A Recombinant Hendra Virus G Glycoprotein Subunit Vaccine Protects Nonhuman Primates against Hendra Virus Challenge
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Chad E. Mire, Joan B. Geisbert, Krystle N. Agans, Yan-Ru Feng, Karla A. Fenton, Katharine N. Bossart, Lianying Yan, Yee-Peng Chan, Christopher C. Broder and Thomas W. Geisbert J. Virol. 2014, 88(9):4624. DOI: 10.1128/JVI.00005-14. Published Ahead of Print 12 February 2014.
A Recombinant Hendra Virus G Glycoprotein Subunit Vaccine Protects Nonhuman Primates against Hendra Virus Challenge Chad E. Mire,a,b Joan B. Geisbert,a,b Krystle N. Agans,a,b Yan-Ru Feng,c Karla A. Fenton,a,b Katharine N. Bossart,d Lianying Yan,c Yee-Peng Chan,c Christopher C. Broder,c Thomas W. Geisberta,b
ABSTRACT
Hendra virus (HeV) is a zoonotic emerging virus belonging to the family Paramyxoviridae. HeV causes severe and often fatal respiratory and/or neurologic disease in both animals and humans. Currently, there are no licensed vaccines or antiviral drugs approved for human use. A number of animal models have been developed for studying HeV infection, with the African green monkey (AGM) appearing to most faithfully reproduce the human disease. Here, we assessed the utility of a newly developed recombinant subunit vaccine based on the HeV attachment (G) glycoprotein in the AGM model. Four AGMs were vaccinated with two doses of the HeV vaccine (sGHeV) containing Alhydrogel, four AGMs received the sGHeV with Alhydrogel and CpG, and four control animals did not receive the sGHeV vaccine. Animals were challenged with a high dose of infectious HeV 21 days after the boost vaccination. None of the eight specifically vaccinated animals showed any evidence of clinical illness and survived the challenge. All four controls became severely ill with symptoms consistent with HeV infection, and three of the four animals succumbed 8 days after exposure. Success of the recombinant subunit vaccine in AGMs provides pivotal data in supporting its further preclinical development for potential human use. IMPORTANCE
A Hendra virus attachment (G) glycoprotein subunit vaccine was tested in nonhuman primates to assess its ability to protect them from a lethal infection with Hendra virus. It was found that all vaccinated African green monkeys were completely protected against subsequent Hendra virus infection and disease. The success of this new subunit vaccine in nonhuman primates provides critical data in support of its further development for future human use.
T
he henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), cause severe and often fatal respiratory disease and encephalitis in horses, pigs, and humans (1–4). In contrast to all other paramyxoviruses, henipaviruses infect a broad range of species spanning six mammalian orders. Because of this broad species tropism, ease of access and propagation, potential for person-toperson transmission, high case fatality rates, and lack of approved countermeasures for human use, HeV and NiV pose significant biosecurity threats and are classified as biosafety level 4 (BSL-4) pathogens. HeV emerged in Australia in 1994 and was identified as the causative agent of an acute respiratory illness in horses (5). HeV is transmitted to horses by pteropid fruit bats, commonly known as flying foxes, with human infection occurring by close contact with infected horses (6, 7). Outbreaks of HeV have occurred in Australia on a nearly annual basis since the initial outbreak, with all episodes involving infection of horses. In total, ⬎80 horses have succumbed to HeV infection, with a case fatality rate of approximately 75%. There have been seven human HeV infections recorded, most recently in 2009, of which four have been fatal (57%) (8). All patients initially presented with influenza-like symptoms after an incubation period of 7 to 16 days. While two of the patients recovered from the influenza-like illness, one developed pneumonitis and succumbed to multiorgan failure. Three different patients developed encephalitic manifestations (mild confusion, ataxia), with two of these cases progressing to seizures and resulting in death (5, 8, 9). There are currently no approved vaccines or antiviral drugs for
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combating human HeV or NiV infection. Regarding treatment options for henipavirus infection, an open-label ribavirin trial was performed with 140 patients during the initial outbreak of NiV in Malaysia in 1998; however, the results of that study remain controversial (10). In addition, three of the seven recorded human HeV cases were treated with ribavirin and one of the patients survived (8). The utility of ribavirin as a countermeasure against HeV infection was subsequently assessed in African green monkeys (AGMs) (11). While there was a small benefit in delaying death, there was no survival benefit in this nonhuman primate model. Currently, the most promising postexposure treatment for henipavirus infection appears to be an experimental human monoclonal antibody (MAb). This MAb, m102.4, targets the ephrin-B2 and -B3 receptor binding domain of the henipavirus envelope attachment (G) glycoprotein (12–16). m102.4 is a potent cross-reactive neutralizing antibody in vitro (17, 18) and had been shown to protect ferrets from a lethal NiV challenge (19) and AGMs from a lethal HeV challenge (20). Importantly, in 2010,
Received 3 January 2014 Accepted 8 February 2014 Published ahead of print 12 February 2014 Editor: R. W. Doms Address correspondence to Christopher C. Broder, [email protected]. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.00005-14 The authors have paid a fee to allow immediate free access to this article.
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Galveston National Laboratorya and Departments of Microbiology and Immunology,b University of Texas Medical Branch, Galveston, Texas, USA; Department of Microbiology and Immunology, Uniformed Services University, Bethesda, Maryland, USAc; Integrated Research Associates, LLC, San Francisco, California, USAd
Henipavirus G Glycoprotein Subunit Vaccine
MATERIALS AND METHODS Statistics. Conducting animal studies at BSL-4 severely restricts the number of animals, the volume of biological samples that can be obtained, and the ability to repeat assays independently and thus limits statistical analysis. Consequently, data are presented as the mean or median values calculated from replicate samples, not replicate assays, and error bars represent the standard deviations across replicates. Statistics were calculated for serum neutralizing antibody titers with GraphPad Prism 5 software by using a two-way analysis of variance comparing treatments and times between all groups and using the Bonferroni method posttest. Viruses and cells. HeV was kindly provided by Tom Ksiazek and was obtained from a patient who was part of the 1994 outbreak in Australia. The virus was propagated on Vero E6 cells in Eagle’s minimal essential medium supplemented with 10% fetal calf serum. The titer of the HeV stock used was ⬃1 ⫻ 107 PFU/ml. The HeV challenge virus stock was assessed for the presence of endotoxin with the Endosafe-PTS portable test system (Charles River, Wilmington, MA). Virus preparations were diluted 1:10 in Limulus amebocyte lysate (LAL) reagent water in accordance with the manufacturer’s directions, and endotoxin levels were tested in LAL Endosafe-PTS cartridges as directed by the manufacturer. Each preparation was found to be below the limit of detection, while positive controls showed that the tests were valid. Vaccine formulation. Production and purification of sGHeV were done as previously described (26). Two vaccines containing sGHeV and Alhydrogel (Accurate Chemical & Scientific Corporation, Westbury, NY) at a weight ratio of 1:25 were prepared with one formulation also containing CpG oligodeoxynucleotide (ODN) 2006 (InvivoGen, San Diego, CA) with a fully phosphorothioate-modified backbone (sGHeV-alum-CpG). The vaccine without CpG was formulated with 100 g of sGHeV and 2.5 mg of aluminum ion (sGHeV-alum) per vaccinated animal. The vaccine with CpG was formulated with 100 g of sGHeV, 2.5 mg of aluminum ion, and 150 mg of ODN 2006 per vaccinated animal. For the vaccine containing both Alhydrogel and CpG, the Alhydrogel and sGHeV were mixed
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before ODN 2006 was added. Each vaccine dose was adjusted to 1 ml with phosphate-buffered saline (PBS), and mixtures were incubated on a rotating wheel at room temperature for at least 2 to 3 h before injection. Each AGM received the same dose of 1 ml for prime and boost vaccinations, and all vaccine doses were given via intramuscular (i.m.) injection. The 100-g dose used in this study was based on the protective efficacy against NiV and the prolonged level of IgG in AGMs vaccinated with this dose rather than 10 or 50 g (26). Animals. Animal studies were performed in BSL-4 biocontainment at the Galveston National Laboratory at the University of Texas Medical Branch (UTMB) at Galveston and were approved by the UTMB Institutional Animal Care and Use Committee. Animal research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adhered to the principles stated in reference 29. The facility where this research was conducted is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Twelve adult AGMs weighing 3 to 6 kg (PreLabs, Hines, IL) were used in this study. AGMs were anesthetized by i.m. injection with ketamine and vaccinated with sGHeV by i.m. injection on day ⫺42 (prime vaccination) and day ⫺21 (boost vaccination) (Fig. 1, black and gray arrows, respectively). The vaccine formulations used are described above. Four AGMs received two doses of vaccine containing sGHeV and Alhydrogel; four animals received two doses of vaccine containing sGHeV, Alhydrogel, and CpG; one animal received two doses of Alhydrogel only; one animal received two doses of CpG only; and two animals were not vaccinated. Animals were inoculated intratracheally (i.t.) with ⬃5 ⫻ 105 PFU of HeV in 5 ml of Dulbecco’s minimal essential medium (DMEM; Sigma-Aldrich, St. Louis, MO) 21 days after the boost vaccination (Fig. 1, *). Animals were anesthetized for clinical examination, including temperature, respiration quality, blood collection, and swabs of nasal, oral, and rectal mucosae on days 0, 3, 5, 7, 10, 15, and 30 postchallenge. AGMs in the vaccine cohorts were euthanized on day 30 postchallenge, whereas three of the four control AGMs had to be euthanized according to approved humane endpoints on day 8 postchallenge. All of the other AGMs survived until the end of the study. Measurement of serum or plasma HeV G-specific antibodies. AGM serum samples collected at the time points indicated were tested for immunoglobulin (Ig) antibodies against HeV G with previously developed multiplexed microsphere assays (11). In brief, 96-well filter plates were primed with PBS. Test serum samples were diluted in PBS at 1:10 at prevaccination time points and at 1:10,000 at postvaccination time points. Biotinylated goat anti-human IgM/IgG and streptavidin-phycoerythrin (strep-PE) were also diluted in PBS. Coupled microspheres (sG-HeV) were prepared by sonication for 1 min, followed by vortex mixing for 1 min each, and then diluted in PBS. Priming liquid was removed from the plates with a Bio-Plex Pro II Wash Station (Bio-Rad Laboratories, Hercules, CA), and 100 l containing 1,500 coupled microspheres was added to each well. The microsphere mixture was removed by vacuum, 100 l of diluted test serum was added to appropriate wells, and the mixture was incubated at room temperature for 30 min while shaking in the dark. Diluted test samples were removed by vacuum, 100 l of diluted biotinylated goat anti-human antibody (1:500; Pierce Protein Biology Products, ThermoFisher Scientific, Waltham, MA) was added to each well, and the mixture was incubated as described above. Liquid was removed by vacuum, 100 l of strep-PE (1:1,000; Qiagen Inc., Valencia, CA) was added to each well, and the mixture was incubated for 30 min. All of the liquid was removed from the plates with a vacuum manifold and washed twice with 300 l of PBS, and the liquid was removed between wash steps. Finally, 125 l of PBS was added to each well and incubated for 2 min as described above. Samples were assayed for mean fluorescence intensity (MFI) across at least a 100 bead region performed on the BioPlex-200 machine and analyzed with Bio-Plex Manager Software (v 6.1) (Bio-Rad). MFI and the standard deviation (SD) of fluorescence intensity across 100 beads were determined for each sample and plotted.
