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Accepted Manuscript 1 The Author 2015 Published by Oxford University Press on behalf of the Infectious Diseases Society of America All rights reserved For Permissions please e mail journals permissions Protective effect of intranasal regimens containing peptidic MERS CoV fusion inhibitor against MERS CoV infection Rudragouda Channappanavar1 a Lu Lu2 a Shuai Xia2 Lanying Du3 David K Meyerholz4 Stanley Perlman1 5 Shibo Jiang2 3 1Departments of 1Microbiology 4Pathology and 5Pediatrics University of Iowa Iowa City IA 52242 USA 2Key Laboratory of Medical Molecular Virology of Ministries of Education and Health Shanghai Medical College and Shanghai Public Health Clinical Center Fudan University Shanghai 200032 China 3Lindsley F Kimball Research Institute New York Blood Center New York NY 10065 USA Correspondence Shibo Jiang M D Key Laboratory of Medical Molecular Virology of Ministries of Education and Health Shanghai Medical College Fudan University 130 Dong An Road Building 13 Xuhui District Shanghai 200032 China shibojiang or Stanley Perlman M D Ph D Department of Microbiology University of Iowa Iowa City IA 52242 USA stanley perlman uiowa edu aRC and LL contributed equally to this work Journal of Infectious Diseases Advance Access published June 8 2015 at University of California Santa Barbara on June 23 2015 http jid oxfordjournals org Downloaded from Accepted Manuscript 2 Abstract To gain entry into the target cell Middle East respiratory syndrome coronavirus MERS CoV utilizes its spike S protein S2 subunit to fuse with the plasma or endosomal membrane We previously identified a peptide derived from the HR2 domain in S2 subunit HR2P which potently blocked MERS CoV S protein mediated membrane fusion Here we tested an HR2P analogue with improved pharmaceutical property HR2P M2 for its inhibitory activity against MERS CoV infection in vitro and in vivo HR2P M2 was highly effective in inhibiting MERS CoV S protein mediated cell cell fusion and infection by pseudoviruses expressing MERS CoV S protein with or without mutation in the HR1 region It interacted with the HR1 peptide to form stable helical complex and blocked the 6 HB formation between the HR1 and HR2 domains in the viral S protein Intranasally administered HR2P M2 effectively protected Ad5 hDPP4 transduced mice from infection by MERS CoV strains with or without mutations in the HR1 region of S protein with 1 000 fold reduction of viral titers in lung and the protection was enhanced by combining HR2P M2 with interferon IFN These results indicate that this combinational regimen merits further development to prevent MERS in high risk populations including healthcare workers and patient family members and to treat MERS CoV infected patients at University of California Santa Barbara on June 23 2015 http jid oxfordjournals org Downloaded from Accepted Manuscript 3 INTRODUCTION Severe human respiratory infection caused by the Middle East Respiratory Syndrome MERS coronavirus MERS CoV was first identified in 2012 37 As of May 25 2015 1 139 laboratory confirmed cases and 431 deaths had been reported to the WHO http www who int csr don 25 may 2015 mers saudi arabia en The high case fatality rate among patients with MERS CoV infection has caused widespread fear because the mode of transmission from zoonotic sources is not well understood and coronaviruses have the potential to mutate becoming more pathogenic and transmissible 4 25 29 37 Bats and dromedary camels are considered to be the natural reservoir and intermediate hosts for MERS CoV respectively but most community acquired cases are not associated with camel contact 2 17 28 33 Cumulative evidence from healthcare settings suggest that person to person transmission of MERS CoV can occur through close contact 4 31 In the absence of an effective vaccine it is essential to develop strategies to prevent camel to human and human to human transmission among the