Research ArticleInfectious Disease

A Neutralizing Human Monoclonal Antibody Protects African Green Monkeys from Hendra Virus Challenge

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Science Translational Medicine  19 Oct 2011:
Vol. 3, Issue 105, pp. 105ra103
DOI: 10.1126/scitranslmed.3002901

Abstract

Hendra virus (HeV) is a recently emerged zoonotic paramyxovirus that can cause a severe and often fatal disease in horses and humans. HeV is categorized as a biosafety level 4 agent, which has made the development of animal models and testing of potential therapeutics and vaccines challenging. Infection of African green monkeys (AGMs) with HeV was recently demonstrated, and disease mirrored fatal HeV infection in humans, manifesting as a multisystemic vasculitis with widespread virus replication in vascular tissues and severe pathologic manifestations in the lung, spleen, and brain. Here, we demonstrate that m102.4, a potent HeV-neutralizing human monoclonal antibody (hmAb), can protect AGMs from disease after infection with HeV. Fourteen AGMs were challenged intratracheally with a lethal dose of HeV, and 12 subjects were infused twice with a 100-mg dose of m102.4 beginning at either 10, 24, or 72 hours after infection and again about 48 hours later. The presence of viral RNA, infectious virus, and HeV-specific immune responses demonstrated that all subjects were infected after challenge. All 12 AGMs that received m102.4 survived infection, whereas the untreated control subjects succumbed to disease on day 8 after infection. Animals in the 72-hour treatment group exhibited neurological signs of disease, but all animals started to recover by day 16 after infection. These results represent successful postexposure in vivo efficacy by an investigational drug against HeV and highlight the potential impact a hmAb can have on human disease.

Introduction

In the middle to late 1990s, two new paramyxoviruses capable of causing severe lethal disease in both animals and humans were identified: Hendra virus (HeV) and Nipah virus (NiV). The first two outbreaks of HeV occurred in Queensland, Australia, in 1994 and were associated with fatalities in horses and humans. In total, 15 horses and two of three infected humans succumbed to fatal HeV disease (1). Infection manifested as a severe respiratory disease in horses; in humans, one fatality was associated with respiratory failure, whereas the other developed encephalitic complications that manifested some 13 months after recovering from a mild meningitic illness that was later found to have been caused by HeV. NiV appeared a few years later in peninsular Malaysia in 1998, causing a widespread outbreak among farmed pigs along with numerous cases of human infection. By mid-1999, more than 265 human cases of encephalitis, including 105 deaths, had been reported in Malaysia, and 11 cases of either encephalitis or respiratory illness with 1 fatality were reported in Singapore (1). More than 1 million pigs were culled to control the disease outbreak, which caused significant economic and social impacts, which are still felt to this day. Upon further biological, molecular, and serological characterization, HeV and NiV were discovered to be closely related viruses that had emerged independently and are now grouped together in the new genus Henipavirus (1, 2), and both are classified as select viral agents in the United States by the Centers for Disease Control and Prevention and require biosafety level 4 (BSL-4) containment worldwide.

Pteropus fruit bats, commonly known as flying foxes, are the natural reservoirs for both viruses. As a group, they are wide-ranging and can be found throughout the Asia-Pacific, as far west as Africa, and as far north as India, Pakistan, and the Philippines (3, 4). The persistence of HeV and NiV in an animal reservoir, their broad species tropism (5), and the severe disease they cause in a wide variety of mammalian hosts including humans distinguish them from all other known paramyxoviruses. NiV outbreaks have occurred nearly every year since its initial discovery (69), and in all outbreaks, severe disease in humans has occurred with fatality rates ranging from 40 to 75%. Notably, from 2001 to 2007, more than half of the identified NiV cases resulted from person-to-person transmission (7). Conversely, HeV has appeared more sporadically in Australia since its initial emergence, with horse fatalities recorded in 1999, 2004, and 2006 and one mildly ill, seroconverting, human case reported in 2004 (10, 11). However, since 2006, HeV has appeared in horses annually along with two severe human cases: one fatality in 2008 and another in 2009 (12, 13). A spillover of HeV occurred in May 2010 (14), with one horse fatality and 11 humans with potential virus exposure (15). Just before the 2010 episode, unusual large-scale flying fox movements were reported and a HeV warning was issued (16). Most recently, there has been a flurry of HeV spillover in Queensland and New South Wales, Australia, which began on June 20, 2011 (17). Seventeen separate occurrences have been reported as of August 30, 2011, including numerous horse deaths and cases of human exposure with multiple properties quarantined and under surveillance.

