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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">A subcutaneously injected UV-inactivated SARS coronavirus vaccine elicits systemic humoral immunity in mice</title><author><name sortKey="Takasuka, Naomi" sort="Takasuka, Naomi" uniqKey="Takasuka N" first="Naomi" last="Takasuka">Naomi Takasuka</name></author><author><name sortKey="Fujii, Hideki" sort="Fujii, Hideki" uniqKey="Fujii H" first="Hideki" last="Fujii">Hideki Fujii</name></author><author><name sortKey="Takahashi, Yoshimasa" sort="Takahashi, Yoshimasa" uniqKey="Takahashi Y" first="Yoshimasa" last="Takahashi">Yoshimasa Takahashi</name></author><author><name sortKey="Kasai, Masataka" sort="Kasai, Masataka" uniqKey="Kasai M" first="Masataka" last="Kasai">Masataka Kasai</name></author><author><name sortKey="Morikawa, Shigeru" sort="Morikawa, Shigeru" uniqKey="Morikawa S" first="Shigeru" last="Morikawa">Shigeru Morikawa</name></author><author><name sortKey="Itamura, Shigeyuki" sort="Itamura, Shigeyuki" uniqKey="Itamura S" first="Shigeyuki" last="Itamura">Shigeyuki Itamura</name></author><author><name sortKey="Ishii, Koji" sort="Ishii, Koji" uniqKey="Ishii K" first="Koji" last="Ishii">Koji Ishii</name></author><author><name sortKey="Sakaguchi, Masahiro" sort="Sakaguchi, Masahiro" uniqKey="Sakaguchi M" first="Masahiro" last="Sakaguchi">Masahiro Sakaguchi</name></author><author><name sortKey="Ohnishi, Kazuo" sort="Ohnishi, Kazuo" uniqKey="Ohnishi K" first="Kazuo" last="Ohnishi">Kazuo Ohnishi</name></author><author><name sortKey="Ohshima, Masamichi" sort="Ohshima, Masamichi" uniqKey="Ohshima M" first="Masamichi" last="Ohshima">Masamichi Ohshima</name></author><author><name sortKey="Hashimoto, Shu Ichi" sort="Hashimoto, Shu Ichi" uniqKey="Hashimoto S" first="Shu-Ichi" last="Hashimoto">Shu-Ichi Hashimoto</name></author><author><name sortKey="Odagiri, Takato" sort="Odagiri, Takato" uniqKey="Odagiri T" first="Takato" last="Odagiri">Takato Odagiri</name></author><author><name sortKey="Tashiro, Masato" sort="Tashiro, Masato" uniqKey="Tashiro M" first="Masato" last="Tashiro">Masato Tashiro</name></author><author><name sortKey="Yoshikura, Hiroshi" sort="Yoshikura, Hiroshi" uniqKey="Yoshikura H" first="Hiroshi" last="Yoshikura">Hiroshi Yoshikura</name></author><author><name sortKey="Takemori, Toshitada" sort="Takemori, Toshitada" uniqKey="Takemori T" first="Toshitada" last="Takemori">Toshitada Takemori</name></author><author><name sortKey="Tsunetsugu Yokota, Yasuko" sort="Tsunetsugu Yokota, Yasuko" uniqKey="Tsunetsugu Yokota Y" first="Yasuko" last="Tsunetsugu-Yokota">Yasuko Tsunetsugu-Yokota</name></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">15314040</idno><idno type="pmc">7108621</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7108621</idno><idno type="RBID">PMC:7108621</idno><idno type="doi">10.1093/intimm/dxh143</idno><date when="2004">2004</date><idno type="wicri:Area/Pmc/Corpus">000B67</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000B67</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">A subcutaneously injected UV-inactivated SARS coronavirus vaccine elicits systemic humoral immunity in mice</title><author><name sortKey="Takasuka, Naomi" sort="Takasuka, Naomi" uniqKey="Takasuka N" first="Naomi" last="Takasuka">Naomi Takasuka</name></author><author><name sortKey="Fujii, Hideki" sort="Fujii, Hideki" uniqKey="Fujii H" first="Hideki" last="Fujii">Hideki Fujii</name></author><author><name sortKey="Takahashi, Yoshimasa" sort="Takahashi, Yoshimasa" uniqKey="Takahashi Y" first="Yoshimasa" last="Takahashi">Yoshimasa Takahashi</name></author><author><name sortKey="Kasai, Masataka" sort="Kasai, Masataka" uniqKey="Kasai M" first="Masataka" last="Kasai">Masataka Kasai</name></author><author><name sortKey="Morikawa, Shigeru" sort="Morikawa, Shigeru" uniqKey="Morikawa S" first="Shigeru" last="Morikawa">Shigeru Morikawa</name></author><author><name sortKey="Itamura, Shigeyuki" sort="Itamura, Shigeyuki" uniqKey="Itamura S" first="Shigeyuki" last="Itamura">Shigeyuki Itamura</name></author><author><name