high risk populations as well as to treat MERS CoV infected patients MERS CoV an enveloped positive sense single stranded RNA virus binds to the target cell through interaction between the receptor binding domain in its spike S protein S1 subunit 8 13 24 30 34 and its receptor dipeptidyl peptidase 4 DPP4 also known as CD26 32 After binding and proteolytic cleavage a fusion peptide at the N terminus of S2 is exposed and is inserted into the plasma or endosomal membrane The heptad repeat 2 HR2 binds to the heptad repeat 1 HR1 in S2 to form a six bundle 6 HB fusion core which brings viral and cell membranes into close apposition for fusion 26 Peptides derived from the HR2 region such as HR2P can also interact with HR1 domain in the viral S protein to form heterogeneous 6 HB and at University of California Santa Barbara on June 23 2015 http jid oxfordjournals org Downloaded from Accepted Manuscript 4 thus block viral fusion with host cell membranes 26 as previously described in the context of HIV and SARS CoV infections 5 20 22 35 In the present study we evaluated an HR2P analogue designated HR2P M2 for its in vitro and in vivo efficacy against infection by the MERS CoV EMC 2012 strain and strains with mutations in the HR1 domain of the S protein Most notably we determined the prophylactic and therapeutic protective activity of HR2P M2 when used alone or in combination with interferon in human DPP4 receptor transduced mice challenged with MERS CoV MATERIALS AND METHODS Cells and Viruses 293T cells and Huh 7 cells were obtained from ATCC Manassas VA USA and the Cell Bank of the Chinese Academy of Science Shanghai China respectively The EMC 2012 strain of MERS CoV passage 8 designated MERS CoV provided by Drs Bart Haagmans and Ron Fouchier Erasmus Medical Center was passaged once on Vero 81 cells Ad5 hDPP4 was developed and propagated by the University of Iowa Gene Transfer Vector Core Circular Dichroism CD Spectroscopic Analysis CD spectroscopy was used to determine the secondary structure of the peptides and their complexes 7 23 26 27 as previously described After CD analysis thermal denaturation of peptide complexes was immediately monitored using the same sample from 4 C to 100 C at 222 nm with a thermal gradient of 5 C min 1 The 222 value of 33 000 deg cm2 dmol 1 was taken as 100 helical content 5 7 26 Data were further processed using Jasco software at University of California Santa Barbara on June 23 2015 http jid oxfordjournals org Downloaded from Accepted Manuscript 5 Native Polyacrylamide Gel Electrophoresis N PAGE N PAGE was performed as previously described 22 23 Peptide HR1P HR1P Q1020R HR1P Q1020R 40 M in PBS was incubated with peptide HR2P M2 40 M at 37oC for 30 min respectively using PBS as control followed by loading on an 18 Tris glycine gel with Tricine glycine running buffer pH 8 3 After staining with Coomassie Blue images were acquired on a FluorChem Imaging System Alpha Innotech ProteinSimple Santa Clara CA USA Fluorescence Native Polyacrylamide Gel Electrophoresis FN PAGE FN PAGE was carried out in the same manner as N PAGE described above except for using FITC conjugated peptide HR2P F Peptide HR1P HR1P Q1020R HR1P Q1020R 40 M in PBS was incubated with peptide HR2P F 40 M at 37oC for 30 min respectively to form 6 HB or incubated with increasing concentrations 40 60 and 80 M of HR2P M2 in PBS at 37oC for 30 min before addition of HR2P F followed by incubation with HR2P F 40 M at 37oC for another 30 min to inhibit 6HB formation Gels were imaged using a FluorChem 8800 Imaging system with excitation wavelength at 302 nm and emission wavelength at 520 nm After imaging the gel was stained with Coomassie Blue and re imaged Inhibition of MERS CoV S protein mediated cell cell fusion MERS CoV S protein mediated cell cell fusion was tested as previously described 26 293T cells transfected with plasmid pAAV IRES MERS EGFP 293T MERS EGFP pAAV IRES MERS Q1020H EGFP 293T MERS Q1020H EGFP or pAAV IRES MERS