【病毒外文文獻】2006 Therapy with a Severe Acute Respiratory Syndrome_Associated Coronavirus_Neutralizing Human Monoclonal Antibody Redu

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MAb Therapy Reduces SARS CoV Disease Severity JID 2006 193 1 March 685 MAJOR ARTICLE Therapy with a Severe Acute Respiratory Syndrome Associated Coronavirus Neutralizing Human Monoclonal Antibody Reduces Disease Severity and Viral Burden in Golden Syrian Hamsters Anjeanette Roberts 1 William D Thomas 2 Jeannette Guarner 4 Elaine W Lamirande 1 Gregory J Babcock 2 Thomas C Greenough 3 Leatrice Vogel 1 Norman Hayes 4 John L Sullivan 3 Sherif Zaki 4 Kanta Subbarao 1 and Donna M Ambrosino 2 1 Laboratory of Infectious Diseases National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda Maryland 2 Massachusetts Biologic Laboratories University of Massachusetts Medical School Jamaica Plain and 3 Departments of Pediatrics and Molecular Medicine University of Massachusetts Medical School Worcester 4 Infectious Disease Pathology Activity National Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta Georgia Background Immunotherapy with monoclonal antibodies MAbs offers safe interventions for the prevention of infection in patients after organ transplantation and for the treatment of cancers and autoimmune diseases MAb 201 is a severe acute respiratory syndrome associated coronavirus SARS CoV specific MAb that prevents establishment of viral replication in vitro and prevents viral replication in vivo when administered prophylactically The efficacy of MAb 201 in the treatment of SARS was evaluated in golden Syrian hamsters an animal model that supports SARS CoV replication to high levels and displays severe pathological changes associated with infection including pneumonitis and pulmonary consolidation Methods Golden Syrian hamsters that were intranasally inoculated with SARS CoV were treated with various doses of MAb 201 or an irrelevant MAb 24 h after inoculation Two to 7 days after infection the hamsters were killed and their lungs were collected for evaluation of viral titers and pathological findings Results Postexposure treatment with MAb 201 can alleviate the viral burden and associated pathological findings in a golden Syrian hamster model of SARS CoV infection After a hamster is treated with MAb 201 its viral burden is reduced by 50 tissue culture infectious doses per gram of tissue and the severity of 2 4 3 9 10 10 associated pathological findings including interstitial pneumonitis and consolidation is also remarkably reduced Conclusions The demonstration of successful postexposure MAb 201 therapy in an animal model that dem onstrates viral replication and associated pulmonary pathological findings suggests that MAb 201 may be useful in the arsenal of tools to combat SARS In late 2002 severe acute respiratory syndrome asso ciated coronavirus SARS CoV was identified as the Received 25 August 2005 accepted 1 December 2005 electronically published 27 January 2006 Presented in part Xth International Nidovirus Symposium Colorado Springs Colorado 25 30 June 2005 abstract S11 3 Potential conflicts of interests none reported Financial support National Institute of Allergy and Infectious Diseases contract NO1 AI65315 Massachusetts Biologic Laboratories Medarex All authors are full time US Government or University of Massachusetts Medical School employees and the work presented in this manuscript was conducted as part of the fulfillment of the job descriptions of the authors Reprints or correspondence Dr Donna M Ambrosino Massachusetts Biologic Laboratories University of Massachusetts Medical School 305 South St Jamaica Plain MA 02130 donna ambrosino umassmed edu The Journal of Infectious Diseases 2006 193 685 92 H17050 2006 by the Infectious Diseases Society of America All rights reserved 0022 1899 2006 19305 0010 15 00 cause of an outbreak of nearly 8100 cases of severe acute respiratory infection with an associated fatality rate of 9 5 The initial outbreak of SARS CoV infection rap idly disseminated through the human population reach ing nearly 30 countries by the middle of 2003 A second widespread outbreak has not yet occurred and rigorous public health interventions are one likely factor con tributing to the absence of a second outbreak Although no animal reservoir for SARS CoV has yet been con firmed many investigators believe that the masked palm civet is a likely candidate and that there remains the potential for the development of a second SARS outbreak as a result of natural or laboratory exposures to SARS CoV Prevention of a sustained second SARS outbreak at New Jersey Institute of Technology on March 26 2015 http jid oxfordjournals org Downloaded from 686 JID 2006 193 1 March Roberts et al would necessitate the development of safe and efficacious vac cines antiviral drugs and immunotherapies Proof of concept studies of several SARS CoV candidate vaccines have been re ported including DNA vectored vaccines recombinant pro tein subunit vaccines whole