Currently, there are no approved vaccines or therapeutics against HeV or NiV (18). However, in the 2010 HeV outbreak, an experimental human monoclonal antibody (hmAb) was used to treat two individuals who had a significant exposure risk (19). To date, both of these individuals have no evidence of HeV infection. The experimental hmAb, m102.4, which targets the ephrin-B2 and ephrin-B3 receptor binding domain of the henipavirus G envelope glycoprotein (2023), is a potent cross-reactive neutralizing antibody in vitro (24, 25) and had been shown capable of protecting ferrets from lethal NiV challenge (26). Recently, we reported on the development of successful nonhuman primate (NHP) models of NiV and HeV infection and disease in the African green monkey (AGM) (27, 28). For both viruses, infection of AGMs is uniformly lethal and disease essentially mirrors the severe clinical symptoms seen in humans, with widespread systemic vasculitis and disease manifestations observed in multiple organ systems. The lungs and brain were the main targets of infection, and development of clinical signs was directly associated with damage of these organs. Here, we report the pharmacokinetic characteristics and efficacy of hmAb m102.4 using the lethal HeV infection AGM model. We present data demonstrating that m102.4 is highly efficacious in preventing lethal disease in HeV-infected AGMs when administered 10, 24, or 72 hours after virus challenge. The exceptional potency and half-life of m102.4 in AGMs demonstrate its tremendous postexposure therapeutic potential for future use in humans. Indeed, the recent decision to administer m102.4 to two humans with high-risk HeV exposure (19) was significantly influenced by some elements of the data now reported here.

Results

In vivo pharmacokinetics of hmAb m102.4 in AGMs

The hmAb m102.4 was originally isolated from a bacteriophage recombinant antibody library derived from a pool of naïve human peripheral blood mononuclear cells (24). Biochemical analysis indicated that m102.4 bound the receptor binding domain of the HeV and NiV attachment glycoprotein (24). After conversion to an immunoglobulin G1 (IgG1) format, m102.4 demonstrated exceptional cross-reactive neutralizing capability in vitro (25) and could also completely protect ferrets from lethal NiV-mediated disease when administered 10 hours after virus challenge (26). The first objective of the present study was to determine the half-life of m102.4 in AGMs. Because AGMs are about two to three times the weight of ferrets, absolute doses of the antibody were doubled compared to what was done previously (26); however, the approximate dose per kilogram was similar. Four subjects were given m102.4 intravenously, either 50 mg (AGM 1 and AGM 2) or 10 mg (AGM 3 and AGM 4) per subject. The subjects were monitored physically for respiration, allergic reaction, eating and drinking abnormalities, and lethargy, both during and after the antibody infusion and no adverse reactions were observed in any subject. Serum was collected at various time points after infusion, and m102.4 concentrations were determined as previously described (26). As indicated in Table 1, the serum levels of m102.4 were above 0.5 mg/ml in the two high-dose subjects and about 0.1 mg/ml in the low-dose subjects immediately after antibody infusion. Serum m102.4 concentration decreased over time, and concentrations correlated with antibody dosage. On day 12, the high-dose subjects had serum m102.4 of >50 μg/ml, and the low-dose subjects had serum m102.4 of >10 μg/ml. A distribution half-time (t11/2) of ~1 day and the elimination half-time (t21/2) of ~11 days of m102.4 were calculated and are typical for human IgG in NHPs (29). The area under the curve (AUC), which is a measure for the exposure to the hmAb, is also shown. As demonstrated, the average AUC values for the two treatment groups are about fivefold different, reflecting the difference in the m102.4 dose.

Table 1

m102.4 concentrations and half-life in AGMs. t11/2, distribution half-life time; t21/2, elimination half-life time; AUC, area under the curve.

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Postexposure protection of AGMs from lethal HeV-mediated disease by m102.4