sortKey="Ishii, Koji" sort="Ishii, Koji" uniqKey="Ishii K" first="Koji" last="Ishii">Koji Ishii</name></author><author><name sortKey="Sakaguchi, Masahiro" sort="Sakaguchi, Masahiro" uniqKey="Sakaguchi M" first="Masahiro" last="Sakaguchi">Masahiro Sakaguchi</name></author><author><name sortKey="Ohnishi, Kazuo" sort="Ohnishi, Kazuo" uniqKey="Ohnishi K" first="Kazuo" last="Ohnishi">Kazuo Ohnishi</name></author><author><name sortKey="Ohshima, Masamichi" sort="Ohshima, Masamichi" uniqKey="Ohshima M" first="Masamichi" last="Ohshima">Masamichi Ohshima</name></author><author><name sortKey="Hashimoto, Shu Ichi" sort="Hashimoto, Shu Ichi" uniqKey="Hashimoto S" first="Shu-Ichi" last="Hashimoto">Shu-Ichi Hashimoto</name></author><author><name sortKey="Odagiri, Takato" sort="Odagiri, Takato" uniqKey="Odagiri T" first="Takato" last="Odagiri">Takato Odagiri</name></author><author><name sortKey="Tashiro, Masato" sort="Tashiro, Masato" uniqKey="Tashiro M" first="Masato" last="Tashiro">Masato Tashiro</name></author><author><name sortKey="Yoshikura, Hiroshi" sort="Yoshikura, Hiroshi" uniqKey="Yoshikura H" first="Hiroshi" last="Yoshikura">Hiroshi Yoshikura</name></author><author><name sortKey="Takemori, Toshitada" sort="Takemori, Toshitada" uniqKey="Takemori T" first="Toshitada" last="Takemori">Toshitada Takemori</name></author><author><name sortKey="Tsunetsugu Yokota, Yasuko" sort="Tsunetsugu Yokota, Yasuko" uniqKey="Tsunetsugu Yokota Y" first="Yasuko" last="Tsunetsugu-Yokota">Yasuko Tsunetsugu-Yokota</name></author></analytic><series><title level="j">International Immunology</title><idno type="ISSN">0953-8178</idno><idno type="eISSN">1460-2377</idno><imprint><date when="2004">2004</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><title>Abstract</title><p>The recent emergence of severe acute respiratory syndrome (SARS) was caused by a novel coronavirus, SARS-CoV. It spread rapidly to many countries and developing a SARS vaccine is now urgently required. In order to study the immunogenicity of UV-inactivated purified SARS-CoV virion as a vaccine candidate, we subcutaneously immunized mice with UV-inactivated SARS-CoV with or without an adjuvant. We chose aluminum hydroxide gel (alum) as an adjuvant, because of its long safety history for human use. We observed that the UV-inactivated SARS-CoV virion elicited a high level of humoral immunity, resulting in the generation of long-term antibody secreting and memory B cells. With the addition of alum to the vaccine formula, serum IgG production was augmented and reached a level similar to that found in hyper-immunized mice, though it was still insufficient to elicit serum IgA antibodies. Notably, the SARS-CoV virion itself was able to induce long-term antibody production even without an adjuvant. Anti-SARS-CoV antibodies elicited in mice recognized both the spike and nucleocapsid proteins of the virus and were able to neutralize the virus. Furthermore, the UV-inactivated virion induced regional lymph node T-cell proliferation and significant levels of cytokine production (IL-2, IL-4, IL-5, IFN-γ and TNF-α) upon restimulation with inactivated SARS-CoV virion <italic>in vitro</italic>. Thus, a whole killed virion could serve as a candidate antigen for a SARS vaccine to elicit both humoral and cellular immunity.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct></listBibl></div1></back></TEI><pmc article-type="other"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Int Immunol</journal-id><journal-id journal-id-type="iso-abbrev">Int. Immunol</journal-id><journal-id journal-id-type="hwp">intimm</journal-id><journal-id journal-id-type="publisher-id">intimm</journal-id><journal-title-group><journal-title>International Immunology</journal-title></journal-title-group><issn pub-type="ppub">0953-8178</issn><issn pub-type="epub">1460-2377</issn><publisher><publisher-name>Oxford University Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">15314040</article-id><article-id pub-id-type="pmc">7108621</article-id><article-id pub-id-type="other">dxh143</article-id><article-id pub-id-type="doi">10.1093/intimm/dxh143</article-id><article-categories><subj-group