Journal: PLoS Pathogens
Article Title: A cross-reactive antibody protects against Ross River virus musculoskeletal disease despite rapid neutralization escape in mice
doi: 10.1371/journal.ppat.1008743
Figure Lengend Snippet: ( A ) 293T cells were transfected with the RRV structural genes, stained using CHK-265 human IgG1, CHIKV immune IgG, or IgG isolated from healthy controls (healthy control IgG), and analyzed by flow cytometry. An anti-RRV mAb (RRV-130; 10 μg/ml) and human isotype control (WNV hE16; 10 μg/ml) were included as positive and negative controls, respectively. ( B ) Neutralization assay. Purified polyclonal CHIKV-immune IgG was preincubated with 10 2 FFU of RRV or CHIKV-LR then added to Vero cells for 20 or 18 h, respectively. Viral foci were counted and compared to wells without mAb to determine relative infection. Purified IgG from healthy individuals (healthy control IgG) was included as a negative control. ( C - E ) Three-week-old male and female WT mice were administered 300 μg of purified human CHIKV immune IgG or healthy control IgG one day prior to infection with 10 3 FFU RRV. ( C ) Weights were followed for 15 dpi. Graph shows mean ±SEM (n = 5–6 per group; two experiments; student’s t-test of AUC analysis: **** P < 0.0001). ( D ) Serum was collected 1 and 3 dpi and titered by qRT-PCR. ( E ) Viral RNA was quantified from spleen, ipsilateral (i.) and contralateral (c.) quadriceps muscles (quad), and ipsilateral and contralateral ankle at 15 dpi using qRT-PCR. ( D-E ) Bars represent the median (n = 5–6 per group; two experiments; Mann-Whitney test; * P < 0.05, ** P < 0.01). Colored circles indicate CHIKV immune IgG treated mouse with identified RRV variant at 3 dpi: orange circle: E2-K189Q mutation; green circle: E2-D214A mutation. ( F , G ) 293T cells were transfected with the WT, E2-D214A, or E2-K189Q RRV structural genes, stained with ( F ) CHK-265 human IgG1, anti-RRV mAb [RRV-130; 0.04 μg/ml (EC 80 )], human isotype control (WNV hE16), ( G ) CHIKV immune IgG, or healthy control IgG at 30 μg/ml unless otherwise noted, and analyzed by flow cytometry. Relative integrated mean fluorescence intensity (iMFI) was calculated by multiplying the percent positive cells and the median fluorescence intensity for each RRV variant and comparing it to the RRV WT control. Bars indicate mean (four experiments performed in duplicate; One-way ANOVA with a Holm-Sidak’s post-test comparing RRV variants to WT; * P < 0.05, **** P < 0.0001). Ab: antibody.
Article Snippet: Cells were fixed at 20 to 22 hpi and stained with mouse RRV immune ascites fluid (ATCC) followed by an anti-mouse IgG conjugated to HRP.
Techniques: Transfection, Staining, Isolation, Control, Flow Cytometry, Neutralization, Purification, Infection, Negative Control, Quantitative RT-PCR, Muscles, MANN-WHITNEY, Variant Assay, Mutagenesis, Fluorescence
ATCC is a verified supplier 
ATCC manufactures this product 
![( A ) 293T cells were transfected with the <t>RRV</t> structural genes, stained using CHK-265 human IgG1, CHIKV <t>immune</t> <t>IgG,</t> or IgG isolated from healthy controls (healthy control IgG), and analyzed by flow cytometry. An anti-RRV mAb (RRV-130; 10 μg/ml) and human isotype control (WNV hE16; 10 μg/ml) were included as positive and negative controls, respectively. ( B ) Neutralization assay. Purified polyclonal CHIKV-immune IgG was preincubated with 10 2 FFU of RRV or CHIKV-LR then added to Vero cells for 20 or 18 h, respectively. Viral foci were counted and compared to wells without mAb to determine relative infection. Purified IgG from healthy individuals (healthy control IgG) was included as a negative control. ( C - E ) Three-week-old male and female WT mice were administered 300 μg of purified human CHIKV immune IgG or healthy control IgG one day prior to infection with 10 3 FFU RRV. ( C ) Weights were followed for 15 dpi. Graph shows mean ±SEM (n = 5–6 per group; two experiments; student’s t-test of AUC analysis: **** P < 0.0001). ( D ) Serum was collected 1 and 3 dpi and titered by qRT-PCR. ( E ) Viral RNA was quantified from spleen, ipsilateral (i.) and contralateral (c.) quadriceps muscles (quad), and ipsilateral and contralateral ankle at 15 dpi using qRT-PCR. ( D-E ) Bars represent the median (n = 5–6 per group; two experiments; Mann-Whitney test; * P < 0.05, ** P < 0.01). Colored circles indicate CHIKV immune IgG treated mouse with identified RRV variant at 3 dpi: orange circle: E2-K189Q mutation; green circle: E2-D214A mutation. ( F , G ) 293T cells were transfected with the WT, E2-D214A, or E2-K189Q RRV structural genes, stained with ( F ) CHK-265 human IgG1, anti-RRV mAb [RRV-130; 0.04 μg/ml (EC 80 )], human isotype control (WNV hE16), ( G ) CHIKV immune IgG, or healthy control IgG at 30 μg/ml unless otherwise noted, and analyzed by flow cytometry. Relative integrated mean fluorescence intensity (iMFI) was calculated by multiplying the percent positive cells and the median fluorescence intensity for each RRV variant and comparing it to the RRV WT control. Bars indicate mean (four experiments performed in duplicate; One-way ANOVA with a Holm-Sidak’s post-test comparing RRV variants to WT; * P < 0.05, **** P < 0.0001). Ab: antibody.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_3899/pmc07433899/pmc07433899__ppat.1008743.g006.jpg)