Monoclonal antibodies protect aged rhesus macaques from SARS-CoV-2-induced immune activation and neuroinflammation

Abstract

Anti-viral monoclonal antibody (mAb) treatments may provide immediate but short-term immunity from coronavirus disease 2019 (COVID-19) in high-risk populations, such as people with diabetes and the elderly; however, data on their efficacy in these populations are limited. We demonstrate that prophylactic mAb treatment blocks viral replication in both the upper and lower respiratory tracts in aged, type 2 diabetic rhesus macaques. mAb infusion dramatically curtails severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-mediated stimulation of interferon-induced chemokines and T cell activation, significantly reducing development of interstitial pneumonia. Furthermore, mAb infusion significantly dampens the greater than 3-fold increase in SARS-CoV-2-induced effector CD4 T cell influx into the cerebrospinal fluid. Our data show that neutralizing mAbs administered preventatively to high-risk populations may mitigate the adverse inflammatory consequences of SARS-CoV-2 exposure.

Keywords: NeuroCOVID, inflammation; SARS-CoV-2; cerebrospinal fluid; effector CD4 T cells; interstitial pneumonia; lymph node; neuroinflammation; pathogenesis; rhesus macaques.

Graphical abstract

Figure 1

Monoclonal antibodies (mAbs) block SARS-CoV-2 replication in the upper respiratory tract, limit interstitial pneumonia, and prevent T cell activation (A) Study schematic: rhesus macaques were infused intravenously (i.v.) with RBD-specific C135 and C144 mAb (RBD mAb, n = 4) or HIV-specific 3BNC117 mAb (control mAb [C], n = 4) 3 days prior to SARS-CoV-2 (2.5 × 10⁶ PFUs) challenge. Nasal swabs (NSs), bronchoalveolar lavage (BAL) fluid (BALF), cerebrospinal fluid (CSF) taps, and blood collections were performed on indicated days. Necropsies were performed on day 7 post-SARS-CoV-2. (B) Kinetics of serum neutralizing titer 50% (NT₅₀) determined by SARS-CoV-2 pseudotyped neutralization assay. Data shown are average of two independent technical replicates. (C and D) Detection of SARS-CoV-2 subgenomic viral RNA (sgRNA), genomic RNA (gRNA), and viral RNA (vRNA) copies per NS (C) and BAL on indicated days during SARS-CoV-2 infection (D). Data in (C) and (D) are from single technical replicates. (E and F) Quantification of vRNA copies in right or left caudal lobes (E) and PFUs per lobe from lung biopsy samples (F). (G) Quantification of vRNA copies in mediastinal lymph nodes (Med LNs). Data in (E)–(G) are average of technical duplicates. (H) Representative immunofluorescence staining of caudal lung lobes from a control mAb-treated animal. Large arrows denote SARS-CoV-2 N protein, small arrows denote CD68⁺ pneumocytes, and ball/stick indicate CD3⁺ T cells. Scale bar (left) 100μm; (right) 25μm (I) Representative H&E images from the right caudal lung lobe harvested at necropsy, 7 days post-infection (pi) of (A) control mAb-treated animals illustrating region of moderately severe interstitial pneumonia (arrow) and perivascular inflammatory cell cuffing (asterisk). (B) RBD mAb-treated animals illustrating the lack of significant interstitial cellular infiltrates. Scale bar: 200 μm. An average of 16 H&E-stained sections of the right caudal lung lobe from all eight animals were examined for histological changes. (J) Representative flow diagram identifying Ki-67⁺PD-1⁺ populations among CD95⁺ CD4 and CD8 T cells (left) and their frequencies (right) in Med LNs. (K) Median fluorescence intensity (left) or frequency (right) of naive or Ki-67⁺PD-1⁺ CD4 T cells expressing CXCR3, ICOS, CD69, or CD28 from day 7 Med LNs of control mAb-treated animals. Flow data represent a single technical replicate. (L) Serum IgM against SARS-CoV-2 RBD and S2 from a single technical replicate. Data points represent individual animals, with bars indicating medians. Gray bars indicate limit of detection (C–G and L). ^∗p < 0.05, ^∗∗p < 0.01, by two-tailed Mann-Whitney U test.

Figure 2

mAbs limit SARS-CoV-2-induced systemic immune activation (A and B) Gating strategy and frequencies of (A) neutrophils (CD3/20⁻HLA-DR⁻CD66⁺) at days −3 and 0 (pre) versus days 3 and 7 (post) and (B) Ki-67⁺PD-1⁺ T_fh cells (CD95⁺CXCR5⁺) in whole blood (n = 4 in each group). (C) Gating strategy and frequency of CD8⁺CD69⁺ T cells expressing Ki-67, PD-1, or granzyme B (GzmB) in liver and spleen 7 days pi. (D) Heatmap displaying log2 normalized protein expression (NPX) values from plasma inflammatory proteins (top) and kinetics of NPX for plasma CXCL9, CXCL10, and CXCL11 (bottom). n = 4 for each experimental group/sample time point. (E) Serum levels of anti-nuclear antibodies (ANAs) and anti-phospholipid antibodies (aPLs) in aged and young animals on days 0 and 7 pi (young, n = 4; aged, n = 8). (F and G) Comparison of sgRNA and vRNA in (F) NSs and (G) BAL in aged and young animals on days 3 and 7 pi (young, n = 4; aged, n = 4). Data points represent individual animals, with bars indicating medians. Gray bars indicate limit of detection. Flow data and NPX data represent a single technical replicate. ^∗p < 0.05 by two-tailed (A and B) or one-tailed Mann-Whitney U test (C–G).

Figure 3

Immune activation in the CNS following mAb infusion (A) Heatmap displaying log2 median values of NPX of inflammatory proteins in the CSF of control mAb-treated animals on indicated days (n = 4 for each time point). Color intensity represents standard deviation from zero mean; all values were within −2 and +2 standard deviations from zero mean. (B) Comparison of pre-study/day −3 (median of pre-study and day −3) and day 7 NPX values of CXCL6, CXCL11, CX3CL1, MCP-4, CXCL10, CXCL11, CXCL19, and 4E-BP1 in the CSF of control and RBD mAb-treated animals. Data points represent individual animals, with bars indicating medians. NPX data represent a single technical replicate. Gray bars indicate limit of detection. ^∗p < 0.05 by one-tailed Mann-Whitney U test.

Figure 4

mAbs limit effector CD4 T cell influx in the CSF (A) Gating strategy and flow cytometry analysis of Ki-67 expression in CD28⁺CD95⁺ (central memory), CD28⁻CD95⁺ (effector memory), and CD28⁺CD95⁻ (naive) CD4 and CD8 T cell subsets in the CSF (green) and whole blood (red). (B) Frequency of Ki-67⁺ CD4 and CD8 T cells in the CSF. (C) Median fluorescent intensity (MFI) and frequency of Ki-67⁺/Ki-67⁻ CD4 T cell subsets expressing activation markers (CD25, CD69, PD-1, and TIGIT) in the CSF. (D) Spearman correlation shows frequencies of activated CD4 T cells in CSF correlate with activated CD4 T cells in mediastinal lymph node at day 7 (r = 0.85; p < 0.05; n = 8). Data points represent individual animals, with bars indicating medians. Flow data represent a single technical replicate. ^∗p < 0.05 by two-tailed Mann-Whitney U test (B).