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simultaneously These rDIs elicited SARS CoV specific serum IgG antibody and T cell responses in vaccinated mice following intranasal or statistics SARS is now known to result from infection with a novel coronavirus SARS CoV Drosten et al 2003 Ksiazek Structural characterization of SARS CoV and characteriza tionofitscompleteRNAgenome Marraetal 2003 Rotaetal 2003 Ruan et al 2003 have provided us with the opportunity to develop a SARS vaccine Like other coronaviruses SARS Virology 351 2006 368 et al 2003 Peiris et al 2003 Evidence that SARS CoVis the subcutaneous administration Mice that were subcutaneously vaccinated with rDIs expressing S protein with or without other structural proteins induced a high level of serum neutralizing IgG antibodies and demonstrated marked protective immunity against SARS CoV challenge in the absence of a mucosal IgA response These results indicate that the potent immune response elicited by subcutaneous injection of rDIs containing S is able to control mucosal infection by SARS CoV Thus replication deficient DIs constructs hold promise for the development of a safe and potent SARS vaccine 2006 Elsevier Inc All rights reserved Keywords Vaccinia virus Vaccine SARS Introduction Severe acute respiratory syndrome SARS has become a priority for healthcare agencies around the world given its communicability associated mortality and the potential for pandemic spread As of 31 July 2003 8 098 SARS cases had been identified worldwide resulting in 774 deaths and a mortality rate of about 9 6 World Health Organization etiologic agent of SARS follows an experimental infection of macaques Macaca fascicularis fulfilling Koch s postulates Fouchier et al 2003 The clinical manifestations of SARS are hardly distinct from other common respiratory viral infections including influenza Because influenza epidemics might occur simultaneously with the eventual re emergence of SARS an effective SARS vaccine is urgently required as well as more sensitive diagnostic tests specific for SARS Department of Pathology National Institute of Infectious Diseases Gakuen Musashimurayama shi Tokyo 208 001 Japan c Department of Virology I National Institute of Infectious Diseases Gakuen Musashimurayama shi Tokyo 208 001 Japan d Department of Virology III National Institute of Infectious Diseases Gakuen Musashimurayama shi Tokyo 208 001 Japan e Department of Immunology National Institute of Infectious Diseases Toyama Shinjuku ku Tokyo 162 8640 Japan Received 22 December 2005 returned to author for revision 9 March 2006 accepted 10 March 2006 Available online 6 May 2006 Abstract SARS coronavirus SARS CoV has recently been identified as the causative agent of SARS We constructed a series of recombinant DIs rDIs a highly attenuated vaccinia strain expressing a gene encoding four structural proteins E M N and S of SARS CoV individually or Department of Virology II National Institute of Infectious b Tetsuro Suzuki Fumihiro Taguchi Masato Tashiro Toshitada Takemori Tatsuo Miyamura Yasuko Tsunetsugu Yokota e a Diseases Toyama Shinjuku ku Tokyo 162 8640 Japan Induction of protective immunity syndrome coronavirus SARS CoV attenuated recombinant Koji Ishii a Hideki Hasegawa b Noriyo Nagata a d Corresponding author Fax 81 3 5285 1150 E mail address yyokota nih go jp Y Tsunetsugu Yokota 0042 6822 see front matter 2006 Elsevier Inc All rights reserved doi 10 1016 j virol 2006 03 020 against severe acute respiratory infection using highly vaccinia virus DIs b Tetsuya Mizutani c Shigeru Morikawa c d e a 380 CoV is a plus stranded RNA virus with a 30 kb genome encoding replicase gene products and the 4 structural proteins i e spike S envelope E membrane M and nucleocapsid results are consistent with data reported by Xiao et al 2003 who expressed the full length S glycoprotein of SARS CoV Tor2 strain in 293 cells and demonstrated a protein approach ing 180 200 kDa by SDS gel electrophoresis Concerning the M protein only a smear band in the stacking gel was detected using a polyclonal antibody against synthetic peptide of the M protein Mizutani et al 2004 presumably because it formed large oligomers with SDS resistance in cells Fig 2A Similar result was mentioned by the analysis of the M protein of SARS CoV Buchholz et al 2004 and infectious bronchitis virus Weisz et al 1993 The subcellular localization of S M and N proteins was analyzed by immunofluorescence staining Cells infected with rDIsSARS M demonstrated M proteins primarily co localized with the Golgi marker GM 130 Fig 2B which is consistent with the results of the recent study Nal et al 2005 Individually expressed SARS CoV N protein could be detected partially with Golgi apparatus but remained princi pally localized to the cytoplasm Fig 2B Overexpressed recombinant SARS S glycoprotein could be detected partially with Golgi apparatus but also be detected throughout the cytoplasm Fig 2B These results indicate that cells