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m102.4 was administered to two individuals in Australia who had a significant risk of exposure to HeV under a compassionate-use protocol. It was also used in 2012 to treat an individual with possible exposure to HeV in Australia and in 2013 to treat an individual with possible laboratory exposure in the United States. To date, all four of these individuals have no evidence of henipavirus infection. In addition to the postexposure treatments, two experimental preventive vaccines against henipaviruses have been evaluated in animal models. A recombinant adeno-associated virus vaccine expressing the NiV G protein completely protected hamsters against a homologous NiV challenge and protected 50% of the animals against a heterologous HeV infection (21). In addition, a recombinant subunit vaccine based on the HeV G protein (sGHeV) completely protected small animals against lethal HeV and NiV infections (22–25) and more recently was shown to be efficacious in the robust AGM model of NiV infection (26). sGHeV (amino acids 73 to 604) is an engineered secreted version of the full ectodomain of the G glycoprotein in which the transmembrane and cytoplasmic tail domains have been deleted from the N terminus (27). Importantly, this vaccine has also been shown to protect horses from lethal HeV infection and has been licensed for use by the equine industry in Australia (28). However, there has been no study to date assessing the performance of this vaccine in a nonhuman primate model of HeV infection, which is a necessary prerequisite for the licensure of such a vaccine for use in humans. Here, we report for the first time the prophylactic efficacy of the sGHeV vaccine in a lethal HeV AGM model that faithfully recapitulates human HeV infection.
Mire et al.
g)-adjuvant or adjuvant alone. Gray arrow; boost vaccination with sGHeV-adjuvant or adjuvant alone. The asterisk shows the day of i.t. HeV challenge with 5 ⫻ 105 PFU (B) Kaplan-Meier survival curves of the control group (black, n ⫽ 4; 2 nonvaccinated, 1 alum only, and 1 alum-CpG only) and the vaccinated groups (sGHeV-alum [teal, n ⫽ 4] and sGHeV-alum-CpG [orange, n ⫽ 4]).
HeV serum neutralization assays. Neutralization titers were determined by a conventional serum neutralization assay. Briefly, serum samples were serially diluted 2-fold and incubated with ⬃100 PFU of HeV for 1 h at 37°C. Virus and antibodies were then added to individual wells of six-well plates of Vero cells. Plates were stained with neutral red 2 days after infection, and plaques were counted 24 h after staining. The 50% neutralization titer was determined as the serum dilution at which at there was a 50% reduction in plaque counts versus the control wells. Specimen collection and processing in HeV-infected AGMs. Blood was collected in EDTA or serum Vacutainers (Becton, Dickinson, Franklin Lakes, NJ). Nasal, oral, and rectal swabs were collected in 1 ml of DMEM (Sigma-Aldrich) and vortexed for 30 s. Immediately following sampling, 100 l of blood or 100 l of DMEM from individual swab samples was added to 600 l of AVL viral lysis buffer (Qiagen) for RNA extraction. For tissues, approximately 100 mg was stored in 1 ml of RNAlater (Qiagen) for 7 days to stabilize the RNA. RNAlater was completely removed, and tissues were homogenized in 600 l of RLT buffer (Qiagen) in a 2-ml cryovial with a TissueLyser (Qiagen) and stainless steel beads. The tissues sampled included conjunctiva, tonsil, oro- and nasopharynx, nasal mucosa, trachea, right bronchus, left bronchus, right lung upper lobe, right lung middle lobe, right lung lower lobe, right lung upper lobe, right lung middle lobe, right lung lower lobe, bronchial lymph node (LN), heart, liver, spleen, kidney, adrenal gland, pancreas, jejunum, colon transversum, brain (frontal and cerebellum), brain stem, cervical spinal cord, pituitary gland, mandibular LN, salivary gland LN, inguinal LN, axillary LN, mesenteric LN, urinary bladder, testes or ovaries, and femoral bone marrow. All blood samples and swabs were inactivated in AVL viral lysis buffer, and tissue samples were homogenized and inactivated in RLT buffer prior to removal from the BSL-4 laboratory. Subsequently, RNA was isolated from blood and swabs with the QIAamp viral RNA kit (Qiagen) and from tissues by using the RNeasy minikit (Qiagen) according to the manufacturer’s instructions supplied with each kit. Hematology and serum biochemistry. Total white blood cell (WBC) counts, differential WBC counts, red blood cell counts, platelet counts, hematocrit values, total hemoglobin concentrations, mean cell volumes, mean corpuscular volumes, and mean corpuscular hemoglobin concentrations were analyzed in blood collected in tubes containing EDTA with a laser-based hematologic analyzer (Beckman Coulter, Brea, CA). Serum samples were tested for concentrations of albumin, amylase, alanine aminotransferase, aspartate aminotransferase (AST), alkaline phosphatase, gamma-glutamyltransferase, glucose, cholesterol, total protein, total bilirubin, blood urea nitrogen (BUN), creatinine (CRE), and C-reactive protein (CRP) by with a Piccolo point-of-care analyzer and Biochemistry Panel Plus analyzer discs (Abaxis, Sunnyvale, CA).
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Histopathology and immunohistochemistry analyses. Necropsy was performed on all AGMs. Tissue samples of all major organs were collected for histopathologic and immunohistochemical examinations and immersion fixed in 10% neutral buffered formalin for at least 21 days at BSL-4. Subsequently, the formalin was changed; specimens were removed from BSL-4, processed at BSL-2 by conventional methods, embedded in paraffin, and sectioned at a 5-m thickness. For immunohistochemistry analysis, specific anti-HeV immunoreactivity was detected with an anti-HeV N protein rabbit primary antibody at a 1:5,000 dilution for 30 min. The tissue sections were processed for immunohistochemistry analysis with the Dako Autostainer (Dako, Carpinteria, CA). The secondary antibody used was biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) at 1:200 for 30 min, followed by Dako LSAB2 streptavidinhorseradish peroxidase (Dako) for 15 min. Slides were developed with Dako 3,3=-diaminobenzidine chromogen (Dako) for 5 min and counterstained with hematoxylin for 1 min. Nonimmune rabbit IgG was used as a negative staining control. Detection of HeV loads. RNA was isolated from nasal, oral, and rectal swabs; blood; or tissues and analyzed with primers/probe targeting the N gene of HeV for quantitative real-time PCR (qRT-PCR) (11), with the probe used here being 6-carboxyfluorescein–5=-TCCTGAGTCTGCCTG AGGGCGGT-3=-– 6-carboxytetramethylrhodamine (Life Technologies, Carlsbad, CA). HeV RNA was detected with the CFX96 detection system (Bio-Rad) in One-Step Probe qRT-PCR kits (Qiagen) under the following cycling conditions: 50°C for 10 min, 95°C for 10 s, and 40 cycles of 95°C for 10 s and 59°C for 30 s. Threshold cycle values representing HeV genomes were analyzed with CFX Manager Software, and data are shown in genome equivalents (GEq). To create the GEq standard, RNA was extracted from HeV challenge stocks and the number of HeV genomes was calculated by using Avogadro’s number and the molecular weight of the HeV genome. Virus titration was performed by plaque assay with Vero cells from all blood samples. Briefly, increasing 10-fold dilutions of the samples were adsorbed to Vero cell monolayers in duplicate wells (200 l); the limit of detection was 25 PFU/ml.
RESULTS
HeV challenge of vaccinated AGMs. We previously described the development of a lethal AGM model for HeV infection with observed clinical signs and pathology highly consistent with the HeV-mediated disease reported in humans (11). Clinical signs in this model include severe depression, respiratory disease leading to acute respiratory distress, neurologic disease, reduced activity, and a time to death of 8 to 10 days. The purpose of the present
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FIG 1 (A) Diagram of the sGHeV vaccination schedule and sampling days after a HeV challenge of AGMs. Black arrow; prime vaccination with sGHeV (100
Henipavirus G Glycoprotein Subunit Vaccine
TABLE 1 Clinical descriptions and outcomes of HeV-challenged AGMs Sex
Group
Clinical illness
Clinical and gross pathology
R335
Male
Control
Fever (day 7), depression (day 8), lethargy (day 8), loss of appetite (days 7, 8), labored breathing (days 5–8), dehydration (days 5, 7), death (day 8)
R372
Female
Control
O7498
Male
Control
O7521
Male
Control
Fever (days 3, 5), Depression (days 10–13), lethargy (days 10–13), loss of appetite (days 4–8), labored breathing (days 7, 8), dehydration (days 7, 8), chills (day 8), hind limb paresis (day 8), euthanasia (day 8) Depression (day 8), lethargy (day 8), loss of appetite (days 8–15), labored breathing (days 7–13), dehydration (day 10), splenomegaly (day 7) survival Fever (day 7), depression (day 8), lethargy (day 8), loss of appetite (days 7, 8), labored breathing (days 5–8), death (day 8)
Thrombocytopenia (day 8), ⬎2-fold increase in WBC count, ⬎3-fold increase in BUN (day 8), ⬎2-fold increase in CRE (day 8), ⬎4-fold increase in AST (day 8), ⬎10-fold increase in CRP (days 7, 8), excess blood-tinged pleural fluid, severely inflated enlarged lungs with severe congestion and hemorrhage of all lobes, darkened liver Moderately inflated enlarged lungs with multifocal areas of congestion
O7500 O7503 O7499 O7501
Male Male Male Male
sG-Alum sG-Alum sG-Alum sG-Alum
None None None None
None None None None
O7515 O7494 O7506 O7477
Male Male Male Male
sG-Alum-CpG sG-Alum-CpG sG-Alum-CpG sG-Alum-CpG
None None None None
None None None None
study was to determine whether vaccination with sGHeV could prevent HeV infection and illness in AGMs. Additionally, we tested whether protection from HeV infection requires vaccination with sGHeV plus two adjuvants (sGHeV-alum-CpG) or a single adjuvant (sGHeV-alum). A time line of the vaccination regimen, HeV challenge, and specimen collection is shown in Fig. 1A. All four control AGMs showed disease consistent with historical controls (11), including loss of appetite, depression, decreased activity, and labored breathing (Table 1). Three of the four control AGMs (R335, O7521, R372) developed acute respiratory distress, and one also developed hind limb paresis; all three of these animals succumbed on day 8 after exposure (Fig. 1B and Table 1). While the remaining control animal (O7498) was clinically ill for a prolonged period of time, it began to recover on postchallenge day 14 and survived until the study endpoint. In contrast, none of the vaccinated animals showed any evidence of clinical illness and all survived (Fig. 1B and Table 1). HeV loads. To determine if there was HeV replication in animals postchallenge, virus shedding was assessed by qRT-PCR of nasal, oral, and rectal swabs (Fig. 2A, B, and C, respectively) and viremia was also screened for by qRT-PCR of whole blood samples (Fig. 2D). HeV GEq were observed in all swab and blood samples from two of the control animals (R335 and O7521, Fig. 2), while we detected HeV GEq in oral swabs from another control animal (R372) on day 7 postchallenge (Fig. 2B, red). The surviving con-
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None
Thrombocytopenia (day 7), ⬎2-fold increase in WBC count (day 8), ⬎2-fold increase in BUN (day 8), 5-fold increase in CRE (day 8), ⬎2-fold increase in AST (day 8), ⬎10-fold increase in CRP (day 8), excess blood-tinged pleural fluid, inflated enlarged lungs with multifocal areas of congestion and hemorrhage particularly of the lower and middle right lobes, darkened liver
trol animal (O7498) had no detectable HeV RNA in any sample. Likewise, none of the specifically vaccinated animals had any detectable HeV RNA in any sample (negative data not shown). HeV RNA was also detected systemically in the tissues of control animals R335 and O7521 (Fig. 3, green and black, respectively), whereas HeV RNA was detected only in the respiratory tissues, the axillary LN, and femoral bone marrow of control animal O7521 (Fig. 3, red). HeV RNA was not detected in tissues of control animal O7498 or in any of the tissues of any of the specifically vaccinated animals (negative data not shown). Detection of HeV RNA in tissues (Fig. 3), swabs, and blood (Fig. 2) correlated with each animal’s outcome and gross pathology (Fig. 1B and Table 1). Histopathological and immunohistochemical analysis of HeV-infected AGMs. Histopathologic analysis of samples from the three control animals that succumbed to a HeV challenge was mostly consistent with previous findings on henipavirus-infected AGMs (11, 20, 26, 30), and lesions from control animals R335 and O7521 were more prominent than those from control animal R372. Noteworthy lesions included interstitial pneumonia, necrosis and hemorrhage of the splenic white pulp, and variable syncytial cell formation in lymphoid tissues. Alveolar spaces were filled with edema fluid, fibrin, foamy alveolar macrophages, and cellular debris. Glomerular tufts were hypercellular and congested, and syncytial cells were occasionally noted within the endothelium of the glomerular tufts. Strong immunoreactivity to HeV antigen
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AGM
Mire et al.
was present within the alveolar capillary endothelium and alveolar macrophages and also within scattered mononuclear cells in the subcapsular and medullary sinuses of various LNs (Fig. 4). Strong immunoreactivity to HeV antigen was also present in the kidneys
within segmental regions of the endothelium of the glomerular tufts (Fig. 4). Neuropathology was not as prominent in the control animals as in HeV-infected AGMs in a previous study (11). Nonetheless, HeV antigen was a notable finding within the endothelium
FIG 3 Viral loads of the AGM control cohort (all vaccinated; negative data not shown) as detected by GEq/mg by qRT-PCR assay of tissues. R335 and R372 (green and red, respectively), nonvaccinated controls; O7498 (white), alum-only control; O7521 (black), alum-CpG only control. R.U., right upper; R.M., right middle; R.L., right lower; L.U., left upper; L.M., left middle; L.L., left lower. Error bars are SDs.
