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Accepted Manuscript Efficacy of antibody based therapies against Middle East respiratory syndrome coronavirus MERS CoV in common marmosets Neeltje van Doremalen Darryl Falzarano Tianlei Ying Emmie de Wit Trenton Bushmaker Friederike Feldmann Atsushi Okumura Yanping Wang Dana P Scott Patrick W Hanley Heinz Feldmann Dimiter S Dimitrov Vincent J Munster PII S0166 3542 16 30538 1 DOI 10 1016 j antiviral 2017 03 025 Reference AVR 4045 To appear in Antiviral Research Received Date 30 September 2016 Accepted Date 29 March 2017 Please cite this article as van Doremalen N Falzarano D Ying T de Wit E Bushmaker T Feldmann F Okumura A Wang Y Scott D P Hanley P W Feldmann H Dimitrov D S Munster V J Efficacy of antibody based therapies against Middle East respiratory syndrome coronavirus MERS CoV in common marmosets Antiviral Research 2017 doi 10 1016 j antiviral 2017 03 025 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting typesetting and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content and all legal disclaimers that apply to the journal pertain M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT Title Efficacy of antibody based therapies against Middle East respiratory syndrome coronavirus MERS CoV in common marmosets Authors Neeltje van Doremalen1 Darryl Falzarano2 Tianlei Ying3 Emmie de Wit2 Trenton Bushmaker1 Friederike Feldmann4 Atsushi Okumura2 5 Yanping Wang3 Dana P Scott4 Patrick W Hanley4 Heinz Feldmann2 Dimiter S Dimitrov3 Vincent J Munster1 Affiliations 1 Virus Ecology Unit Laboratory of Virology Division of Intramural Research National Institute of Allergy and Infectious Diseases National Institutes of Health Rocky Mountain Laboratories Hamilton MT USA 2 Disease Modeling and Transmission Laboratory of Virology Division of Intramural Research National Institute of Allergy and Infectious Diseases National Institutes of Health Rocky Mountain Laboratories Hamilton MT USA 3 Protein Interactions Section Cancer and Inflammation Program Center for Cancer Research National Cancer Institute National Institutes of Health Frederick MD USA 4 Rocky Mountain Veterinary Branch Division of Intramural Research National Institute of Allergy and Infectious Diseases National Institutes of Health Rocky Mountain Laboratories Hamilton MT USA 5 Department of Microbiology University of Washington Seattle WA USA These authors contributed equally to this work To whom correspondence should be addressed Vincent J Munster Rocky Mountains Laboratories 903 S 4th street Hamilton MT USA munstervj niaid nih gov M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 2 Current affiliation VIDO InterVac University of Saskatchewan Saskatoon SK Canada Current affiliation Key Laboratory of Medical Molecular Virology of Ministries of Education and Health School of Basic Medical Sciences and Institute of Medical Microbiology Fudan University Shanghai 200032 China M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 3 Abstract Cases of Middle East respiratory syndrome coronavirus MERS CoV continue to be identified and with a lack of effective clinical treatment and no preventative strategies treatment using convalescent plasma or monoclonal antibodies mAbs is a potential quick route to an intervention Passive immunotherapy via either convalescent plasma or mAbs has proven to be effective for other infectious agents Following infection with MERS CoV common marmosets were treated with high titer hyperimmune plasma or the mAb m336 at 6 and 48 hours post inoculation Both treatments reduced signs of clinical disease but reduction in viral loads in the respiratory tract were only found in the hyperimmune plasma group A decrease in gross pathology was found only in the mAb treated group but no histological differences were observed between treated and control animals While both hyperimmune plasma and the m336 treatments reduced the severity of disease in the common marmoset neither treatment resulted in full protection against disease Keywords MERS CoV treatment monoclonal antibodies hyperimmune plasma common marmoset immunotherapy M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 4 1 Introduction Middle East respiratory syndrome coronavirus MERS CoV was first detected in 2012 in a resident of Saudi Arabia and has since resulted in 1800 cases with a case fatality rate of 36 WHO 2015 The severity and the epidemic potential of MERS CoV highlights the importance of the development of treatment options As of yet no specific vaccine or antiviral treatment against MERS CoV is available Few studies have been published investigating the effectiveness of existing antiviral treatments and no treatments have been thoroughly assessed in clinical trials as of yet Convalescent plasma has been identified by the World Health Organization WHO and the International Severe Acute Respiratory and Emerging Infection Consortium ISARIC