Previously, we demonstrated that intratracheal inoculation of AGMs with 4 × 105 TCID50 (median tissue culture infectious dose) of HeV caused a uniformly lethal outcome (28). Rapidly progressive clinical illness was noted in these studies; clinical signs included severe depression, respiratory disease leading to acute respiratory distress, and severely reduced mobility; and time to reach approved humane endpoint criteria for euthanasia ranged from 7 to 9 days. Here, we sought to evaluate the therapeutic benefit of m102.4 using this HeV AGM model. The first m102.4 efficacy study conducted in ferrets demonstrated complete protection against a lethal NiV challenge when a single 50-mg dose was administered intravenously, at ~25 mg/kg, 10 hours after oral-nasal NiV challenge (26). Notably, surviving ferrets had the highest levels of serum m102.4 on day 3 after infection, suggesting that day 3 was a potentially important therapeutic window. Here, in the AGM model, we chose to use a 100-mg dose of m102.4 administered intravenously (~20 mg/kg) twice, with the first dose given 10 hours after infection and the second dose 3 days after virus challenge. To determine whether the m102.4 therapeutic window could be extended, we also included two additional treatment groups: one group received m102.4 (100 mg per dose) beginning at 24 hours after infection and again at 3 days after infection, and the third treatment group received m102.4 (100 mg per dose) at 72 hours after infection and again at 5 days after virus challenge. In total, 14 subjects were challenged with a uniformly lethal dose of HeV (4 × 105 TCID50) by intratracheal inoculation. After challenge, two control subjects received infusions of saline, and for each treatment group, four subjects received m102.4 intravenously. No adverse reactions were observed upon m102.4 infusion in any animal. The control subjects (AGM 13 and AGM 17), consistent with historical controls, showed elevated temperatures (102.2 to 104°F), severe sustained behavior changes (depression, decreased activity), a gradual increase in respiratory rate (>50/min with open mouth breathing), and a significant decrease in platelet count at end-stage disease (table S1). Both subjects developed respiratory disease and were euthanized with respiratory distress on day 8 after infection. AGM 17 showed radiological changes consistent with interstitial pneumonia. In contrast, subjects treated with m102.4 at 10 or 24 hours after infection and again 3 days later (10 hr/d3 and 24 hr/d3) (AGM 14, 15, 16, 18, 19, 20, 21, and 22) showed either mild (mildly depressed) or no clinical signs of disease (table S1); no radiological changes were noted (fig. S1); and hematology and clinical chemistry assays were normal. One of the four subjects (AGM 23) that received m102.4 therapy at 72 hours after infection and again on day 5 after infection (72 hr/d5) started to show radiological changes on day 6 in the lung fields (mild interstitial pneumonia); however, changes were transient and not as severe as those noted in the control subject AGM 17. Between days 6 and 13, all four subjects in this group (AGM 23, 24, 25, and 26) showed a transient decrease in platelet counts, and from days 7 to 11, they developed temporal moderate to severe neurological signs including depression, imbalance, tremor/twitching (mainly in legs), head tilting, and seizure (only AGM 23) (table S1). By days 17 to 18, all subjects in the 72 hr/d5 treatment group began to improve clinically, and between days 24 and 27, all subjects appeared healthy and neurological signs were no longer present. A Kaplan-Meier survival graph with two additional historic control subjects is shown in Fig. 1.

Fig. 1

Survival curve of HeV-infected monkeys. Data from control subjects and m102.4-treated subjects were used to generate the Kaplan-Meier survival curve. The control included data from two additional historical control subjects (28). Subjects received m102.4 infusions at 10 hours and 3 days after infection (10 hr/d3), 24 hours and 3 days after infection (24 hr/d3), or 72 hours and 5 days after infection (72 hr/d5). Each group contained four subjects (n = 4). Average time to end-stage disease was 8 days in control subjects, whereas all m102.4-treated subjects survived until euthanasia at the end of the study.

Upon necropsy, gross pathological changes in the control subjects were consistent with those demonstrated in historical controls (28), whereas HeV-specific pathological changes were not observed in any of the m102.4-treated animals (table S2). Immunohistochemistry revealed high levels of HeV antigen in the lung and brain of the control subjects (Fig. 2A). Further molecular analysis revealed a tissue tropism and HeV RNA load that was similar to that of historic controls (28), with significant evidence of virus in the lung, spleen, lymph nodes (LNs), and brain (Fig. 2B). Virus isolation was attempted from all tissues collected from the control subjects, and positive tissues are indicated with a (+) in Fig. 2B. As expected, HeV was recovered from numerous tissues, highlighting the extensive dissemination of HeV within the body. In previous studies, low amounts of viral RNA were detected, HeV was isolated from AGM blood after HeV infection, and viral RNA loads increased as disease manifested (28). Here, blood samples collected over the course of infection were assayed for infectious HeV and HeV RNA, and positive samples are shown in Fig. 2C. Consistent with previous findings, a gradual rise in viral RNA over time was evident in the control subjects, and HeV was isolated from blood of both control subjects as indicated (+). Only very low levels of viral RNA could be detected on days 6 and 10 in some m102.4-treated subjects, and all blood samples from all m102.4-treated subjects were negative for HeV isolation. Tissue samples were collected from all m102.4-treated subjects upon necropsy and assayed for the presence of HeV RNA and infectious virus. Occasionally, TaqMan polymerase chain reaction (PCR) detected only very low levels of viral RNA in some tissues (spleen, lung, and brain), the major target organs of HeV and NiV infection in vivo and predominantly only in the late treatment cohort (72 hr/d5), unlike the control subjects where significant levels of HeV RNA were detected in every sampled tissue (Fig. 2B). Infectious HeV could not be recovered from any of the tissues collected from the m102.4-treated subjects. Together, these data demonstrate that m102.4 therapy prevented widespread HeV dissemination in the challenged subjects. Spleen, lung, and brain tissues from surviving subjects were also assayed for the presence of HeV antigen. All tissue architecture appeared normal, all survivor tissues examined were negative for HeV antigen (fig. S2), and control subject tissues showed significant HeV antigen staining. In the spleen, occasional cells were found to be antigen-positive in two subjects, AGM 18 (10 hr/d3) and AGM 19 (24 hr/d3) (fig. S3), but this was markedly lower in comparison to the antigen staining in the control animal (fig. S3C) and most likely represents residual viral antigen.

