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1 Scientific RepoRts 6 31629 DOI 10 1038 srep31629 Passive T ransfer of A Germline like Neutralizing Human Monoclonal Antibody Protects T ransgenic Mice Against Lethal Middle East Respiratory Syndrome Coronavirus Infection Anurodh Shankar Agrawal 1 Tianlei Ying 2 Xinrong Tao 1 T ania Garron 1 Abdullah Algaissi 1 3 Yanping Wang 4 Lili Wang 4 Bi Hung Peng 5 Shibo Jiang 3 Dimiter S Dimitrov 4 Chien Te K Tseng 1 Middle East Respiratory Syndrome coronavirus MERS CoV has repeatedly caused outbreaks in the Arabian Peninsula To date no approved medical countermeasures MCM are available to combat MERS CoV infections Several neutralizing human monoclonal antibodies mAbs including m336 a germline like human mAb have been chosen as promising MCM for MERS CoV However their clinical development has been hindered by the lack of a robust animal model that recapitulate the morbidity and mortality of human infections We assessed the prophylactic and therapeutic efficacy of m336 by using well characterized transgenic mice shown to be highly sensitive to MERS CoV infection and disease We found that mice treated with m336 prior to or post lethal MERS CoV challenging were fully protected compared to control mice which sufferered from profound weight loss and uniform death within days after infection Taken together these results support further development of m336 and other human monoclonal antibodies as potential therapeutics for MERS CoV infection Middle East Respiratory Syndrome coronavirus MERS CoV a recently identified novel coronavirus that causes fatal acute respiratory illness in human was initially isolated from a Saudi Arabian patient with acute pneumo nia and renal failure in June 2012 1 As of July 6th 2016 1 782 cases with 634 deaths have been confirmed in 27 countries http www who int emergencies mers cov en While the clinical presentations of MERS CoV are very similar to those of SARS CoV phylogenetic analysis has revealed that MERS CoV is genetically more closely related to bat coronaviruses than to SARS CoV suggesting that it may have originated from bats before evolving into a human pathogen 2 8 Of note MERS CoV has been detected in dromedary camels and a high prevalence of MERS CoV specific antibodies can be found in camels from some regions in the Middle East and Africa 9 14 A recent study revealed the co circulation of several human coronavirus species and MERS CoV lineages in dromedary camels in Saudi Arabia including a recombinant strain which has been a dominant isolate from patients since December 2014 and subsequently led to human outbreaks in 2015 15 This study suggested that the dromedary camel may serve as an important reservoir and that MERS CoV may represent a continuous 1 Department of Microbiology and Immunology University of Texas Medical Branch Galveston TX 77555 USA 2 Key Laboratory of Medical Molecular Virology of Ministries of Education and Health School of Basic Medical Sciences Fudan University Shanghai 200032 China 3 Department of Medical Laboratories Technology College of Applied Medical Sciences Jazan University Jazan Saudi Arabia 4 Protein Interactions Section Cancer and Inflammation Program Center for Cancer Research National Cancer Institute National Institutes of Health Frederick MD 21702 USA 5 Neuroscience Cell Biology University of Texas Medical Branch Galveston TX 77555 USA These authors contributed equally to this work Correspondence and requests for materials should be addressed to C T K T email sktseng utmb edu Received 13 May 2016 Accepted 22 July 2016 Published 19 August 2016 OPEN 2 Scientific RepoRts 6 31629 DOI 10 1038 srep31629 and long term threat to people particularly those who interact closely with camels in the Arabian Peninsula Even though MERS CoV presently has limited human to human transmission 2 16 the high mortality rate of this virus and limited information on the mechanism able to confer increased human to human transmission have raised concerns of a potential MERS pandemic Indeed the recent outbreaks in Korea and the appearance of super spreading events indicate that MERS CoV has the ability to cause large outbreaks outside of the Arabian Peninsula 17 19 Currently no approved vaccines or drugs are available to treat this viral infection These facts high light an urgent need to develop potent prophylactic and therapeutic agents to fight this lethal virus Similar to other coronaviruses MERS CoV uses the envelope spike S glycoprotein a class I transmembrane protein for interaction with its cellular receptor for binding fusion and entry into the target cell 20 The recep tor binding domain RBD located in the S1 domain of the MERS CoV spike is responsible for binding to the well characterized cellular receptor identified as DPP4 CD26 and is therefore critical for binding and entry of the virus 20 22 Therefore neutralizing antibodies capable of blocking such interaction could be promising pre ventive and or therapeutic candidates