I) MdFI of Vidu-AF647 + CD45 + cells or pDCs after PBMC from healthy donors (n=6) were treated with IC (–) or anti-BDCA2 (+) followed by a 2-hour culture with anti-Qβ and Vidu-AF647. (J) Frequency and (K) MdFI of Vidu-AF647 + CD45 + cells or pDCs after PBMC from healthy donors (n=12) were treated with IC (–) or anti-BDCA2 (+) followed by a 2-hour culture with anti-Qβ and Vidu-AF647. (L) Contour plots from one representative donor showing Vidu-AF647 + pDCs and (M) frequency of Vidu-AF647 + CD45 + or pDCs (n= 3 donors) after IC, anti-CD32, anti-BDCA2 or anti-CD32/BDCA2 treatment of PBMCs followed by a 20-hour culture with Vidu-AF647 and anti-Qβ. Antibody pre-treatment was done at a final concentration of 1 μg/ml for 15–30 minutes; anti-Qβ and Vidu-AF647 were each used at a final concentration of 5 μg/ml. Statistical significance was determined using a paired t-test (B, C) or a two-way ANOVA with Sidak’s multiple comparisons test (D-K, M) : *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant.
    Figure Legend Snippet: The interaction of Vidu-AF647 with pDCs, as determined by flow cytometry, is dependent on anti-Qβ and is mediated through both CD32 and BDCA2. (A-C) PBMCs from healthy donors were cultured for 2 hours in medium (No Tx), Vidu-AF647 alone or Vidu-AF647 and anti-Qβ. (D-M) PBMCs from healthy donors were cultured for 2 hours or 20 hours with Vidu-AF647 and anti-Qβ with or without receptor blocking. IC or receptor-specific antibodies (anti-CD32 or anti-BDCA2) were added to PBMC cultures prior to Vidu-AF647 and anti-Qβ. Vidu-AF647 signal associated with CD45 + cells or BDCA4 + pDCs was determined by flow cytometry. (A) Gating on Vidu-AF647 + pDCs in PBMCs from one representative donor. (B) Frequency and (C) MdFI of Vidu-AF647 + pDCs (n=9 donors). (D) Frequency and (E) MdFI of Vidu-AF647 + CD45 + or pDCs after PBMC from healthy donors (n=6) were treated with IC (–) or anti-CD32 (+) followed by a 2-hour culture with anti-Qβ and Vidu-AF647. (F) Frequency and (G) MdFI of Vidu-AF647 + CD45 + cells or pDCs after PBMC from healthy donors (n=5) were treated with IC (–) or anti-CD32 (+) followed by a 20-hour culture with anti-Qβ and Vidu-AF647. (H) Frequency and (I) MdFI of Vidu-AF647 + CD45 + cells or pDCs after PBMC from healthy donors (n=6) were treated with IC (–) or anti-BDCA2 (+) followed by a 2-hour culture with anti-Qβ and Vidu-AF647. (J) Frequency and (K) MdFI of Vidu-AF647 + CD45 + cells or pDCs after PBMC from healthy donors (n=12) were treated with IC (–) or anti-BDCA2 (+) followed by a 2-hour culture with anti-Qβ and Vidu-AF647. (L) Contour plots from one representative donor showing Vidu-AF647 + pDCs and (M) frequency of Vidu-AF647 + CD45 + or pDCs (n= 3 donors) after IC, anti-CD32, anti-BDCA2 or anti-CD32/BDCA2 treatment of PBMCs followed by a 20-hour culture with Vidu-AF647 and anti-Qβ. Antibody pre-treatment was done at a final concentration of 1 μg/ml for 15–30 minutes; anti-Qβ and Vidu-AF647 were each used at a final concentration of 5 μg/ml. Statistical significance was determined using a paired t-test (B, C) or a two-way ANOVA with Sidak’s multiple comparisons test (D-K, M) : *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant.

    Techniques Used: Flow Cytometry, Cell Culture, Blocking Assay, Concentration Assay

    Anti-BDCA2 blocks anti-Qβ-coated Vidu induced pDC activation and differentiation. (A, B) IFNα levels detected by ELISA in culture supernatants obtained 20 hours after PBMC from healthy donors (n=6) were treated with IC (white bars), anti-CD32 (orange bar) or anti-BDCA2 (green bar) prior to addition of anti-Qβ and Vidu-AF647. (C) Flow cytometry contour plots from one representative donor and (D) frequency of PDL1 + CD80 - P1 pDC detected across multiple donors (n=6) showing the impact of IC, anti-CD32 or anti-BDCA2 treatment on the expression of PD-L1 and CD80 on BDCA4 + pDCs after PBMC were cultured for 20 hours with anti-Qβ and Vidu-AF647 (subsets of pDCs are referred to as P0, P1, P2 or P3); PBMC cultured alone were stained to show background expression levels (No Treatment). Antibody pre-treatment was done at a final concentration of 1 μg/ml for 15–30 minutes; anti-Qβ and Vidu-AF647 were used at final concentrations of 5 μg/ml for 20 hours. Statistical significance was determined using a paired t-test (A, B) or one-way ANOVA with Dunnett’s multiple-comparisons test (D) : *p<0.05, **p<0.01, ns, not significant.
    Figure Legend Snippet: Anti-BDCA2 blocks anti-Qβ-coated Vidu induced pDC activation and differentiation. (A, B) IFNα levels detected by ELISA in culture supernatants obtained 20 hours after PBMC from healthy donors (n=6) were treated with IC (white bars), anti-CD32 (orange bar) or anti-BDCA2 (green bar) prior to addition of anti-Qβ and Vidu-AF647. (C) Flow cytometry contour plots from one representative donor and (D) frequency of PDL1 + CD80 - P1 pDC detected across multiple donors (n=6) showing the impact of IC, anti-CD32 or anti-BDCA2 treatment on the expression of PD-L1 and CD80 on BDCA4 + pDCs after PBMC were cultured for 20 hours with anti-Qβ and Vidu-AF647 (subsets of pDCs are referred to as P0, P1, P2 or P3); PBMC cultured alone were stained to show background expression levels (No Treatment). Antibody pre-treatment was done at a final concentration of 1 μg/ml for 15–30 minutes; anti-Qβ and Vidu-AF647 were used at final concentrations of 5 μg/ml for 20 hours. Statistical significance was determined using a paired t-test (A, B) or one-way ANOVA with Dunnett’s multiple-comparisons test (D) : *p<0.05, **p<0.01, ns, not significant.