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FIG 2 Viral loads of the AGM control cohort (all vaccinated; negative data not shown) expressed in GEq/ml and determined by qRT-PCR assay of nasal swabs (A), oral swabs (B), rectal swabs (C), and blood (D). R335 and R372 (green and red, respectively), nonvaccinated controls; O7498 (white), alum-only control; O7521 (black), alum-CpG-only control. Error bars are SDs.
Henipavirus G Glycoprotein Subunit Vaccine
FIG 4 Lack of HeV antigen in representative sGHeV-vaccinated tissues and localization of HeV antigen in representative control tissues by immunohistochemical staining. Lung, spleen, kidney, and brain stem samples were labeled with an N protein-specific polyclonal rabbit antibody, and images were taken. Magnifications: lung, ⫻20; spleen, ⫻20; kidney, ⫻40; brain stem, ⫻60. sG-CpG-Alum, all tissues from O7515; sG-Alum, all tissues from O7500. Controls: lung tissue from O7521, spleen tissue from O7521, kidney tissue from R335, brain stem tissue from R335.
of the brain stem (Fig. 4). Representative tissue sections from the control animals are shown in Fig. 4. Examination of tissue sections from vaccinated AGMs revealed only normal tissue architecture. Importantly, HeV antigen was not detected in any tissue of any sGHeV-vaccinated AGM by immunohistochemical techniques (Fig. 4). At the study endpoint, the tissue architecture was also normal in the control AGM that survived (O7498); HeV antigen was also undetectable in tissues from this animal. Humoral immune response to HeV G. The AGMs used in this study were vaccinated with sGHeV with adjuvant; therefore, we were interested in measuring the humoral immune responses to HeV G and serum neutralizing titers induced in all of the animals. Circulating Ig antibodies specific for HeV G in serum were measured by microsphere assay as done previously (11). As expected, we did not detect HeV G-specific Ig in serum samples before prime vaccination (day ⫺42) but we were able to detect Ig directed against HeV G in serum samples from the sGHeV-vaccinated cohorts on the day of boost vaccination (day ⫺21) along with an increase in titer after the boost vaccination (day 0), as well as detectable HeV G-specific Ig after the challenge (Fig. 5, teal and orange). This pattern was also seen when we tested the circulating neutralizing antibody titers in serum samples against HeV, with both specifically vaccinated groups producing good neutralizing antibody titers with higher levels detected after the boost vaccination (Table 2).
est findings, which have since repeated, have had a major impact on equine veterinary practices (33). Specifically, Australian veterinarians reduced or ceased equine medicine in order to avoid contracting HeV. In order to address this problem, the Australian Government supported the commercial development of the sGHeV vaccine Equivac HeV for use in horses (28, 33). The Equivac HeV vaccine was launched late in 2012 and is currently in use in Australia, with more than 150,000 horses vaccinated to date. While significant progress on a veterinary vaccine for HeV has been made, the development of effective human vaccines and antiviral drugs for high-consequence pathogens such as HeV and NiV has been a much slower and complicated process. In particular, the restriction of infectious HeV work to BSL-4 containment has hampered vaccine development progress. In addition, conventional clinical trials with vaccines or antiviral therapies against viruses such as HeV are not practical or possible. To address the development of countermeasures for exotic pathogens such as HeV, the U.S. Food and Drug Administration implemented the Animal Efficacy Rule in 2002. This rule specifically applies to the development of countermeasures when human efficacy studies are not possible or ethical. In brief, this rule permits the evaluation of vaccines or therapeutics with data generated from studies performed in animal models that faithfully recapitulate human disTABLE 2 Serum HeV neutralizing antibody titers of vaccinated AGMs Serum HeV neutralizing antibody titera Vaccine
AGM
Day ⫺42b
Day ⫺21b
Day 0b
Day 30b
sG-Alum
O7500 O7503 O7499 O7501
⬍20 ⬍20 ⬍20 ⬍20
160 80 160 160
1,280 640 640 1,280
640 320 160 320
sG-Alum-CpG
O7515 O7494 O7506 O7477
⬍20 ⬍20 ⬍20 ⬍20
160 320 320 640
1,280 1,280 2,560 1,280
640 640 640 640
Alum only
O7498
⬍20
⬍20
20
40
CpG only
O7521
⬍20
⬍20
20
NSc
DISCUSSION
None
A substantial increase in the number of independent HeV spillover events between bats and horses occurred in Australia in 2011 (31). Notably, the first case of HeV seroconversion in a dog in Australia was also reported during these episodes (32). These lat-
R335 R372
⬍20 ⬍20
⬍20 ⬍20
⬍20 ⬍20
NS NS
a Reciprocal serum dilution at which 50% of virus was neutralized. Boldface values highlight moderate to good antibody titers. b Day after HeV challenge. c NS, not sampled.
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FIG 5 Detection of HeV G-specific antibodies from sGHeV-adjuvant-vaccinated AGMs. MFI is shown on the y axis and represents the binding of specific Ig (IgG and IgM) to sGHeV. Each error bar represents the SD of fluorescence intensity across 100 beads for each sample.
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ACKNOWLEDGMENTS We thank the staff of the UTMB Animal Resources Center for animal care. This study was supported in part by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health, and in part by the Department of Health and Human Services, National Institutes of Health (grants AI082121 to T.W.G. and AI054715 and AI077995 to C.C.B.). K.N.B. and C.C.B. are coinventors on pending U.S. patents and Australian patent 2005327194, pertaining to soluble forms of Hendra and Nipah G glycoproteins; assignees are the United States of America as represented by the Department of Health and Human Services (Washington,
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DC) and the Henry M. Jackson Foundation for the Advancement of Military Medicine Inc. (Bethesda, MD). The rest of us have no competing interests. All of the opinions, interpretations, conclusions, and recommendations presented here are ours and are not necessarily endorsed by UTMB or the Department of Defense.
REFERENCES 1. Eaton BT, Broder CC, Middleton D, Wang LF. 2006. Hendra and Nipah viruses: different and dangerous. Nat. Rev. Microbiol. 4:23–35. http://dx .doi.org/10.1038/nrmicro1323. 2. Broder CC. 2012. Henipavirus outbreaks to antivirals: the current status of potential therapeutics. Curr. Opin. Virol. 2:176 –187. http://dx.doi.org /10.1016/j.coviro.2012.02.016. 3. Marsh GA, Wang LF. 2012. Hendra and Nipah viruses: why are they so deadly? Curr. Opin. Virol. 2:242–247. http://dx.doi.org/10.1016/j.coviro .2012.03.006. 4. Mahalingam S, Herrero LJ, Playford EG, Spann K, Herring B, Rolph MS, Middleton D, McCall B, Field H, Wang LF. 2012. Hendra virus: an emerging paramyxovirus in Australia. Lancet Infect. Dis. 12:799 – 807. http://dx.doi.org/10.1016/S1473-3099(12)70158-5. 5. Selvey LA, Wells RM, McCormack JG, Ansford AJ, Murray K, Rogers RJ, Lavercombe PS, Selleck P, Sheridan JW. 1995. Infection of humans and horses by a newly described morbillivirus. Med. J. Aust. 162:642– 645. 6. Field HE, Mackenzie JS, Daszak P. 2007. Henipaviruses: emerging paramyxoviruses associated with fruit bats. Curr. Top. Microbiol. Immunol. 315:133–159. http://dx.doi.org/10.1007/978-3-540-70962-6_7. 7. Middleton DJ, Weingartl HM. 2012. Henipaviruses in their natural animal hosts. Curr. Top. Microbiol. Immunol. 359:105–121. http://dx.doi .org/10.1007/82_2012_210. 8. Playford EG, McCall B, Smith G, Slinko V, Allen G, Smith I, Moore F, Taylor C, Kung YH, Field H. 2010. Human Hendra virus encephalitis associated with equine outbreak, Australia, 2008. Emerg. Infect. Dis. 16: 219 –223. http://dx.doi.org/10.3201/eid1602.090552. 9. Wong KT, Robertson T, Ong BB, Chong JW, Yaiw KC, Wang LF, Ansford AJ, Tannenberg A. 2009. Human Hendra virus infection causes acute and relapsing encephalitis. Neuropathol. Appl. Neurobiol 35:296 – 305. http://dx.doi.org/10.1111/j.1365-2990.2008.00991.x. 10. Chong HT, Kamarulzaman A, Tan CT, Goh KJ, Thayaparan T, Kunjapan SR, Chew NK, Chua KB, Lam SK. 2001. Treatment of acute Nipah encephalitis with ribavirin. Ann. Neurol. 49:810 – 813. http://dx.doi.org /10.1002/ana.1062. 11. Rockx B, Bossart KN, Feldmann F, Geisbert JB, Hickey AC, Brining D, Callison J, Safronetz D, Marzi A, Kercher L, Long D, Broder CC, Feldmann H, Geisbert TW. 2010. A novel model of lethal Hendra virus infection in African green monkeys and the effectiveness of ribavirin treatment. J. Virol. 84:9831–9839. http://dx.doi.org/10.1128/JVI.01163-10. 12. Bonaparte MI, Dimitrov AS, Bossart KN, Crameri G, Mungall BA, Bishop KA, Choudhry V, Dimitrov DS, Wang LF, Eaton BT, Broder CC. 2005. Ephrin-B2 ligand is a functional receptor for Hendra virus and Nipah virus. Proc. Natl. Acad. Sci. U. S. A. 102:10652–10657. http://dx.doi .org/10.1073/pnas.0504887102. 13. Negrete OA, Levroney EL, Aguilar HC, Bertolotti-Ciarlet A, Nazarian R, Tajyar S, Lee B. 2005. EphrinB2 is the entry receptor for Nipah virus, an emergent deadly paramyxovirus. Nature 436:401– 405. http://dx.doi .org/10.1038/nature03838. 14. Negrete OA, Wolf MC, Aguilar HC, Enterlein S, Wang W, Muhlberger E, Su SV, Bertolotti-Ciarlet A, Flick R, Lee B. 2006. Two key residues in ephrinB3 are critical for its use as an alternative receptor for Nipah virus. PLoS Pathog. 2:e7. http://dx.doi.org/10.1371/journal.ppat.0020007. 15. Bishop KA, Stantchev TS, Hickey AC, Khetawat D, Bossart KN, Krasnoperov V, Gill P, Feng YR, Wang L, Eaton BT, Wang LF, Broder CC. 2007. Identification of Hendra virus G glycoprotein residues that are critical for receptor binding. J. Virol. 81:5893–5901. http://dx.doi.org/10 .1128/JVI.02022-06. 16. Xu K, Rockx B, Xie Y, DeBuysscher BL, Fusco DL, Zhu Z, Chan YP, Xu Y, Luu T, Cer RZ, Feldmann H, Mokashi V, Dimitrov DS, Bishop-Lilly KA, Broder CC, Nikolov DB. 2013. Crystal structure of the Hendra virus attachment G glycoprotein bound to a potent cross-reactive neutralizing human monoclonal antibody. PLoS Pathog. 9:e1003684. http://dx.doi.org /10.1371/journal.ppat.1003684. 17. Zhu Z, Dimitrov AS, Bossart KN, Crameri G, Bishop KA, Choudhry V, Mungall BA, Feng YR, Choudhary A, Zhang MY, Feng Y, Wang LF,