Q1020R EGFP 293T MERS Q1020R EGFP respectively co expressing the MERS CoV S protein and EGFP on cell surface were used as effector cells whereas 293T EGFP cells at University of California Santa Barbara on June 23 2015 http jid oxfordjournals org Downloaded from Accepted Manuscript 6 expressing only EGFP were used as negative control cells 293T MERS EGFP or 293T MERS Q1020H EGFP or 293T MERS Q1020R EGFP and 293T EGFP cells 1 104 were incubated with Huh 7 cells expressing the MERS CoV receptor DPP4 5 104 as target cells in the absence or presence of test peptides at the indicated concentrations for 2 4 hrs at 37 C 293T MERS EGFP and 293T EGFP cells fused or unfused with Huh 7 cells were counted under an inverted fluorescence microscope Nikon Eclipse Ti S and the concentration for 50 inhibition IC50 was calculated using the CalcuSyn software 9 Inhibition of pseudotyped MERS CoV infection Pseudoviruses carrying MERS CoV S protein with or without mutations were produced as previously described 19 38 Briefly pseudovirus was incubated with a test peptide at graded concentration at 37 C for 1 h followed by addition of the virus peptide mixture to Huh 7 cells Cultures were re fed with fresh medium 12 hrs post infection and incubated for an additional 48 hrs at 37 C followed by addition of luciferase substrate Promega Fluorescence was assessed using a luciferase kit Promega and an Ultra 384 luminometer Tecan San Jose CA USA Mice Specific pathogen free 6 to 12 wk old C57BL 6 mice and RAG1 mice were purchased from the National Cancer Institute and the Jackson Laboratory Bar Harbor ME USA respectively and bred at the University of Iowa All studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health Animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Iowa Protocol 4041009 at University of California Santa Barbara on June 23 2015 http jid oxfordjournals org Downloaded from Accepted Manuscript 7 Transduction and Infection of Mice C57BL 6 and RAG1 mice were transduced intranasally with 2 5 108 PFU of Ad5 hDPP4 in 75 L of DMEM Five days post transduction mice were infected intranasally with wild type or mutant MERS CoV 105 PFU 50 L of DMEM and treated intranasally with 50 L of PBS or HR2P M2 200 g peptide alone or in combination with IFN All work with MERS CoV was conducted in the University of Iowa Biosafety Level 3 BSL3 Laboratory Virus Titers Lung virus titers were determined using Vero 81 cells as previously described 39 Virus titers are expressed as PFU g lung tissue Construction of Recombinant Viruses Recombinant BACs with Q1020R H mutations were engineered using the KanR I SceI marker cassette for positive and negative selection as previously described 16 pBAC MERS CoV a generous gift from Luis Enjuanes was transformed into GS1783 E coli cells a generous gift from Greg Smith containing an arabinose inducible I SceI restriction enzyme A PCR product was generated and transformed into GS1783 cells and recombined with pBAC MERS CoV Recombinant BACs were selected on LB chloramphenicol kanamycin plates and verified by restriction enzyme digestion and PCR The resulting BAC clone was termed pBAC MERS CoV Q1020R H at University of California Santa Barbara on June 23 2015 http jid oxfordjournals org Downloaded from Accepted Manuscript 8 RESULTS HR2P M2 inhibits MERS CoV S protein mediated cell cell fusion and suppresses infection by pseudoviruses carrying MERS CoV S protein with mutations in HR1 domain We tested the inhibitory activity of peptide HR2P M2 a variant of HR2P Figure 1A on MERS CoV S protein mediated cell cell fusion using peptides MERS HR2P and SARS CP 1 derived from the HR2 domain of MERS CoV and SARS CoV S proteins respectively 22 26 as controls Both HR2P M2 and HR2P strongly inhibited S protein mediated cell cell fusion in a dose dependent manner with IC50 values of 0 55 and 0 97 M respectively Figure 1Ba indicating that HR2P M2 has improved membrane fusion inhibitory activity compared to HR2P SARS CP 