Fig 1.
ELISA analysis of mAbs Gn3 and Gn32 using recombinant glycoprotein Gn as antigen.
Experiments were repeated three times.
Fig 2.
Detection of mAb using native indirect immunofluorescence assay and a 3D-model of Gn32 derived epitope.
Binding of glycoprotein Gn by monoclonal antibodies Gn3 (A) and Gn32 (B) on surface of MP-12 infected cells was visualized with Cy3-labeled secondary antibody. Mock infected cells (C). Cell nuclei were stained with DAPI (magnification 40x). (D) Epitope of Gn32 (displayed in yellow) is located on the surface of glycoprotein Gn (PDB 5Y0Y) in a spacefill 3D-model (Geneious Prime, Auckland, New Zealand, Version 2019.2.3).
Fig 3.
mAb Gn3 and Gn32 neutralize RVFV in two serum neutralization tests.
Data are shown as percentage neutralization and a regression model is fitted using 4-Parameter Logistic. (A) SNT using RVFV- strain MP-12. (B) SNT using RVFV- strain 35/74 Experiments were performed in duplicate.
Fig 4.
Survival curves and cumulative mean clinical scores of tested groups.
(A) Efficacy of antibody protection was assessed by survival analysis. (A) Kaplan-Meier log rank test was performed; n.s. between mAb-Gn3-treated (T1/T2) and PBS control, p<0.001 between mAb-Gn3-Gn32-treated (T1) and PBS control, p<0.001 between mAb-Gn3-Gn32-treated (T2) and PBS control, n.s. between mAb-Gn3-Gn32-treated (T1/T2) and Gn3 (T1/T2). (B) Clinical scores were monitored and recorded daily after infection. n.s. not significant.
Fig 5.
PCR results of brain, liver and cruor and ELISA results of serum from heart blood of individual mice.
(A) Virus loads were assessed by quantitative real-time RT-PCR with RVFV specific primers and quantified with a synthetic calibrator in samples from liver, brain and cruor. The results represent the virus loads [log10 copies (mg tissue)] of each group in a box blot diagram. Significance was analyzed by ANOVA (Kruskal-Wallis H) test (*p<0.05) (B) Antibodies in mice serum samples at day of death were determined by a commercial competition ELISA kit targeting antibodies against nucleocapsid protein. Results are shown as sample-to-positive ratio for each individual mouse. Horizontal line marks ELISA cut off.
Fig 6.
Histopathological and immunohistochemical findings in different tissues of three individual mice.
Light microscopy revealed three principally different patterns of lesions and antigen distribution in the livers (first row, A-F), brains (second row, G-L), spleens (third row, M-R), and lungs (fourth row, S-X) of mice which succumbed due to the infection within the first 6 days (left column: P18-865, mouse #25, 3 dpi, A, B, G, H, M, N, S, T) later in the time course of the disease (middle column: P18-870, mouse #30, 8 dpi, C, D, I, J, O, P, U, V) and those which survived until the end of the observation period (right column, P18-878, mouse #78, 13 dpi, E, F, K, L, W, X and P18-851, mouse #68, 13 dpi Q,R). A.) The mice dying until 6 dpi display mainly a severe, acute, diffuse, necrotizing hepatitis with Councilman corpuscles (arrows), suggestive of hepatocellular apoptosis. B.) These mice show coalescing to diffuse antigen within hepatocytes. M.) There are few apoptotic or necrotic lymphocytes in the white matter of the spleen (arrow). C.) The few mice dying later than 6 dpi display multifocal infiltrations of macrophages and lymphocytes (arrow) and hepatocellular vacuolar degeneration within the livers. D.) There is no antigen present within the liver. I.) There are oligofocal necrotic neurons and glia (arrows) within the brain, interpreted as necrotizing polioencephalitis. J.) There is multifocal, neuronal antigen accumulation. O.) There are many apoptotic or necrotic lymphocytes (arrow) leading to lymphatic depletion within the spleen. Q.) There is follicular hyperplasia with enlarged follicles with a pale lymphoblast-rich center and a darker peripheral zone of more differentiated lymphocytes. A, C, E, G, I, K, M, O, Q, S, U, W.) hematoxylin-eosin. B, D, F, H, J, L, N, P, R, T, V, X.) Immunohistochemistry employing the avidin-biotin-peroxidase-complex method for RVFV nucleoprotein with AEC as chromogen (red-brown) and hematoxylin counterstain (blue). A-X.) Bars = 50 μm.
Table 1.
The frequency distribution of the lesion patterns in the different experimental groups shows a trend to a more favourable outcome (pattern C) in the combined treatment groups.