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ease. At this time, the ferret and AGM appear to be the models that most accurately reflect human HeV infection. The protective efficacy of sGHeV has been evaluated in the ferret (24) and AGM (26) models of NiV-mediated infection and in the ferret (25) and now in the AGM model of HeV-mediated disease. Additionally, the durability of the sGHeV vaccine has been tested in ferrets 1 year postvaccination against a NiV challenge with promising results (24). In these studies, all vaccinated animals developed high levels of antigen-specific serum Ig (Fig. 5) and neutralizing antibodies before the challenge (Table 2) which were comparable to the levels of antigen-specific Ig and neutralizing antibody against HeV seen in a previous study (26). While both of the vaccine formulations in the present study resulted in HeV G-specific Ig and significant titers of neutralizing antibody against HeV, the cohort of AGMs receiving the sGHeV-alum-CpG vaccine formulation had a significantly (P ⬍ 0.05) higher neutralizing antibody titer by Bonferroni posttest on day 0 (Table 2). Nevertheless, the present findings have clearly demonstrated that sGHeV-alum alone is also capable of providing complete protection from a HeV challenge. As HeV and NiV replicate and cause severe pathology in the lung and CpG motifs have been shown to elicit Th1 and mucosal immunity regardless of the immunization route used (34–36), the most recent sG vaccine studies have used and explored several CpG adjuvants. Additionally, several CpG motifs have also entered into human clinical trials, which could facilitate future regulatory processes for this adjuvant in general. Host responses to CpG are highly sequence and species specific. The CpG adjuvant used in feline and ferret sG vaccine trials had been used previously in humans (37–39) and cats. Mucosal IgA was detected in vaccinated and protected animals (23), suggesting that CpG may have contributed to mucosal immunity. As vaccine studies transitioned to nonhuman primates, a slightly different CpG motif (26) was selected that was shown to be an effective adjuvant in humans and nonhuman primates (40, 41). As demonstrated here and previously, AGMs vaccinated with sG and CpG (ODN 2006) mount robust immune responses and are protected from a lethal HeV or NiV challenge. Interestingly, all sG-vaccinated animals were protected from a lethal HeV challenge in the present study, but the animals that received sG with CpG had significantly higher antibody titers. Importantly, all specifically vaccinated animals were protected from HeV disease (Fig. 1B; Table 1). In addition, there was no evidence of clinical illness or infectious HeV in any vaccinated animal across all of these efficacy studies. The efficacy of the sGHeV vaccine against HeV infection described in this report provides further critical evidence that this vaccine should be moved toward further development as a human use vaccine against HeV and NiV with alum alone and alum plus CpG as adjuvant choices based on the neutralizing antibody observations in this study.
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28. 29. 30.
31. 32. 33. 34.
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by a soluble G glycoprotein of Hendra virus. J. Virol. 79:6690 – 6702. http: //dx.doi.org/10.1128/JVI.79.11.6690-6702.2005. Richmond R. 2012. The Hendra vaccine has arrived. Aust. Vet. J. 90:N2. http://dx.doi.org/10.1111/j.1751-0813.2012.00087.GRP.x. National Research Council. 2013. Guide for the care and use of laboratory animals, eighth edition. National Academies Press, Washington, DC. Geisbert TW, Daddario-DiCaprio KM, Hickey AC, Smith MA, Chan YP, Wang LF, Mattapallil JJ, Geisbert JB, Bossart KN, Broder CC. 2010. Development of an acute and highly pathogenic nonhuman primate model of Nipah virus infection. PLoS One 5:e10690. http://dx.doi.org/10 .1371/journal.pone.0010690. Field H, Crameri G, Kung NY, Wang LF. 2012. Ecological aspects of Hendra virus. Curr. Top. Microbiol. Immunol. 359:11–23. http://dx.doi .org/10.1007/82_2012_214. Anonymous. 2011. Hendra virus, equine—Australia (21): (QL) canine. ProMED Mail archive number 20110802.2324. International Society for Infectious Diseases, Brookline, MA. Mendez DH, Judd J, Speare R. 2012. Unexpected result of Hendra virus outbreaks for veterinarians, Queensland, Australia. Emerg. Infect. Dis. 18:83– 85. http://dx.doi.org/10.3201/eid1801.111006. McCluskie MJ, Weeratna RD, Payette PJ, Davis HL. 2002. Parenteral and mucosal prime-boost immunization strategies in mice with hepatitis B surface antigen and CpG DNA. FEMS Immunol. Med. Microbiol. 32: 179 –185. http://dx.doi.org/10.1111/j.1574-695X.2002.tb00551.x. Chu JH, Chiang CC, Ng ML. 2007. Immunization of flavivirus West Nile recombinant envelope domain III protein induced specific immune response and protection against West Nile virus infection. J. Immunol. 178: 2699 –2705. http://www.jimmunol.org/content/178/5/2699.full. Knight JB, Huang YY, Halperin SA, Anderson R, Morris A, Macmillan A, Jones T, Burt DS, Van Nest G, Lee SF. 2006. Immunogenicity and protective efficacy of a recombinant filamentous haemagglutinin from Bordetella pertussis. Clin. Exp. Immunol. 144:543–551. http://dx.doi.org /10.1111/j.1365-2249.2006.03097.x. Halperin SA, Van Nest G, Smith B, Abtahi S, Whiley H, Eiden JJ. 2003. A phase I study of the safety and immunogenicity of recombinant hepatitis B surface antigen co-administered with an immunostimulatory phosphorothioate oligonucleotide adjuvant. Vaccine 21:2461–2467. http://dx.doi .org/10.1016/S0264-410X(03)00045-8. Broide DH. 2006. Prospects for CpG based immunotherapy in asthma and allergy. Arb. Paul Ehrlich Inst. Bundesamt Sera Impfstoffe Frankf. A. M. 2006:217–220; discussion 220 –212. Anonymous. 2006. CpG 7909: PF 3512676, PF-3512676. Drugs R. D. 7:312–316. http://dx.doi.org/10.2165/00126839-200607050-00004. Conforti VA, de Avila DM, Cummings NS, Wells KJ, Ulker H, Reeves JJ. 2007. The effectiveness of a CpG motif-based adjuvant (CpG ODN 2006) for LHRH immunization. Vaccine 25:6537– 6543. http://dx.doi.org /10.1016/j.vaccine.2007.05.056. Hartmann G, Weeratna RD, Ballas ZK, Payette P, Blackwell S, Suparto I, Rasmussen WL, Waldschmidt M, Sajuthi D, Purcell RH, Davis HL, Krieg AM. 2000. Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo. J. Immunol. 164:1617–1624. http://www.jimmunol .org/content/164/3/1617.
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20.
Xiao X, Eaton BT, Broder CC, Dimitrov DS. 2006. Potent neutralization of Hendra and Nipah viruses by human monoclonal antibodies. J. Virol. 80:891– 899. http://dx.doi.org/10.1128/JVI.80.2.891-899.2006. Zhu Z, Bossart KN, Bishop KA, Crameri G, Dimitrov AS, McEachern JA, Feng Y, Middleton D, Wang LF, Broder CC, Dimitrov DS. 2008. Exceptionally potent cross-reactive neutralization of Nipah and Hendra viruses by a human monoclonal antibody. J. Infect. Dis. 197:846 – 853. http://dx.doi.org/10.1086/528801. Bossart KN, Zhu Z, Middleton D, Klippel J, Crameri G, Bingham J, McEachern JA, Green D, Hancock TJ, Chan YP, Hickey AC, Dimitrov DS, Wang LF, Broder CC. 2009. A neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute Nipah virus infection. PLoS Pathog. 5:e1000642. http://dx.doi.org/10 .1371/journal.ppat.1000642. Bossart KN, Geisbert TW, Feldmann H, Zhu Z, Feldmann F, Geisbert JB, Yan L, Feng YR, Brining D, Scott D, Wang Y, Dimitrov AS, Callison J, Chan YP, Hickey AC, Dimitrov DS, Broder CC, Rockx B. 2011. A neutralizing human monoclonal antibody protects African green monkeys from Hendra virus challenge. Sci. Transl. Med. 3:105ra103. http://dx .doi.org/10.1126/scitranslmed.3002901. Ploquin A, Szecsi J, Mathieu C, Guillaume V, Barateau V, Ong KC, Wong KT, Cosset FL, Horvat B, Salvetti A. 2013. Protection against henipavirus infection by use of recombinant adeno-associated virusvector vaccines. J. Infect. Dis. 207:469 – 478. http://dx.doi.org/10.1093 /infdis/jis699. McEachern JA, Bingham J, Crameri G, Green DJ, Hancock TJ, Middleton D, Feng YR, Broder CC, Wang LF, Bossart KN. 2008. A recombinant subunit vaccine formulation protects against lethal Nipah virus challenge in cats. Vaccine 26:3842–3852. http://dx.doi.org/10.1016/j.vaccine .2008.05.016. Mungall BA, Middleton D, Crameri G, Bingham J, Halpin K, Russell G, Green D, McEachern J, Pritchard LI, Eaton BT, Wang LF, Bossart KN, Broder CC. 2006. Feline model of acute Nipah virus infection and protection with a soluble glycoprotein-based subunit vaccine. J. Virol. 80: 12293–12302. http://dx.doi.org/10.1128/JVI.01619-06. Pallister JA, Klein R, Arkinstall R, Haining J, Long F, White JR, Payne J, Feng YR, Wang LF, Broder CC, Middleton D. 2013. Vaccination of ferrets with a recombinant G glycoprotein subunit vaccine provides protection against Nipah virus disease for over 12 months. Virol. J. 10:237. http://dx.doi.org/10.1186/1743-422X-10-237. Pallister J, Middleton D, Wang LF, Klein R, Haining J, Robinson R, Yamada M, White J, Payne J, Feng YR, Chan YP, Broder CC. 2011. A recombinant Hendra virus G glycoprotein-based subunit vaccine protects ferrets from lethal Hendra virus challenge. Vaccine 29:5623–5630. http: //dx.doi.org/10.1016/j.vaccine.2011.06.015. Bossart KN, Rockx B, Feldmann F, Brining D, Scott D, LaCasse R, Geisbert JB, Feng YR, Chan YP, Hickey AC, Broder CC, Feldmann H, Geisbert TW. 2012. A Hendra virus G glycoprotein subunit vaccine protects African green monkeys from Nipah virus challenge. Sci. Transl. Med. 4:146ra107. http://dx.doi.org/10.1126/scitranslmed.3004241. Bossart KN, Crameri G, Dimitrov AS, Mungall BA, Feng YR, Patch JR, Choudhary A, Wang LF, Eaton BT, Broder CC. 2005. Receptor binding, fusion inhibition, and induction of cross-reactive neutralizing antibodies
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Chad E. Mire, Joan B. Geisbert, Krystle N. Agans, Yan-Ru Feng, Karla A. Fenton, Katharine N. Bossart, Lianying Yan, Yee-Peng Chan, Christopher C. Broder and Thomas W. Geisbert J. Virol. 2014, 88(9):4624. DOI: 10.1128/JVI.00005-14. Published Ahead of Print 12 February 2014.