    Techniques Used: Activation Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Cell Culture, Staining, Concentration Assay

    Antibody against BDCA2 reduces internalization of anti-Qβ-coated Vidu-AF647 by pDCs, as determined by multicolor imaging flow cytometry. (A-E) PBMCs from healthy donors were cultured for 20 hours with Vidu-AF647 and anti-Qβ. IC, anti-CD32 or anti-BDCA2 antibodies were added to PBMC cultures prior to Vidu-AF647 and anti-Qβ. Localization of Vidu-AF647 signal associated with pDCs was visualized and analyzed with IDEAS software. (A) Representative images of pDCs from healthy donors. Individual pDC morphology (BF), surface staining of CD45 (purple), BDCA2 (green) and BDCA4 (yellow) are shown for samples treated with anti-CD32 (or IC) or anti-BDCA2. (B) Representative gating and images of Vidu-AF647 + and Vidu-AF647 - pDCs; ‘Merge’ image includes CD45 and Vidu (red) signal. (C) Representative images of Vidu-AF647 + pDCs reflecting the range of Vidu Internalization Scores calculated with IDEAS software. (D, E) Vidu-AF647 Internalization Scores signal from individual pDCs collected from matched donors (n=4) treated with IC, anti-CD32 or anti-BDCA2 prior to culture with anti-Qβ and Vidu-AF647. Antibody pre-treatment was done at a final concentration of 1 μg/ml for 15–30 minutes; anti-Qβ and Vidu-AF647 were each used at a final concentration of 5 μg/ml. Samples were acquired at 40X magnification on Amnis ImageStream MkII. Statistical significance was determined using a paired t-test: ****p<0.0001, ns, not significant.
    Figure Legend Snippet: Antibody against BDCA2 reduces internalization of anti-Qβ-coated Vidu-AF647 by pDCs, as determined by multicolor imaging flow cytometry. (A-E) PBMCs from healthy donors were cultured for 20 hours with Vidu-AF647 and anti-Qβ. IC, anti-CD32 or anti-BDCA2 antibodies were added to PBMC cultures prior to Vidu-AF647 and anti-Qβ. Localization of Vidu-AF647 signal associated with pDCs was visualized and analyzed with IDEAS software. (A) Representative images of pDCs from healthy donors. Individual pDC morphology (BF), surface staining of CD45 (purple), BDCA2 (green) and BDCA4 (yellow) are shown for samples treated with anti-CD32 (or IC) or anti-BDCA2. (B) Representative gating and images of Vidu-AF647 + and Vidu-AF647 - pDCs; ‘Merge’ image includes CD45 and Vidu (red) signal. (C) Representative images of Vidu-AF647 + pDCs reflecting the range of Vidu Internalization Scores calculated with IDEAS software. (D, E) Vidu-AF647 Internalization Scores signal from individual pDCs collected from matched donors (n=4) treated with IC, anti-CD32 or anti-BDCA2 prior to culture with anti-Qβ and Vidu-AF647. Antibody pre-treatment was done at a final concentration of 1 μg/ml for 15–30 minutes; anti-Qβ and Vidu-AF647 were each used at a final concentration of 5 μg/ml. Samples were acquired at 40X magnification on Amnis ImageStream MkII. Statistical significance was determined using a paired t-test: ****p<0.0001, ns, not significant.

    Techniques Used: Imaging, Flow Cytometry, Cell Culture, Software, Staining, Concentration Assay

    Anti-Qβ dose impacts on Vidu-induced BDCA2 internalization and on the IFNα response to TLR9 stimulation. (A-F) PBMCs from healthy donors were cultured for 20 hours with IC, anti-BDCA2, G10 and anti-BDCA2, or Vidu and anti-Qβ. Localization of the BDCA2 signal in pDCs was detected by both surface and intracellular staining and visualized by multicolor imaging flow cytometry. BDCA2 Internalization Scores were calculated using IDEAS software; cells shown represent the average score for the treatment. IFNα was measured by ELISA in cell culture supernatants. (A) Representative images of CD45 + (purple) BDCA2 + (green) pDCs left untreated (Media), treated with IC or anti-BDCA2 (1 μg/ml). (B) Average BDCA2 Internalization Scores calculated from samples (3 donors) treated as described in (A) . (C) Representative images of CD45 + BDCA2 + pDCs treated with G10 CpG-A (2.5 μg/ml) and varying doses of anti-BDCA2. (D) Average BDCA2 Internalization Scores (left y-axis, green bars) and IFNα levels (right y-axis, grey bars) from samples (4 donors) treated as described in (C) . (E) Representative images of CD45 + BDCA2 + pDCs after being cultured with a fixed amount of Vidu and varying concentrations of anti-Qβ. (F) Average BDCA2 Internalization Scores (left y-axis, green bars) and IFNα levels (right y-axis, grey bars) from PBMC (3 donors) treated as described in (E) . G10 was used at a final concentration of 2.5 μg/ml and Vidu was used at a final concentration of 5 μg/ml. Samples were acquired at 60X magnification on Amnis ImageStream MkII. Statistical significance was determined using a one-way ANOVA with a Dunnett’s multiple comparison test: *p<0.05, **p<0.01, ns, not significant.
    Figure Legend Snippet: Anti-Qβ dose impacts on Vidu-induced BDCA2 internalization and on the IFNα response to TLR9 stimulation. (A-F) PBMCs from healthy donors were cultured for 20 hours with IC, anti-BDCA2, G10 and anti-BDCA2, or Vidu and anti-Qβ. Localization of the BDCA2 signal in pDCs was detected by both surface and intracellular staining and visualized by multicolor imaging flow cytometry. BDCA2 Internalization Scores were calculated using IDEAS software; cells shown represent the average score for the treatment. IFNα was measured by ELISA in cell culture supernatants. (A) Representative images of CD45 + (purple) BDCA2 + (green) pDCs left untreated (Media), treated with IC or anti-BDCA2 (1 μg/ml). (B) Average BDCA2 Internalization Scores calculated from samples (3 donors) treated as described in (A) . (C) Representative images of CD45 + BDCA2 + pDCs treated with G10 CpG-A (2.5 μg/ml) and varying doses of anti-BDCA2. (D) Average BDCA2 Internalization Scores (left y-axis, green bars) and IFNα levels (right y-axis, grey bars) from samples (4 donors) treated as described in (C) . (E) Representative images of CD45 + BDCA2 + pDCs after being cultured with a fixed amount of Vidu and varying concentrations of anti-Qβ. (F) Average BDCA2 Internalization Scores (left y-axis, green bars) and IFNα levels (right y-axis, grey bars) from PBMC (3 donors) treated as described in (E) . G10 was used at a final concentration of 2.5 μg/ml and Vidu was used at a final concentration of 5 μg/ml. Samples were acquired at 60X magnification on Amnis ImageStream MkII. Statistical significance was determined using a one-way ANOVA with a Dunnett’s multiple comparison test: *p<0.05, **p<0.01, ns, not significant.

    Techniques Used: Cell Culture, Staining, Imaging, Flow Cytometry, Software, Enzyme-linked Immunosorbent Assay, Concentration Assay, Comparison

    The “Goldilocks Effect” of anti-Qβ concentration, BDCA2 internalization and the IFNα response to Vidu. (A) Suboptimal response: At low anti-Qβ concentrations, minimal Vidu uptake occurs, resulting in weak TLR9 pathway activation and low IFNα production. (B) Optimal response: Moderate anti-Qβ concentrations facilitate peak IFNα production by maximizing Vidu uptake while maintaining low levels of BDCA2 internalization. (C) Inhibitory response: High anti-Qβ concentrations induce significant BDCA2 internalization following Vidu uptake, which suppresses the TLR9-mediated IFNα response.
    Figure Legend Snippet: The “Goldilocks Effect” of anti-Qβ concentration, BDCA2 internalization and the IFNα response to Vidu. (A) Suboptimal response: At low anti-Qβ concentrations, minimal Vidu uptake occurs, resulting in weak TLR9 pathway activation and low IFNα production. (B) Optimal response: Moderate anti-Qβ concentrations facilitate peak IFNα production by maximizing Vidu uptake while maintaining low levels of BDCA2 internalization. (C) Inhibitory response: High anti-Qβ concentrations induce significant BDCA2 internalization following Vidu uptake, which suppresses the TLR9-mediated IFNα response.