as a potential treatment against MERS CoV to reduce clinical consequences of MERS CoV infection 2013 WHO 2014 and recently a study protocol was developed to investigate the feasibility of convalescent plasma treatment in MERS patients Arabi et al 2015 In vivo the administration of convalescent sera obtained from dromedary camels resulted in dose dependent decreased lung viral titers and disease severity in an adenovirus hDPP4 mouse model Zhao et al 2015 Several monoclonal antibodies mAbs have been developed against MERS CoV which show neutralizing capacity in vitro Jiang et al 2014 Tang et al 2014 Ying et al 2014 Efficacy of mAbs has been assessed in several MERS CoV mouse models generally showing reduction in virus replication Corti et al 2015 Li et al 2015 Pascal et al 2015 Luke et al 2016 These studies suggest that mAbs have potential as MERS CoV treatment The mAb m336 identified from a large phage displayed antibody library panned against recombinant MERS CoV spike protein receptor binding domain inhibited 90 MERS CoV M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 5 pseudovirus infection at a concentration of 0 039 g ml Ying et al 2014 m336 was shown to almost completely overlap with the binding site of DPP4 and mimic critical interactions between DPP4 and the MERS CoV spike protein Modjarrad et al 2016 It has therefore been speculated that the potential for viral escape mutants might be limited by the requirement of the spike protein to bind to DPP4 Ying et al 2015 Prophylactic treatment with m336 resulted in significantly reduced viral titer in rabbit lung tissue Houser et al 2016 and both prophylactic and therapeutic treatment with m336 protected mice against lethality by MERS CoV infection Agrawal et al 2016 Here we assess the effect of treatment with marmoset derived hyperimmune plasma as well as the human mAb m336 on disease outcome in the recently developed marmoset MERS CoV infection model which recapitulates severe respiratory disease Falzarano et al 2014 2 Materials and Methods 2 1 Ethics statement Approval of animal experiments was obtained from the Institutional Animal Care and Use Committee at Rocky Mountain Laboratories All experiments were performed in an Association for Assessment and Accreditation of Laboratory Animal Care approved facility by certified staff following the guidelines and basic principles in the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals the NIH Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act United States Department of Agriculture The Institutional Biosafety Committee IBC approved work with infectious MERS CoV strains M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 6 under BSL3 conditions Sample inactivation was performed according to IBC approved standard operating procedures for removal of specimens from high containment 2 2 Generation of MERS CoV hyperimmune sera Hyperimmune plasma was obtained from a convalescent common marmoset Callithrix jacchus from a previous experiment Falzarano et al 2014 inoculated with 5 2x106 TCID50 MERS CoV via the intratracheal intranasal ocular and oral route then inoculated with 5 2x106 TCID50 MERS CoV on 20 dpi via the intratracheal route and finally inoculated with 5 2x106 TCID50 MERS CoV adjuvated with Titermax Gold Sigma Aldrich on 41 dpi via the intramuscular route The final virus neutralizing VN titer was 3840 This method was chosen as sera collected after the initial infection did not contain sufficient neutralizing antibodies VN titer 40 As a control plasma was obtained from an uninfected common marmoset internal collection VN titer 20 2 3 Study design The common marmoset MERS CoV infection model was used MERS CoV infection results in the development of more severe respiratory disease than observed in the rhesus macaque model de Wit et al 2013 Munster et al 2013 Falzarano et al 2014 Common marmosets were procured from an USDA approved source Worldwide Primates Inc Animals were monitored for the presence of disease by clinical observation and serology for the presence of disease at Worldwide Primates Inc Additionally when animals arrived at Rocky Mountain Laboratories M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 7 they were placed in quarantine and clinically evaluated by serum chemistry complete blood counts and thoracic radiography to confirm absence of previous infection Three different groups were created the hyperimmune plasma group H the monoclonal antibody group M and the control group C Three animals were randomly assigned per group and inoculated as described previously Falzarano et al 2014 Briefly inoculation with MERS CoV strain EMC 2012 was performed intranasally 100 l per nare orally 500 l intratracheally 500 l and ocular 50 l per eye with DMEM containing 4x106 TCID50 MERS CoV ml total dose 5 2x106 TCID50 The hyperimmune plasma and monoclonal antibody groups consisting of one female and two male common