A Recombinant Hendra Virus G Glycoprotein Subunit Vaccine Protects Nonhuman Primates against Hendra Virus Challenge Chad E. Mire,a,b Joan B. Geisbert,a,b Krystle N. Agans,a,b Yan-Ru Feng,c Karla A. Fenton,a,b Katharine N. Bossart,d Lianying Yan,c Yee-Peng Chan,c Christopher C. Broder,c Thomas W. Geisberta,b
ABSTRACT
Hendra virus (HeV) is a zoonotic emerging virus belonging to the family Paramyxoviridae. HeV causes severe and often fatal respiratory and/or neurologic disease in both animals and humans. Currently, there are no licensed vaccines or antiviral drugs approved for human use. A number of animal models have been developed for studying HeV infection, with the African green monkey (AGM) appearing to most faithfully reproduce the human disease. Here, we assessed the utility of a newly developed recombinant subunit vaccine based on the HeV attachment (G) glycoprotein in the AGM model. Four AGMs were vaccinated with two doses of the HeV vaccine (sGHeV) containing Alhydrogel, four AGMs received the sGHeV with Alhydrogel and CpG, and four control animals did not receive the sGHeV vaccine. Animals were challenged with a high dose of infectious HeV 21 days after the boost vaccination. None of the eight specifically vaccinated animals showed any evidence of clinical illness and survived the challenge. All four controls became severely ill with symptoms consistent with HeV infection, and three of the four animals succumbed 8 days after exposure. Success of the recombinant subunit vaccine in AGMs provides pivotal data in supporting its further preclinical development for potential human use. IMPORTANCE
A Hendra virus attachment (G) glycoprotein subunit vaccine was tested in nonhuman primates to assess its ability to protect them from a lethal infection with Hendra virus. It was found that all vaccinated African green monkeys were completely protected against subsequent Hendra virus infection and disease. The success of this new subunit vaccine in nonhuman primates provides critical data in support of its further development for future human use.
T
he henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), cause severe and often fatal respiratory disease and encephalitis in horses, pigs, and humans (1–4). In contrast to all other paramyxoviruses, henipaviruses infect a broad range of species spanning six mammalian orders. Because of this broad species tropism, ease of access and propagation, potential for person-toperson transmission, high case fatality rates, and lack of approved countermeasures for human use, HeV and NiV pose significant biosecurity threats and are classified as biosafety level 4 (BSL-4) pathogens. HeV emerged in Australia in 1994 and was identified as the causative agent of an acute respiratory illness in horses (5). HeV is transmitted to horses by pteropid fruit bats, commonly known as flying foxes, with human infection occurring by close contact with infected horses (6, 7). Outbreaks of HeV have occurred in Australia on a nearly annual basis since the initial outbreak, with all episodes involving infection of horses. In total, ⬎80 horses have succumbed to HeV infection, with a case fatality rate of approximately 75%. There have been seven human HeV infections recorded, most recently in 2009, of which four have been fatal (57%) (8). All patients initially presented with influenza-like symptoms after an incubation period of 7 to 16 days. While two of the patients recovered from the influenza-like illness, one developed pneumonitis and succumbed to multiorgan failure. Three different patients developed encephalitic manifestations (mild confusion, ataxia), with two of these cases progressing to seizures and resulting in death (5, 8, 9). There are currently no approved vaccines or antiviral drugs for
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combating human HeV or NiV infection. Regarding treatment options for henipavirus infection, an open-label ribavirin trial was performed with 140 patients during the initial outbreak of NiV in Malaysia in 1998; however, the results of that study remain controversial (10). In addition, three of the seven recorded human HeV cases were treated with ribavirin and one of the patients survived (8). The utility of ribavirin as a countermeasure against HeV infection was subsequently assessed in African green monkeys (AGMs) (11). While there was a small benefit in delaying death, there was no survival benefit in this nonhuman primate model. Currently, the most promising postexposure treatment for henipavirus infection appears to be an experimental human monoclonal antibody (MAb). This MAb, m102.4, targets the ephrin-B2 and -B3 receptor binding domain of the henipavirus envelope attachment (G) glycoprotein (12–16). m102.4 is a potent cross-reactive neutralizing antibody in vitro (17, 18) and had been shown to protect ferrets from a lethal NiV challenge (19) and AGMs from a lethal HeV challenge (20). Importantly, in 2010,
Received 3 January 2014 Accepted 8 February 2014 Published ahead of print 12 February 2014 Editor: R. W. Doms Address correspondence to Christopher C. Broder, [email protected]. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.00005-14 The authors have paid a fee to allow immediate free access to this article.
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Galveston National Laboratorya and Departments of Microbiology and Immunology,b University of Texas Medical Branch, Galveston, Texas, USA; Department of Microbiology and Immunology, Uniformed Services University, Bethesda, Maryland, USAc; Integrated Research Associates, LLC, San Francisco, California, USAd
Henipavirus G Glycoprotein Subunit Vaccine
MATERIALS AND METHODS Statistics. Conducting animal studies at BSL-4 severely restricts the number of animals, the volume of biological samples that can be obtained, and the ability to repeat assays independently and thus limits statistical analysis. Consequently, data are presented as the mean or median values calculated from replicate samples, not replicate assays, and error bars represent the standard deviations across replicates. Statistics were calculated for serum neutralizing antibody titers with GraphPad Prism 5 software by using a two-way analysis of variance comparing treatments and times between all groups and using the Bonferroni method posttest. Viruses and cells. HeV was kindly provided by Tom Ksiazek and was obtained from a patient who was part of the 1994 outbreak in Australia. The virus was propagated on Vero E6 cells in Eagle’s minimal essential medium supplemented with 10% fetal calf serum. The titer of the HeV stock used was ⬃1 ⫻ 107 PFU/ml. The HeV challenge virus stock was assessed for the presence of endotoxin with the Endosafe-PTS portable test system (Charles River, Wilmington, MA). Virus preparations were diluted 1:10 in Limulus amebocyte lysate (LAL) reagent water in accordance with the manufacturer’s directions, and endotoxin levels were tested in LAL Endosafe-PTS cartridges as directed by the manufacturer. Each preparation was found to be below the limit of detection, while positive controls showed that the tests were valid. Vaccine formulation. Production and purification of sGHeV were done as previously described (26). Two vaccines containing sGHeV and Alhydrogel (Accurate Chemical & Scientific Corporation, Westbury, NY) at a weight ratio of 1:25 were prepared with one formulation also containing CpG oligodeoxynucleotide (ODN) 2006 (InvivoGen, San Diego, CA) with a fully phosphorothioate-modified backbone (sGHeV-alum-CpG). The vaccine without CpG was formulated with 100 g of sGHeV and 2.5 mg of aluminum ion (sGHeV-alum) per vaccinated animal. The vaccine with CpG was formulated with 100 g of sGHeV, 2.5 mg of aluminum ion, and 150 mg of ODN 2006 per vaccinated animal. For the vaccine containing both Alhydrogel and CpG, the Alhydrogel and sGHeV were mixed
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before ODN 2006 was added. Each vaccine dose was adjusted to 1 ml with phosphate-buffered saline (PBS), and mixtures were incubated on a rotating wheel at room temperature for at least 2 to 3 h before injection. Each AGM received the same dose of 1 ml for prime and boost vaccinations, and all vaccine doses were given via intramuscular (i.m.) injection. The 100-g dose used in this study was based on the protective efficacy against NiV and the prolonged level of IgG in AGMs vaccinated with this dose rather than 10 or 50 g (26). Animals. Animal studies were performed in BSL-4 biocontainment at the Galveston National Laboratory at the University of Texas Medical Branch (UTMB) at Galveston and were approved by the UTMB Institutional Animal Care and Use Committee. Animal research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adhered to the principles stated in reference 29. The facility where this research was conducted is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Twelve adult AGMs weighing 3 to 6 kg (PreLabs, Hines, IL) were used in this study. AGMs were anesthetized by i.m. injection with ketamine and vaccinated with sGHeV by i.m. injection on day ⫺42 (prime vaccination) and day ⫺21 (boost vaccination) (Fig. 1, black and gray arrows, respectively). The vaccine formulations used are described above. Four AGMs received two doses of vaccine containing sGHeV and Alhydrogel; four animals received two doses of vaccine containing sGHeV, Alhydrogel, and CpG; one animal received two doses of Alhydrogel only; one animal received two doses of CpG only; and two animals were not vaccinated. Animals were inoculated intratracheally (i.t.) with ⬃5 ⫻ 105 PFU of HeV in 5 ml of Dulbecco’s minimal essential medium (DMEM; Sigma-Aldrich, St. Louis, MO) 21 days after the boost vaccination (Fig. 1, *). Animals were anesthetized for clinical examination, including temperature, respiration quality, blood collection, and swabs of nasal, oral, and rectal mucosae on days 0, 3, 5, 7, 10, 15, and 30 postchallenge. AGMs in the vaccine cohorts were euthanized on day 30 postchallenge, whereas three of the four control AGMs had to be euthanized according to approved humane endpoints on day 8 postchallenge. All of the other AGMs survived until the end of the study. Measurement of serum or plasma HeV G-specific antibodies. AGM serum samples collected at the time points indicated were tested for immunoglobulin (Ig) antibodies against HeV G with previously developed multiplexed microsphere assays (11). In brief, 96-well filter plates were primed with PBS. Test serum samples were diluted in PBS at 1:10 at prevaccination time points and at 1:10,000 at postvaccination time points. Biotinylated goat anti-human IgM/IgG and streptavidin-phycoerythrin (strep-PE) were also diluted in PBS. Coupled microspheres (sG-HeV) were prepared by sonication for 1 min, followed by vortex mixing for 1 min each, and then diluted in PBS. Priming liquid was removed from the plates with a Bio-Plex Pro II Wash Station (Bio-Rad Laboratories, Hercules, CA), and 100 l containing 1,500 coupled microspheres was added to each well. The microsphere mixture was removed by vacuum, 100 l of diluted test serum was added to appropriate wells, and the mixture was incubated at room temperature for 30 min while shaking in the dark. Diluted test samples were removed by vacuum, 100 l of diluted biotinylated goat anti-human antibody (1:500; Pierce Protein Biology Products, ThermoFisher Scientific, Waltham, MA) was added to each well, and the mixture was incubated as described above. Liquid was removed by vacuum, 100 l of strep-PE (1:1,000; Qiagen Inc., Valencia, CA) was added to each well, and the mixture was incubated for 30 min. All of the liquid was removed from the plates with a vacuum manifold and washed twice with 300 l of PBS, and the liquid was removed between wash steps. Finally, 125 l of PBS was added to each well and incubated for 2 min as described above. Samples were assayed for mean fluorescence intensity (MFI) across at least a 100 bead region performed on the BioPlex-200 machine and analyzed with Bio-Plex Manager Software (v 6.1) (Bio-Rad). MFI and the standard deviation (SD) of fluorescence intensity across 100 beads were determined for each sample and plotted.