    Techniques Used: Concentration Assay, Activation Assay

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    The interaction of Vidu-AF647 with pDCs, as determined by flow cytometry, is dependent on anti-Qβ and is mediated through both CD32 and <t>BDCA2.</t> (A-C) PBMCs from healthy donors were cultured for 2 hours in medium (No Tx), Vidu-AF647 alone or Vidu-AF647 and anti-Qβ. (D-M) PBMCs from healthy donors were cultured for 2 hours or 20 hours with Vidu-AF647 and anti-Qβ with or without receptor blocking. IC or receptor-specific antibodies (anti-CD32 or anti-BDCA2) were added to PBMC cultures prior to Vidu-AF647 and anti-Qβ. Vidu-AF647 signal associated with CD45 + cells or BDCA4 + pDCs was determined by flow cytometry. (A) Gating on Vidu-AF647 + pDCs in PBMCs from one representative donor. (B) Frequency and (C) MdFI of Vidu-AF647 + pDCs (n=9 donors). (D) Frequency and (E) MdFI of Vidu-AF647 + CD45 + or pDCs after PBMC from healthy donors (n=6) were treated with IC (–) or anti-CD32 (+) followed by a 2-hour culture with anti-Qβ and Vidu-AF647. (F) Frequency and (G) MdFI of Vidu-AF647 + CD45 + cells or pDCs after PBMC from healthy donors (n=5) were treated with IC (–) or anti-CD32 (+) followed by a 20-hour culture with anti-Qβ and Vidu-AF647. (H) Frequency and (I) MdFI of Vidu-AF647 + CD45 + cells or pDCs after PBMC from healthy donors (n=6) were treated with IC (–) or anti-BDCA2 (+) followed by a 2-hour culture with anti-Qβ and Vidu-AF647. (J) Frequency and (K) MdFI of Vidu-AF647 + CD45 + cells or pDCs after PBMC from healthy donors (n=12) were treated with IC (–) or anti-BDCA2 (+) followed by a 2-hour culture with anti-Qβ and Vidu-AF647. (L) Contour plots from one representative donor showing Vidu-AF647 + pDCs and (M) frequency of Vidu-AF647 + CD45 + or pDCs (n= 3 donors) after IC, anti-CD32, anti-BDCA2 or anti-CD32/BDCA2 treatment of PBMCs followed by a 20-hour culture with Vidu-AF647 and anti-Qβ. Antibody pre-treatment was done at a final concentration of 1 μg/ml for 15–30 minutes; anti-Qβ and Vidu-AF647 were each used at a final concentration of 5 μg/ml. Statistical significance was determined using a paired t-test (B, C) or a two-way ANOVA with Sidak’s multiple comparisons test (D-K, M) : *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant.
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    antibodies targeting mpxv antigens  (Sino Biological)
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    Sino Biological antibodies targeting mpxv antigens
    Preparation and validation of rVSV-expressing <t>MPXV</t> antigens. ( a ) Design of rVSVs. The codon sequences of MPXV A35R (546 bp), A29L (397 bp), <t>B6R</t> (986 bp), and <t>M1R</t> (753 bp) were inserted into the VSV backbone between the M and G proteins. Additionally, an mCherry protein was fused at the N-terminus of the P protein. ( b ) Fluorescence signal of the rescued rVSVs. Viruses obtained through reverse genetics, with images captured 72–96 h after suspension transfection. Five fields of view were analyzed for each virus. ( c ) Expression of MPXV antigen proteins by the VSV vector. BHK-21 cells were infected with each rVSV at an MOI of 1.0. At 24 h post-infection, cells were lysed in 500 μL of lysis buffer. Subsequently, 10 μL of the lysate was analyzed by Western blot using anti-MPXV A35R, A29L, B6R, and M1R <t>monoclonal</t> <t>antibodies</t> to confirm the expression of the MPXV antigens; the sizes of each protein were A35R −20.0 kDa, A29L −14.5 kDa, B6R −36.2 kDa, M1R-27.6 kDa, respectively. ( d ) One-step growth curve validation of the rVSVs. BHK-21 cells were infected with each recombinant virus at an MOI of 0.01. The virus was collected at various time points (0, 12, 24, 36, 48, 60, and 72 h), and the virus titer was measured by PFU assay. Each sample was tested in triplicate.
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    target antigen 476 expression  (ATCC)
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    Preparation and validation of rVSV-expressing <t>MPXV</t> antigens. ( a ) Design of rVSVs. The codon sequences of MPXV A35R (546 bp), A29L (397 bp), <t>B6R</t> (986 bp), and <t>M1R</t> (753 bp) were inserted into the VSV backbone between the M and G proteins. Additionally, an mCherry protein was fused at the N-terminus of the P protein. ( b ) Fluorescence signal of the rescued rVSVs. Viruses obtained through reverse genetics, with images captured 72–96 h after suspension transfection. Five fields of view were analyzed for each virus. ( c ) Expression of MPXV antigen proteins by the VSV vector. BHK-21 cells were infected with each rVSV at an MOI of 1.0. At 24 h post-infection, cells were lysed in 500 μL of lysis buffer. Subsequently, 10 μL of the lysate was analyzed by Western blot using anti-MPXV A35R, A29L, B6R, and M1R <t>monoclonal</t> <t>antibodies</t> to confirm the expression of the MPXV antigens; the sizes of each protein were A35R −20.0 kDa, A29L −14.5 kDa, B6R −36.2 kDa, M1R-27.6 kDa, respectively. ( d ) One-step growth curve validation of the rVSVs. BHK-21 cells were infected with each recombinant virus at an MOI of 0.01. The virus was collected at various time points (0, 12, 24, 36, 48, 60, and 72 h), and the virus titer was measured by PFU assay. Each sample was tested in triplicate.
    Target Antigen 476 Expression, supplied by ATCC, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    target antigens  (Servicebio Inc)
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    Servicebio Inc target antigens
    Preparation and validation of rVSV-expressing <t>MPXV</t> antigens. ( a ) Design of rVSVs. The codon sequences of MPXV A35R (546 bp), A29L (397 bp), <t>B6R</t> (986 bp), and <t>M1R</t> (753 bp) were inserted into the VSV backbone between the M and G proteins. Additionally, an mCherry protein was fused at the N-terminus of the P protein. ( b ) Fluorescence signal of the rescued rVSVs. Viruses obtained through reverse genetics, with images captured 72–96 h after suspension transfection. Five fields of view were analyzed for each virus. ( c ) Expression of MPXV antigen proteins by the VSV vector. BHK-21 cells were infected with each rVSV at an MOI of 1.0. At 24 h post-infection, cells were lysed in 500 μL of lysis buffer. Subsequently, 10 μL of the lysate was analyzed by Western blot using anti-MPXV A35R, A29L, B6R, and M1R <t>monoclonal</t> <t>antibodies</t> to confirm the expression of the MPXV antigens; the sizes of each protein were A35R −20.0 kDa, A29L −14.5 kDa, B6R −36.2 kDa, M1R-27.6 kDa, respectively. ( d ) One-step growth curve validation of the rVSVs. BHK-21 cells were infected with each recombinant virus at an MOI of 0.01. The virus was collected at various time points (0, 12, 24, 36, 48, 60, and 72 h), and the virus titer was measured by PFU assay. Each sample was tested in triplicate.
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    human endogenous antigen positive target cell line h226  (ATCC)
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    Preparation and validation of rVSV-expressing <t>MPXV</t> antigens. ( a ) Design of rVSVs. The codon sequences of MPXV A35R (546 bp), A29L (397 bp), <t>B6R</t> (986 bp), and <t>M1R</t> (753 bp) were inserted into the VSV backbone between the M and G proteins. Additionally, an mCherry protein was fused at the N-terminus of the P protein. ( b ) Fluorescence signal of the rescued rVSVs. Viruses obtained through reverse genetics, with images captured 72–96 h after suspension transfection. Five fields of view were analyzed for each virus. ( c ) Expression of MPXV antigen proteins by the VSV vector. BHK-21 cells were infected with each rVSV at an MOI of 1.0. At 24 h post-infection, cells were lysed in 500 μL of lysis buffer. Subsequently, 10 μL of the lysate was analyzed by Western blot using anti-MPXV A35R, A29L, B6R, and M1R <t>monoclonal</t> <t>antibodies</t> to confirm the expression of the MPXV antigens; the sizes of each protein were A35R −20.0 kDa, A29L −14.5 kDa, B6R −36.2 kDa, M1R-27.6 kDa, respectively. ( d ) One-step growth curve validation of the rVSVs. BHK-21 cells were infected with each recombinant virus at an MOI of 0.01. The virus was collected at various time points (0, 12, 24, 36, 48, 60, and 72 h), and the virus titer was measured by PFU assay. Each sample was tested in triplicate.
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    target antigen  (ATCC)
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    Preparation and validation of rVSV-expressing <t>MPXV</t> antigens. ( a ) Design of rVSVs. The codon sequences of MPXV A35R (546 bp), A29L (397 bp), <t>B6R</t> (986 bp), and <t>M1R</t> (753 bp) were inserted into the VSV backbone between the M and G proteins. Additionally, an mCherry protein was fused at the N-terminus of the P protein. ( b ) Fluorescence signal of the rescued rVSVs. Viruses obtained through reverse genetics, with images captured 72–96 h after suspension transfection. Five fields of view were analyzed for each virus. ( c ) Expression of MPXV antigen proteins by the VSV vector. BHK-21 cells were infected with each rVSV at an MOI of 1.0. At 24 h post-infection, cells were lysed in 500 μL of lysis buffer. Subsequently, 10 μL of the lysate was analyzed by Western blot using anti-MPXV A35R, A29L, B6R, and M1R <t>monoclonal</t> <t>antibodies</t> to confirm the expression of the MPXV antigens; the sizes of each protein were A35R −20.0 kDa, A29L −14.5 kDa, B6R −36.2 kDa, M1R-27.6 kDa, respectively. ( d ) One-step growth curve validation of the rVSVs. BHK-21 cells were infected with each recombinant virus at an MOI of 0.01. The virus was collected at various time points (0, 12, 24, 36, 48, 60, and 72 h), and the virus titer was measured by PFU assay. Each sample was tested in triplicate.
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    Image Search Results