infected with rDIsSARS expressed significant levels of SARS CoV N Marra et al 2003 Rota et al 2003 The S protein is thought to be involved in receptor binding while the E protein has a role in viral assembly the M protein is important for virus budding and the N protein has a role in viral RNA packaging for review see reference Holmes 2003 Recently angioten sin converting enzyme 2 ACE2 has been identified as a cellular receptor for SARS CoV Li et al 2003 Thus the first step of infection likely involves binding of S protein to the ACE2 receptor In a model of MHV infection S protein is known to contain important virus neutralizing epitopes that elicit neutralizing antibody responses in mice Collins et al 1982 Therefore the S protein of coronavirus might be manipulated to induce immunity However S M and N proteins are also known to contribute to the host immune response Antonetal 1996 Jackwood andHilt 1995 ADNA vaccine encoding the S glycoprotein of the SARS CoV induces Tcell and neutralizing antibody responses as well as protective immunity in a mouse model Yang et al 2004 Vaccination with a plasmid expressing N protein is capable of generating strong N specific humoral and T cell mediated immune responses in vaccinated C57BL 6 mice Kim et al 2004 Zhao et al 2005 Zhu et al 2004 In addition N specific CD8 T cells provide protective immunity against some coronaviruses Collisson et al 2000 Seo et al 1997 The DIs strain is a highly restricted host range mutant of the vaccinia virus isolated by successive 1 day egg passage of the DIE vaccinia strain an authorized strain for smallpox vaccine and actually used in Japan until 1981 DIs does not replicate and is not pathogenic in mice guinea pigs or rabbits Furthermore the DIs does not replicate in various mammalian cell lines Tagaya et al 1961 Recently we established a system for foreign gene expression by inserting target genes into this strain after which expression of i bacteriophage T7 polymerase and ii the full length HIV 1 NL432 gag gene was observed Ishii et al 2002 thus demonstrating the usefulness of this system In the present study we constructed a 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 These rDIs vaccines were administered to mice either subcutaneously or intranasal ly and the humoral and cellular immunity against SARS CoV in vaccinated mice were analyzed We demonstrated here that replication deficient DIs constructs expressing S protein alone or in combination with other components but not N alone elicited strong protective immune responses against SARS CoV infection Results Expression of SARS CoV structural proteins by rDIs The structures of transfer vectors used in this study pDIsSARS E pDIsSARS M pDIsSARS N pDIsSARS S K Ishii et al Virology pDIsSARS E M pDIsSARS E M S and pDIsSARS E M N S were summarized in Fig 1 Expression of SARS CoV N and S proteins in chick embryo fibroblast CEF cells infected with rDIsSARS was detected by Western blotting using monoclonal antibodies Fig 2A Ohnishi et al 2005 Purified SARS CoV virion was used as a positive control Fig 2A lane PC A robust signal was detected at 50 kDa corresponding to the N protein of SARS CoV as predicted by its genomic size Marra et al 2003 Rota et al 2003 A band approaching 200 kDa likely corresponds to the S protein which is known to be heavily glycosylated Fig 2A Our Fig 1 Schematic diagram of rDIs constructs expressing SARS CoV structural proteins DNAfragmentsencodingE M NandSproteinswereinsertedintothe location of the 15 4 kb deletion in DIs using the vaccinia virus transfer vector pDIsgptmH5 Six rDIs constructs are shown 369351 2006 368 380 proteins under the control of mH5 promoter with an expected post translational processing Nal et al 2005 You et al 2005 370 K Ishii et al Virology rDIsSARS induces serum IgG antibody responses specific for SARS CoV To examine the anti SARS CoV response in mice after inoculation with rDIsSARS four mice in each group were subcutaneously or intranasally inoculated three times with 10 pfu of rDIsSARS N rDIsSARS M rDIsSARS S rDIs SARS E M S or rDIsSARS E M N S Ten days after the final inoculation vaccinated mice were observed to have high levels of anti SARS CoV IgG antibodies in their sera Fig 3 In order to prove effective vaccination we next examined whether neutralizing antibodies against SARS CoV were elicited in these mice Neutralizing antibodies against Fig 2 Western blot analysis and indirect immunofluorescence analysis A CEF proteins M N and S respectively Purified SARS CoV virion 0 5 g was used antibodies N and S or polyclonal antibodies M Detection of bound antibodies was antibody and visualized by chemiluminescence B CEF cells were infected with respectively To detect SARS CoV proteins the cells were incubated with rabbit with FITC conjugated goat anti rabbit IgG To analyze subcellular localization of rhodamine conjugated goat anti mouse IgG were used SARS proteins are shown in is shown in yellow 351 2006 368 380 SARS CoV