subj-group-type="heading"><subject>Original Research Papers</subject></subj-group></article-categories><title-group><article-title>A subcutaneously injected UV-inactivated SARS coronavirus vaccine elicits systemic humoral immunity in mice</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Takasuka</surname><given-names>Naomi</given-names></name><xref ref-type="corresp" rid="d36961e153"></xref></contrib><contrib contrib-type="author"><name><surname>Fujii</surname><given-names>Hideki</given-names></name></contrib><contrib contrib-type="author"><name><surname>Takahashi</surname><given-names>Yoshimasa</given-names></name></contrib><contrib contrib-type="author"><name><surname>Kasai</surname><given-names>Masataka</given-names></name></contrib><contrib contrib-type="author"><name><surname>Morikawa</surname><given-names>Shigeru</given-names></name></contrib><contrib contrib-type="author"><name><surname>Itamura</surname><given-names>Shigeyuki</given-names></name></contrib><contrib contrib-type="author"><name><surname>Ishii</surname><given-names>Koji</given-names></name></contrib><contrib contrib-type="author"><name><surname>Sakaguchi</surname><given-names>Masahiro</given-names></name></contrib><contrib contrib-type="author"><name><surname>Ohnishi</surname><given-names>Kazuo</given-names></name></contrib><contrib contrib-type="author"><name><surname>Ohshima</surname><given-names>Masamichi</given-names></name></contrib><contrib contrib-type="author"><name><surname>Hashimoto</surname><given-names>Shu-ichi</given-names></name></contrib><contrib contrib-type="author"><name><surname>Odagiri</surname><given-names>Takato</given-names></name></contrib><contrib contrib-type="author"><name><surname>Tashiro</surname><given-names>Masato</given-names></name></contrib><contrib contrib-type="author"><name><surname>Yoshikura</surname><given-names>Hiroshi</given-names></name></contrib><contrib contrib-type="author"><name><surname>Takemori</surname><given-names>Toshitada</given-names></name></contrib><contrib contrib-type="author"><name><surname>Tsunetsugu-Yokota</surname><given-names>Yasuko</given-names></name></contrib></contrib-group><author-notes><p>1Department of Immunology, 2First, 3Second and 4Third Departments of Virology, 5National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan</p><corresp id="d36961e153"><italic>Correspondence to</italic>: Y. Tsunetsugu-Yokota; E-mail: <email>yyokota@nih.go.jp</email></corresp></author-notes><pub-date pub-type="ppub"><month>10</month><year>2004</year></pub-date><pub-date pub-type="epub" iso-8601-date="2004-08-16"><day>16</day><month>8</month><year>2004</year></pub-date><volume>16</volume><issue>10</issue><fpage>1423</fpage><lpage>1430</lpage><history><date date-type="accepted"><day>15</day><month>7</month><year>2004</year></date><date date-type="received"><day>6</day><month>5</month><year>2004</year></date></history><permissions><copyright-statement>© 2004 The Japanese Society for Immunology</copyright-statement><copyright-year>2004</copyright-year><license><license-p>This article is made available via the PMC Open Access Subset for unrestricted re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the COVID-19 pandemic or until permissions are revoked in writing. Upon expiration of these permissions, PMC is granted a perpetual license to make this article available via PMC and Europe PMC, consistent with existing copyright protections.</license-p></license></permissions><self-uri xlink:href="dxh143.pdf"></self-uri><abstract><title>Abstract</title><p>The recent emergence of severe acute respiratory syndrome (SARS) was caused by a novel coronavirus, SARS-CoV. It spread rapidly to many countries and developing a SARS vaccine is now urgently required. In order to study the immunogenicity of UV-inactivated purified SARS-CoV virion as a vaccine candidate, we subcutaneously immunized mice with UV-inactivated SARS-CoV with or without an adjuvant. We chose aluminum hydroxide gel (alum) as an adjuvant, because of its long safety history for human use. We observed that the