    The interaction of Vidu-AF647 with pDCs, as determined by flow cytometry, is dependent on anti-Qβ and is mediated through both CD32 and BDCA2. (A-C) PBMCs from healthy donors were cultured for 2 hours in medium (No Tx), Vidu-AF647 alone or Vidu-AF647 and anti-Qβ. (D-M) PBMCs from healthy donors were cultured for 2 hours or 20 hours with Vidu-AF647 and anti-Qβ with or without receptor blocking. IC or receptor-specific antibodies (anti-CD32 or anti-BDCA2) were added to PBMC cultures prior to Vidu-AF647 and anti-Qβ. Vidu-AF647 signal associated with CD45 + cells or BDCA4 + pDCs was determined by flow cytometry. (A) Gating on Vidu-AF647 + pDCs in PBMCs from one representative donor. (B) Frequency and (C) MdFI of Vidu-AF647 + pDCs (n=9 donors). (D) Frequency and (E) MdFI of Vidu-AF647 + CD45 + or pDCs after PBMC from healthy donors (n=6) were treated with IC (–) or anti-CD32 (+) followed by a 2-hour culture with anti-Qβ and Vidu-AF647. (F) Frequency and (G) MdFI of Vidu-AF647 + CD45 + cells or pDCs after PBMC from healthy donors (n=5) were treated with IC (–) or anti-CD32 (+) followed by a 20-hour culture with anti-Qβ and Vidu-AF647. (H) Frequency and (I) MdFI of Vidu-AF647 + CD45 + cells or pDCs after PBMC from healthy donors (n=6) were treated with IC (–) or anti-BDCA2 (+) followed by a 2-hour culture with anti-Qβ and Vidu-AF647. (J) Frequency and (K) MdFI of Vidu-AF647 + CD45 + cells or pDCs after PBMC from healthy donors (n=12) were treated with IC (–) or anti-BDCA2 (+) followed by a 2-hour culture with anti-Qβ and Vidu-AF647. (L) Contour plots from one representative donor showing Vidu-AF647 + pDCs and (M) frequency of Vidu-AF647 + CD45 + or pDCs (n= 3 donors) after IC, anti-CD32, anti-BDCA2 or anti-CD32/BDCA2 treatment of PBMCs followed by a 20-hour culture with Vidu-AF647 and anti-Qβ. Antibody pre-treatment was done at a final concentration of 1 μg/ml for 15–30 minutes; anti-Qβ and Vidu-AF647 were each used at a final concentration of 5 μg/ml. Statistical significance was determined using a paired t-test (B, C) or a two-way ANOVA with Sidak’s multiple comparisons test (D-K, M) : *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant.

    Journal: Frontiers in Immunology

    Article Title: BDCA2 plays a central role in the binding, internalization and response of plasmacytoid dendritic cells to vidutolimod