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m102.4 was administered to two individuals in Australia who had a significant risk of exposure to HeV under a compassionate-use protocol. It was also used in 2012 to treat an individual with possible exposure to HeV in Australia and in 2013 to treat an individual with possible laboratory exposure in the United States. To date, all four of these individuals have no evidence of henipavirus infection. In addition to the postexposure treatments, two experimental preventive vaccines against henipaviruses have been evaluated in animal models. A recombinant adeno-associated virus vaccine expressing the NiV G protein completely protected hamsters against a homologous NiV challenge and protected 50% of the animals against a heterologous HeV infection (21). In addition, a recombinant subunit vaccine based on the HeV G protein (sGHeV) completely protected small animals against lethal HeV and NiV infections (22–25) and more recently was shown to be efficacious in the robust AGM model of NiV infection (26). sGHeV (amino acids 73 to 604) is an engineered secreted version of the full ectodomain of the G glycoprotein in which the transmembrane and cytoplasmic tail domains have been deleted from the N terminus (27). Importantly, this vaccine has also been shown to protect horses from lethal HeV infection and has been licensed for use by the equine industry in Australia (28). However, there has been no study to date assessing the performance of this vaccine in a nonhuman primate model of HeV infection, which is a necessary prerequisite for the licensure of such a vaccine for use in humans. Here, we report for the first time the prophylactic efficacy of the sGHeV vaccine in a lethal HeV AGM model that faithfully recapitulates human HeV infection.
Mire et al.
g)-adjuvant or adjuvant alone. Gray arrow; boost vaccination with sGHeV-adjuvant or adjuvant alone. The asterisk shows the day of i.t. HeV challenge with 5 ⫻ 105 PFU (B) Kaplan-Meier survival curves of the control group (black, n ⫽ 4; 2 nonvaccinated, 1 alum only, and 1 alum-CpG only) and the vaccinated groups (sGHeV-alum [teal, n ⫽ 4] and sGHeV-alum-CpG [orange, n ⫽ 4]).
HeV serum neutralization assays. Neutralization titers were determined by a conventional serum neutralization assay. Briefly, serum samples were serially diluted 2-fold and incubated with ⬃100 PFU of HeV for 1 h at 37°C. Virus and antibodies were then added to individual wells of six-well plates of Vero cells. Plates were stained with neutral red 2 days after infection, and plaques were counted 24 h after staining. The 50% neutralization titer was determined as the serum dilution at which at there was a 50% reduction in plaque counts versus the control wells. Specimen collection and processing in HeV-infected AGMs. Blood was collected in EDTA or serum Vacutainers (Becton, Dickinson, Franklin Lakes, NJ). Nasal, oral, and rectal swabs were collected in 1 ml of DMEM (Sigma-Aldrich) and vortexed for 30 s. Immediately following sampling, 100 l of blood or 100 l of DMEM from individual swab samples was added to 600 l of AVL viral lysis buffer (Qiagen) for RNA extraction. For tissues, approximately 100 mg was stored in 1 ml of RNAlater (Qiagen) for 7 days to stabilize the RNA. RNAlater was completely removed, and tissues were homogenized in 600 l of RLT buffer (Qiagen) in a 2-ml cryovial with a TissueLyser (Qiagen) and stainless steel beads. The tissues sampled included conjunctiva, tonsil, oro- and nasopharynx, nasal mucosa, trachea, right bronchus, left bronchus, right lung upper lobe, right lung middle lobe, right lung lower lobe, right lung upper lobe, right lung middle lobe, right lung lower lobe, bronchial lymph node (LN), heart, liver, spleen, kidney, adrenal gland, pancreas, jejunum, colon transversum, brain (frontal and cerebellum), brain stem, cervical spinal cord, pituitary gland, mandibular LN, salivary gland LN, inguinal LN, axillary LN, mesenteric LN, urinary bladder, testes or ovaries, and femoral bone marrow. All blood samples and swabs were inactivated in AVL viral lysis buffer, and tissue samples were homogenized and inactivated in RLT buffer prior to removal from the BSL-4 laboratory. Subsequently, RNA was isolated from blood and swabs with the QIAamp viral RNA kit (Qiagen) and from tissues by using the RNeasy minikit (Qiagen) according to the manufacturer’s instructions supplied with each kit. Hematology and serum biochemistry. Total white blood cell (WBC) counts, differential WBC counts, red blood cell counts, platelet counts, hematocrit values, total hemoglobin concentrations, mean cell volumes, mean corpuscular volumes, and mean corpuscular hemoglobin concentrations were analyzed in blood collected in tubes containing EDTA with a laser-based hematologic analyzer (Beckman Coulter, Brea, CA). Serum samples were tested for concentrations of albumin, amylase, alanine aminotransferase, aspartate aminotransferase (AST), alkaline phosphatase, gamma-glutamyltransferase, glucose, cholesterol, total protein, total bilirubin, blood urea nitrogen (BUN), creatinine (CRE), and C-reactive protein (CRP) by with a Piccolo point-of-care analyzer and Biochemistry Panel Plus analyzer discs (Abaxis, Sunnyvale, CA).
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Histopathology and immunohistochemistry analyses. Necropsy was performed on all AGMs. Tissue samples of all major organs were collected for histopathologic and immunohistochemical examinations and immersion fixed in 10% neutral buffered formalin for at least 21 days at BSL-4. Subsequently, the formalin was changed; specimens were removed from BSL-4, processed at BSL-2 by conventional methods, embedded in paraffin, and sectioned at a 5-m thickness. For immunohistochemistry analysis, specific anti-HeV immunoreactivity was detected with an anti-HeV N protein rabbit primary antibody at a 1:5,000 dilution for 30 min. The tissue sections were processed for immunohistochemistry analysis with the Dako Autostainer (Dako, Carpinteria, CA). The secondary antibody used was biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) at 1:200 for 30 min, followed by Dako LSAB2 streptavidinhorseradish peroxidase (Dako) for 15 min. Slides were developed with Dako 3,3=-diaminobenzidine chromogen (Dako) for 5 min and counterstained with hematoxylin for 1 min. Nonimmune rabbit IgG was used as a negative staining control. Detection of HeV loads. RNA was isolated from nasal, oral, and rectal swabs; blood; or tissues and analyzed with primers/probe targeting the N gene of HeV for quantitative real-time PCR (qRT-PCR) (11), with the probe used here being 6-carboxyfluorescein–5=-TCCTGAGTCTGCCTG AGGGCGGT-3=-– 6-carboxytetramethylrhodamine (Life Technologies, Carlsbad, CA). HeV RNA was detected with the CFX96 detection system (Bio-Rad) in One-Step Probe qRT-PCR kits (Qiagen) under the following cycling conditions: 50°C for 10 min, 95°C for 10 s, and 40 cycles of 95°C for 10 s and 59°C for 30 s. Threshold cycle values representing HeV genomes were analyzed with CFX Manager Software, and data are shown in genome equivalents (GEq). To create the GEq standard, RNA was extracted from HeV challenge stocks and the number of HeV genomes was calculated by using Avogadro’s number and the molecular weight of the HeV genome. Virus titration was performed by plaque assay with Vero cells from all blood samples. Briefly, increasing 10-fold dilutions of the samples were adsorbed to Vero cell monolayers in duplicate wells (200 l); the limit of detection was 25 PFU/ml.
RESULTS
HeV challenge of vaccinated AGMs. We previously described the development of a lethal AGM model for HeV infection with observed clinical signs and pathology highly consistent with the HeV-mediated disease reported in humans (11). Clinical signs in this model include severe depression, respiratory disease leading to acute respiratory distress, neurologic disease, reduced activity, and a time to death of 8 to 10 days. The purpose of the present
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FIG 1 (A) Diagram of the sGHeV vaccination schedule and sampling days after a HeV challenge of AGMs. Black arrow; prime vaccination with sGHeV (100
Henipavirus G Glycoprotein Subunit Vaccine
TABLE 1 Clinical descriptions and outcomes of HeV-challenged AGMs Sex
Group
Clinical illness
Clinical and gross pathology
R335
Male
Control
Fever (day 7), depression (day 8), lethargy (day 8), loss of appetite (days 7, 8), labored breathing (days 5–8), dehydration (days 5, 7), death (day 8)
R372
Female
Control
O7498
Male
Control
O7521
Male
Control
Fever (days 3, 5), Depression (days 10–13), lethargy (days 10–13), loss of appetite (days 4–8), labored breathing (days 7, 8), dehydration (days 7, 8), chills (day 8), hind limb paresis (day 8), euthanasia (day 8) Depression (day 8), lethargy (day 8), loss of appetite (days 8–15), labored breathing (days 7–13), dehydration (day 10), splenomegaly (day 7) survival Fever (day 7), depression (day 8), lethargy (day 8), loss of appetite (days 7, 8), labored breathing (days 5–8), death (day 8)
Thrombocytopenia (day 8), ⬎2-fold increase in WBC count, ⬎3-fold increase in BUN (day 8), ⬎2-fold increase in CRE (day 8), ⬎4-fold increase in AST (day 8), ⬎10-fold increase in CRP (days 7, 8), excess blood-tinged pleural fluid, severely inflated enlarged lungs with severe congestion and hemorrhage of all lobes, darkened liver Moderately inflated enlarged lungs with multifocal areas of congestion
O7500 O7503 O7499 O7501
Male Male Male Male
sG-Alum sG-Alum sG-Alum sG-Alum
None None None None
None None None None
O7515 O7494 O7506 O7477
Male Male Male Male
sG-Alum-CpG sG-Alum-CpG sG-Alum-CpG sG-Alum-CpG
None None None None
None None None None
study was to determine whether vaccination with sGHeV could prevent HeV infection and illness in AGMs. Additionally, we tested whether protection from HeV infection requires vaccination with sGHeV plus two adjuvants (sGHeV-alum-CpG) or a single adjuvant (sGHeV-alum). A time line of the vaccination regimen, HeV challenge, and specimen collection is shown in Fig. 1A. All four control AGMs showed disease consistent with historical controls (11), including loss of appetite, depression, decreased activity, and labored breathing (Table 1). Three of the four control AGMs (R335, O7521, R372) developed acute respiratory distress, and one also developed hind limb paresis; all three of these animals succumbed on day 8 after exposure (Fig. 1B and Table 1). While the remaining control animal (O7498) was clinically ill for a prolonged period of time, it began to recover on postchallenge day 14 and survived until the study endpoint. In contrast, none of the vaccinated animals showed any evidence of clinical illness and all survived (Fig. 1B and Table 1). HeV loads. To determine if there was HeV replication in animals postchallenge, virus shedding was assessed by qRT-PCR of nasal, oral, and rectal swabs (Fig. 2A, B, and C, respectively) and viremia was also screened for by qRT-PCR of whole blood samples (Fig. 2D). HeV GEq were observed in all swab and blood samples from two of the control animals (R335 and O7521, Fig. 2), while we detected HeV GEq in oral swabs from another control animal (R372) on day 7 postchallenge (Fig. 2B, red). The surviving con-
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None
Thrombocytopenia (day 7), ⬎2-fold increase in WBC count (day 8), ⬎2-fold increase in BUN (day 8), 5-fold increase in CRE (day 8), ⬎2-fold increase in AST (day 8), ⬎10-fold increase in CRP (day 8), excess blood-tinged pleural fluid, inflated enlarged lungs with multifocal areas of congestion and hemorrhage particularly of the lower and middle right lobes, darkened liver
trol animal (O7498) had no detectable HeV RNA in any sample. Likewise, none of the specifically vaccinated animals had any detectable HeV RNA in any sample (negative data not shown). HeV RNA was also detected systemically in the tissues of control animals R335 and O7521 (Fig. 3, green and black, respectively), whereas HeV RNA was detected only in the respiratory tissues, the axillary LN, and femoral bone marrow of control animal O7521 (Fig. 3, red). HeV RNA was not detected in tissues of control animal O7498 or in any of the tissues of any of the specifically vaccinated animals (negative data not shown). Detection of HeV RNA in tissues (Fig. 3), swabs, and blood (Fig. 2) correlated with each animal’s outcome and gross pathology (Fig. 1B and Table 1). Histopathological and immunohistochemical analysis of HeV-infected AGMs. Histopathologic analysis of samples from the three control animals that succumbed to a HeV challenge was mostly consistent with previous findings on henipavirus-infected AGMs (11, 20, 26, 30), and lesions from control animals R335 and O7521 were more prominent than those from control animal R372. Noteworthy lesions included interstitial pneumonia, necrosis and hemorrhage of the splenic white pulp, and variable syncytial cell formation in lymphoid tissues. Alveolar spaces were filled with edema fluid, fibrin, foamy alveolar macrophages, and cellular debris. Glomerular tufts were hypercellular and congested, and syncytial cells were occasionally noted within the endothelium of the glomerular tufts. Strong immunoreactivity to HeV antigen
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AGM
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was present within the alveolar capillary endothelium and alveolar macrophages and also within scattered mononuclear cells in the subcapsular and medullary sinuses of various LNs (Fig. 4). Strong immunoreactivity to HeV antigen was also present in the kidneys
within segmental regions of the endothelium of the glomerular tufts (Fig. 4). Neuropathology was not as prominent in the control animals as in HeV-infected AGMs in a previous study (11). Nonetheless, HeV antigen was a notable finding within the endothelium
FIG 3 Viral loads of the AGM control cohort (all vaccinated; negative data not shown) as detected by GEq/mg by qRT-PCR assay of tissues. R335 and R372 (green and red, respectively), nonvaccinated controls; O7498 (white), alum-only control; O7521 (black), alum-CpG only control. R.U., right upper; R.M., right middle; R.L., right lower; L.U., left upper; L.M., left middle; L.L., left lower. Error bars are SDs.