inactivated virus vaccines and live attenuated vectored vaccines 1 6 Prototypes of each of these candidate vaccines have been examined in animal mod els and the vast majority have been shown to be highly im munogenic and efficacious in preventing infection resulting from subsequent challenge with SARS CoV Several antiviral drugs have demonstrated inhibition of viral replication in vitro 7 and type I interferon has been shown to reduce the severity of SARS associated disease in a nonhuman primate model 8 In addition human IgG1 antibodies specific to SARS have been generated by a variety of techniques and passive transfer of these antibodies has been shown to prevent infection in animal models 9 12 These strategies will be likely components in the prevention of a second widespread outbreak of SARS Although several candidate vaccines have been shown to be safe and efficacious in a variety of animal models a licensed vaccine for SARS CoV is not imminent Concerns that SARS vaccines will cause disease enhancement after reexposure to SARS CoV will likely need to be addressed before licensure occurs This concern has arisen because cats immunized against or infected with feline infectious peritonitis virus can develop an accelerated and fatal illness upon reexposure to feline in fectious peritonitis virus 13 14 In a recent study a Canadian group demonstrated potential disease enhancement after chal lenge with SARS CoV in ferrets vaccinated with modified vac cinia virus Ankara expressing the SARS CoV spike protein 15 In this study modified vaccinia virus Ankara SARS vectors expressing nucleocapsid and spike proteins of SARS CoV the TOR2 strain were poorly immunogenic compared with a sim ilar vector administered to mice 1 2 because only low levels of humoral immunity were measured before challenge with SARS CoV and immunized ferrets developed moderately se vere hepatitis after homologous challenge with the TOR2 strain of SARS CoV 15 In a primate model of SARS hepatitis was also observed in unvaccinated SARS CoV infected animals 16 These observations necessitate closer scrutiny of any potential SARS vaccine Although prevention of SARS CoV infection by vaccination would be ideal additional interventions for the prevention of SARS CoV associated disease such as postexposure therapies with protective antibodies would be of great use in outbreaks of SARS or exposures to SARS CoV in unvaccinated popula tions Postexposure treatment with interferons may also hold some promise but immunotherapy with a SARS specific MAb would expand the treatment repertoire available and might be better tolerated than would treatment with interferons Im munotherapy with humanized MAbs has been established for several years and offers safe interventions for infectious disease various cancers and autoimmune disorders 17 18 Use of a humanized antibody avoids the potential for the development of human anti murine antibody responses after multiple ex posures 19 and clinical use of a virus specific humanized monoclonal antibody i e palivizumab has been demon strated for years to safely and efficaciously prevent respira tory disease associated with respiratory syncytial virus infec tions 20 Previously MAb 201 a human monoclonal anti body generated from transgenic mice expressing human im munoglobulin genes Medarex was shown to specifically bind to the angiotensin converting enzyme 2 receptor binding do main of the SARS CoV spike protein neutralize virus entry in in vitro assays and provide protection from SARS CoV rep lication in the respiratory tissues of mice when administered prophylactically 11 The viral burden in the lungs of mice that received MAb 201 treatment 24 h before intranasal infec tion with TCID 50 of SARS CoV mouse was reduced 1 mil 5 10 lion fold to a level below the limit of detection 11 The golden Syrian hamster provides a better model for spe cific evaluation of therapeutic effects than does the mouse Like the mouse the hamster supports high levels of viral replication in pulmonary tissues however unlike the mouse which dem onstrates few to no remarkable pathological findings hamsters show moderate to severe interstitial inflammation and pul monary consolidation in association with replication of SARS CoV 21 In the hamster model immunotherapy may there fore be examined on 2 levels the ability to alleviate viral burden and the ability to reduce associated pathological findings Ex amination of pulmonary tissues after SARS CoV infection and subsequent MAb therapy will establish whether a decrease in the viral titer would be accompanied by a decrease in the se verity of associated pathological findings Therefore to explore the immunotherapeutic potential of MAb 201 TCID 50 of 3 10 SARS CoV was administered intranasally and 24 h later ham sters were treated either with various doses of MAb 201 or with an irrelevant human MAb administered intraperitoneally We found that treatment with SARS CoV specific human MAb i e MAb 201 can significantly reduce viral replication and