    doi: 10.3389/fimmu.2026.1769287

    Figure Lengend Snippet: The interaction of Vidu-AF647 with pDCs, as determined by flow cytometry, is dependent on anti-Qβ and is mediated through both CD32 and BDCA2. (A-C) PBMCs from healthy donors were cultured for 2 hours in medium (No Tx), Vidu-AF647 alone or Vidu-AF647 and anti-Qβ. (D-M) PBMCs from healthy donors were cultured for 2 hours or 20 hours with Vidu-AF647 and anti-Qβ with or without receptor blocking. IC or receptor-specific antibodies (anti-CD32 or anti-BDCA2) were added to PBMC cultures prior to Vidu-AF647 and anti-Qβ. Vidu-AF647 signal associated with CD45 + cells or BDCA4 + pDCs was determined by flow cytometry. (A) Gating on Vidu-AF647 + pDCs in PBMCs from one representative donor. (B) Frequency and (C) MdFI of Vidu-AF647 + pDCs (n=9 donors). (D) Frequency and (E) MdFI of Vidu-AF647 + CD45 + or pDCs after PBMC from healthy donors (n=6) were treated with IC (–) or anti-CD32 (+) followed by a 2-hour culture with anti-Qβ and Vidu-AF647. (F) Frequency and (G) MdFI of Vidu-AF647 + CD45 + cells or pDCs after PBMC from healthy donors (n=5) were treated with IC (–) or anti-CD32 (+) followed by a 20-hour culture with anti-Qβ and Vidu-AF647. (H) Frequency and (I) MdFI of Vidu-AF647 + CD45 + cells or pDCs after PBMC from healthy donors (n=6) were treated with IC (–) or anti-BDCA2 (+) followed by a 2-hour culture with anti-Qβ and Vidu-AF647. (J) Frequency and (K) MdFI of Vidu-AF647 + CD45 + cells or pDCs after PBMC from healthy donors (n=12) were treated with IC (–) or anti-BDCA2 (+) followed by a 2-hour culture with anti-Qβ and Vidu-AF647. (L) Contour plots from one representative donor showing Vidu-AF647 + pDCs and (M) frequency of Vidu-AF647 + CD45 + or pDCs (n= 3 donors) after IC, anti-CD32, anti-BDCA2 or anti-CD32/BDCA2 treatment of PBMCs followed by a 20-hour culture with Vidu-AF647 and anti-Qβ. Antibody pre-treatment was done at a final concentration of 1 μg/ml for 15–30 minutes; anti-Qβ and Vidu-AF647 were each used at a final concentration of 5 μg/ml. Statistical significance was determined using a paired t-test (B, C) or a two-way ANOVA with Sidak’s multiple comparisons test (D-K, M) : *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant.

    Article Snippet: Monoclonal REAfinity ® recombinant human antibodies targeting pDC surface antigens BDCA2 (Miltenyi, cat. #130-124-317) and BDCA4 (Miltenyi, cat. #130-124-318) were evaluated for their ability to inhibit binding and/or uptake of anti-Qβ-coated Vidu.

    Techniques: Flow Cytometry, Cell Culture, Blocking Assay, Concentration Assay

    Anti-BDCA2 blocks anti-Qβ-coated Vidu induced pDC activation and differentiation. (A, B) IFNα levels detected by ELISA in culture supernatants obtained 20 hours after PBMC from healthy donors (n=6) were treated with IC (white bars), anti-CD32 (orange bar) or anti-BDCA2 (green bar) prior to addition of anti-Qβ and Vidu-AF647. (C) Flow cytometry contour plots from one representative donor and (D) frequency of PDL1 + CD80 - P1 pDC detected across multiple donors (n=6) showing the impact of IC, anti-CD32 or anti-BDCA2 treatment on the expression of PD-L1 and CD80 on BDCA4 + pDCs after PBMC were cultured for 20 hours with anti-Qβ and Vidu-AF647 (subsets of pDCs are referred to as P0, P1, P2 or P3); PBMC cultured alone were stained to show background expression levels (No Treatment). Antibody pre-treatment was done at a final concentration of 1 μg/ml for 15–30 minutes; anti-Qβ and Vidu-AF647 were used at final concentrations of 5 μg/ml for 20 hours. Statistical significance was determined using a paired t-test (A, B) or one-way ANOVA with Dunnett’s multiple-comparisons test (D) : *p<0.05, **p<0.01, ns, not significant.

    Journal: Frontiers in Immunology

    Article Title: BDCA2 plays a central role in the binding, internalization and response of plasmacytoid dendritic cells to vidutolimod

    doi: 10.3389/fimmu.2026.1769287

    Figure Lengend Snippet: Anti-BDCA2 blocks anti-Qβ-coated Vidu induced pDC activation and differentiation. (A, B) IFNα levels detected by ELISA in culture supernatants obtained 20 hours after PBMC from healthy donors (n=6) were treated with IC (white bars), anti-CD32 (orange bar) or anti-BDCA2 (green bar) prior to addition of anti-Qβ and Vidu-AF647. (C) Flow cytometry contour plots from one representative donor and (D) frequency of PDL1 + CD80 - P1 pDC detected across multiple donors (n=6) showing the impact of IC, anti-CD32 or anti-BDCA2 treatment on the expression of PD-L1 and CD80 on BDCA4 + pDCs after PBMC were cultured for 20 hours with anti-Qβ and Vidu-AF647 (subsets of pDCs are referred to as P0, P1, P2 or P3); PBMC cultured alone were stained to show background expression levels (No Treatment). Antibody pre-treatment was done at a final concentration of 1 μg/ml for 15–30 minutes; anti-Qβ and Vidu-AF647 were used at final concentrations of 5 μg/ml for 20 hours. Statistical significance was determined using a paired t-test (A, B) or one-way ANOVA with Dunnett’s multiple-comparisons test (D) : *p<0.05, **p<0.01, ns, not significant.

    Article Snippet: Monoclonal REAfinity ® recombinant human antibodies targeting pDC surface antigens BDCA2 (Miltenyi, cat. #130-124-317) and BDCA4 (Miltenyi, cat. #130-124-318) were evaluated for their ability to inhibit binding and/or uptake of anti-Qβ-coated Vidu.

    Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Cell Culture, Staining, Concentration Assay

    Antibody against BDCA2 reduces internalization of anti-Qβ-coated Vidu-AF647 by pDCs, as determined by multicolor imaging flow cytometry. (A-E) PBMCs from healthy donors were cultured for 20 hours with Vidu-AF647 and anti-Qβ. IC, anti-CD32 or anti-BDCA2 antibodies were added to PBMC cultures prior to Vidu-AF647 and anti-Qβ. Localization of Vidu-AF647 signal associated with pDCs was visualized and analyzed with IDEAS software. (A) Representative images of pDCs from healthy donors. Individual pDC morphology (BF), surface staining of CD45 (purple), BDCA2 (green) and BDCA4 (yellow) are shown for samples treated with anti-CD32 (or IC) or anti-BDCA2. (B) Representative gating and images of Vidu-AF647 + and Vidu-AF647 - pDCs; ‘Merge’ image includes CD45 and Vidu (red) signal. (C) Representative images of Vidu-AF647 + pDCs reflecting the range of Vidu Internalization Scores calculated with IDEAS software. (D, E) Vidu-AF647 Internalization Scores signal from individual pDCs collected from matched donors (n=4) treated with IC, anti-CD32 or anti-BDCA2 prior to culture with anti-Qβ and Vidu-AF647. Antibody pre-treatment was done at a final concentration of 1 μg/ml for 15–30 minutes; anti-Qβ and Vidu-AF647 were each used at a final concentration of 5 μg/ml. Samples were acquired at 40X magnification on Amnis ImageStream MkII. Statistical significance was determined using a paired t-test: ****p<0.0001, ns, not significant.