were induced in mice following subcutaneous or intranasal injection of rDIsSARS S rDIsSARS E M S or rDIsSARS E M N S but not in mice immunized with rDIsSARS N or rDIsSARS M These results of ELISA data were incorporated into Fig 3 by depicting the neutralization positive serum as closed circles Thus our results consistent with others Bisht et al 2004 Buchholz et al 2004 Yang et al 2004 indicate that the S protein is a prerequisite for eliciting a sufficient IgG antibody response for neutralization Similar neutralizing activity was obtained in mice receiving S alone or in combination with other components Therefore we expected that the rDIsSARS expressing E M N S proteins in combination could be the best vaccine candidate among others cells were infected with rDIs constructs expressing SARS CoV structural as a positive control SARS CoV proteins were detected using monoclonal done with horseradish peroxidase conjugated goat anti mouse or anti rabbit rDIs constructs expressing SARS CoV structural proteins M N and S polyclonal antibodies against these proteins The cells were further incubated these proteins monoclonal antibody against GM 130 Golgi marker and green Golgi apparatus is shown in red and co localization where it occurs Intranasal inoculation of rDIsSARS expressing E M N S induces SARS CoV specific IgA in nasal mucosa and a high Fig 3 Detection of anti SARS CoV IgG in vaccinated mice IgG antibody levels against SARS CoV were determined as described in Materials and methods SARS CoV specific IgG titers were calculated as follows SARS specific IgG titer ELISA units ml the unit value obtained for wells coated with virus infected cell lysate the unit value obtained for wells coated with non infected cell lysate P 0 1 P 0 05 P 0 01 vs DIs administered group The data for neutralizing sera are represented by closed circles and the data for non neutralizing sera are represented by open circles K Ishii et al Virology level of mucosal IgG in parallel with that of serum IgG Mucosal IgA response is believed to be crucial for the protective immunity against various pathogens Meeusen et al 2004 We next examined mucosal immunity in the respiratory tracts of mice inoculated with rDIsSARS either subcutaneously or intranasally The level of anti SARS CoV IgA within nasal wash fluid of vaccinated mice was determined by enzyme linked immunosorbent assay ELISA As shown in Fig 4A substantial levels of anti SARS CoV IgAwere detected only in mice received intranasal inoculation of rDIsSARS E M N S compared to those inoculated with parental DIs P 0 0010 The level of IgA detected in intranasally rDIsSARS E M N S inoculated mice was similar to that observed following intranasal immunization with UV inactivated purified SARS CoV virion positive control On the other hand subcutaneous injection of all forms of rDIsSARS produced only slightly higher levels of IgA than those observed in DIs injected control mice Therefore the results indicated that the subcutaneous route of injection is inefficient especially when mucosal IgA response is required Since neutralizing activity was nevertheless detected in the nasal washes of mice following subcutaneous immunization data not shown we also measured anti SARS CoV IgG levels in the nasal washes of these mice Fig 4B High levels of IgG were detected in the nasal washes of mice following nasal immunization which were observed to correspond well with IgG levels in the serum Fig 4C A similar trend was observed in mice following subcutaneous immunization despite at a lower level than in mice immunized intranasally These results suggest that neutralizing IgG antibodies are capable of reaching the mucosal surface if plasma levels are high enough Protection of rDIsSARS immunized mice from nasal SARS CoV challenge is achieved without mucosal IgA response The level ofprotection against SARS CoVchallenge in mice following inoculation with rDIsSARS is a critical issue for the vaccine development We inoculated three times with 10 pfu of rDIsSARS N rDIsSARS E M S or rDIsSARS E M N S into four mice in each group either subcutaneously or intranasally One week after final inoculation the mice were challenged intranasally with 10 4 tissue culture 50 infectious dose TCID 50 of SARS CoV The results were shown in Fig 4D In mice inoculated with saline 10 3 TCID 50 ml of SARS CoV were recovered from lung wash fluid on day 3 In contrast titers of SARS CoV from the lungs of mice subcutaneously immunized with rDIsSARS E M S or rDIsSARS E M N S were below the limits of detection The same was true for mice intranasally immunized with rDIsSARS E M N S where as the virus was recovered in mice similarly immunized with rDIsSARS E M S Taken into consideration of a relatively low or marginal level of mucosal IgA antibody in mice intranasally immunized with rDIsSARS E M N S or rDIsSARS E M S or even no IgA response by subcutaneous route as described above it was suggested that mucosal IgG antibody but not IgA