UV-inactivated SARS-CoV virion elicited a high level of humoral immunity, resulting in the generation of long-term antibody secreting and memory B cells. With the addition of alum to the vaccine formula, serum IgG production was augmented and reached a level similar to that found in hyper-immunized mice, though it was still insufficient to elicit serum IgA antibodies. Notably, the SARS-CoV virion itself was able to induce long-term antibody production even without an adjuvant. Anti-SARS-CoV antibodies elicited in mice recognized both the spike and nucleocapsid proteins of the virus and were able to neutralize the virus. Furthermore, the UV-inactivated virion induced regional lymph node T-cell proliferation and significant levels of cytokine production (IL-2, IL-4, IL-5, IFN-γ and TNF-α) upon restimulation with inactivated SARS-CoV virion <italic>in vitro</italic>. Thus, a whole killed virion could serve as a candidate antigen for a SARS vaccine to elicit both humoral and cellular immunity.</p></abstract><kwd-group kwd-group-type="KWD"><kwd>alum</kwd><kwd>cellular immunity</kwd><kwd>neutralizing antibody</kwd><kwd>parenteral administration</kwd><kwd>vaccination</kwd></kwd-group><kwd-group kwd-group-type="ABR"><kwd>ACE2 angiotensin-converting enzyme 2</kwd><kwd>ASC antibody-secreting cell</kwd><kwd>E envelope</kwd><kwd>M membrane</kwd><kwd>N nucleocapsid protein</kwd><kwd>SARS severe acute respiratory syndrome</kwd><kwd>SARS-CoV SARS-associated coronavirus</kwd><kwd>S spike protein</kwd></kwd-group></article-meta></front><body><sec><title>Introduction</title><p>A new disease called severe acute respiratory syndrome (SARS) originated in China in late 2002 and spread rapidly to many countries. Upon this outbreak, a global collaboration network was coordinated by WHO. As a result of this unprecedented international effort, a novel type of coronavirus (SARS-CoV) was identified as the etiologic agent of SARS (<xref rid="BIB1">1</xref>,<xref rid="BIB2">2</xref>) in March 2003. The genomic sequence of SARS-CoV was completed and we now know that SARS-CoV has all the features and characteristics of other coronaviruses, but it is quite different from all previously known coronaviruses (groups I∼III), representing a new group (group IV) (<xref rid="BIB3">3</xref>,<xref rid="BIB4">4</xref>). It is assumed that SARS-CoV is a mutant coronavirus transmitted from a wild animal that developed the ability to productively infect humans (<xref rid="BIB3">3</xref>,<xref rid="BIB5">5</xref>). The genome of SARS-CoV is a single-stranded plus-sense RNA ∼30 kb in length and containing five major open reading frames that encode non-structural replicase polyproteins and structural proteins: the spike (S), envelope (E), membrane (M) and nucleocapsid protein (N), in the same order and of approximately the same sizes as those of other coronaviruses (<xref rid="BIB5">5</xref>).</p><p>The reason why SARS-CoV induces severe respiratory distress in some, but not all, infected individuals is still unclear. In patients with SARS and probable SARS cases, virus is detected in sputum, stool and plasma by RT–PCR (<xref rid="BIB1">1</xref>,<xref rid="BIB2">2</xref>). These patients developed serum antibodies against SARS-CoV and high antibody titers against N protein were maintained for more than 5 months after infection (<xref rid="BIB6">6</xref>). Because of their generally poor pathogenicity and difficulty of propagation <italic>in vitro</italic>, there have been few studies regarding immunity to human coronaviruses OC43 and 229E. In the veterinary field, however, coronaviruses have been known for many years to cause a variety of lung, liver and gut diseases in animals. As we learned from these animal models, both humoral and cellular immune responses may contribute to protection against coronavirus diseases, including SARS [for review see (<xref rid="BIB7">7</xref>)].