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FIG 2 Viral loads of the AGM control cohort (all vaccinated; negative data not shown) expressed in GEq/ml and determined by qRT-PCR assay of nasal swabs (A), oral swabs (B), rectal swabs (C), and blood (D). R335 and R372 (green and red, respectively), nonvaccinated controls; O7498 (white), alum-only control; O7521 (black), alum-CpG-only control. Error bars are SDs.
Henipavirus G Glycoprotein Subunit Vaccine
FIG 4 Lack of HeV antigen in representative sGHeV-vaccinated tissues and localization of HeV antigen in representative control tissues by immunohistochemical staining. Lung, spleen, kidney, and brain stem samples were labeled with an N protein-specific polyclonal rabbit antibody, and images were taken. Magnifications: lung, ⫻20; spleen, ⫻20; kidney, ⫻40; brain stem, ⫻60. sG-CpG-Alum, all tissues from O7515; sG-Alum, all tissues from O7500. Controls: lung tissue from O7521, spleen tissue from O7521, kidney tissue from R335, brain stem tissue from R335.
of the brain stem (Fig. 4). Representative tissue sections from the control animals are shown in Fig. 4. Examination of tissue sections from vaccinated AGMs revealed only normal tissue architecture. Importantly, HeV antigen was not detected in any tissue of any sGHeV-vaccinated AGM by immunohistochemical techniques (Fig. 4). At the study endpoint, the tissue architecture was also normal in the control AGM that survived (O7498); HeV antigen was also undetectable in tissues from this animal. Humoral immune response to HeV G. The AGMs used in this study were vaccinated with sGHeV with adjuvant; therefore, we were interested in measuring the humoral immune responses to HeV G and serum neutralizing titers induced in all of the animals. Circulating Ig antibodies specific for HeV G in serum were measured by microsphere assay as done previously (11). As expected, we did not detect HeV G-specific Ig in serum samples before prime vaccination (day ⫺42) but we were able to detect Ig directed against HeV G in serum samples from the sGHeV-vaccinated cohorts on the day of boost vaccination (day ⫺21) along with an increase in titer after the boost vaccination (day 0), as well as detectable HeV G-specific Ig after the challenge (Fig. 5, teal and orange). This pattern was also seen when we tested the circulating neutralizing antibody titers in serum samples against HeV, with both specifically vaccinated groups producing good neutralizing antibody titers with higher levels detected after the boost vaccination (Table 2).
est findings, which have since repeated, have had a major impact on equine veterinary practices (33). Specifically, Australian veterinarians reduced or ceased equine medicine in order to avoid contracting HeV. In order to address this problem, the Australian Government supported the commercial development of the sGHeV vaccine Equivac HeV for use in horses (28, 33). The Equivac HeV vaccine was launched late in 2012 and is currently in use in Australia, with more than 150,000 horses vaccinated to date. While significant progress on a veterinary vaccine for HeV has been made, the development of effective human vaccines and antiviral drugs for high-consequence pathogens such as HeV and NiV has been a much slower and complicated process. In particular, the restriction of infectious HeV work to BSL-4 containment has hampered vaccine development progress. In addition, conventional clinical trials with vaccines or antiviral therapies against viruses such as HeV are not practical or possible. To address the development of countermeasures for exotic pathogens such as HeV, the U.S. Food and Drug Administration implemented the Animal Efficacy Rule in 2002. This rule specifically applies to the development of countermeasures when human efficacy studies are not possible or ethical. In brief, this rule permits the evaluation of vaccines or therapeutics with data generated from studies performed in animal models that faithfully recapitulate human disTABLE 2 Serum HeV neutralizing antibody titers of vaccinated AGMs Serum HeV neutralizing antibody titera Vaccine
AGM
Day ⫺42b
Day ⫺21b
Day 0b
Day 30b
sG-Alum
O7500 O7503 O7499 O7501
⬍20 ⬍20 ⬍20 ⬍20
160 80 160 160
1,280 640 640 1,280
640 320 160 320
sG-Alum-CpG
O7515 O7494 O7506 O7477
⬍20 ⬍20 ⬍20 ⬍20
160 320 320 640
1,280 1,280 2,560 1,280
640 640 640 640
Alum only
O7498
⬍20
⬍20
20
40
CpG only
O7521
⬍20
⬍20
20
NSc
DISCUSSION
None
A substantial increase in the number of independent HeV spillover events between bats and horses occurred in Australia in 2011 (31). Notably, the first case of HeV seroconversion in a dog in Australia was also reported during these episodes (32). These lat-
R335 R372
⬍20 ⬍20
⬍20 ⬍20
⬍20 ⬍20
NS NS
a Reciprocal serum dilution at which 50% of virus was neutralized. Boldface values highlight moderate to good antibody titers. b Day after HeV challenge. c NS, not sampled.
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FIG 5 Detection of HeV G-specific antibodies from sGHeV-adjuvant-vaccinated AGMs. MFI is shown on the y axis and represents the binding of specific Ig (IgG and IgM) to sGHeV. Each error bar represents the SD of fluorescence intensity across 100 beads for each sample.
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ACKNOWLEDGMENTS We thank the staff of the UTMB Animal Resources Center for animal care. This study was supported in part by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health, and in part by the Department of Health and Human Services, National Institutes of Health (grants AI082121 to T.W.G. and AI054715 and AI077995 to C.C.B.). K.N.B. and C.C.B. are coinventors on pending U.S. patents and Australian patent 2005327194, pertaining to soluble forms of Hendra and Nipah G glycoproteins; assignees are the United States of America as represented by the Department of Health and Human Services (Washington,
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DC) and the Henry M. Jackson Foundation for the Advancement of Military Medicine Inc. (Bethesda, MD). The rest of us have no competing interests. All of the opinions, interpretations, conclusions, and recommendations presented here are ours and are not necessarily endorsed by UTMB or the Department of Defense.
REFERENCES 1. Eaton BT, Broder CC, Middleton D, Wang LF. 2006. Hendra and Nipah viruses: different and dangerous. Nat. Rev. Microbiol. 4:23–35. http://dx .doi.org/10.1038/nrmicro1323. 2. Broder CC. 2012. Henipavirus outbreaks to antivirals: the current status of potential therapeutics. Curr. Opin. Virol. 2:176 –187. http://dx.doi.org /10.1016/j.coviro.2012.02.016. 3. Marsh GA, Wang LF. 2012. Hendra and Nipah viruses: why are they so deadly? Curr. Opin. Virol. 2:242–247. http://dx.doi.org/10.1016/j.coviro .2012.03.006. 4. Mahalingam S, Herrero LJ, Playford EG, Spann K, Herring B, Rolph MS, Middleton D, McCall B, Field H, Wang LF. 2012. Hendra virus: an emerging paramyxovirus in Australia. Lancet Infect. Dis. 12:799 – 807. http://dx.doi.org/10.1016/S1473-3099(12)70158-5. 5. Selvey LA, Wells RM, McCormack JG, Ansford AJ, Murray K, Rogers RJ, Lavercombe PS, Selleck P, Sheridan JW. 1995. Infection of humans and horses by a newly described morbillivirus. Med. J. Aust. 162:642– 645. 6. Field HE, Mackenzie JS, Daszak P. 2007. Henipaviruses: emerging paramyxoviruses associated with fruit bats. Curr. Top. Microbiol. Immunol. 315:133–159. http://dx.doi.org/10.1007/978-3-540-70962-6_7. 7. Middleton DJ, Weingartl HM. 2012. Henipaviruses in their natural animal hosts. Curr. Top. Microbiol. Immunol. 359:105–121. http://dx.doi .org/10.1007/82_2012_210. 8. Playford EG, McCall B, Smith G, Slinko V, Allen G, Smith I, Moore F, Taylor C, Kung YH, Field H. 2010. Human Hendra virus encephalitis associated with equine outbreak, Australia, 2008. Emerg. Infect. Dis. 16: 219 –223. http://dx.doi.org/10.3201/eid1602.090552. 9. Wong KT, Robertson T, Ong BB, Chong JW, Yaiw KC, Wang LF, Ansford AJ, Tannenberg A. 2009. Human Hendra virus infection causes acute and relapsing encephalitis. Neuropathol. Appl. Neurobiol 35:296 – 305. http://dx.doi.org/10.1111/j.1365-2990.2008.00991.x. 10. Chong HT, Kamarulzaman A, Tan CT, Goh KJ, Thayaparan T, Kunjapan SR, Chew NK, Chua KB, Lam SK. 2001. Treatment of acute Nipah encephalitis with ribavirin. Ann. Neurol. 49:810 – 813. http://dx.doi.org /10.1002/ana.1062. 11. Rockx B, Bossart KN, Feldmann F, Geisbert JB, Hickey AC, Brining D, Callison J, Safronetz D, Marzi A, Kercher L, Long D, Broder CC, Feldmann H, Geisbert TW. 2010. A novel model of lethal Hendra virus infection in African green monkeys and the effectiveness of ribavirin treatment. J. Virol. 84:9831–9839. http://dx.doi.org/10.1128/JVI.01163-10. 12. Bonaparte MI, Dimitrov AS, Bossart KN, Crameri G, Mungall BA, Bishop KA, Choudhry V, Dimitrov DS, Wang LF, Eaton BT, Broder CC. 2005. Ephrin-B2 ligand is a functional receptor for Hendra virus and Nipah virus. Proc. Natl. Acad. Sci. U. S. A. 102:10652–10657. http://dx.doi .org/10.1073/pnas.0504887102. 13. Negrete OA, Levroney EL, Aguilar HC, Bertolotti-Ciarlet A, Nazarian R, Tajyar S, Lee B. 2005. EphrinB2 is the entry receptor for Nipah virus, an emergent deadly paramyxovirus. Nature 436:401– 405. http://dx.doi .org/10.1038/nature03838. 14. Negrete OA, Wolf MC, Aguilar HC, Enterlein S, Wang W, Muhlberger E, Su SV, Bertolotti-Ciarlet A, Flick R, Lee B. 2006. Two key residues in ephrinB3 are critical for its use as an alternative receptor for Nipah virus. PLoS Pathog. 2:e7. http://dx.doi.org/10.1371/journal.ppat.0020007. 15. Bishop KA, Stantchev TS, Hickey AC, Khetawat D, Bossart KN, Krasnoperov V, Gill P, Feng YR, Wang L, Eaton BT, Wang LF, Broder CC. 2007. Identification of Hendra virus G glycoprotein residues that are critical for receptor binding. J. Virol. 81:5893–5901. http://dx.doi.org/10 .1128/JVI.02022-06. 16. Xu K, Rockx B, Xie Y, DeBuysscher BL, Fusco DL, Zhu Z, Chan YP, Xu Y, Luu T, Cer RZ, Feldmann H, Mokashi V, Dimitrov DS, Bishop-Lilly KA, Broder CC, Nikolov DB. 2013. Crystal structure of the Hendra virus attachment G glycoprotein bound to a potent cross-reactive neutralizing human monoclonal antibody. PLoS Pathog. 9:e1003684. http://dx.doi.org /10.1371/journal.ppat.1003684. 17. Zhu Z, Dimitrov AS, Bossart KN, Crameri G, Bishop KA, Choudhry V, Mungall BA, Feng YR, Choudhary A, Zhang MY, Feng Y, Wang LF,
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ease. At this time, the ferret and AGM appear to be the models that most accurately reflect human HeV infection. The protective efficacy of sGHeV has been evaluated in the ferret (24) and AGM (26) models of NiV-mediated infection and in the ferret (25) and now in the AGM model of HeV-mediated disease. Additionally, the durability of the sGHeV vaccine has been tested in ferrets 1 year postvaccination against a NiV challenge with promising results (24). In these studies, all vaccinated animals developed high levels of antigen-specific serum Ig (Fig. 5) and neutralizing antibodies before the challenge (Table 2) which were comparable to the levels of antigen-specific Ig and neutralizing antibody against HeV seen in a previous study (26). While both of the vaccine formulations in the present study resulted in HeV G-specific Ig and significant titers of neutralizing antibody against HeV, the cohort of AGMs receiving the sGHeV-alum-CpG vaccine formulation had a significantly (P ⬍ 0.05) higher neutralizing antibody titer by Bonferroni posttest on day 0 (Table 2). Nevertheless, the present findings have clearly demonstrated that sGHeV-alum alone is also capable of providing complete protection from a HeV challenge. As HeV and NiV replicate and cause severe pathology in the lung and CpG motifs have been shown to elicit Th1 and mucosal immunity regardless of the immunization route used (34–36), the most recent sG vaccine studies have used and explored several CpG adjuvants. Additionally, several CpG motifs have also entered into human clinical trials, which could facilitate future regulatory processes for this adjuvant in general. Host responses to CpG are highly sequence and species specific. The CpG adjuvant used in feline and ferret sG vaccine trials had been used previously in humans (37–39) and cats. Mucosal IgA was detected in vaccinated and protected animals (23), suggesting that CpG may have contributed to mucosal immunity. As vaccine studies transitioned to nonhuman primates, a slightly different CpG motif (26) was selected that was shown to be an effective adjuvant in humans and nonhuman primates (40, 41). As demonstrated here and previously, AGMs vaccinated with sG and CpG (ODN 2006) mount robust immune responses and are protected from a lethal HeV or NiV challenge. Interestingly, all sG-vaccinated animals were protected from a lethal HeV challenge in the present study, but the animals that received sG with CpG had significantly higher antibody titers. Importantly, all specifically vaccinated animals were protected from HeV disease (Fig. 1B; Table 1). In addition, there was no evidence of clinical illness or infectious HeV in any vaccinated animal across all of these efficacy studies. The efficacy of the sGHeV vaccine against HeV infection described in this report provides further critical evidence that this vaccine should be moved toward further development as a human use vaccine against HeV and NiV with alum alone and alum plus CpG as adjuvant choices based on the neutralizing antibody observations in this study.
