【病毒外文文獻(xiàn)】2009 Neutralizing antibody against severe acute respiratory syndrome (SARS)-coronavirus spike is highly effective for th

Microbiol Immunol 2009 53 75 82 doi 10 1111 j 1348 0421 2008 00097 x ORIGINAL ARTICLE Neutralizing antibody against severe acute respiratory syndrome SARS coronavirus spike is highly effective for the protection of mice in the murine SARS model Koji Ishii 1 Hideki Hasegawa 2 Noriyo Nagata 2 Yasushi Ami 3 Shuetsu Fukushi 4 Fumihiro Taguchi 5 and Yasuko Tsunetsugu Yokota 6 4 Department of Virology I 1 Department of Virology II 5 Department of Virology III 2 Department of Pathology 3 Division of Experimental Animals Research and 6 Department of Immunology National Institute of Infectious Diseases Toyama Shinjuku ku Tokyo 162 8640 Japan ABSTRACT We evaluated the efficacy of three SARS vaccine candidates in a murine SARS model utilizing low virulence Ppand SARS CoVcoinfection Vaccinated mice were protected from severerespiratorydisease in parallel with a low virus titer in the lungs and a high neutralizing antibody titer in the plasma Importantly the administration of spike protein specific neutralizing monoclonal antibody protected mice from the disease indicating that the neutralization is sufficient for protection Moreover a high level of IL 6 and MCP 1 production but not other 18 cytokines tested on days 2 and 3 after SARS CoV infection was closely linked to the virus replication and disease severity suggesting the importance of these cytokines in the lung pathogenicity of SARS CoV infection Key words coronavirus Pasteurella pneumotropica severe acute respiratory syndrome SARS A new disease called SARS originated in China in late 2002 and spread rapidly throughout a number of countries Structural characterization of the SARS CoV and charac terization of its complete RNA genome 1 3 have pro vided us with the opportunity to develop a SARS vaccine Like other coronaviruses SARS CoV is a plus stranded RNA virus with a 30 kb genome encoding replicase gene products and four structural proteins i e spike S en velope E membrane M and nucleocapsid N 1 2 The S protein is a type I fusion protein with an approximate molecular weight of 180 kDa The angiotensin converting enzyme 2 ACE2 has been reported to function as a re ceptor for SARS CoV 4 and amino acids 270 510 of the S protein are required for interaction with the receptor 5 suggesting that the S protein would be an ideal tar get for a vaccine In fact passive transfer of neutralizing antibody can prevent replication of the SARS CoV in the Correspondence Yasuko Tsunetsugu Yokota Department of Immunology National Institute of Infectious Diseases Shinjuku ku Tokyo 162 8640 Japan Tel 81 3 5285 1111 ext 2133 fax 81 3 5285 1150 email yyokota nih go jp Received 2 September 2008 revised 5 October 2008 accepted 22 October 2008 List of Abbreviations CoV coronavirus IFN interferon Ig immunoglobulin IL interleukin IP IFN inducible protein MCP monocyte chemotactic protein Pp Pasteurella pneumotropica SARS severe acute respiratory syndrome s c subcutaneously TNF tumor necrosis factor mouse respiratory tract 6 7 and many vaccine studies of SARS CoV have identified the S protein among other SARS CoV structural proteins as a major determinant of neutralization 8 Developing an animal model is crucial for evaluating the vaccine efficacy on SARS CoV induced lung patho geneses SARS CoV infection occurs transiently in the mouse and the virus is cleared by day 7 postinfection 7 although an age related susceptibility to lung disease in old mice has been shown 9 In the course of studying the cell entry mechanism of SARS CoV we found that some proteases such as trypsin and elastase produced in the host animals enhance SARS CoV infection in cultured cells 10 In a previous study we examined whether weak inflammation in the lungs induced by low pathogenicity bacterial infection which could induce elastase enhances SARS CoV infection and we showed that low virulence c 2008 The Societies and Blackwell Publishing Asia Pty Ltd 75 K Ishii et al Pp infection as well as administration of lipopolysaccha ride derived from Escherichia coli induced elastase in the lungs and enhanced the replication of SARS CoV which resulted in the exacerbated respiratory disease caused by SARS CoV infection with a high mortality rate These results indicate that coinfection of SARS CoV with low virulence microorganisms induces exacerbated pneumo nia and suggest the possibility that elastase is involved in the pathogenesis of exacerbated pneumonia caused by SARS CoV infection 11 In our research group UV inactivated and UV and formalin inactivated whole virion vaccines were produced and both were shown to be effective on the elicita tion of persistent neutralizing antibodies accompanied by T cell responses 12 We also constructed a replication deficient recombinant vaccinia virus DIs expressing one or more SARS CoV structural proteins E M N and S or a combination of E M and S E M S or E M N and S E M N S 13 When these