marmosets each received 1 ml hyperimmune plasma or m336 diluted in PBS 5 mg ml intravenous I V at 6 hpi and 1 ml hyperimmune plasma or m336 subcutaneous S C at 2 dpi marmosets H1 3 M1 3 Two out of three animals in the control group all male common marmosets received 1 ml control plasma I V 6 hpi and 1 ml control plasma S C 2 dpi marmosets C1 2 The third animal received 1 ml of PBS diluent of the mAb via the same routes marmoset C3 The animals were observed twice daily for clinical signs of disease using a scoring system as described previously Falzarano et al 2014 Breathing was scored as normal 100 minute Based on the scoring sheet euthanasia was indicated at a clinical score of 35 or more Clinical exams were performed on 0 2 5 and 7 dpi on anaesthetized animals using isoflurane and ketamine X rays were taken and nasal oral fecal and urogenital swabs were collected in 1 ml DMEM with 50 U ml penicillin and 50 g ml streptomycin Blood samples were collected on 5 2 and 7 dpi and examined using the Piccolo Xpress chemistry analyzer Abaxis The blood sample collected on 2 dpi was obtained before M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 8 treatment was administered Temperature was monitored with IPTT 300 temperature probes BMDS that were injected interscapularly prior to the start of the experiment All animals were euthanized at 7 dpi Fig 1A Terminal blood samples were obtained and samples of the following tissues were collected conjunctiva nasal mucosa tonsil trachea four lung lobes mediastinal lymph node liver spleen kidney and bladder Gross pathology surface area of the lung which was either consolidated and or hyperemic per lung lobe was documented as percentage area affected by lesions 2 4 Radiography Radiographic images acquired included ventrodorsal right lateral and left lateral thoracic images Thoracic radiographs were obtained using a mobile digital radiography unit with a flat panel digital detector Sound Technologies tru DR Sound Eklin Carlsbad CA Each set of radiographs was graded according to a published scoring paradigm Brining et al 2010 as follows 0 normal examination 1 mild interstitial pulmonary infiltrates 2 moderate interstitial infiltrates perhaps with partial cardiac border effacement and small areas of pulmonary consolidation alveolar patterns and air bronchograms 3 pulmonary consolidation as the primary lung pathology seen as a progression from grade 2 lung pathology Grading per animal was done independently and blinded by two veterinarians 2 5 Virus and cells HCoV EMC 2012 was provided by the Erasmus Medical Center Rotterdam The Netherlands Virus propagation was performed in VeroE6 cells provided by the Bowen laboratory Colorado M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 9 State University in DMEM supplemented with 2 fetal calf serum 1 mM L glutamine 50 U ml penicillin and 50 g ml streptomycin 2 DMEM VeroE6 cells were maintained in DMEM supplemented with 10 fetal calf serum 1 mM L glutamine 50 U ml penicillin and 50 g ml streptomycin 2 6 Histopathology and immunohistochemistry Marmoset tissues were evaluated for pathology and the presence of viral antigen All tissues were fixed for a minimum of 7 days in 10 neutral buffered formalin and subsequently embedded in paraffin Lungs were perfused with 10 formalin and processed for histologic review The lung is divided into right upper right lower left upper and left lower lobe Each of these four sections are then sampled at the hilus at mid lobe and at the periphery of the lobe for a minimum of 12 sections per animal This method is used for all non human primate studies at Rocky Mountain Laboratories Hereafter tissue sections were stained with hematoxylin and eosin For the detection of viral antigen immunohistochemistry was performed using an in house produced rabbit polyclonal antiserum against HCoV EMC 2012 1 1000 Grading was done blinded by a board certified veterinary pathologist To obtain morphometrical data of immunohistochemistry staining stained sections were scanned with an Aperio ScanScope XT Aperio Technologies Inc Vista CA and analyzed using the ImageScope Positive Pixel Count algorithm version 9 1 Between 30 and 105 millimeters squared were evaluated at 2x magnification The default parameters of the Positive Pixel Count hue of 0 1 and width of 0 5 detected antigen adequately M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 10 2 7 RNA extraction Tissue for RNA analysis was collected in triplicate Lung tissue was obtained from the hilar and mid lobe region of the lung Samples were analyzed independently in duplicate Tissues 30 mg were homogenized in RLT buffer and RNA was extracted using the RNeasy method Qiagen according to the manufacturer s instructions RNA was extracted from swabs using the QiaAmp Viral RNA kit on the QIAxtractor 2 8 Quantitative PCR The UpE MERS CoV detection assay was used for the detection of MERS CoV viral RNA