</p><p>The clinical manifestation of SARS is hardly distinct from other common respiratory viral infections including influenza. Because an influenza epidemic may occur simultaneously with the re-emergence of SARS, it is urgently required that we develop effective SARS vaccines as well as sensitive diagnostic tests specific for SARS. Recently, the angiotensin-converting enzyme 2 (ACE2) was identified as a cellular receptor for SARS-CoV (<xref rid="BIB8">8</xref>). The first step in viral infection is presumably the binding of S protein to its receptor, ACE2. In the murine MHV model, S proteins are known to contain important virus-neutralizing epitopes that elicit neutralizing antibodies in mice (<xref rid="BIB9">9</xref>,<xref rid="BIB10">10</xref>). Therefore, the S protein would be the first candidate coronavirus protein for induction of immunity. However, the S, M and N proteins are also known to contribute to generating the host immune response (<xref rid="BIB11">11</xref>,<xref rid="BIB12">12</xref>).</p><p>Following an established vaccine protocol is one of the best ways to shorten the time and cost of new vaccine development. Most of the currently available vaccines for humans are inactivated and applied cutaneously, except oral polio vaccine, and adjuvant usage is mostly limited to aluminum hydroxide gel (alum). In order to know the immunogenicity of inactivated SARS-CoV as a vaccine candidate, we immunized mice with UV-inactivated SARS-CoV either with or without alum. We report here the evaluation of humoral and cellular immunity elicited by UV-inactivated SARS-CoV administered subcutaneously.</p></sec><sec><title>Methods</title><sec><title>Preparation of UV-inactivated purified SARS-CoV</title><p>SARS-CoV (HKU39849) was kindly supplied by Dr J.S.M. Peiris, Department of Microbiology, The University of Hong Kong. The virus was amplified in Vero E6 cells and purified by sucrose density gradient centrifugation. Concentrated virus was then exposed to UV light (4.75 J/cm<sup>2</sup>) in order to inactivate the virus. We confirmed that the virus completely lost its infectivity by this method.</p></sec><sec><title>Immunization of mice</title><p>Female BALB/c mice were purchased from Nippon SLC Inc. (Shizuoka, Japan) and were housed under specific pathogen-free conditions. All experimental procedures were carried out under NIID-recommended guidelines. Mice were subcutaneously injected via their back or right and left hind leg footpads with 10 μg of UV-inactivated purified SARS-CoV with or without 2 mg of alum, and boosted by the same procedure 7 weeks after priming.</p></sec><sec><title>Detection of immunoglobulins in the serum samples</title><p>Blood was obtained from the tail vein and allowed to clot overnight at 4°C. Sera were then collected by centrifugation. For ELISA, microtiter plates (Dynatech, Chantily, VA) were coated overnight at 4°C with SARS-CoV-infected or mock-infected Vero E6 cell lysates, which had been treated with 1% NP40 followed by UV-inactivation. To detect S or N protein, the plates were coated with 1% NP40 lysates of chick embryo fibroblasts that had been infected with S or N protein-expressing DIs (attenuated vaccinia virus) (<xref rid="BIB13">13</xref>). The plates were blocked with 1% OVA in PBS–Tween (0.05%) and then incubated with the sera serially diluted at 1:25–1:10<sup>5</sup> for 1 h at room temperature. Plates were incubated with either peroxidase-conjugated anti-mouse IgG (1:2000, Zymed, San Francisco, CA), IgM or IgA (1:2000, Southern Biotechnology, Birmingham, AL) antibody. For detection of IgG subclasses, either peroxidase-conjugated anti-mouse IgG<sub>1</sub>, IgG<sub>2a</sub>, IgG<sub>2b</sub> (1:2000, Zymed) or IgG<sub>3</sub> (1:2000, Southern Biotechnology) was used. Plates were washed three times with PBS–Tween at each step. Antibodies were detected by <italic>O</italic>-phenylenediamine (Zymed), and the absorbance of each well was read at 490 nm using a model 680 microplate reader (Bio-Rad, Hercules, CA). As a standard for IgG detection, serum was obtained from a hyper-immunized mouse; the OD490nm value of 100 U/ml standard was ∼3 in all assays. SARS-CoV-specific IgG titer was calculated as follows: SARS-specific IgG titer (U/ml) = (the unit value obtained at wells coated with virus-infected cell lysates) − (the unit value obtained at wells coated with non-infected cell lysates).