recombinant DIs vaccines were given to mice either s c or intranasally the humoral and cellular immunities against SARS CoV were elicited We showed that a high level of serum neutralizing IgG antibody elicited by subcutaneous injection of these vac cinias strongly suppressed SARS CoV replication in the lungs and that the neutralizing IgA type antibody elicited only by mucosal intranasal immunization was not abso lutely required Furthermore we demonstrated that DIs expressing the S protein alone or in combination with other components but not N alone elicited strong neu tralizing antibody and T cell responses against SARS CoV infection Although we and others demonstrated that the vaccine expressing the S protein alone was able to inhibit SARS CoV replication efficiently in mice the role of the N specific T cell response for protection had not been for mally excluded because the SARS CoV infection in mice is usually cleared rapidly without causing any pulmonary disease Utilizing a murine model system of severe respiratory disease caused by the coinfection of Pp and SARS CoV we here evaluated the protective efficacy of our SARS vaccine candidates UV or UV plus formalin inactivated whole virion and recombinant DIs expressing the S protein only We have monitored the bodyweight and analyzed the virus replication and cytokine production in the lung lavage of vaccinated and na ve mice after SARS CoV infection By giving SKOT 20 monoclonal neutralizing antibody recog nizing the S protein just before Pp infection the mortality of mice coinfected with Pp and SARS CoV was dramati cally reduced in a dose dependent manner These results clearly show that the neutralizing antibody against S pro tein is highly effective and sufficient to prevent SARS de velopment Furthermore we found that IL 6 and MCP 1 production was associated with high titers of SARS CoV suggesting the possible link between the increased produc tion of these cytokines and the lung pathogenicity caused by Pp and SARS CoV coinfection MATERIALS AND METHODS Virus and virus titration Fr mo the mouse adapted Frankfurt 1 Fr 1 strain cre ated by passaging 10 times through the mice and finally grown in VeroE6 cells was propagated and plaque assayed with VeroE6 cells as previously described 10 VeroE6 cells were grown and maintained in Dulbecco s modified mini mal essential medium DMEM Nissui Tokyo Japan and virus infectivity was determined by plaque assay as previ ously described 10 Preparation of UV or UV and formalin inactivated purified SARS CoV UV inactivated purified SARS CoV UV V was pre pared as previously described 14 In brief SARS CoV HKU29849 was amplified in Vero E6 cells exposed to UV light 4 75 J cm 2 and then purified by sucrose density gradient centrifugation A portion of the UV inactivated purified virions was further treated overnight with 0 02 formalin UV F V to assure the safety of a whole virion vaccine 12 Vaccination Animal studies were carried out under a protocol approved by the Animal Care and Use Committee of the National In stitute of Infectious Diseases Japan Six week old BALB c male mice were purchased from SLC Hamamatsu Japan or Charles River Japan CRJ Tokyo Japan These mice areserologicallycheckedtobefreefrominfectionswith pathogenic microorganisms including Pp For vaccina tion with recombinant DIs mice were s c immunized with 10 6 plaque forming units p f u of rDIs SARS S or DIs as a negative control After 4 weeks identical titers of viruses were re administered For vaccination with a whole inactivated virion mice were s c injected into the back with 10 g UV V or UV F V with 2 mg alum and boosted by the same procedure 7 weeks after priming One week later mice were anesthetized with xylazine and ketamine by intraperitoneal i p administration and in tranasally inoculated with 6 10 6 colony forming units c f u of Pp MaM strain 11 suspended in 20 L PBS and kept in globe box isolators in a BSL 3 laboratory in our in stitute during the experimental period One day later the mice were intranasally challenged with 0 8 10 6 TCID 50 of SARS CoV in 20 L saline as previously described 11 Three 5 and 7 days after challenge by SARS CoV serum 76 c 2008 The Societies and Blackwell Publishing Asia Pty Ltd Neutralizing Ab against SARS CoV spike nasal and lung lavage fluids were collected to measure viral titers and antibodies against SARS CoV from mice that were killed under anesthesia with chloroform The bodyweight of these mice was measured every day Detection of SARS CoV specific antibodies IgG titers against SARS CoV were determined by ELISA as previously described 14 Neutralization antibody titers were determined as previously described 15 Briefly samples were heat inactivated and diluted twofold from 1 80 to 1 5120 with DMEM containing 5 fetal bovine serum and 3000 infectious units of vesicular stomati tis virus VSV pseudotype bearing SARS CoV S protein VSV SARS St19 The mixture was incubated for 1 hr at 37 C for neutralization After incubation the mixture was inoculated onto Vero E6 