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. 2009 Oct;5(10):e1000642.
doi: 10.1371/journal.ppat.1000642. Epub 2009 Oct 30.

A neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection

Katharine N Bossart  1 , Zhongyu Zhu, Deborah Middleton, Jessica Klippel, Gary Crameri, John Bingham, Jennifer A McEachern, Diane Green, Timothy J Hancock, Yee-Peng Chan, Andrew C Hickey, Dimiter S Dimitrov, Lin-Fa Wang, Christopher C Broder
Affiliations

A neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection

Katharine N Bossart et al. PLoS Pathog. 2009 Oct.

Abstract

Nipah virus is a broadly tropic and highly pathogenic zoonotic paramyxovirus in the genus Henipavirus whose natural reservoirs are several species of Pteropus fruit bats. Nipah virus has repeatedly caused outbreaks over the past decade associated with a severe and often fatal disease in humans and animals. Here, a new ferret model of Nipah virus pathogenesis is described where both respiratory and neurological disease are present in infected animals. Severe disease occurs with viral doses as low as 500 TCID(50) within 6 to 10 days following infection. The underlying pathology seen in the ferret closely resembles that seen in Nipah virus infected humans, characterized as a widespread multisystemic vasculitis, with virus replicating in highly vascular tissues including lung, spleen and brain, with recoverable virus from a variety of tissues. Using this ferret model a cross-reactive neutralizing human monoclonal antibody, m102.4, _targeting the henipavirus G glycoprotein was evaluated in vivo as a potential therapeutic agent. All ferrets that received m102.4 ten hours following a high dose oral-nasal Nipah virus challenge were protected from disease while all controls died. This study is the first successful post-exposure passive antibody therapy for Nipah virus using a human monoclonal antibody.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. NiV in ferret tissues.
Immunohistochemistry was done using a rabbit antiserum against Nipah virus nucleoprotein with haematoxylin counterstain as previously described . (A) ferret 7–50000, lung tissue with severe acute necrotizing alveolitis and vasculitis. Antigen present in syncytia (long arrow) and blood vessel wall (short arrow). (B) ferret 4–500, antigen staining in arachnoid membrane of the meninges, with non-suppurative inflammation. (C) ferret 7–50000, kidney with antigen staining in necrotic glomerular tuft and tubular epithelium; syncytia present in the epithelium of Bowman's capsule (arrow). (D) ferret 4–500, inflammation of the peri-tracheal tissues. Antigen present in blood vessel walls, syncytial cell (arrow). Scale bars = 50 µm.
Figure 2
Figure 2. NiV RNA and virus isolation from ferrets post-challenge.
Samples were collected in duplicate, RNA was extracted from one replicate and assayed using Taqman PCR detecting NiV nucleoprotein (N) RNA . Samples were assayed in triplicate. Ct values were analyzed against a known NiV cDNA control and results are shown as the average relative NiV N gene expression (Y-axis). Error bars represent the standard deviation. Day of sampling or tissue is indicated (X-axis). Ferret # and NiV dose (TCID50) are indicated (inset). (A) Blood, pharyngeal swabs, rectal swabs. (B) Vascular and lymphoid tissues (top panel), other tissues (bottom panel); diaph: diaphragmatic; bron-LN: bronchial-lymph node; occ: occipital; olf: olfactory. For tissue samples with relative NiV N gene expression above 10, virus isolation was performed on duplicate samples. The presence of infectious virus is indicated by the (+) above individual data bars. Virus isolation was not attempted on blood or swabs.
Figure 3
Figure 3. Gross pathology of the lung post-mortem in ferrets treated with m102.4 prior to NiV challenge.
(A) Ferret 21-pre; 20 days post infection (dpi): no signs of illness, disease or lesions: healthy ferret lungs. (B) Ferret 23-pre; 13 dpi: delayed disease onset, scattered small pinpoint lesions. (C) Ferret 24-pre; 13 dpi: delayed disease onset, scattered small pinpoint lesions. (D) NiV-infected control ferret; 8 dpi: typical disease onset, extensive pinpoint lesions.
Figure 4
Figure 4. Immunohistochemistry demonstrating NiV antigen in brain tissue in ferrets treated with m102.4 prior to NiV challenge.
(A) Ferret 23-pre; 13 dpi: viral antigen in ependymal epithelium of the lateral ventricle, associated with focal hemorrhage. (B) Ferret 24-pre; 13 dpi: single focus of viral antigen in the cerebellum. Scale bar = 50 µm.
Figure 5
Figure 5. Viral loads in ferrets from m102.4 efficacy trial.
All samples were collected in duplicate. RNA was extracted from one replicate and assayed using Taqman PCR assay detecting NiV nucleoprotein (N) RNA. RNA samples were assayed in triplicate. The Ct values were analyzed against a NiV cDNA control and results are shown as the average relative NiV N gene expression (Y-axis). Error bars represent the standard deviation. Day of sampling or tissue sample is indicated on the X-axis. Ferret # and treatment group (control (con), pre- or post-challenge) are indicated (inset). (A) Blood, pharyngeal swabs, rectal swabs. (B) Tissues from diseased ferrets (top panel) or healthy ferrets (bottom panel); diaph: diaphragmatic; bron-LN: bronchial-lymph node; occ: occipital; olf: olfactory. Virus isolation was attempted from duplicate samples where relative NiV N gene expression levels were above 10. The presence of infectious virus is indicated by the (+) above individual data bars.
Figure 6
Figure 6. In vivo m102.4 concentrations in ferrets following NiV challenge.
Blood was collected at various time points post-challenge (X-axis). Serum was collected and frozen at −80°C until the completion of the study. All sera were γ-irradiated to facilitate removal from the BSL4 laboratory. Sera was diluted and assayed using a NiV G-specific microsphere assay described previously ,. An m102.4 standard curve ranging from 500 ng/ml to 0.5 ng/ml and all sera samples were assayed in duplicate simultaneously. Serum m102.4 concentrations were extrapolated from the standard curve using non-linear regression analysis and results are shown as the average concentration. Error bars represent the standard deviation. Ferret # and treatment group (pre- or post-challenge) are indicated (inset). (A) m102.4 administered 24 hrs pre-challenge. (B) m102.4 administered ten hours after post-challenge.
Figure 7
Figure 7. Ferret immune responses to NiV G.
Blood was collected at various time points post-challenge, as indicated by the X-axis. Serum was collected and frozen at −80°C until the completion of the study. All sera were γ-irradiated to facilitate removal from the BSL-4 laboratory. Ferret sera were diluted and assayed using the NiV G-specific microsphere assay described previously . Ferret # and corresponding treatment group (control (con), pre- or post-challenge) are indicated in the legend. Median fluorescence intensities (M.F.I.) are shown on the Y-axis. All assays were performed in duplicate and the mean M.F.I. is shown. Error bars represent the range of M.F.I. for each sample.
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