</p></sec><sec><title>ELISPOT assay for antibody-secreting cells (ASCs)</title><p>Recombinant N protein (amino acids 1–49 and 340–390) of SARS-CoV (Biodesign, Saco, ME) was diluted to 10 μg/ml in PBS, and then added at 100 μl per well to plates supported by a nitrocellulose filter (Millipore, Bedford, MA). After overnight incubation at 4°C, the plates were washed with PBS three times and then blocked at 4°C overnight with 1% OVA in PBS–Tween (0.05%). After erythrocyte lysis, single cell suspensions from BMs were suspended in RPMI supplemented with 10% FCS, 5 × 10<sup>−5</sup> M 2ME, 2 mM <sc>L</sc>-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin, and then applied to the plates at a concentration of 3 × 10<sup>5</sup> cells per well. After 24 h cultivation, the plates were recovered and stained with alkaline phosphatase-conjugated anti-mouse IgG<sub>1</sub> antibody (Southern Biotechnologies). Alkaline phosphatase activity was visualized using 3-amino-ethyl carbozole and napthol AS-MX phosphate/fast blue BB (Sigma). The frequency of plasma cells specific for N protein was determined from the N protein-coated plates after background on the uncoated plates was subtracted.</p></sec><sec><title>Coronavirus neutralizing assay</title><p>Serum was inactivated by incubation at 56°C for 30 min. The known tissue culture infectious dose (TCID) of SARS-CoV was incubated for 1 h in the presence or absence of serum antibodies serially diluted 5-fold, and then added to Vero E6 cell culture grown confluently in a 96-well microtiter plate. After 48 h, cells were fixed with 10% formaldehyde and stained with crystal violet to visualize the cytopathic effect induced by the virus (<xref rid="BIB14">14</xref>). Neutralization antibody titers were expressed as the minimum dilution number of serum that inhibited the cytopathic effect.</p></sec><sec><title>Western blotting</title><p>Purified SARS-CoV virion (0.5 μg) was fractionated on SDS–PAGE under reduced conditions. Proteins were transferred to PVDF membrane (Genetics, Tokyo, Japan) and reacted with the diluted sera (1:1000) that had been obtained from mice inoculated with UV-irradiated SARS-CoV. After washing, the membrane was reacted with HRP-conjugated F(ab′)<sub>2</sub> fragment anti-mouse IgG (H+L) (1:20 000 Jackson Immuno Research, West Grove, PA), followed by visualization of the bands on X-ray film (Kodak, Rochester, NY) using chemiluminescent regents (Amasham Biosciences, Piscataway, NJ).</p></sec><sec><title>Regional T cell response</title><p>Popliteal and inguinal lymph nodes and spleens were harvested from mice 1 week after the boost vaccination. After the preparation of a single cell suspension, T cells were purified by depletion of B220<sup>+</sup>, Gr1<sup>+</sup>, CD11b<sup>+</sup>, IgD<sup>+</sup> and IgM<sup>+</sup> cells using a magnetic cell sort system (MACS: Miltenyi Biotec, Bergisch Gladbach, Germany). To prepare antigen-presenting cells (APC), normal BALB/c mouse splenocytes were depleted of CD3<sup>+</sup> T cells by MACS and irradiated at 2000 cGy.</p><p>Purified T cells taken from lymph nodes (1 × 10<sup>5</sup> cells/well) were cultured with irradiated APC (5 × 10<sup>5</sup> cells/well) in the presence or absence of UV-irradiated purified SARS-CoV virion (1 or 10 μg/ml). Four days after the cultivation, the level of cytokine concentration in the culture supernatant was measured by flow cytometry using a mouse Th1/Th2 cytokine cytometric bead array kit (Becton Dickinson, San Jose, CA). T-cell proliferation was monitored by the incorporation of [<sup>3</sup>H]thymidine (18.5 kBq/well, ICN Biomedicals, Costa Mesa, CA) added 8 h prior to cell harvest. The cells were harvested on a 96-well microplate bonded with a GF/B filter (Packard Instruments, Meriden, CT). Incorporated radioactivity was counted by a microplate scintillation counter (Packard Instruments).