cells seeded on 96 well plates The infectivity of VSV SARS St19 was determined by counting the number of green fluorescence protein GFP positive cells The Nab titer was defined as the reciprocal of the highest dilution at which more than 50 inhibition of infectivity was observed Analysis of cytokines and chemokines Cytokines and chemokines were assayed either using a mouse inflammatory cytokine IL 6 IL 10 MCP 1 IFN TNF and IL 12p70 cytometric bead array kit Becton Dickinson San Jose CA USA 12 or using the Luminex 200 system Luminex Co Austin TX USA as previously reported 16 Lung homogenates prepared as described above were diluted 1 10 with a lysis buffer and viruses included in the materials were completely inac tivated by UV irradiation for 10 min In the Luminex system the following cytokines and chemokines were measured by a mouse cytokine 20 plex antibody bead kit Bioscience International Inc Camarillo CA USA fibroblast growth factor basic granulocyte macrophage colony stimulating factor IFN IL 10 IL 12 IL 13 IL 17 IL 1 IL 1 IL 2 IL 4 IL 5 IL 6 IL 10 keratinocyte derived chemokine KC MCP 1 monokine induced by IFN IFN inducible protein IP 10 TNF andvascu lar endothelial growth factor VEGF RESULTS Efficacy of a whole inactivated SARS CoV virion vaccine and recombinant DIs expressing S protein We studied the SARS vaccine efficacy using a recently established murine SARS model 11 In this model a low virulence Pp infection exacerbates lung pathogenesis associated with SARS CoV infection We first examined the protective efficacy of UV and UV plus formalin inactivated whole virion UV V and UV F V respectively Twelve mice in each group were s c inoculated with 10 g UV V or UV F V with alum or alum in PBS alum only as a control booster immu nized with the same vaccines at 7 weeks and then infected with Pp intranasally 1 week later One day after the infec tion of Pp mice were intranasally infected with a mouse adapted strain of SARS CoV Fr mo Although Pp is of low virulence and causes only a mild respiratory disease Pp infected mice showed a transient loss of bodyweight and ruffled hair from 1 to 3 days postinfection and then gradually recovered However mice coinfected with SARS CoV 1 day after Pp infection had severe weight loss and showed high mortality with exacerbated pneumonia 11 In this murine model of SARS both control and UV V or UV F V vaccinated mice showed a transient decrease in bodyweight until day 2 after SARS CoV infection Fig 1 Then most of the vaccinated mice recovered to their orig inal weight before SARS CoV infection whereas control mice alum only continuously lost weight and 90 died at day 7 postinfection In a second set of experiments mice were immunized with recombinant DIs S or empty DIs as a negative con trol DIs cont 5 weeks and 1 week before Pp infection and infected with SARS CoV the next day Similar to the results of whole virion vaccines as described above mice vaccinated with DIs showed transient and minimal clin ical symptoms such as ruffled hair and weight loss until 3 days after Fr mo infection Fig 2 left Severe symp toms continued during the observation period in control mice On days 4 7 after virus infection these mice suf fered the loss of 40 or more of their bodyweight and more than 80 of those mice died after exhibiting severe respiratory disease whereas none of the mice vaccinated with recombinant DIs expressing S proteins died Fig 2 right To further evaluate the vaccine effect we mea sured serum neutralizing antibody and SARS CoV titer in the lung lavage of vaccinated mice Before Pp and SARS CoV coinfection vaccinated mice developed a high level of anti SARS CoV IgG with neutralizing activity data not shown As shown in Figure 3a a high level of neutralizing antibody was maintained during coinfection experiments at day 3 until day 7 postinfection in all vaccinated mouse groups The SARS CoV titer in the lung lavage was high at day 3 postinfection decreased at day 5 Fig 3b blank column alum only and DIs cont and virus became un detectable at day 7 data not shown In contrast the virus titers were significantly reduced in parallel with a high titer of plasma neutralizing antibody in all vaccinated groups Fig 3a Therefore both whole virion vaccines and vac cinia vector expressing spike protein were protective from a highly pathogenic pulmonary infection of SARS CoV in c 2008 The Societies and Blackwell Publishing Asia Pty Ltd 77 K Ishii et al Fig 1 Left Bodyweights of mice vaccinated with inactivated virions in a murine model system of severe respiratory disease caused by the coinfection of Pp and SARS CoV Mice were s c injected on the back with 10 g UV V or UV F V with 2 mg alum and boosted by the same procedure 7 weeks after priming One week later mice were infected intranasally with Pp 6 10 6 c f u day 1 and 1 day later with SARS CoV 0 8 10 6 p f u Fr mo day 0 and were weighed daily after Pp infection Mice were vaccinated with UV inactivated virions with alum UV and formalin inactivated virions with alum or