Fig. 2

HeV antigen, infectious virus, and viral RNA in untreated control AGM tissues and infectious HeV and viral RNA in blood samples from the m102.4 efficacy trial. (A) Localization of HeV antigen by immunohistochemical stain in the lung and brainstem of AGM 13. Sections were stained with a NiV nucleoprotein–specific rabbit polyclonal antibody, and images were obtained at an original magnification of ×40; however, one panel was photographed at ×200 as indicated. (B) Detection of HeV and viral RNA in tissues collected from AGM 13 and AGM 17. (C) Detection of HeV and viral RNA in blood samples. RNA samples were assayed in triplicate using TaqMan PCR. Blood and tissue Ct values were analyzed against Ct values generated from a standard curve of HeV RNA, as described in Materials and Methods, and a relative HeV n gene expression value was calculated for each blood replicate. Results are shown as average relative HeV n gene expression levels. The data are means ± SD. In (C), data from individual subjects are shown and indicated as none or different hatched patterns, and bar coloration (inset legend) indicates the different treatment groups. Virus isolation was attempted on all tissue and blood samples, and positive samples are indicated by (+). The blood sample (*) from one control subject was a terminal sample taken on day 8.

To evaluate whether the host immune response contributed to the mechanism of protection from disease, we assayed plasma samples from HeV-infected AGM plasma for the presence of antibodies directed against the HeV fusion (F) glycoprotein, the other major antigenic target of virus-neutralizing antibodies, and results are shown in Fig. 3. Anti-F antibodies were detected in m102.4-treated subjects on day 13 after infection and gradually rose over a 3-week period in all treated subjects. The control subjects did not seroconvert, which is not unexpected given the severe disease observed and that the subjects had to be euthanized because of irreversible clinical signs on day 8 after infection. However, for the treated subjects, seroconversion to HeV F and a subsequent rise in titer over time suggest that all treated subjects became infected with HeV, including the subjects in groups 10 hr/d3 and 24 hr/d3, where no clinical signs were evident. Additionally, anti-F antibodies increased markedly on day 20 after infection and likely played a role in recovery from disease in the 72 hr/d5–treated animals. Plasma m102.4 levels were measured in HeV-infected AGMs, and results are shown in Table 2. As expected, in the 10 hr/d3 and 24 hr/d3 groups, m102.4 levels increased on day 6, after the second dose of m102.4 infused on day 3. All subjects in the 72 hr/d5 group had high levels of m102.4 by day 6 after the two doses administered on days 3 and 5. Although m102.4 levels remained high on day 10 after infection, the 72 hr/d5 subjects presented with the first signs of neurological disease as early as day 7 after infection, which progressed to moderate/severe disease over the following days. HeV-mediated disease began to abate around day 17/18 and started to resolve around day 20, coinciding with the significant rise in anti-F antibody titer demonstrated in Fig. 3. Together, these data strongly suggest that m102.4 therapy did not prevent dissemination but instead significantly reduced viral loads in vivo, which, in effect, allowed the host an extended period to mount a protective immune response. Finally, data generated from both the uninfected and the HeV-infected AGMs demonstrate that the half-life of m102.4 in AGMs of about 10 to 12 days (Table 1) is far superior to the m102.4 half-life previously observed in NiV-infected ferrets (26), which is not unexpected because m102.4 is human in origin and hypothesized to be more homologous to antibodies from NHPs.