    Journal: Frontiers in Immunology

    Article Title: BDCA2 plays a central role in the binding, internalization and response of plasmacytoid dendritic cells to vidutolimod

    doi: 10.3389/fimmu.2026.1769287

    Figure Lengend Snippet: Antibody against BDCA2 reduces internalization of anti-Qβ-coated Vidu-AF647 by pDCs, as determined by multicolor imaging flow cytometry. (A-E) PBMCs from healthy donors were cultured for 20 hours with Vidu-AF647 and anti-Qβ. IC, anti-CD32 or anti-BDCA2 antibodies were added to PBMC cultures prior to Vidu-AF647 and anti-Qβ. Localization of Vidu-AF647 signal associated with pDCs was visualized and analyzed with IDEAS software. (A) Representative images of pDCs from healthy donors. Individual pDC morphology (BF), surface staining of CD45 (purple), BDCA2 (green) and BDCA4 (yellow) are shown for samples treated with anti-CD32 (or IC) or anti-BDCA2. (B) Representative gating and images of Vidu-AF647 + and Vidu-AF647 - pDCs; ‘Merge’ image includes CD45 and Vidu (red) signal. (C) Representative images of Vidu-AF647 + pDCs reflecting the range of Vidu Internalization Scores calculated with IDEAS software. (D, E) Vidu-AF647 Internalization Scores signal from individual pDCs collected from matched donors (n=4) treated with IC, anti-CD32 or anti-BDCA2 prior to culture with anti-Qβ and Vidu-AF647. Antibody pre-treatment was done at a final concentration of 1 μg/ml for 15–30 minutes; anti-Qβ and Vidu-AF647 were each used at a final concentration of 5 μg/ml. Samples were acquired at 40X magnification on Amnis ImageStream MkII. Statistical significance was determined using a paired t-test: ****p<0.0001, ns, not significant.

    Article Snippet: Monoclonal REAfinity ® recombinant human antibodies targeting pDC surface antigens BDCA2 (Miltenyi, cat. #130-124-317) and BDCA4 (Miltenyi, cat. #130-124-318) were evaluated for their ability to inhibit binding and/or uptake of anti-Qβ-coated Vidu.

    Techniques: Imaging, Flow Cytometry, Cell Culture, Software, Staining, Concentration Assay

    Anti-Qβ dose impacts on Vidu-induced BDCA2 internalization and on the IFNα response to TLR9 stimulation. (A-F) PBMCs from healthy donors were cultured for 20 hours with IC, anti-BDCA2, G10 and anti-BDCA2, or Vidu and anti-Qβ. Localization of the BDCA2 signal in pDCs was detected by both surface and intracellular staining and visualized by multicolor imaging flow cytometry. BDCA2 Internalization Scores were calculated using IDEAS software; cells shown represent the average score for the treatment. IFNα was measured by ELISA in cell culture supernatants. (A) Representative images of CD45 + (purple) BDCA2 + (green) pDCs left untreated (Media), treated with IC or anti-BDCA2 (1 μg/ml). (B) Average BDCA2 Internalization Scores calculated from samples (3 donors) treated as described in (A) . (C) Representative images of CD45 + BDCA2 + pDCs treated with G10 CpG-A (2.5 μg/ml) and varying doses of anti-BDCA2. (D) Average BDCA2 Internalization Scores (left y-axis, green bars) and IFNα levels (right y-axis, grey bars) from samples (4 donors) treated as described in (C) . (E) Representative images of CD45 + BDCA2 + pDCs after being cultured with a fixed amount of Vidu and varying concentrations of anti-Qβ. (F) Average BDCA2 Internalization Scores (left y-axis, green bars) and IFNα levels (right y-axis, grey bars) from PBMC (3 donors) treated as described in (E) . G10 was used at a final concentration of 2.5 μg/ml and Vidu was used at a final concentration of 5 μg/ml. Samples were acquired at 60X magnification on Amnis ImageStream MkII. Statistical significance was determined using a one-way ANOVA with a Dunnett’s multiple comparison test: *p<0.05, **p<0.01, ns, not significant.

    Journal: Frontiers in Immunology

    Article Title: BDCA2 plays a central role in the binding, internalization and response of plasmacytoid dendritic cells to vidutolimod

    doi: 10.3389/fimmu.2026.1769287

    Figure Lengend Snippet: Anti-Qβ dose impacts on Vidu-induced BDCA2 internalization and on the IFNα response to TLR9 stimulation. (A-F) PBMCs from healthy donors were cultured for 20 hours with IC, anti-BDCA2, G10 and anti-BDCA2, or Vidu and anti-Qβ. Localization of the BDCA2 signal in pDCs was detected by both surface and intracellular staining and visualized by multicolor imaging flow cytometry. BDCA2 Internalization Scores were calculated using IDEAS software; cells shown represent the average score for the treatment. IFNα was measured by ELISA in cell culture supernatants. (A) Representative images of CD45 + (purple) BDCA2 + (green) pDCs left untreated (Media), treated with IC or anti-BDCA2 (1 μg/ml). (B) Average BDCA2 Internalization Scores calculated from samples (3 donors) treated as described in (A) . (C) Representative images of CD45 + BDCA2 + pDCs treated with G10 CpG-A (2.5 μg/ml) and varying doses of anti-BDCA2. (D) Average BDCA2 Internalization Scores (left y-axis, green bars) and IFNα levels (right y-axis, grey bars) from samples (4 donors) treated as described in (C) . (E) Representative images of CD45 + BDCA2 + pDCs after being cultured with a fixed amount of Vidu and varying concentrations of anti-Qβ. (F) Average BDCA2 Internalization Scores (left y-axis, green bars) and IFNα levels (right y-axis, grey bars) from PBMC (3 donors) treated as described in (E) . G10 was used at a final concentration of 2.5 μg/ml and Vidu was used at a final concentration of 5 μg/ml. Samples were acquired at 60X magnification on Amnis ImageStream MkII. Statistical significance was determined using a one-way ANOVA with a Dunnett’s multiple comparison test: *p<0.05, **p<0.01, ns, not significant.

    Article Snippet: Monoclonal REAfinity ® recombinant human antibodies targeting pDC surface antigens BDCA2 (Miltenyi, cat. #130-124-317) and BDCA4 (Miltenyi, cat. #130-124-318) were evaluated for their ability to inhibit binding and/or uptake of anti-Qβ-coated Vidu.

    Techniques: Cell Culture, Staining, Imaging, Flow Cytometry, Software, Enzyme-linked Immunosorbent Assay, Concentration Assay, Comparison

    The “Goldilocks Effect” of anti-Qβ concentration, BDCA2 internalization and the IFNα response to Vidu. (A) Suboptimal response: At low anti-Qβ concentrations, minimal Vidu uptake occurs, resulting in weak TLR9 pathway activation and low IFNα production. (B) Optimal response: Moderate anti-Qβ concentrations facilitate peak IFNα production by maximizing Vidu uptake while maintaining low levels of BDCA2 internalization. (C) Inhibitory response: High anti-Qβ concentrations induce significant BDCA2 internalization following Vidu uptake, which suppresses the TLR9-mediated IFNα response.

    Journal: Frontiers in Immunology

    Article Title: BDCA2 plays a central role in the binding, internalization and response of plasmacytoid dendritic cells to vidutolimod

    doi: 10.3389/fimmu.2026.1769287

    Figure Lengend Snippet: The “Goldilocks Effect” of anti-Qβ concentration, BDCA2 internalization and the IFNα response to Vidu. (A) Suboptimal response: At low anti-Qβ concentrations, minimal Vidu uptake occurs, resulting in weak TLR9 pathway activation and low IFNα production. (B) Optimal response: Moderate anti-Qβ concentrations facilitate peak IFNα production by maximizing Vidu uptake while maintaining low levels of BDCA2 internalization. (C) Inhibitory response: High anti-Qβ concentrations induce significant BDCA2 internalization following Vidu uptake, which suppresses the TLR9-mediated IFNα response.