alum only The number of mice used in this study was 17 UV V 12 UV F V and 17 alum respectively Bar shows the standard error of the mean SEM Right Survival curves of mice vaccinated with inactivated virions Fig 2 Left Bodyweights of mice vaccinated with recombinant DIs in a murine model system of severe respiratory disease caused by the coinfection of Pp and SARS CoV Mice were i p injected with 10 6 p f u of rDIs SARS S or DIs and boosted by the same procedure 4 weeks after priming One week later mice were infected intranasally with Pp day 1 and 1 day later with SARS CoV day 0 and were weighed daily after Pp infection Mice were vaccinated with rDIs SARS S or DIs only Bar shows the SEM n 18 Right Survival curves of mice vaccinated with recombinant vaccinia virus the presence of opportunistic infection by Pp in this case Furthermore the results by DIs S vaccine suggest that the neutralizing antibody against SARS CoV spike pro tein alone is highly effective for the prevention of SARS development Inflammatory cytokines are suppressed in vaccinated mice Ami et al 11 demonstrated previously that the levels of IP 10 and IFN are significantly high in SARS CoV infected and Pp SARS CoV coinfected mouse lung ho mogenates at 2 days after SARS CoV infection However these cytokines became undetectable at day 4 postinfec tion To better understand the involvement of cytokines in the lung pathogenesis of SARS we simultaneously mea sured six cytokines IL 6 IL 10 MCP 1 IFN TNF and IL 12p70 in the lung lavage of vaccinated or unvac cinated mice at day 3 after SARS CoV infection when viruses replicate most extensively and vaccinated mice experienced maximum weight loss As shown in Figure 4 the increased production of MCP 1 and IL 6 in the lungs of the control mouse group alum and DIs cont was strongly suppressed in vaccinated mice which were able to control virus replication Increased production of other cytokines including TNF was not observed Therefore it is possible that IL 6 and MCP 1 play a role in the es tablishment of lung pathology in the Pp and SARS CoV coinfection model Neutralizing monoclonal antibody against S protein is highly effective for protection It is known that many of the neutralizing antibod ies against SARS CoV recognize a receptor binding do main RBD in S1 of the spike protein although other N terminal domains of the S1 and S2 domains also have neutralizing epitopes 8 Previously we established four S protein specific monoclonal antibodies with potent in vitro neutralization activity 17 Among them a major 78 c 2008 The Societies and Blackwell Publishing Asia Pty Ltd Neutralizing Ab against SARS CoV spike Fig 3 a Neutralizing titers in the plasma of vaccinated mice Titers were calculated as previously described 15 b SARS CoV titers in the lung lavage of vaccinated mice Bar shows the SEM n 3 Fig 4 Concentrations of TNF IL 6 and MCP 1 in the lung lavage of vaccinated mice Cytokines and chemokines in the lung lavage of vaccinated and control mice on day 3 after SARS CoV infection were assayed by flow cytometry using a mouse inflammatory cytokine IL 6 IL 10 MCP 1 IFN TNF and IL 12p70 cytometric bead array kit Bar shows the SEM n 3 epitope of SKOT 20 localizes to the RBD of the S protein and exhibits the most potent neutralizing activity 18 To demonstrate that the anti spike neutralizing antibody is mainly responsible for the protective efficacy we ob served in SARS model mice we administered SKOT 20 i p once just before Pp infection As shown in Figure 5a the mice treated with SKOT 20 recovered from the loss of bodyweight and were resistant to the fatal outcome as we observed in vaccinated mice When we measured a virus titer in the lung lavage at day 3 after SARS CoV infection it was dramatically decreased in accordance with the concentration of neutralizing an tibody given Fig 5b The level of serum neutralizing an tibody in these mice was proportionally high at this time point The neutralizing activity was low but still detectable in the lung lavage These results clearly show that the neu tralizing antibody against S protein is highly effective to prevent SARS development Kinetics of cytokine and chemokine production We have observed a high level of IL 6 and MCP 1 produc tion in Pp and SARS CoV coinfected mouse lung lavage at day 3 after SARS CoV infection Fig 4 However the cytokine profiles in these mice may be variably modi fied by Pp infection alone SARS CoV infection or both Therefore we measured 20 cytokines in lung lavages of these protected and unprotected mice by using a mouse cytokine 20 plex antibody bead kit at day 0 after Pp in fection before SARS CoV infection day 1 day 2 and day 3 after SARS CoV infection as previously reported 16 The production of two cytokines IL 6 and MCP 1 were increased at days 2 and 3 after SARS CoV infection in asso ciation with high titers of SARS CoV as shown in Figure 4 Interestingly the production of IL 6 was increased by Pp infection alone decreasing on day 1 and increasing again whereas that of MCP 1 in