antibody likely contributed to the protective immunity especially in mice simultaneously immunized with recombinant rDIsSARS E M N S On the other hand titers of SARS CoV from the lung wash fluid of mice intranasally or subcutaneously immunized with rDIsSARS N were similar or slightly lower than the titers of negative controls suggesting that intranasal or subcutaneous administration of rDIsSARS N does not protect mice from SARS CoV challenge which is highly reflected by the non neutralizing nature of anti SARS CoV N antibodies Cellular immunity induced by rDIsSARS Although now we know that the systemic neutralizing IgG antibody against SARS CoV S protein is a major component of protective immunity T cell responses are also important to protect hosts from various viral infection In a previous study of coronaviruses S protein was shown to play an important role in viral pathogenesis as well as induction of protective immunity Holmes 2003 In order to assess the ability of rDIsSARS to induce SARS CoV S specific T cells T cells from axillary lymph nodes ALN superficial cervical lymph nodes CLN and spleens of mice subcutaneously immunized with rDIs SARS S or DIs were isolated and stimulated in vitro with UV inactivated purified SARS CoV virion Culture supernatant was collected 4 days later and the levels of interferon IFN interleukin IL 2 IL 4 IL 5 and tumor necrosis factor 371351 2006 368 380 TNF were measured T cells in ALN produced the greatest cytokine levels Fig 5 and data not shown This is not surprising in light of the subcutaneous route of immunization 372 K Ishii et al Virology 351 2006 368 380 Notably mice immunized with rDIsSARS S produced a high level of IFN upon in vitro stimulation with UV inactivated purified SARS CoV virion The production of TNF an inflammatory cytokine was significantly elevated in T cells in Fig 4 Mucosallysecretedanti SARS CoVIgG butnotIgA antibodiesareprotective antibodies against SARS CoV were determined as described in Materials and methods Error bars representthe mean SD B Titers of anti SARS CoV IgG in the nasal washings SARS CoV IgG in the sera of vaccinated mice Error bars represent the mean SD later with 10 4 TCID 50 of SARS CoV Virus titers are expressed as log 10 TCID 50 Error administered group Fig 5 InvitroresponseofSARS CoV specificTcellsinmicesubcutaneouslyimmunized after the third vaccination of either DIs control or rDIsSARS S After preparation of single cell depleted normal BALB c mouse splenocytes as APCs in the presence or absence and TNF concentrations in the culture supernatant were measured ALN of rDIsSARS S immunized mice after in vitro stimulation with virion antigens However TNF production was observed also in mice immunized with parental DIs without in vitro stimulation with virion antigens Since T cells from the fromnasalSARS CoVchallengeinvaccinatedmice ThelevelsofIgAandIgG A Titers of anti SARS CoV IgA in the nasal washings of vaccinated mice of vaccinated mice Error bars represent the mean SD C Titers of anti D The titers of SARS CoVin the lungs of vaccinated mice challenged 1 week bars represent the mean SD P 0 1 P 0 05 P 0 01 vs DIs withrDIsSARS S CLN ALNandspleenswereobtainedfrommice1 week cell suspensions Tcells were purified and cultivated with irradiated and T of 10 g ml of purified UV irradiated SARS CoV virion Four days later IFN lymph nodes of na ve mice did not produce cytokines even after in vitro stimulation with virion antigens data not shown it is possible that injection of DIs induces mild local inflammation even when viral proliferation does not occur at the injection site The pattern of IL 2 IL 5 and IL 4 production were similar to that of IFN and the maximum level of these cytokines in ALN Tcells from rDIsSARS S immunized mice were 254 227 and 88 ng ml respectively Next we analyzed the antigenic epitopes of SARS CoV specific T cells in the spleen We carried out IFN enzyme linked immunospot ELISPOT analysis using four 20 mer peptides corresponding to the ACE2 binding region of the S protein selected using the SYFPEITHI score S44 47 as well as overlapping 20 mer peptides pool covering a whole N protein When the splenic T cells of mice were analyzed following intranasal or subcutaneous immunization with the most potent vaccine rDIsSARS E M N S a high level of reactivity against S46 was observed especially in the T cells of clone as an antigen presenting cells APC Expression of S protein on the S6 2 clone was confirmed by FACS analysis using anti SARS S monoclonal antibody Fig 7A An empty vector transfectant BOS 5 was used asanegativecontrolAPC Subcutaneous and intranasal immunization with the most potent rDIsSARS E M N S generated a significant level of S specific T cells Fig 7B and a dramatic decrease in S specific T cells was observed following partial depletion of CD8 T cells Fig 7C Therefore rDIsSARS E M N S was able to induce both SARS CoV reactive CD4 and CD8 T cells Histopathological findings The immunogenicity of rDI