Figure 5.

Antigenic specificity of CSF- and PBMC-derived monoclonal antibodies

(A) Bar graph depicting the frequency of the top five most expanded clones in PBMCs and CSF of affected individual 1.

(B) Graph depicting overlap of clones found in CSF and PBMCs of an affected individual. Green indicates clones only found in CSF, orange clones shared between the CSF and PBMC, and red clones unique to PBMCs. The yellow box indicates clones that would fall under the top 10 most frequent clones in each compartment.

(C) Heatmap showing CSF-derived (mAbs C1–C5) and PBMC-derived (mAbs P1–P3 and P5) mAb binding to nine peptides from immunogenic regions of S, N, and ORF3a as well as whole S and N protein along with the RBD of the S protein. mAb numbers correspond to the clone numbers from (A) and (B); PBMC clone 4 (mAb P4) did not express well as a mAb and was not used for subsequent studies. mAbs were screened in technical replicates. Heatmap values are mean fold change of the fluorescent anti-IgG antibody signal over intra-assay negative controls.

(D) Sagittal mouse brain sections were immunostained with mAbs 1–9, and a representative whole-brain sagittal image is shown for PBMC-derived mAbs (mAb 7) and CSF-derived mAbs (mAb 4). An anti-hemagglutinin (anti-HA) antibody in the same IgG1 backbone was used as a negative control. Scale bars, 500 μm.

(E) Select regions of immunostaining from mAbs 1–4. (i) mAb 1 immunostaining of cerebellar Purkinje cells (arrow) and the overlying molecular layer. (ii) mAb C2 immunostaining of cortical neuropil and occasional staining of neuron-like somata (arrow). (iii) mAb C3 immunostaining of large cells within the hilus of the hippocampus. (iv) mAb C4 immunostaining of mitral-like cells of the olfactory bulb (arrow). (v) mAb C4 immunostaining of pyramidal neurons (arrow) in CA3 of the hippocampus. (vi) mAb C4 immunostaining of neuronal cell bodies in layer II of the cortex (arrow). Scale bars, 10 μm.