    Article Snippet: Monoclonal REAfinity ® recombinant human antibodies targeting pDC surface antigens BDCA2 (Miltenyi, cat. #130-124-317) and BDCA4 (Miltenyi, cat. #130-124-318) were evaluated for their ability to inhibit binding and/or uptake of anti-Qβ-coated Vidu.

    Techniques: Concentration Assay, Activation Assay

    Preparation and validation of rVSV-expressing MPXV antigens. ( a ) Design of rVSVs. The codon sequences of MPXV A35R (546 bp), A29L (397 bp), B6R (986 bp), and M1R (753 bp) were inserted into the VSV backbone between the M and G proteins. Additionally, an mCherry protein was fused at the N-terminus of the P protein. ( b ) Fluorescence signal of the rescued rVSVs. Viruses obtained through reverse genetics, with images captured 72–96 h after suspension transfection. Five fields of view were analyzed for each virus. ( c ) Expression of MPXV antigen proteins by the VSV vector. BHK-21 cells were infected with each rVSV at an MOI of 1.0. At 24 h post-infection, cells were lysed in 500 μL of lysis buffer. Subsequently, 10 μL of the lysate was analyzed by Western blot using anti-MPXV A35R, A29L, B6R, and M1R monoclonal antibodies to confirm the expression of the MPXV antigens; the sizes of each protein were A35R −20.0 kDa, A29L −14.5 kDa, B6R −36.2 kDa, M1R-27.6 kDa, respectively. ( d ) One-step growth curve validation of the rVSVs. BHK-21 cells were infected with each recombinant virus at an MOI of 0.01. The virus was collected at various time points (0, 12, 24, 36, 48, 60, and 72 h), and the virus titer was measured by PFU assay. Each sample was tested in triplicate.

    Journal: mBio

    Article Title: A cocktail vaccine with monkeypox virus antigens confers protection without selecting mutations in potential immune evasion genes in the vaccinia WR strain challenge

    doi: 10.1128/mbio.03200-25

    Figure Lengend Snippet: Preparation and validation of rVSV-expressing MPXV antigens. ( a ) Design of rVSVs. The codon sequences of MPXV A35R (546 bp), A29L (397 bp), B6R (986 bp), and M1R (753 bp) were inserted into the VSV backbone between the M and G proteins. Additionally, an mCherry protein was fused at the N-terminus of the P protein. ( b ) Fluorescence signal of the rescued rVSVs. Viruses obtained through reverse genetics, with images captured 72–96 h after suspension transfection. Five fields of view were analyzed for each virus. ( c ) Expression of MPXV antigen proteins by the VSV vector. BHK-21 cells were infected with each rVSV at an MOI of 1.0. At 24 h post-infection, cells were lysed in 500 μL of lysis buffer. Subsequently, 10 μL of the lysate was analyzed by Western blot using anti-MPXV A35R, A29L, B6R, and M1R monoclonal antibodies to confirm the expression of the MPXV antigens; the sizes of each protein were A35R −20.0 kDa, A29L −14.5 kDa, B6R −36.2 kDa, M1R-27.6 kDa, respectively. ( d ) One-step growth curve validation of the rVSVs. BHK-21 cells were infected with each recombinant virus at an MOI of 0.01. The virus was collected at various time points (0, 12, 24, 36, 48, 60, and 72 h), and the virus titer was measured by PFU assay. Each sample was tested in triplicate.

    Article Snippet: Commercial antibodies targeting MPXV antigens (A35:40886-M0026; A29: 40891-M0036; M1R: 40904-T62; B6R: 40902-R007) were obtained from Sino Biological.

    Techniques: Biomarker Discovery, Expressing, Fluorescence, Suspension, Transfection, Virus, Plasmid Preparation, Infection, Lysis, Western Blot, Bioprocessing, Recombinant

    Humoral immune response levels in mice immunized with single-component rVSV vaccines. ( a ) Immunization schedule for the BALB/C mice. ( b ) Daily body weight changes of mice post-challenge. Body weight changes were calculated and compared with the initial weight. n = 5 biologically independent mice. ( c ) Specific IgG analysis. IgG responses in the sera on days 7, 14, 21, 28, 35, and 42 of vaccinated mice were evaluated by ELISA for binding to individually MPXV antigen proteins (A35R, A29L, B6R, and M1R). n = 5 per group. Threshold was set at the mean OD 450 of negative controls + 3 SD; antigen-specific IgG remained detectable at serum dilutions up to 1: 640,000. ( d ) Neutralizing antibody analysis. Serum was tested for neutralizing antibodies against VACV using a plaque reduction neutralization test (PRNT 50 ). n = 5 biologically independent mice. Data are presented as mean values ± SEM. Statistical significance is indicated by asterisks: *** P < 0.001 and * P < 0.05.

    Journal: mBio

    Article Title: A cocktail vaccine with monkeypox virus antigens confers protection without selecting mutations in potential immune evasion genes in the vaccinia WR strain challenge

    doi: 10.1128/mbio.03200-25

    Figure Lengend Snippet: Humoral immune response levels in mice immunized with single-component rVSV vaccines. ( a ) Immunization schedule for the BALB/C mice. ( b ) Daily body weight changes of mice post-challenge. Body weight changes were calculated and compared with the initial weight. n = 5 biologically independent mice. ( c ) Specific IgG analysis. IgG responses in the sera on days 7, 14, 21, 28, 35, and 42 of vaccinated mice were evaluated by ELISA for binding to individually MPXV antigen proteins (A35R, A29L, B6R, and M1R). n = 5 per group. Threshold was set at the mean OD 450 of negative controls + 3 SD; antigen-specific IgG remained detectable at serum dilutions up to 1: 640,000. ( d ) Neutralizing antibody analysis. Serum was tested for neutralizing antibodies against VACV using a plaque reduction neutralization test (PRNT 50 ). n = 5 biologically independent mice. Data are presented as mean values ± SEM. Statistical significance is indicated by asterisks: *** P < 0.001 and * P < 0.05.

    Article Snippet: Commercial antibodies targeting MPXV antigens (A35:40886-M0026; A29: 40891-M0036; M1R: 40904-T62; B6R: 40902-R007) were obtained from Sino Biological.

    Techniques: Vaccines, Enzyme-linked Immunosorbent Assay, Binding Assay, Plaque Reduction Neutralization Test

    Cellular immune response levels in mice immunized with single-component rVSV vaccines. All spleen cells were isolated from mice 14 days post-triple immunization and subjected to in vitro stimulation with MPXV antigens (A29L, A35R, M1R, and B6R) prior to analysis. ( a ) Lymphocyte proliferation induced by single-component rVSV vaccines ( n = 5). The positive control (PC) group stimulated mouse lymphocytes with Concanavalin A (ConA), while the negative control (NC) group stimulated lymphocytes from negative mice with the respective antigen. Each sample was tested three times. ( b–d ) Differentiation levels of specific T cells. Flow cytometry was used to assess the proportions of CD4+, CD8+, and CD3+ positive specific T cells induced by single-component rVSV vaccines in mice ( n = 3). The data were processed and presented as percentages using the CytExpert software. ( e–h ) Cytokine detection by ELISA. Measurement of IFN-γ ( e ), TNF-α ( f ), IL-2 ( g ), and IL-4 ( h ) levels in the supernatant of stimulated lymphocytes ( n = 5). Each sample was tested two times. Data are presented as mean ± SEM, and statistical significance was determined by Student’s t- test. Statistical significance is indicated by asterisks: **** P < 0.0001, *** P < 0.001, ** P < 0.01, and * P < 0.05.