Fig. 3

Detection of F-specific antibodies in m102.4-treated AGMs. Median fluorescence intensities (MFI) are shown on the y axis and represent binding to soluble F. Error bars represent the SDs of fluorescence intensity across 100 beads for each sample. Three of four subjects in the 10 hr/d3 group were sampled on days 24 and 30 instead of days 27 and 35; the remaining animal (diagonal left stripes) in this group was sampled on days 27 and 35. No sample (*) was collected for one animal (small diagonal right stripes) on day 24.

Table 2

m102.4 plasma levels (μg/ml) in HeV-challenged AGMs. NS, not sampled.

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Our findings demonstrate that m102.4, a henipavirus-neutralizing hmAb that is specific for the ephrin receptor binding site of the viral attachment G envelope glycoprotein, is capable of preventing lethal disease in HeV-infected NHPs. The exceptional antiviral potency and pharmacological properties of m102.4 highlight its therapeutic potential for future approved use in humans.

Discussion

Human case fatality rates are about 60% for HeV and as high as 75% for NiV, and during the past decade, nearly annual spillover occurrences of both viruses have been recorded. Because of these repeated episodes, together with an ever-increasing number of research facilities working with infectious HeV and NiV, the development of effective therapies against these pathogens has become a critical need. An increasing amount of scientific evidence supports the notion that antibody can be sufficient to protect against lethal HeV- and NiV-mediated disease (26, 3033) after virus exposure. With the exception of the m102.4 trial in ferrets, all previous experiments that have examined the effectiveness of antibody therapy against henipaviruses have used either polyclonal hamster serum or mouse monoclonal antibody (mAb), neither of which would be suitable for use in humans.

The goal of the present study was to evaluate the protective efficacy of m102.4 in a model system in which both the disease pathogenic processes and the animal host closely reflected the human condition. For these reasons, we examined the efficacy of m102.4, a recombinant hmAb, using a lethal NHP model of HeV infection. In these studies, a high dose of HeV was inoculated by an intratracheal route and hmAb was administered by intravenous infusion, mimicking a mucosal challenge and paralleling a systemic treatment scenario. We found that HeV-mediated disease and its associated pathogenic processes in AGMs essentially mirrored the outcomes observed in HeV-infected humans. In addition, the pharmacokinetics of m102.4 in the AGM was similar to previously published human immunoglobulin half-life data generated in NHPs. Of the utmost importance, we were able to demonstrate that m102.4 protected all subjects from illness (10 hr/d3 and 24 hr/d3) and fatal disease (72 hr/d5) when administered after a lethal HeV challenge. The data presented here were generated from experiments that were designed in a way to reflect as close as possible those circumstances that might be expected to be encountered in the management of human cases of HeV exposure and possible infection, and we believe that the results are highly relevant to the potential postexposure treatment of human cases.

In August 2009, a limited amount of m102.4 was administered on a compassionate-use basis to treat a HeV-infected individual while in a coma (E. G. Playford, personal communication). At that time, only the potent in vitro neutralization activity of m102.4 and its pharmacokinetic properties in ferrets had been known. Unfortunately, the available antibody dose was low (100 mg, corresponding to about 1 to 2 mg/kg) and intravenous administration of the hmAb occurred well after the onset of encephalitis, and the individual died shortly thereafter. The efficacy of m102.4 in NiV-infected ferrets has since been reported, demonstrating a viable therapeutic treatment option against NiV (26). During the 2010 HeV spillover occurrence, 11 people had potential exposure and 2 of those individuals were considered to be at high risk for HeV exposure (15). In this recent episode, m102.4 was requested by Australian health authorities early as a compassionate-use therapeutic option, even though no human safety testing has been performed and it was not recommended for use in humans. However, a preliminary m102.4 pharmacokinetic and efficacy study in HeV-infected AGMs had been recently completed and a portion of those findings now included here were available at that time. In this instance, m102.4 was administered to two individuals before any HeV diagnosis or onset of clinical disease (19), with doses (~19 mg/kg) sufficient to achieve a high serum concentration, and to date, both individuals remain healthy and no evidence of HeV infection was ever detected (E. G. Playford, personal communication). The antibody appeared well tolerated when administered, which was not unexpected because m102.4 is a fully human mAb. As we continue to evaluate the efficacy of m102.4 in NHPs, it will be critical to establish acceptable therapeutic guidelines pertinent to either accidental or natural exposure to HeV. In addition, both the therapeutic window and the appropriate dose of m102.4 need to be defined and its efficacy evaluated in NiV-infected AGMs.