that this reduction in viral replication correlates with a reduc tion in the severity of observed pathological findings in the pulmonary tissues of a SARS CoV susceptible host MATERIALS AND METHODS All work with infectious virus and with infected animals was performed in biosafety level 3 facilities by personnel wearing positive pressure air purifying respirators HEPA AirMate 3M All animal protocols used in these studies have been approved by the Animal Care and Use Committee of the National In stitute of Allergy and Infectious Diseases at New Jersey Institute of Technology on March 26 2015 http jid oxfordjournals org Downloaded from MAb Therapy Reduces SARS CoV Disease Severity JID 2006 193 1 March 687 Table 1 Viral titers in lungs obtained from hamsters treated with monoclonal antibody 201 MAb 201 after challenge with severe acute respiratory syndrome associated coronavirus SARS CoV Experiment treatment group dose Hamsters no Serum titer of SARS CoV specific antibodies mean H11506 SE Lung titer of SARS CoV c mean H11506 SENeutralizing a IgG ELISA b 1 MAb 201 4 mg kg 2 10 H11506 011H11506 34 3H11506 0 4 40 mg kg 5 35 H11506 2 156 H11506 24 3 8 H11506 0 9 d Subneutralizing e 4 40 mg kg 5 8 H11506 012H11506 44 3H11506 0 7 Control f 40 mg kg 6 8 H11506 00H11506 061H11506 0 5 2 MAb 201 40 mg kg 4 27 H11506 5 125 H11506 34 8H11506 0 3 g 80 mg kg 4 53 H11506 9 278 H11506 28 4 1 H11506 0 3 g Control f 40 80 mg kg 8 8 H11506 00H11506 08 0H11506 0 2 3 MAb 201 40 mg kg 4 45 H11506 20 178 H11506 52 5 8 H11506 0 3 d Subneutralizing e 40 mg kg 2 8 H11506 03H11506 27 3H11506 0 3 Control f 40 mg kg 4 8 H11506 00H11506 080H11506 0 2 a Titers were measured using microneutralization assays performed on Vero cell monolayers and are expressed as reciprocal titers Lowest dilution tested 1 8 b SARS CoV specific IgG antibodies detected by ELISA c Viral titers were measured on days 3 and 5 after infection in experiment 1 and on day 2 after infection peak titer in experiments 2 and 3 Kruskal Wallis test across all treatment groups P 0001 d Mann Whitney U test MAb 201 treated groups vs control group P 045 e Hamsters that had no measurable neutralizing antibody titer 1 8 24 h after MAb 201 treatment were analyzed separately f Palivizumab an irrelevant humanized MAb g Mann Whitney U test MAb 201 treated groups vs control group P 008 Animal studies Female golden Syrian hamsters LVG SYR were obtained from Charles River Laboratories and were pair housed in individually ventilated microisolator rodent cages Hamsters were rested for H110913 days before initiation of the fol lowing experiments In experiment 1 golden Syrian hamsters age 43 days were lightly anesthetized by inhalation of isoflurane USP Baxter Healthcare and were inoculated intranasally with TCID 50 of 3 10 SARS CoV in a total volume of 100 mL The hamsters were treated 24 h later with intraperitoneal injections of 4 mg kg or 40 mg kg MAb 201 or with 40 mg kg palivizumab an irrelevant humanized MAb 22 which was used as a control total volume 1 0 mL 12 hamsters group One day after MAb treatment ham sters were bled and sera were assayed for SARS CoV specific ELISA IgG antibodies hereafter referred to as IgG ELISA an tibodies and neutralizing antibodies Hamsters were killed at 3 and 5 days after SARS CoV infection 6 hamsters group were killed on each of these days and lungs were harvested for viral titer determination or histopathological evaluation Lungs ob tained from 3 hamsters from each group on each of these days were either 1 homogenized 10 wt vol and assayed in serial 10 fold dilutions on Vero cell monolayers for determination of viral titers limit of detection TCID 50 of SARS CoV g of 1 5 10 tissue or 2 inflated with and stored in 10 formalin and then processed for histopathological examination In experiments 2 and 3 golden Syrian hamsters age 45 48 days anesthetized by inhalation of isoflurane were inoculated intranasally with TCID 50 of SARS CoV in a total volume 3 10 of 100 mL After 24 h the hamsters were treated with various doses of MAb 201 or an irrelevant human MAb total volume 0 5 mL One day after MAb treatment hamsters were bled and serum samples were assayed for SARS CoV specific IgG ELISA and neutralizing antibodies Hamsters were killed 2 days after SARS CoV infection lungs were harvested and 10 lung homogenates were assayed for determination of viral titers Similarly SARS CoV inoculated and MAb treated hamsters were killed 5 or 7 days after infection and lungs were inflated with and stored in 10 formalin for histopathological exam ination and then processed Treatment strategies the number of hamsters evaluated IgG ELISA and neutralizing antibody titers viral titers in lung homogenates and pathological find ings are summarized in tables 1 and 2 ELISAs The presence of SARS specific IgG ELISA anti bodies was determined by coating 96 well plates at 4H11034C over night with 1 mg mL S270 510 SARS spike protein in PBS as reported elsewhere 11 A standard curve was generated from at New Jersey Institute