Journal: medRxiv

Article Title: Cross-binding antibodies capable of neutralizing diverse orthohantaviruses are produced in response to Puumala virus infection

doi: 10.1101/2025.03.04.25323331

Figure Lengend Snippet: Following an incubation period of 1-6 weeks, HFRS symptoms manifest as five distinct phases: febrile, hypotensive, oliguric, polyuric, and convalescent. The average time for which each stage lasts is shown, in addition to the symptoms associated with each phase. Serum creatinine, a measure of kidney injury, is displayed, as is urine output as a marker of kidney function. IgM and IgG titers are also shown. The four time points at which patient sera were sampled are also shown in relation to each disease phase. Time point 1 (T1) was sampled six days post-symptom onset (PSO), range: 2-11 days PSO). Time point 2 (T2) was sampled 12 days PSO (range: 8-20 days PSO). Time point 3 (T3) was sampled 28 PSO (range: 21-64 days PSO). Time point 4 (T4) was sampled 197 days PSO (range: 180-256 days PSO). Adapted from Avsic-Zupanc et al. . Created in BioRender. Clark, J. (2025) https://BioRender.com/p36u208

Article Snippet: When assaying other immunoglobulin subtypes, anti-human IgA HRP (Sigma-Aldrich, A0295, 1:3000), anti-human IgM HRP (SouthernBiotech, 2020-05, 1:3000), anti-human IgG1 (Southern Biotech, 9054-05, 1:5000), anti-human IgG2 (Southern Biotech, 9060-05,1:5000), anti-human IgG3 (Southern Biotech, 9210-0, 1:5000), or anti-human IgG4 (Southern Biotech, 9200-05, 1:5000) were used.

Techniques: Incubation, Marker

Journal: medRxiv

Article Title: Cross-binding antibodies capable of neutralizing diverse orthohantaviruses are produced in response to Puumala virus infection

doi: 10.1101/2025.03.04.25323331

Figure Lengend Snippet: ELISAs were carried out using patient sera and plates coated with ultracentrifuge purified rVSV expressing the GnGc of A) PUUV, B) SEOV, C) DOBV, D) HTNV, E) SNV, or F) ANDV. Wild-type VSV G) was used as a negative control. IgG titers are shown as area under the curve (AUC) with the geometric mean and geometric standard deviation denoted by error bars. 11 age and sex matched healthy control sera were included as negative controls and an in-house, anti-PUUV monoclonal antibody was used as a positive control. AUC values were log10 transformed and statistical significance was interrogated via ordinary one-way ANOVA using a Tukey’s multiple comparisons test. Only comparisons with p ≤ 0.05 are shown. Limits of detection (LOD) were calculated for each antigen using the average + 3x standard deviations of the AUC of the negative control sera and are shown as dotted lines on each graph. The percentage of serum samples with LODs above the limit of detection are shown as pie charts, with the percentage of positive samples displayed in green. The n for each time point is displayed below each pie chart.

Article Snippet: When assaying other immunoglobulin subtypes, anti-human IgA HRP (Sigma-Aldrich, A0295, 1:3000), anti-human IgM HRP (SouthernBiotech, 2020-05, 1:3000), anti-human IgG1 (Southern Biotech, 9054-05, 1:5000), anti-human IgG2 (Southern Biotech, 9060-05,1:5000), anti-human IgG3 (Southern Biotech, 9210-0, 1:5000), or anti-human IgG4 (Southern Biotech, 9200-05, 1:5000) were used.

Techniques: Purification, Expressing, Negative Control, Standard Deviation, Control, Positive Control, Transformation Assay

Journal: medRxiv

Article Title: Cross-binding antibodies capable of neutralizing diverse orthohantaviruses are produced in response to Puumala virus infection

doi: 10.1101/2025.03.04.25323331

Figure Lengend Snippet: ELISAs were carried out using patient sera and plates coated with recombinant truncated, Gn of A) PUUV and B) SEOV. IgG titers are shown as area under the curve (AUC) with the geometric mean and geometric standard deviation denoted by error bars. 11 age and sex matched healthy control sera were included as negative controls and an in-house, anti-PUUV monoclonal antibody was used as a positive control. AUC values were log10 transformed and statistical significance was interrogated via ordinary one-way ANOVA using a Tukey’s multiple comparisons test. Only comparisons with p ≤ 0.05 are shown. Limits of detection (LOD) were calculated for each antigen using the average + 3x standard deviations of the AUC of the negative control sera and are shown as dotted lines on each graph. The percentage of serum samples with LODs above the limit of detection are shown as pie charts, with the percentage of positive samples displayed in green. The n for each time point is displayed below each pie chart.

Article Snippet: When assaying other immunoglobulin subtypes, anti-human IgA HRP (Sigma-Aldrich, A0295, 1:3000), anti-human IgM HRP (SouthernBiotech, 2020-05, 1:3000), anti-human IgG1 (Southern Biotech, 9054-05, 1:5000), anti-human IgG2 (Southern Biotech, 9060-05,1:5000), anti-human IgG3 (Southern Biotech, 9210-0, 1:5000), or anti-human IgG4 (Southern Biotech, 9200-05, 1:5000) were used.

Techniques: Recombinant, Standard Deviation, Control, Positive Control, Transformation Assay, Negative Control

Journal: medRxiv

Article Title: Cross-binding antibodies capable of neutralizing diverse orthohantaviruses are produced in response to Puumala virus infection

doi: 10.1101/2025.03.04.25323331

Figure Lengend Snippet: ELISAs were carried out using patient sera to investigate A) anti-PUUV IgM, B) anti-PUUV IgA, C) anti-SEOV IgM, and D) anti-SEOV IgA. IgG subtype ELISAs were performed to investigate the quantities of anti-PUUV E) IgG1, F) IgG2, G) IgG3, or H) IgG4. I) A schematic of the luciferase-based reporter assay, which was utilized to characterize the effector activity promoted by patient sera. RVSV-PUUV infected Huh.75 cells were incubated with patient sera and reporter cells that express the FcγRIIIa receptor, coupled to a gene expression pathway that results in the production of luciferase. J ) The FcγRIIIa activity of the sera re shown as AUCs, with the mean and standard deviation denoted by error bars. 11 age and sex matched healthy control sera were included as negative controls, and an in-house, anti-PUUV monoclonal antibody was used as a positive control. AUC values were log10 transformed and statistical significance was interrogated via ordinary one-way ANOVA using a Tukey’s multiple comparisons test. Only comparisons with p ≤ 0.05 are shown. Limits of detection (LOD) are shown as dotted lines on each graph. The percentage of serum samples with LODs above the limit of detection are shown as pie charts with the percentage of positive samples displayed in green. The n for each time point is displayed below each pie chart.

Article Snippet: When assaying other immunoglobulin subtypes, anti-human IgA HRP (Sigma-Aldrich, A0295, 1:3000), anti-human IgM HRP (SouthernBiotech, 2020-05, 1:3000), anti-human IgG1 (Southern Biotech, 9054-05, 1:5000), anti-human IgG2 (Southern Biotech, 9060-05,1:5000), anti-human IgG3 (Southern Biotech, 9210-0, 1:5000), or anti-human IgG4 (Southern Biotech, 9200-05, 1:5000) were used.

Techniques: Luciferase, Reporter Assay, Activity Assay, Infection, Incubation, Gene Expression, Standard Deviation, Control, Positive Control, Transformation Assay