Corman et al 2012 5 l RNA was tested with the Rotor GeneTM probe kit Qiagen according to instructions of the manufacturer Dilutions of MERS CoV with known titer were run in parallel 2 9 Determination of limit of detection quantitative PCR Dilutions of in vitro transcribed UpE MERS CoV RNA were run on the digital droplet PCR Biorad in quadruplicate to determine genome copies Hereafter dilutions were run on the Rotor GeneTM in quadruplicate The last dilution to give a Ct value in all replicates was defined as the limit of detection LOD in genome copies Finally the number of genome copies was determined in the MERS CoV dilutions with known titer and LOD was calculated in TCID50 equivalent M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 11 2 10 Infectious virus titration Small tissue samples up to 100 mg in 1 ml of 2 DMEM were homogenized Hereafter MERS CoV was titrated in quadruplicate in VeroE6 cells cells were inoculated with ten fold serial dilutions of tissue homogenate incubated 1h at 37 C washed twice with PBS and scored for cytopathic effect 5 days later TCID50 was adjusted for tissue weight and calculated by the method of Spearman Karber 2 11 Virus neutralization assay Two fold serial dilutions of heat inactivated 30 minutes 56 C marmoset sera were prepared in 2 DMEM after which 100 TCID50 of MERS CoV virus was added After 1hr incubation at 37 C virus was added to VeroE6 cells At 5 dpi cytopathic effect was scored The virus neutralization titer was expressed as the reciprocal value of the highest dilution of the serum which still inhibited MERS CoV virus replication 2 12 Statistical analysis Student s t test unpaired one tailed was used to test for significance P values of 0 05 were considered significant All values are reported as mean SD 3 Results 3 1 Neutralizing antibody levels in the serum of treated and untreated marmosets The final VN titer of hyperimmune plasma was 3840 control plasma was 60 breaths minute was observed in all other animals and progressed to labored breathing 100 breaths minute in all three control animals C1 C3 and one treated animal H1 This was accompanied by open mouth breathing in all control animals but not H1 All radiographs were independently and blindly graded by clinical veterinarians Brining et al 2010 and in all views were normal score 0 prior to inoculation on day 0 Within the control M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 13 group all three animals were graded at 2 at 5 dpi with severe interstitial infiltrates seen early that progressed to pulmonary consolidation within the caudal and middle lung lobes on 7 dpi score 3 Both the hyperimmune plasma treatment and the mAb treatment group on average had lower graded radiographs in comparison to the control group On 7 dpi one hyperimmune plasma treated animal was characterized as having grade 1 H2 mild interstitial pulmonary infiltrates in the left caudal lung lobes while the remaining two hyperimmune plasma treated animals had either moderate H1 grade 2 or severe H3 grade 3 radiographic signs One mAb treated animal had normal radiographic findings M2 grade 0 throughout the duration of the study whereas the other two animals M1 M3 were scored as presenting with mild radiographic findings grade 1 Fig 2B 2C and Fig S1 Blood chemistry values were investigated using the Piccolo Xpress chemistry analyzer on 5 2 and 7 days post inoculation Overall no apparent differences in measured clinical chemistries were noted between the control and the treated groups The values of the investigated parameters BUN creatinine ALT AST ALP GGT total protein glucose and calcium were found to be within the normal ranges for marmosets and no consistent patterns or trends were noted between groups Fig S2 3 3 Respiratory tract pathology in treated and untreated marmosets Gross pathology of the lungs was scored by a board certified veterinary pathologist for each lung lobe both dorsal and ventral For the control animals the median percent lesions was 32 5 C3 55 C1 and 67 5 C2 No abnormal pathological findings were observed in one of the hyperimmune plasma treated animals H2 whereas the median of gross pathology of the lungs M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 14 in the other two treated animals was 25 H1 and 77 5 H3 Animals treated with m336 showed relatively little gross pathology median 0 M1 2 5 M2 and 22 5 M3 This difference between control and treated animals was found to be significantly different using an unpaired one tail Student s t test Hyperimmune plasma treated p 0 0352 MAb treated p 0 0007 Fig 2c and 3A As a measure of pulmonary edema and inflammation lung to body weight ratios were calculated for each animals No significant differences were observed between the control and hyperimmune plasma treated group however the mAb treated group had a significantly lower lung to body weight ratio than the control group p 0 0039 Fig 3B No