Monoclonal antibody therapy protects nonhuman primates against mucosal exposure to Lassa virus

Abstract

Lassa fever (LF) is an acute viral illness that causes thousands of deaths annually in West Africa. There are currently no Lassa virus (LASV) vaccines or antivirals approved for human use. Recently, we showed that combinations of broadly neutralizing human monoclonal antibodies (BNhuMAbs) known as Arevirumab-2 or Arevirumab-3 protected up to 100% of cynomolgus macaques against challenge with diverse lineages of LASV when treatment was initiated at advanced stages of disease. This previous work assessed efficacy against parenteral exposure. However, transmission of LASV to humans occurs primarily by mucosal exposure to virus shed from Mastomys rodents. Here, we describe the development of a lethal intranasal exposure macaque model of LF. This model is employed to show that Arevirumab cocktails rescue 100% of macaques from lethal LASV infection when treatment is initiated 8 days after LASV exposure. Our work demonstrates BNhuMAbs have utility in treating LASV infection acquired through mucosal exposure.

Keywords: Arenavirus; Lassa virus; animal model; antibody; hemorrhagic fever; intranasal; monoclonal; mucosal; neutralizing; nonhuman primate.

Graphical abstract

Figure 1

Survival analysis, clinical scoring, virus replication kinetics, and tissue viral load in macaques challenged by MAD system with LASV lineage II strain 0043/LV/14 (A) Kaplan-Meier survival curves for LASV-challenged macaques. Differences in curves were tested by the Mantel-Cox log rank test. (B) Clinical scoring for macaques challenged with LASV. The dashed line denotes the minimum clinical score by which humane euthanasia criteria were met. (C and D) Viral load was determined by RT-qPCR of vRNA from whole blood (C) or plaque titration of plasma (D) collected at predetermined time points. For both panels, individual data points represent the mean of two technical replicates. To fit on a log-scale axis, zero values (below limit of quantitation [LOQ]) are plotted as “1” (10⁰). (E) Tissue LASV vRNA load from macaques challenged with LASV. For (C), (D), and (E), dashed horizontal lines indicate the LOQ for the assay (1,000 GEq/mL or GEq/g for RT-qPCR; 25 PFU/mL for plaque titration). To fit on a log-scale axis, zero values (below LOQ) are plotted as “1” (10⁰). For (D) and (E), bars represent the mean for the cohort, and symbols within bars represent the mean of two technical replicate assays for each individual animal (for viral quantitation assays only). Error bars represent ± SD. For all relevant panels, reported p values are two-tailed. Abbreviations for tissues are as follows: ALN, axillary lymph node; ILN, inguinal lymph node; Liv, liver; Spl, spleen; Kid, kidney; Adr, adrenal gland; RUL, right upper lung; RML, right middle lung; RLL, right lower lung; LUL, left upper lung; LML, left middle lung; LLL, left lower lung; BrFr, brain frontal cortex; BrSt, brain stem; CSC, cervical spinal cord; MnLN, mandibular lymph node; sMnLN, submandibular lymph node; Ton, tonsil; Hrt, heart; MsLN, mesenteric lymph node; Duo, duodenum; Pan, pancreas; TrCol, transverse colon; Ile, ileum; Uri, urinary bladder; Gon, gonad; Nas, nasal mucosa; Ut/Pro, uterus/prostate; Conj, conjunctiva. See also Table S1.