1 peptide did not inhibit MERS CoV S mediated cell cell fusion at concentrations up to 20 M confirming our previous observation 26 The MERS CoV S protein HR1 domain the target site of HR2 derived peptides e g HR2P and HR2P M2 has largely conserved sequences when different strains are compared Among clinical MERS CoV strains only one amino acid change at position 1020 of the HR1 domain Q1020H or Q1020R Figure 1A was observed and this was present in nearly all strains 1 11 To test whether HR2P M2 was effective against these MERS CoV strains we compared the inhibitory activity of HR2P M2 on cell cell fusion mediated by MERS CoV S protein with and without Q1020H or Q1020R mutations Surprisingly HR2P M2 was about 0 9 to 2 6 fold more potent in inhibiting cell cell fusion mediated by MERS CoV S protein bearing Q1020H and Q1020R respectively than the wild type S protein of the EMC 2012 strain Figure 1Bb Subsequently we compared HR2P M2 with HR2P and SARS CP 1 for their inhibitory activity on the entry of pseudovirus expressing MERS CoV S protein As shown in Figure 1Ca HR2P M2 was also more potent than HR2P with IC50 values of 0 61 and 1 21 M respectively at University of California Santa Barbara on June 23 2015 http jid oxfordjournals org Downloaded from Accepted Manuscript 9 while again CP 1 exhibited no inhibition at concentrations up to 20 M We then constructed pseudoviruses expressing MERS CoV S protein with Q1020H or Q1020R mutations and compared their sensitivity to HR2P M2 Similarly HR2P M2 was about 5 to 8 fold more effective in inhibiting infection by the pseudoviruses carrying mutant S protein Q1020H or Q1020R than that with the wild type S protein while CP 1 displayed no inhibition of either mutant MERS CoV pseudovirus Figure 1Cb c These results suggest that HR2P M2 is expected to be effective against all MERS CoV strains isolated so far HR2P M2 interacted with the HR1 peptide to form a stable helical complex to block 6 HB formation between the HR1 and HR2 peptides We previously showed that peptides derived from the MERS CoV S protein HR1 and HR2 domain HR1P and HR2P respectively could interact together to form stable helical complex with helicity of 76 and Tm value of 87 oC 26 Using circular dichroism CD spectroscopy we compared the secondary structures of HR2P M2 HR1P and its mutants HR1P Q1020H and HR1P Q1020R and the complexes formed between HR1 and HR2 peptides As shown in Figure 2A C HR2P M2 showed low helicity 6 while all the HR1 peptides displayed random structures However all the complexes formed between HR2P M2 and the HR1 peptides exhibited helical structure with helicity in a range of 79 88 Figure 2A 2B and 2C in addition to high Tm value 91 95oC Figure 2D suggesting that HR2P M2 can interact with all these HR1 peptides to form highly stable helical complexes We then used N PAGE and FN PAGE 22 23 26 to further investigate the interaction between HR2P M2 and HR1P or its mutant HR1P alone was not detected in the gel lanes 1 3 Figure 3A as this peptide carries a net positive charge and thus migrated out of the gel under native electrophoresis condition 26 HR2P M2 alone migrated rapidly lane 4 Figure 3A at University of California Santa Barbara on June 23 2015 http jid oxfordjournals org Downloaded from Accepted Manuscript 10 while the mixture of HR2P M2 and HR1P or its mutant migrated more slowly in the gel lanes 5 7 Figure 3A Similarly HR2P F alone exhibited a rapidly migrating band lane 8 Figure 3A while the mixture of HR2P M2 and HR1P or its mutants migrated more slowly in the gel lanes 9 11 Figure 3A These results confirmed that both HR2P M2 and HR2P F interact with HR1P and its mutants to form 6 HB We subsequently assessed the ability of HR2P M2 to inhibit 6 HB formation between MERS CoV HR1 and HR2 peptides using FN PAGE as previously described 22 23 26 As shown in Figure 3B the sample containing only HR2P F lane 2 displayed a single fluorescence band located in the lower portion of the