Together, the results presented here strongly support the further development of m102.4 as a postexposure therapeutic modality for HeV- and NiV-infected humans. m102.4 is a recombinant human mAb and the only one available to demonstrate potent in vivo efficacy against a highly lethal emerging infectious agent in a relevant NHP model of disease. Its success represents a significant technological milestone in the fight against an important emerging viral disease. HeV and NiV cause similar disease in AGMs, and an important next step will be to evaluate the efficacy of m102.4 in NiV-infected AGMs. The importance of such studies is highlighted by the continuing appearance of NiV and HeV with the most recent outbreaks occurring in March (9) and June to July 2011 (17), respectively.

Materials and Methods

Statistics

Conducting animal studies in BSL-4 severely restricts the number of animal subjects, the volume of biological samples that can be obtained, and the ability to repeat assays independently and thus limit statistical analysis. Consequently, data are presented as the mean or median calculated from replicate samples, not replicate assays, and error bars represent the SD across replicates.

Viruses and cells

HeV was propagated and titered on Vero cells as previously described (28). All infectious virus work was performed in a BSL-4 at the Integrated Research Facility, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health of the Rocky Mountain Laboratories (Hamilton, MT).

Animals

m102.4 pharmacokinetic studies. Four young adult AGMs (Chlorocebus aethiops), weighing 4 to 6 kg (Three Springs Scientific Inc.), were caged individually. Purified m102.4 was prepared as previously described (26). Before antibody infusion or phlebotomy, monkeys were sedated using ketamine (5 to 20 mg/kg) injected intramuscularly. Antibody was infused using either the saphenous or the cephalic vein. Antibody dose was 10 or 50 mg of m102.4, and each animal was administered m102.4 as a single intravenous infusion at ~2.5 or ~11 mg/kg, respectively. Subjects were monitored for adverse effects during and after the antibody infusion and during blood withdrawal. Blood was collected in serum tubes from the femoral, saphenous, or cephalic veins on days 0, 1, 3, 6, 12, 24, 36, and 48 after infusion. Serum was frozen at −20°C. Approval for animal experiments was obtained from the Boston University Institutional Animal Care and Use Committee (IACUC).

m102.4 efficacy trials. Fourteen young adult AGMs weighing 4 to 6 kg (Three Springs Scientific Inc.) were caged individually. Subjects were anesthetized by intramuscular injection of ketamine (10 to 15 mg/kg) and inoculated intratracheally with 4 × 105 TCID50 of HeV in 4 ml of Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich). Four subjects were infused with m102.4 beginning 10 hours after challenge and again 3 days after challenge (10 hr/d3); four subjects were infused with m102.4 beginning 24 hours after challenge and again 3 days after challenge (24 hr/d3); four subjects were infused with m102.4 beginning 72 hours after challenge and again 5 days after challenge (72 hr/d5). Control subjects (AGM 13 and AGM 17) were infused with saline. Each dose of m102.4 was 100 mg administered intravenously (~25 mg/kg). Subjects were anesthetized for antibody infusion and clinical examination including temperature, respiration rate, chest radiographs, blood draw, and swabs of nasal, oral, and rectal mucosa on days 0, 1, 3, 6, 9, 13, 16, 20, 27, and 35 after infection. Subjects were euthanized on day 40 after infection except three subjects in the 10 hr/d3 group, where euthanasia was done 88 days after challenge. Both control subjects had to be euthanized according to approved humane endpoints on day 8 after infection. All other subjects survived until the end of the study. Upon necropsy, various tissues were collected for virology and histopathology. Tissues sampled include conjunctiva, tonsil, oropharynx/nasopharynx, nasal mucosa, trachea, right bronchus, left bronchus, right lung upper lobe, right lung middle lobe, right lung lower lobe, left lung upper lobe, left lung middle lobe, left lung lower lobe, bronchial LN, heart, liver, spleen, kidney, adrenal gland, pancreas, jejunum, colon transversum, brain (frontal), brain (cerebellum), brainstem, cervical spinal cord, pituitary gland, mandibular LN, salivary LN, inguinal LN, axillary LN, mesenteric LN, urinary bladder, testes or ovaries, and femoral bone marrow. Experiments were conducted under BSL-4 conditions, and approval for animal experiments was obtained from the Rocky Mountain Laboratories IACUC.