Recently human monoclonal antibodies mAbs capable of neutralizing MERS CoV have been identified and characterized by several research groups 23 28 These antibodies have been isolated from naive human antibody libraries from transgenic humanized mice or from B cells of an infected individual and they recognize different epitopes on MERS CoV RBD One of the most potent mAbs m336 is a germline like antibody identified from a very large 10 11 size phage displayed antibody library derived from B cells of healthy donors This mAb exhibits exceptionally potent neutralizing activity IC 50 0 0 0 5 g m l in vitro 23 Moreover because its epitope almost completely 90 overlaps with the receptor binding site of DPP4 on MERS CoV RBD as is evident by its recently solved crystal structure 29 the probability of generation of resistant mutants may be absent or very low Notably although the functions of these mAbs have been extensively characterized in vitro their further clinical development has been hindered by the lack of an effective animal model of MERS CoV infection MERS CoV cannot infect small laboratory animals e g mice hamsters and ferrets as a consequence of species specific differences in DPP4 while only causing mild to moderate symptoms in rhesus macaques Marmosets which are more susceptible to MERS CoV developed a moderate to severe disease but limited availability and high cost have hampered their use 30 Rabbits can be infected but the infec tious virus is challenging to detect 31 32 It was found that the expression of human DPP4 could overcome the lack of susceptibility in normal mice With prior transduction of adenoviral human DPP4 expressing vectors mice became susceptible to MERS CoV infection without revealing any measurable clinical manifestations 33 In contrast transgenic Tg mice with the human DPP4 gene integrated into the genome readily developed acute morbidity weight loss and uniform death occurred within a week 34 35 making it an ideal preclinical model for the development of vaccines and treatments against MERS Some of the aforementioned human neutralizing monoclonal antibodies have been shown to protect engi neered human DPP4 expressing mice and the naturally permissive rabbits entirely based on their ability to inhibit MERS CoV infection and or alleviate histopathology of the lungs 27 28 36 37 T o further verify the protective efficacy of these human monoclonal antibodies particularly m336 against MERS CoV infection it is highly desirable to use the well characterized human DPP4 Tg mice known to result in acute disease weight loss and death 34 35 By using this highly permissive Tg mouse model we evaluated the prophylactic and therapeutic efficacy of m336 mAb in vivo We report in this study for the first time that treatment of Tg mice with a single dose of m336 antibody prior to or after challenging with 1 000 LD 50 of MERS CoV protected mice from the lethality in a dose dependent manner thereby representing the first antibody tested for its protective efficacy against lethal MERS CoV infection Results Prophylactic efficacy of MERS CoV RBD specific human monoclonal antibody m336 We established a Tg mouse model which is profoundly sensitive and susceptible to MERS CoV infection as deter mined by high viral titers in the lungs as well as a high rate of morbidity and mortality 34 38 Equipped with this small animal model of human MERS CoV we investigated the protective efficacy of mAb m336 To accomplish this each group n 6 of mice was treated via the intraperitoneal i p route with two different doses 0 1 mg and 1 mg per mouse diluted in 100 l PBS and challenged intranasally i n at 12 h post treatment with 10 4 TCID 50 i e 1 000 LD 50 of MERS CoV in a volume of 60 l 38 Challenged mice were monitored daily for clinical manifes tations weight loss and mortality As shown in Fig 1 the group treated with 1 mg mAb survived viral infection without showing any clinical symptoms These mice initially showed either no weight loss or recovered from mild weight loss within three days Fig 1A On the other hand the group treated with 0 1 mg mAb showed a gradual weight loss 15 20 until day 13 just before starting to recover Fig 1A All surviving mice one died on day 13 in mice treated with 0 1 mg of m336 continued to recover and appeared well up to 21 dpi when the experiment was terminated Fig 1B All MERS CoV challenged mice pretreated with a high dose 1 mg of irrelevant mAb m102 4 exhibited profound weight loss 15 and succumbed to infection with 100 mortality by day 8 p i Fig 1A B Therapeutic efficacy of MERS CoV RBD specific human monoclonal antibody m336 T o deter mine the therapeutic potential of this human monoclonal m336 antibody groups of mice N 6 per group were challenged i n with 10 4 TCID 50 of MERS CoV i e 1 000 LD 50 in a volume of 60 l and then treated i p 12 hours later with a single dose of either 1 mg or 0 1 mg of m336 or 1 mg of m102 4 antibody control in 100 l per mouse followed by monitoring daily for wellbeing