    Journal: mBio

    Article Title: A cocktail vaccine with monkeypox virus antigens confers protection without selecting mutations in potential immune evasion genes in the vaccinia WR strain challenge

    doi: 10.1128/mbio.03200-25

    Figure Lengend Snippet: Cellular immune response levels in mice immunized with single-component rVSV vaccines. All spleen cells were isolated from mice 14 days post-triple immunization and subjected to in vitro stimulation with MPXV antigens (A29L, A35R, M1R, and B6R) prior to analysis. ( a ) Lymphocyte proliferation induced by single-component rVSV vaccines ( n = 5). The positive control (PC) group stimulated mouse lymphocytes with Concanavalin A (ConA), while the negative control (NC) group stimulated lymphocytes from negative mice with the respective antigen. Each sample was tested three times. ( b–d ) Differentiation levels of specific T cells. Flow cytometry was used to assess the proportions of CD4+, CD8+, and CD3+ positive specific T cells induced by single-component rVSV vaccines in mice ( n = 3). The data were processed and presented as percentages using the CytExpert software. ( e–h ) Cytokine detection by ELISA. Measurement of IFN-γ ( e ), TNF-α ( f ), IL-2 ( g ), and IL-4 ( h ) levels in the supernatant of stimulated lymphocytes ( n = 5). Each sample was tested two times. Data are presented as mean ± SEM, and statistical significance was determined by Student’s t- test. Statistical significance is indicated by asterisks: **** P < 0.0001, *** P < 0.001, ** P < 0.01, and * P < 0.05.

    Article Snippet: Commercial antibodies targeting MPXV antigens (A35:40886-M0026; A29: 40891-M0036; M1R: 40904-T62; B6R: 40902-R007) were obtained from Sino Biological.

    Techniques: Vaccines, Isolation, In Vitro, Positive Control, Negative Control, Flow Cytometry, Software, Enzyme-linked Immunosorbent Assay

    ( a ) The cocktail immunization induced high titers of IgG and neutralizing antibodies in mice. Cocktail immunization regimens and administration routes. ( b ) Weight changes in mice post-immunization, expressed as a percentage of initial body weight in experimental (G1–G6) and control (PBS) groups, with data showing weight monitoring at different time points ( n = 5). ( c ) Serum samples from mice were collected at different times post-immunization, and IgG antibody titers specific to MPXV antigens A29L, A35R, M1R, and B6R were measured by ELISA. ( d ) PRNT 50 titers (log 10 ) in experimental (G1–G6) and control (NC) groups at 21 and 42 days post-immunization. Statistical significance denoted as **** ( P < 0.0001), ** ( P < 0.01), and * ( P < 0.05) (Student’s t -test).

    Journal: mBio

    Article Title: A cocktail vaccine with monkeypox virus antigens confers protection without selecting mutations in potential immune evasion genes in the vaccinia WR strain challenge

    doi: 10.1128/mbio.03200-25

    Figure Lengend Snippet: ( a ) The cocktail immunization induced high titers of IgG and neutralizing antibodies in mice. Cocktail immunization regimens and administration routes. ( b ) Weight changes in mice post-immunization, expressed as a percentage of initial body weight in experimental (G1–G6) and control (PBS) groups, with data showing weight monitoring at different time points ( n = 5). ( c ) Serum samples from mice were collected at different times post-immunization, and IgG antibody titers specific to MPXV antigens A29L, A35R, M1R, and B6R were measured by ELISA. ( d ) PRNT 50 titers (log 10 ) in experimental (G1–G6) and control (NC) groups at 21 and 42 days post-immunization. Statistical significance denoted as **** ( P < 0.0001), ** ( P < 0.01), and * ( P < 0.05) (Student’s t -test).

    Article Snippet: Commercial antibodies targeting MPXV antigens (A35:40886-M0026; A29: 40891-M0036; M1R: 40904-T62; B6R: 40902-R007) were obtained from Sino Biological.

    Techniques: Control, Enzyme-linked Immunosorbent Assay

    The cocktail immunization elicited robust cellular immune responses. All spleen cells were isolated from mice 14 days post-triple immunization and subjected to in vitro stimulation with MPXV mixed antigens (A29L, A35R, M1R, and B6R) prior to analysis. ( a ) Lymphocyte proliferation induced by cocktail vaccines ( n = 5). Each sample was tested in triplicate. The PC group stimulated mouse lymphocytes with ConA, while the NC group stimulated lymphocytes from negative mice with the respective antigen. ( b–d ) Differentiation levels of specific T cells. Flow cytometry was used to assess the proportions of CD4 + , CD8 + , and CD3 + positive cells induced by cocktail vaccines in mice ( n = 3). The data were processed and presented as percentages using the CytExpert software. ( e–h ) Expression levels of cytokines. IFN-γ ( e ), TNF-α ( f ), IL-2 ( g ), and IL-4 ( h ) levels in the supernatant of stimulated lymphocytes were detected by ELISA. ( n = 5). Each sample was tested in triplicate. Data are presented as mean ± SEM, and statistical significance was determined by Student’s t- test. Statistical significance is indicated by asterisks: **** P < 0.0001, *** P < 0.001, ** P < 0.01, and * P < 0.05.

    Journal: mBio

    Article Title: A cocktail vaccine with monkeypox virus antigens confers protection without selecting mutations in potential immune evasion genes in the vaccinia WR strain challenge

    doi: 10.1128/mbio.03200-25

    Figure Lengend Snippet: The cocktail immunization elicited robust cellular immune responses. All spleen cells were isolated from mice 14 days post-triple immunization and subjected to in vitro stimulation with MPXV mixed antigens (A29L, A35R, M1R, and B6R) prior to analysis. ( a ) Lymphocyte proliferation induced by cocktail vaccines ( n = 5). Each sample was tested in triplicate. The PC group stimulated mouse lymphocytes with ConA, while the NC group stimulated lymphocytes from negative mice with the respective antigen. ( b–d ) Differentiation levels of specific T cells. Flow cytometry was used to assess the proportions of CD4 + , CD8 + , and CD3 + positive cells induced by cocktail vaccines in mice ( n = 3). The data were processed and presented as percentages using the CytExpert software. ( e–h ) Expression levels of cytokines. IFN-γ ( e ), TNF-α ( f ), IL-2 ( g ), and IL-4 ( h ) levels in the supernatant of stimulated lymphocytes were detected by ELISA. ( n = 5). Each sample was tested in triplicate. Data are presented as mean ± SEM, and statistical significance was determined by Student’s t- test. Statistical significance is indicated by asterisks: **** P < 0.0001, *** P < 0.001, ** P < 0.01, and * P < 0.05.

    Article Snippet: Commercial antibodies targeting MPXV antigens (A35:40886-M0026; A29: 40891-M0036; M1R: 40904-T62; B6R: 40902-R007) were obtained from Sino Biological.

    Techniques: Isolation, In Vitro, Vaccines, Flow Cytometry, Software, Expressing, Enzyme-linked Immunosorbent Assay