All animal work was performed by certified staff in an Association for Assessment and Accreditation of Laboratory Animal Care–approved facility at Boston University and Rocky Mountain Laboratories.

Measurement of serum or plasma m102.4 and F glycoprotein–specific antibodies

The m102.4 levels were determined using previously published multiplexed microsphere assays (26). Antibodies to the F glycoprotein were measured in HeV-infected subjects simultaneously by including a recombinant soluble F (sF) glycoprotein (34)–coupled microsphere in the assay. Coupling of sF to microsphere #43 (Luminex Corporation) was done as described previously (35). Plasma from HeV-infected AGMs was inactivated by γ irradiation (50 μGy) before testing. Sera and plasma were assayed at 1:5000 and 1:10,000 dilutions. Assays were performed on a Luminex 200 IS machine equipped with Bio-Plex Manager Software (v 5.0) (Bio-Rad Laboratories). Median fluorescence intensity (MFI) and the SD of fluorescence intensity across 100 beads were determined for each sample. For m102.4 concentrations, unknowns were extrapolated from an m102.4 standard curve using nonlinear regression analysis [Bio-Plex Manager Software (v 5.0)]. Each pharmacokinetic data set in Table 1, plotted as ln[C] versus t, was interpolated by two linear regressions: ln[C] = k1 × t + A1 for t ≤ 1 day and ln[C] = k2 × t + A2 for t ≥ 3 days, describing the antibody distribution and elimination phases, respectively. Here, k1 is the distribution rate constant, k2 is the elimination rate constant, and A1 and A2 are measures for the volume of the distribution. Distribution half-life time t11/2 = ln 2/k1 and elimination half-life time t21/2 = ln 2/k2. The AUC, that is, the integral of the plasma concentration on time, was calculated by the trapezoid method for the time ≤48 days. A correction for the time >48 days was calculated using the following formula: exp(A2) × exp(48 × k2)/k2, which was obtained by extrapolation of the elimination phase regression to infinity. AUC values are measures for the antibody presence in vivo, that is, exposure of body to m102.4, and also account for the bioavailability of m102.4.

Specimen collection and processing in HeV-infected AGMs

Blood was collected in EDTA, sodium citrate, or serum Vacutainers (Beckman Dickinson). Immediately after sampling, 140 μl of blood was added to 560 μl of AVL viral lysis buffer (Qiagen Inc.) for RNA extraction. Serum was frozen for chemistry and serological assays. For tissues, about 100 mg was stored in 1 ml of RNAlater (Qiagen Inc.) for a minimum of 24 hours to stabilize RNA, and about 100 mg was stored for virus isolation. For tissues stored in RNAlater, RNAlater was completely removed and tissues were homogenized in 600 μl of RLT buffer in a 2-ml cryovial using Qiagen tissue lyser and stainless steel beads. An aliquot representing about 30 mg was added to fresh RLT buffer (600 μl final volume) (Qiagen Inc.) for RNA extraction. Blood samples in AVL viral lysis buffer and tissue samples in RLT buffer were removed from the BSL-4 laboratory using approved standard operating protocols. RNA was isolated from blood and swabs using the QIAamp viral RNA kit (Qiagen Inc.) and from tissues using the RNeasy Mini kit (Qiagen Inc.) according to the manufacturer’s instructions supplied with each kit.

Virus isolation

Vero cells were seeded in 24-well plates in DMEM containing 10% fetal calf serum (FCS), penicillin (100 U/ml), and streptomycin (100 mg/ml). Tissues were weighed and homogenized [1:10 (w/v) in phosphate-buffered saline (PBS)] in a 2-ml cryovial for 8 min using Qiagen tissue lyser and stainless steel beads. Homogenates were clarified by centrifugation and diluted 1:10 using DMEM containing 1% FCS, penicillin (100 U/ml), and streptomycin (100 mg/ml) (DMEM-1). Duplicate wells were inoculated with 200 μl of a 1% tissue homogenate and incubated at 37°C. One well was incubated for 30 min, and one well was incubated overnight; both were washed once with PBS and cultured in 1 ml of DMEM-1. Cultures were examined for the presence or absence of syncytia/cytopathic effect for 5 days. Negative samples were passaged twice onto new cells before being deemed negative.