Figure 2

Survival analysis and virus replication kinetics in macaques challenged with LASV lineage II strain 0043/LV/14 and treated with Arevirumab-2 or Arevirumab-3 (A) Kaplan-Meier survival curves for LASV-challenged macaques. The curve for the in-study untreated controls animals (C-5–C-7) is shown separately from the curve for the additional identically challenged controls from the initial model development study (n = 4) used for analysis; however, for statistical comparison, all untreated controls were grouped together (n = 7). Differences in curves were tested by the Mantel-Cox log rank test and corrected for multiple comparisons using the Holm-Šídák method. The proportion of survivors was compared using Fisher’s exact test and corrected for multiple comparisons using the Hochberg method. (B–E) Viral load was determined by RT-qPCR of RNA from whole blood (B, D) or plaque titration of plasma (C, E) collected at predetermined time points. (B, D) LASV RNA abundance from macaques treated with Arevirumab-2 (B) or Arevirumab-3 (D). (C, E) Plasma plaque assay titers from macaques treated with Arevirumab-2 (C) or Arevirumab-3 (E). For (B)–(E), individual data points represent the mean of two technical replicates, except for grouped untreated controls, which represent the mean for the cohort ±SD. Dashed horizontal lines indicate the limit of quantitation (LOQ) for the assay (1,000 GEq/mL for RT-qPCR; 25 PFU/mL for plaque titration). To fit on a log-scale axis, zero values (below LOQ) are plotted as “1” (10⁰). For all relevant panels, reported p values are two-tailed. (F) Tissue LASV RNA load from macaques challenged with LASV and treated with either Arevirumab-2 or Arevirumab-3. Values for individual animals (circles within bars) are plotted as the mean of two technical replicate assays; bars represent the mean for the cohort ±SD. Significance was determined Mann-Whitney U-tests correcting for multiple comparisons. ∗p = 0.013; ∗∗p = 0.002. Abbreviations for tissues are as follows: ALN, axillary lymph node; ILN, inguinal lymph node; Liv, liver; Spl, spleen; Kid, kidney; Adr, adrenal gland; RUL, right upper lung; RML, right middle lung; RLL, right lower lung; LUL, left upper lung; LML, left middle lung; LLL, left lower lung; BrFr, brain frontal cortex; BrSt, brain stem; CSC, cervical spinal cord; MnLN, mandibular lymph node; sMnLN, submandibular lymph node; Ton, tonsil; Hrt, heart; MsLN, mesenteric lymph node; Duo, duodenum; Pan, pancreas; TrCol, transverse colon; Ile, ileum; Uri, urinary bladder; Gon, gonad; Nas, nasal mucosa; Ut/Pro, uterus/prostate; Conj, conjunctiva. See also Table S2.

Figure 3

Representative gross, histopathology, and immunohistochemistry for anti-LASV antigen of macaques challenged with LASV 0043/LV/14 (A) Necrotizing hepatitis of an untreated positive control (C-4). (B) Liver of a survivor (Tx-5), no significant lesions, H&E 40×. (C) Liver of survivor (Tx-10), no significant lesions, H&E (40×). (D) Liver of a positive control (C-6) showing hepatitis (black arrow). (E) Liver of a positive control (C-6) showing LASV antigen-positive (brown) hepatocytes (black arrow), sinusoidal lining cells, and endothelium (white arrow) (C-6), IHC (40×). (F) Liver of a survivor (Tx-5) showing no significant immunolabeling, IHC (40×). (G) Liver of a survivor (Tx-10) showing no significant immunolabeling, IHC (40×). (H) Spleen of a positive control (C-6) showing splenitis with tingible body macrophages (black arrow), H&E (20×). (I) Spleen of a positive control showing LASV antigen-positive (brown) mononuclear cells within the red and white pulp (C-6), IHC (20×). (J) Spleen of a survivor (Tx-5) showing no significant immunolabeling, IHC (20×). (K) Spleen of a survivor (Tx-10) showing no significant immunolabeling, IHC (20×). (L) Positive control (C-2) with hemorrhagic interstitial pneumonia. (M) Lung of a positive control (C-6) showing LASV antigen-positive (brown) alveolar macrophages (black arrow), alveolar septal macrophages, and endothelium (white arrow), IHC (20×). (N) Lung of a survivor (Tx-5) showing no significant immunolabeling, IHC (20×). (O) Lung of survivor (Tx-2) showing no significant immunolabeling, IHC (20×). (P) Kidney of a positive control (C-6) with nephritis (black arrow), H&E (20×). (Q) Kidney of a positive control (C-6) showing LASV antigen-positive (brown) tubular epithelium (black arrow), interstitial mononuclear cells, and endothelium of glomerular tuffs and large caliber vessels (white arrows), IHC (20×). (R) Kidney of a survivor (Tx-5) showing LASV antigen-positive (brown) mononuclear cells within the renal medullary interstitium (black arrow), IHC (20×). (S) Kidney of a survivor (Tx-5) showing LASV antigen-positive (brown) mononuclear cells and endothelium within the glomerular tuffs (black arrow), IHC (20×). (T) Brain of a positive control (C-6) showing lymphocytic perivascular cuffs and glial nodule (black arrow), H&E (20×). (U) Brain of a positive control (C-6) with LASV antigen-positive (brown) glial nodule (black arrow) and endothelium (white arrow), IHC (20×). (V) Brain of a survivor (Tx-5) with LASV antigen-positive (brown) positive mononuclear cells in a perivascular cuff (white arrow), IHC (60 ×). (W) Brain of a survivor (Tx-2) with LASV antigen-positive (brown) positive mononuclear cells in a perivascular cuff (white arrow), IHC (60 ×). Scale bars in all panels represent 100 μm.