of Technology on March 26 2015 http jid oxfordjournals org Downloaded from 688 JID 2006 193 1 March Roberts et al Table 2 Findings of histopathological evaluations of lungs obtained from hamsters treated with monoclonal antibody 201 MAb 201 after challenge with severe acute respiratory syndrome associated coronavirus SARS CoV Experiment treatment group dose Hamsters no Serum titer of SARS CoV specific antibodies mean H11506 SE Median severity score for associated pathological finding in the lung a Neutralizing b IgG ELISA c Interstitial pneumonitis Consolidation 2 MAb 201 Day 1 40 mg kg d 338H11506 8 131 H11506 25 1 2 Day 2 40 mg kg e 326H11506 395H11506 61 80 mg kg 4 45 H11506 5 200 H11506 14 1 2 Subneutralizing f 40 80 mg kg 2 8 H11506 01H11506 13 Control g 40 80 mg kg 8 8 H11506 00H11506 03 3 3 MAb 201 40 mg kg 6 35 H11506 8 207 H11506 28 1 1 Subneutralizing f 40 mg kg 3 8 H11506 08H11506 82 Control g 40 mg kg 8 8 H11506 00H11506 03 3 a Severity of interstitial pneumonitis or consolidation observed on day 5 or 7 after infection 0 no finding 1 mild 2 moderately severe and 3 severe b Titers were measured using microneutralization assays performed on Vero cell monolayers and are expressed as reciprocal titers Lowest dilution tested 1 8 c SARS CoV specific IgG antibodies detected by ELISA d Treatment administered 1 day after challenge with SARS CoV e Treatment administered 2 days after challenge with SARS CoV f Hamsters that had no measurable neutralizing antibody titer 1 8 24 h after MAb 201 treatment were analyzed separately g Palivizumab an irrelevant humanized MAb serial dilutions of normal hamster serum samples spiked with SARS spike protein 201 100 mg mL Palivizumab 100 mg mL in PBS 0 1 Tween with 0 5 human serum albumin served as a negative control All serum samples obtained from SARS infected hamsters were heat inactivated for 90 min at 56H11034C to inactivate SARS CoV and all dilutions 3 fold were made in PBS 0 1 Tween 0 5 human serum albumin Plates were washed with PBS 0 05 Tween The limits of detection are reported as 0 4 mg mL and are based on the highest con centration tested dilution 1 100 or 1 200 and the limit of quantitation of the assay 2 ng mL Each sample was run in duplicate and assays were conducted in a blinded fashion The plates were developed with a goat anti human IgG FAb2 al kaline phosphatase conjugate Microneutralization assays for the determination of neu tralizing antibody titers Blood samples were collected by retro orbital bleeding of hamsters that received isoflurane as general anesthesia and 0 5 tetracaine hydrochloride oph thalmic solution Bausch a grade of 2 moderately severe denoted the presence of multiple areas where there was confluence of 2 foci of in terstitial pneumonitis or consolidation and a grade of 3 se vere denoted the presence of large confluent areas of inter stitial pneumonitis or consolidation that involved more than half a lobe Statistical analyses The nonparametric Mann Whitney U test Kruskal Wallis test and Spearman s rank correlation were the statistical methods used for ascertaining the significance of observed differences Statistical significance was denoted by P 05 RESULTS In an initial pilot experiment experiment 1 we examined the potential of MAb 201 in the postexposure treatment of SARS CoV infection Within any single treatment group 4 mg kg MAb 201 40 mg kg MAb 201 or the control treatment no noticeable difference was observed in the levels of viral repli cation in the pulmonary tissues between 3 and 5 days after infection Therefore data from days 3 and 5 afterP 1 05 infection were combined and are presented according to cor responding treatment groups in tables 1 and 2 However we did note significant differences in the virus levels in the lungs and in the degree of associated pathological findings in MAb 201 treated hamsters compared with those in control ham sters table 1 experiment 1 In the group treated with 4 mg kg MAb 201 only 2 of 6 hamsters achieved measurable titers of neutralizing antibody and IgG ELISA antibodies after in traperitoneal administration of the treatment The number of hamsters in this group was therefore too small for relevant sta tistical analysis to be done Of the 6 hamsters in the group treated with 40 mg kg MAb 201 5 had measurable titers of neutralizing and IgG ELISA antibodies In a comparison of this group with the control group statistically significantnp6 reductions in the virus levels were achieved with MAb 201 treatment Mann Whitney U test Furthermore his Pp 04 topathological findings suggested that treatment with MAb 201 administered 24 h after SARS CoV infection reduced the se verity of interstitial pneumonitis observed 3 and 5 days after SARS CoV infection as well as the severity of consolidation observed 5 days after infection data not shown The observations from this initial experiment led to 2 ad ditional experiments in which lungs were collected for deter mination of viral titers 2 days after inoculation with SARS CoV i e at the time