gel while the mixture of HR1P and HR2P F lane 3 migrated more slowly This band corresponded to 6 HB When increasing concentrations of HR2P M2 were incubated with HR1P at 37oC for 30 min before addition of HR2P F lanes 4 6 the fluorescence intensity of the HR2P F band gradually increased while that of bands of the complexes corresponding to 6 HB decreased suggesting that HR2P M2 inhibited 6 HB formation between the HR1P and HR2P F in a dose dependent manner HR2P M2 via intranasal administration inhibited MERS CoV infection in Ad5 hDPP4 transduced mice Since mice are impervious to infection with MERS CoV 10 we sensitized them to infection by transduction with Ad5 hDPP4 39 prior to testing the in vivo efficacy of HR2P M2 against infection by MERS CoV EMC 2012 or recombinant MERS CoV carrying Q1020R or Q1020H mutations in the S protein HR2P M2 200 g or PBS control was intranasally administered to Ad5 hDPP4 transduced mice 6 hrs before challenge with 1 105 PFU of MERS CoV in a total volume of 50 L of DMEM As shown in Figure 4 viral titers in the lungs of PBS treated mice challenged with recombinant MERS CoV carrying WT Q1020R or at University of California Santa Barbara on June 23 2015 http jid oxfordjournals org Downloaded from Accepted Manuscript 11 Q1020H S protein peaked at about 106 105 and 105 PFU gm respectively Strikingly titers in the lungs of mice treated with HR2P M2 and challenged with WT Q1020R and Q1020H MERS CoV were decreased to 103 PFU gm wild type or below the limit of detection Q1020R and Q1020H representing 1 000 fold reduction of viral titers in lung The limit of detection LOD of the viral titers in our assay was 102 PFU gm suggesting that mice intranasally treated with HR2P M2 6 hrs before challenge with MERS CoV or its variants were highly protected from MERS CoV infection Intranasal application of HR2P M2 and interferon IFN in combination before or after viral challenge protected Ad5 hDPP4 transduced mice from MERS CoV infection Studies using the HIV specific peptide enfuvirtide demonstrated the emergence of resistance in the HR1 domain of HIV during prolonged exposure 36 Therefore to diminish the likelihood of this occurrence and to provide an additional layer of protection in the event of any emerging resistant virus we included IFN in our prophylactic and therapeutic regimens IFN potently inhibited MERS CoV infection in cell culture 18 and we previously showed that IFN treatment before or after MERS CoV challenge significantly accelerated the kinetics of virus clearance 39 Initially we tested the in vivo efficacy of IFN HR2P M2 and their combination as prophylaxis in MERS CoV infected C57BL 6 mice Mice were transduced with Ad5 hDPP4 and treated 5 days later by intranasal administration of PBS IFN HR2P M2 or IFN HR2P M2 Six hrs later mice were challenged with MERS CoV and virus titers were measured at 72 hrs post infection p i Virus titers were significantly reduced in all treated groups with complete clearance observed in mice treated with IFN HR2P M2 Figure 5Aa To assess whether these at University of California Santa Barbara on June 23 2015 http jid oxfordjournals org Downloaded from Accepted Manuscript 12 drugs could be used therapeutically MERS CoV infected Ad5 hDPP4 transduced mice were treated at 12 and 36 hrs p i using PBS IFN HR2P M2 or IFN HR2P M2 and virus titers were measured at 96 hrs p i As shown in Figure 5Ab titers were significantly decreased in all inhibitor treated groups p 0 003 compared to PBS treated group with the greatest reduction observed in IFN HR2P M2 treated mice Additionally treatment with HR2P M2 alone resulted in significant reduction in the viral titers in comparison to IFN alone treatment groups Figure 5 A a b Additional cohorts of control mice and mice treated therapeutically were sacrificed at 7 days p i and lungs were examined for histological changes In agreement with our previous report 39 the lungs of MERS CoV infected mice showed perivascular peribr