weight loss and other clinical manifestations and mortality of mice We noted that whereas treatment with 1 mg of m336 antibodies was effective in the protection against the lethality caused by MERS CoV infection it failed to protect mice fully from the onset of clinical illness weight loss Specifically all of the challenged mice treated with 1 mg of m336 antibody suffered an atten uated 10 and transient weight loss until day 9 and gradually recovered to day 21 when the experiment was 3 Scientific RepoRts 6 31629 DOI 10 1038 srep31629 terminated Fig 2 Similarly challenged mice treated with a low dose of 0 1 mg of m336 antibodies suffered from attenuated and transient weight loss until day 7 p i and gradually recovered However we noted a single death at day 9 in this low dose treatment group Fig 2 As expected all mice treated with a single dose of 1 mg of con trol m102 4 antibody exhibited profound weight loss 15 and succumbed to MERS CoV infection with 100 mortality by day 8 p i Fig 2 Taken together these results indicate that this MERS CoV RBD specific human m336 antibody can be highly effective as prophylactic or therapeutic modalities in protecting highly permissive transgenic mice against MERS CoV infection and disease Figure 1 Prophylactic efficacy of mAb m336 in protecting Tg mice against lethal dose MERS CoV challenge Tg mice were treated i p with m336 antibody 12 h before challenge i n with 10 4 TCID 50 of MERS CoV An irrelevant human mAb m102 4 was included as the control Challenged mice were monitored daily for the weight loss A and accumulated mortality B expressed as percent weight loss and survival respectively Figure 2 Therapeutic efficacy of mAbm336 in protecting Tg mice against lethal MERS CoV challenge Tg mice were treated i p with human m336 antibody 12 h after infection i n with 10 4 TCID 50 of MERS CoV An irrelevant mAb m102 4 was also included as the control Challenged mice were monitored daily for the weight loss A and accumulated mortality B expressed as percent weight loss and survival respectively 4 Scientific RepoRts 6 31629 DOI 10 1038 srep31629 Lung virus titers in mice treated with MERS CoV RBD specific human monoclonal antibody m336 We also investigated the protective mechanism of m336 against MERS CoV by determining the lung virus titers in challenged mice at day 2 after treatment Specifically we sacrificed two mice out of 6 in each group as described above for Figs 1 and 2 and their lung specimens were harvested for determining viral titers by using via Vero E6 cell based infectivity assay and quantitative PCR Q PCR based assay targeting the upstream E gene of MERS CoV As shown in Fig 3A we were unable to recover infectious virus from any mouse treated with 1 mg of m336 antibody either before or after challenge with MERS CoV However we were able to detect a barely detectable infectious virus with the limit of detection LOD of 2 3 log TICD 50 g from a single mouse receiving 0 1 mg of m336 prior to viral challenge These results indicated that mAb m336 most likely confers protection from lethal challenge by restricting viral replication within the lungs thereby preventing viral infection in the brains and other organs Titers of viral RNA copy number as shown by qRT PCR assays were also compared among groups having different doses of mAbs Lungs of infected mice were harvested on day 2 post and pre virus challenge group All groups exhibited detectable viral RNA Titers were significantly lower than those in the control group in all m336 treated groups In the pretreatment group mice treated with 1 mg of m336 showed a 2 log reduction in viral RNA detection while a 1 log reduction in viral numbers was seen in mice treated within 0 1 mg m336 when compared to mice receiving control mAb m102 4 In the post treatment group a smaller 1 log dif ference in viral RNA copy number compared to that in the pretreatment group was observed between mice treated with 1 mg antibody compared with those receiving control antibody while a more than 1 log reduction in viral RNA number was seen in mice treated with 0 1 mg m336 when compared to mice receiving control mAb Fig 3B These data indicate that m336 confers significant protection to mice when administered pre or post viral challenge Taken together these results suggest to us that the epitope targeted by this exceptionally potent RBD specific m336 antibody has a great potential for further development as a potent preventive and therapeutic agent in the future Treatment with m336 attenuates lung pathology associated with MERS CoV infection The effect of m336 antibody treatment on the pulmonary pathology associated with MERS CoV infection was eval uated by using formalin fixed paraffin embedded and hematoxylin eosin H E stained lung specimens har vested at day 2 p i Pulmonary pathology was noted in all mice that were treated with different doses of m336 or control m102 4 antibodies either before or after viral infection On a severity scale of 0 to 3 none mild moder ate