Figure 4

Blood transcriptional profiling of NHPs exposed to LASV and treated with Arevirumab (A) Venn diagram depicting overlapping DE transcripts in each group (Control [N = 7]; Treated [N = 10]) at each day p.i. (B) Heatmap depicting the most significant DE transcripts in the blood of LASV-infected NHPs at 11 and 14 days p.i. (multiple hypothesis Benjamini­Hochberg false discovery rate [FDR] corrected p value less than 0.05). Red indicates upregulated transcripts; blue indicates downregulated transcripts; white indicates no change from baseline. (C) Immune cell type profiling based on transcriptional changes in treated groups versus the control cohort at 11 days p.i. (top plot) and 14 days p.i. (bottom plot). Subjects were treated with Arevirumab-2 (N = 5), Arevirumab-3 (N = 5), or a vehicle control (N = 7). Abbreviations: p.i., post infection.

Figure 5

Pathway analysis of blood transcriptomes in treated versus control NHPs exposed to LASV and treated with Arevirumab Topmost downregulated (A and C) and upregulated (B and D) pathways in Arevirumab-2 (N = 5)- and Arevirumab-3 (N = 5)-treated versus control (N = 7) subjects at 11 (A and B) and 14 days (C and D) p.i.. Any differentially expressed transcripts with a Benjamini-Hochberg adjusted p value less than 0.05 were deemed significant for pathway enrichment. Pathways are sorted by Z score. Red indicates upregulated pathways; blue indicates downregulated pathways; white indicates no change. Abbreviations: p.i., post infection.

Figure 6

Transcriptional analysis of brain tissue from NHPs exposed to LASV and treated with Arevirumab or a vehicle control (A) Principal component analysis (PCA) of all normalized transcripts to visualize the relatedness of brain samples via dimensional reduction. Each dot represents an individual brain RNA sample. We compared treated subjects with lymphohistiocytic perivascular infiltrates (N = 7; Tx-1, Tx-2, Tx-3, Tx-5, Tx-6, Tx-8, and Tx-10) and those without lesions (N = 3; Tx-4, Tx-7, and Tx-9) versus control (N = 7; C-1, C-2, C-3, C-4, C-5, C-6, and C-7) and uninfected (N = 6) subjects. Frontal brain tissue was collected at the terminal or study endpoint. (B) Volcano plot displaying overall -logl0(p values) and log2 fold changes for each mRNA target for treated subjects with and without detectable infiltrates. The target highlighted in red indicates a single DE transcript with an adjusted p value < 0.05 and > 1.5 log2 fold change. (C) Venn diagram depicting overlapping DE transcripts in each group (Control [N = 7]; Treated infiltrates [N = 5]; Treated no lesions [N = 5]). (D) Heatmap depicting the most significant DE transcripts in the brain of LASV-infected NHPs at the terminal or study endpoint (multiple hypothesis Benjamini-Hochberg false discovery rate (FDR) corrected p value less than 0.05). Red indicates upregulated transcripts; blue indicates downregulated transcripts; white indicates no change from baseline. (E) Immune cell type profiling based on brain-specific transcriptional changes in each group. Abbreviations: PC1, principal component 1; PC2, principal component 2.

Figure 7

Circulating cytokine and chemokine profiling of Arevirumab-2 and Arevirumab-3 treated and untreated cynomolgus macaques Heatmap depicting fold changes in expression of pro-inflammatory mediators and markers in plasma of treated and untreated LASV-infected NHPs as compared to baseline. Red indicates increased expression, blue indicates decreased expression. Timepoints, individual animals, and groups are also depicted by color scales.