Histopathology and immunohistochemistry

Necropsy was performed on all subjects. Tissue samples of all major organs were collected for histopathologic and immunohistochemical examination and were immersion-fixed in 10% neutral buffered formalin for at least 7 days in BSL-4. Subsequently, formalin was changed; specimens were removed from BSL-4 under approved standard operating protocols, processed in BSL-2 by conventional methods, embedded in paraffin, and sectioned at 5-μm thickness. Tissues for immunohistochemistry were stained on the Discovery XT automated stainer (Ventana Medical Systems Inc.) using an antibody to Nipah-nucleoprotein (1:5000) and the DAB map detection kit (Ventana Medical Systems Inc.). Nonimmune rabbit IgG was used as a negative staining control.

HeV TaqMan PCR

The HeV nucleocapsid (n) gene TaqMan PCR assay has been described previously (28). All reactions contained 2 μl of RNA, master mixes were set up following the manufacturer’s protocols, and each reaction was done in a total volume of 25 μl. For blood, 2 μl of RNA represented 4.7 μl of whole blood, and for tissues, 2 μl of RNA represented 1.2 mg of tissue. All samples were assayed in triplicate. To account for plate-to-plate variation, for each TaqMan plate, we assayed RNA representing 4.7, 47, and 470 TCID50 HeV, derived from the inoculum, in triplicate and set the average Ct to a relative HeV n gene expression value of 1, 10, and 100, respectively. Sample Ct values were analyzed against Ct values generated from the standard curve of HeV RNA, and a relative HeV n gene expression value was calculated for each replicate. Ct value analysis was done using Rotor-Gene 6000 software, and data are shown as the mean relative HeV n gene expression levels ± SD.

Supplementary Material

www.sciencetranslationalmedicine.org/cgi/content/full/3/105/105ra103/DC1

Fig. S1. Radiographic images of lung fields on day 6 after Hendra virus infection of African green monkeys.

Fig. S2. Absence of Hendra virus antigen in the lung and brain of m102.4-treated African green monkeys.

Fig. S3. Hendra virus antigen detection in spleens of African green monkeys that received m102.4 therapy.

Table S1. Clinical scoring and outcome of Hendra virus–infected African green monkeys.

Table S2. Gross pathology summary of Hendra-infected African green monkeys.

Footnotes

  • * These authors contributed equally to this work.

  • Present address: Sealy Center for Vaccine Development, Departments of Pathology and Microbiology & Immunology, The University of Texas Medical Branch, Galveston, TX 77555–0610, USA.

References and Notes

  1. Acknowledgments: We thank the staff of the Rocky Mountain Veterinary Branch for animal care and veterinary support. We thank Y. Feng for help with the m102.4 production. Funding: These studies were supported in part by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, NIH, and in part by the Department of Health and Human Services, NIH, grants AI082121 and AI057159 (to T.W.G.) and AI054715 and AI077995 (to C.C.B.), and the Intramural Biodefense Program of the National Institute of Allergy and Infectious Diseases, the Intramural Program of the National Cancer Institute, Center for Cancer Research, and by federal funds from the National Cancer Institute, NIH, under contract N01-CO-12400. Author contributions: K.N.B., B.R., H.F., C.C.B., and T.W.G. designed the studies. B.R., H.F., T.W.G., F.F., D.B., and J.B.G. contributed to performance of experiments and data collection and analysis. B.R., H.F., and T.W.G. performed gross pathological analysis. B.R., K.N.B., A.C.H., and J.C. performed serological and nucleic acid analysis experiments and data collection. Y.-P.C. provided critical reagents and contributed to assay design. L.Y., Y.-R.F., and Y.W. produced, purified, and quality controlled the m102.4 monoclonal antibody. Z.Z. and D.S.D. provided critical reagents, contributed to study design, and reviewed and edited the manuscript. D.S. performed histological analysis and J.B.G. and D.B. facilitated the conduct of the in vivo animal studies. A.S.D. provided the analysis of the antibody pharmacokinetics studies. K.N.B., C.C.B., B.R., H.F., and T.W.G. wrote and edited the manuscript. C.C.B. prepared the final versions of the manuscript and supplementary material. Competing interests: C.C.B. and D.S.D. are U.S. federal employees, and D.S.D., Z.Z., and C.C.B. are coinventors on U.S. patent 7,988,971, “Human monoclonal antibodies against Hendra and Nipah viruses”; assignees are the United States of America as represented by the Department of Health and Human Services (Washington, DC) and Henry M. Jackson Foundation for the Advancement of Military Medicine Inc. (Bethesda, MD). All other authors declare no competing interests. The opinions or assertions contained herein are the private ones of the author(s) and are not to be construed as official or reflecting the views of the Department of Defense, the Uniformed Services University of Health Sciences, and the National Institutes of Allergy and Infectious Diseases, NIH.
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