severe H E stained samples from mice pretreated with 1 mg and 0 1 mg of m336 antibody were graded 0 and 1 respectively for perivascular and intra alveolar infiltration of mononuclear cells including lymphocytes macrophages monocytes Fig 4 Middle panel whereas those obtained from mice that received post infection Figure 3 Treatment with m336 antibody significantly inhibited MERS CoV infection within the lungs Lung specimens collected at day 2 after viral challenge were processed for assessing the viral titers by using both Vero E6 based infectivity assay and qRT PCR targeting upstream E gene of MERS CoV and expressed as log 10 TCID 50 gram and log 10 TCID 50 equivalent eq gram respectively A Prophylactic and therapeutic efficacy of human m336 antibody treatment in reducing the lung titers of infectious virus B Prophylactic and therapeutic efficacy of human m336 antibody in reducing the titers of viral RNA The data shown are representative of at least two independently conducted assays using the same samples Data is presented as Mean standard error SE P 0 001 as determined by using Student s t test 5 Scientific RepoRts 6 31629 DOI 10 1038 srep31629 treatment with either dose of m336 were graded 1 Fig 4 right panel compared to the grade 2 assigned to mice received control antibody treatment prior to infection Fig 4 left panel Discussion MERS CoV has attracted significant basic research and clinical studies since it was first discovered in early 2012 Even though the transmissibility of MERS CoV among humans remains low at present as a mutation prone RNA virus it could eventually evolve into a highly communicable and more virulent human pathogens This emphasizes the urgent need for the development of an effective antiviral therapy which could restrict the spread of this deadly disease In other viral infections neutralizing antibodies have been shown to protect the host from disease progression and or reduce the severity of clinical symptoms Passive immunotherapy for prophylaxis and treatment of infectious viral diseases has been widely used for many decades 39 43 Passive transfer of neutralizing antibodies is also a promising strategy for both prophylaxis and treatment against MERS CoV infection To this end we and others have successfully demonstrated the protective efficacy of specific human neutralizing mon oclonal antibodies in animal models of MERS CoV infection 23 24 26 28 Among a panel of MERS CoV specific mAbs generated by using a vast phage display library 23 we identified three mAbs which specifically bind to the MERS CoV RBD with very high affinity Among these three identified we noted that mAb m336 exhibited the highest potency in neutralizing live MERS CoV Here we further characterized this novel human mAb in our Tg mouse model of MERS CoV infection and showed prophylactic and therapeutic protection of mice treated with m336 before and after a lethal challenge with the virus respectively Thus mAb m336 is highly promising as a potent inhibitor for urgent prophylaxis in adjunctive treatment for patients infected with MERS CoV In our studies we noted that passively transferred with 1 mg and 0 1 mg of m336 monoclonal antibodies to individual mice 12 h prior to challenge with 1 000 LD 50 of MERS CoV resulted in 100 and 75 protection against lethality respectively Fig 1 suggesting that using 0 1 mg m336 mouse as a prophylaxis is suboptimal to completely neutralize viral infection thereby allowing residual viruses to replicate within lungs during the course of infection These data demonstrate that m336 confers a dose dependent reduction of MERS CoV infec tion corroborating lower viral RNA levels and live virus isolation determined for these mice when compared to control mice Our study also confirmed the therapeutic efficacy of m336 in a dose dependent manner Similar to the prophylactic studies administration of a single dose of m336 antibody at a concentration of either 1 or 0 1 mg per mouse at 12 h after MERS CoV challenge provided 100 and 75 protection respectively against Figure 4 Treatment with human m336 antibody significantly attenuates lung pathology of mice challenged with MERS CoV Lung specimens collected at day 2 post infection were fixed appropriately processed and H E stained for assessing the lung pathology The lung pathology scores were graded from 0 3 none mild moderate and severe based on the extent of mononuclear cell infiltration Left mice treated with 1 mg m102 4 control antibody 12 h prior to viral infection Middle mice treated with 1 or 0 1 mg of m336 antibody 12 h prior to viral infection as indicated Right mice treated with 1 or 0 1 mg of m336 antibody 12 h after viral infection as indicated 6 Scientific RepoRts 6 31629 DOI 10 1038 srep31629 infection induced lethality accompanied by reduced viral loads both infectious virus and viral RNA within the lungs However we also noted the recovery of bodyweight loss and the reduction of viral loads in mice treated with 1 mg of m336 at 12 hrs after infectio