Henipavirus G Glycoprotein Subunit Vaccine
18.
19.
21.
22.
23.
24.
25.
26.
27.
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28. 29. 30.
31. 32. 33. 34.
35.
36.
37.
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by a soluble G glycoprotein of Hendra virus. J. Virol. 79:6690 – 6702. http: //dx.doi.org/10.1128/JVI.79.11.6690-6702.2005. Richmond R. 2012. The Hendra vaccine has arrived. Aust. Vet. J. 90:N2. http://dx.doi.org/10.1111/j.1751-0813.2012.00087.GRP.x. National Research Council. 2013. Guide for the care and use of laboratory animals, eighth edition. National Academies Press, Washington, DC. Geisbert TW, Daddario-DiCaprio KM, Hickey AC, Smith MA, Chan YP, Wang LF, Mattapallil JJ, Geisbert JB, Bossart KN, Broder CC. 2010. Development of an acute and highly pathogenic nonhuman primate model of Nipah virus infection. PLoS One 5:e10690. http://dx.doi.org/10 .1371/journal.pone.0010690. Field H, Crameri G, Kung NY, Wang LF. 2012. Ecological aspects of Hendra virus. Curr. Top. Microbiol. Immunol. 359:11–23. http://dx.doi .org/10.1007/82_2012_214. Anonymous. 2011. Hendra virus, equine—Australia (21): (QL) canine. ProMED Mail archive number 20110802.2324. International Society for Infectious Diseases, Brookline, MA. Mendez DH, Judd J, Speare R. 2012. Unexpected result of Hendra virus outbreaks for veterinarians, Queensland, Australia. Emerg. Infect. Dis. 18:83– 85. http://dx.doi.org/10.3201/eid1801.111006. McCluskie MJ, Weeratna RD, Payette PJ, Davis HL. 2002. Parenteral and mucosal prime-boost immunization strategies in mice with hepatitis B surface antigen and CpG DNA. FEMS Immunol. Med. Microbiol. 32: 179 –185. http://dx.doi.org/10.1111/j.1574-695X.2002.tb00551.x. Chu JH, Chiang CC, Ng ML. 2007. Immunization of flavivirus West Nile recombinant envelope domain III protein induced specific immune response and protection against West Nile virus infection. J. Immunol. 178: 2699 –2705. http://www.jimmunol.org/content/178/5/2699.full. Knight JB, Huang YY, Halperin SA, Anderson R, Morris A, Macmillan A, Jones T, Burt DS, Van Nest G, Lee SF. 2006. Immunogenicity and protective efficacy of a recombinant filamentous haemagglutinin from Bordetella pertussis. Clin. Exp. Immunol. 144:543–551. http://dx.doi.org /10.1111/j.1365-2249.2006.03097.x. Halperin SA, Van Nest G, Smith B, Abtahi S, Whiley H, Eiden JJ. 2003. A phase I study of the safety and immunogenicity of recombinant hepatitis B surface antigen co-administered with an immunostimulatory phosphorothioate oligonucleotide adjuvant. Vaccine 21:2461–2467. http://dx.doi .org/10.1016/S0264-410X(03)00045-8. Broide DH. 2006. Prospects for CpG based immunotherapy in asthma and allergy. Arb. Paul Ehrlich Inst. Bundesamt Sera Impfstoffe Frankf. A. M. 2006:217–220; discussion 220 –212. Anonymous. 2006. CpG 7909: PF 3512676, PF-3512676. Drugs R. D. 7:312–316. http://dx.doi.org/10.2165/00126839-200607050-00004. Conforti VA, de Avila DM, Cummings NS, Wells KJ, Ulker H, Reeves JJ. 2007. The effectiveness of a CpG motif-based adjuvant (CpG ODN 2006) for LHRH immunization. Vaccine 25:6537– 6543. http://dx.doi.org /10.1016/j.vaccine.2007.05.056. Hartmann G, Weeratna RD, Ballas ZK, Payette P, Blackwell S, Suparto I, Rasmussen WL, Waldschmidt M, Sajuthi D, Purcell RH, Davis HL, Krieg AM. 2000. Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo. J. Immunol. 164:1617–1624. http://www.jimmunol .org/content/164/3/1617.
jvi.asm.org 4631
Downloaded from http://jvi.asm.org/ on November 22, 2014 by NORTHERN ILLINOIS UNIV
20.
Xiao X, Eaton BT, Broder CC, Dimitrov DS. 2006. Potent neutralization of Hendra and Nipah viruses by human monoclonal antibodies. J. Virol. 80:891– 899. http://dx.doi.org/10.1128/JVI.80.2.891-899.2006. Zhu Z, Bossart KN, Bishop KA, Crameri G, Dimitrov AS, McEachern JA, Feng Y, Middleton D, Wang LF, Broder CC, Dimitrov DS. 2008. Exceptionally potent cross-reactive neutralization of Nipah and Hendra viruses by a human monoclonal antibody. J. Infect. Dis. 197:846 – 853. http://dx.doi.org/10.1086/528801. Bossart KN, Zhu Z, Middleton D, Klippel J, Crameri G, Bingham J, McEachern JA, Green D, Hancock TJ, Chan YP, Hickey AC, Dimitrov DS, Wang LF, Broder CC. 2009. A neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute Nipah virus infection. PLoS Pathog. 5:e1000642. http://dx.doi.org/10 .1371/journal.ppat.1000642. Bossart KN, Geisbert TW, Feldmann H, Zhu Z, Feldmann F, Geisbert JB, Yan L, Feng YR, Brining D, Scott D, Wang Y, Dimitrov AS, Callison J, Chan YP, Hickey AC, Dimitrov DS, Broder CC, Rockx B. 2011. A neutralizing human monoclonal antibody protects African green monkeys from Hendra virus challenge. Sci. Transl. Med. 3:105ra103. http://dx .doi.org/10.1126/scitranslmed.3002901. Ploquin A, Szecsi J, Mathieu C, Guillaume V, Barateau V, Ong KC, Wong KT, Cosset FL, Horvat B, Salvetti A. 2013. Protection against henipavirus infection by use of recombinant adeno-associated virusvector vaccines. J. Infect. Dis. 207:469 – 478. http://dx.doi.org/10.1093 /infdis/jis699. McEachern JA, Bingham J, Crameri G, Green DJ, Hancock TJ, Middleton D, Feng YR, Broder CC, Wang LF, Bossart KN. 2008. A recombinant subunit vaccine formulation protects against lethal Nipah virus challenge in cats. Vaccine 26:3842–3852. http://dx.doi.org/10.1016/j.vaccine .2008.05.016. Mungall BA, Middleton D, Crameri G, Bingham J, Halpin K, Russell G, Green D, McEachern J, Pritchard LI, Eaton BT, Wang LF, Bossart KN, Broder CC. 2006. Feline model of acute Nipah virus infection and protection with a soluble glycoprotein-based subunit vaccine. J. Virol. 80: 12293–12302. http://dx.doi.org/10.1128/JVI.01619-06. Pallister JA, Klein R, Arkinstall R, Haining J, Long F, White JR, Payne J, Feng YR, Wang LF, Broder CC, Middleton D. 2013. Vaccination of ferrets with a recombinant G glycoprotein subunit vaccine provides protection against Nipah virus disease for over 12 months. Virol. J. 10:237. http://dx.doi.org/10.1186/1743-422X-10-237. Pallister J, Middleton D, Wang LF, Klein R, Haining J, Robinson R, Yamada M, White J, Payne J, Feng YR, Chan YP, Broder CC. 2011. A recombinant Hendra virus G glycoprotein-based subunit vaccine protects ferrets from lethal Hendra virus challenge. Vaccine 29:5623–5630. http: //dx.doi.org/10.1016/j.vaccine.2011.06.015. Bossart KN, Rockx B, Feldmann F, Brining D, Scott D, LaCasse R, Geisbert JB, Feng YR, Chan YP, Hickey AC, Broder CC, Feldmann H, Geisbert TW. 2012. A Hendra virus G glycoprotein subunit vaccine protects African green monkeys from Nipah virus challenge. Sci. Transl. Med. 4:146ra107. http://dx.doi.org/10.1126/scitranslmed.3004241. Bossart KN, Crameri G, Dimitrov AS, Mungall BA, Feng YR, Patch JR, Choudhary A, Wang LF, Eaton BT, Broder CC. 2005. Receptor binding, fusion inhibition, and induction of cross-reactive neutralizing antibodies