</p></sec></sec><sec><title>Results</title><sec><title>Inoculation with UV-inactivated SARS-CoV results in an antigen-specific IgG<sub>1</sub> response, probably by generating long-term ASCs as well as memory cells</title><p>To examine the level of anti-SARS-CoV response in mice after inoculation with vaccine candidates, three mice in each group were subcutaneously inoculated with 10 μg of UV-inactivated purified SARS-CoV with (Virion/Alum) or without alum (Virion), or inoculated with alum alone (Alum) or left untreated (None) as a control (<xref rid="FIG1">Fig. 1</xref>). One month after inoculation, vaccinated mice elicited the anti-SARS CoV IgG antibody in sera at high levels. As expected, the alum adjuvant enhanced the level of IgG antibody response, >10-fold higher than the level without adjuvant (<xref rid="FIG1">Fig. 1C</xref> compared with B). When mice were boosted at 7 weeks, the level of IgG antibody in both groups of mice was further increased ∼10-fold above the primary response (<xref rid="FIG1">Fig. 1B and C</xref>). Notably, the level of serum antibodies induced by a single injection of virion, even in the absence of the alum adjuvant, was maintained at least more than 6 months (<xref rid="FIG1">Fig. 1D</xref>). These results suggest that long-term ASCs can be established by a single shot of UV-inactivated virion administration.</p><fig id="FIG1" orientation="portrait" position="float"><label>Fig. 1.</label><caption><p>The level of SARS-specific IgG in subcutaneously vaccinated mice. Mice were subcutaneously primed with 10 μg of UV-inactivated SARS-CoV virion (B), or virion with 2 mg of alum (C), or alum alone or none (A) and boosted with the same dose in their footpads at 7 weeks after priming. Serum was collected at the indicated time point and subjected to ELISA to detect SARS-specific IgG using SARS-CoV-infected Vero cell lysates as a coating antigen. Circles and bars represent the amount of IgG antibody in the serum of each mouse and the mean, respectively. The amount of IgG was arbitrarily calculated based on the concentration of hyper-immune sera. A representative result of two independent experiments is shown. (D) Mice were vaccinated with 10 μg of UV-inactivated SARS-CoV virion subcutaneously into their backs. Serum was collected from individual mice at the indicated time point and subjected to ELISA to detect SARS-specific IgG.</p></caption><graphic xlink:href="intimmdxh143f01_lw"></graphic></fig><p>Upon restimulation with antigen, memory B cells rapidly differentiate into ASCs and migrate into the bone marrow to establish a long-term ASC pool (<xref rid="BIB15">15</xref>,<xref rid="BIB16">16</xref>). To enumerate the number of plasma cells specific for SARS-CoV, we performed an ELISPOT assay using recombinant N proteins, amino acid numbers 1–49 (N1–49) and 340–390 (N340–390) as coating antigens. Consistent with the serum anti-SARS CoV IgG level, SARS-specific IgG<sub>1</sub> plasma cells were maintained in the bone marrow at day 10 after boost immunization with virion/alum (<xref rid="FIG2">Fig. 2</xref>). In contrast, the number of spots from control mice was below the detection limit (i.e. <1 ASC/9 × 10<sup>5</sup> cells).</p><fig id="FIG2" orientation="portrait" position="float"><label>Fig. 2.</label><caption><p>The number of SARS-specific IgG<sub>1</sub> plasma cells in BM. Mice were primed and boosted by subcutaneous injection into their back with 10 μg of UV-inactivated SARS-CoV virion with 2 mg of alum (VA). BMs were collected at 10 days after boost and subjected to ELISPOT to detect SARS-specific IgG<sub>1</sub> plasma cells. Bars represent the number of plasma cells specific to N1–49 and N340–390 antigen in SARS-vaccinated and control mice, respectively. Data are means of triplicate cultures. The number of spots from control mice was below the detection limit (i.e. <1 ASC/9 × 10<sup>5</sup> cells: dashed line). A representative result of two independent experiments is shown. N.D.: not detected.</p></caption><graphic xlink:href="intimmdxh143f02_lw"></graphic></fig></sec><sec><title>UV-inactivated SARS-CoV induces IgG<sub>1</sub> antibody with neutralizing activity</title><p>We determined the subclass of serum anti-SARS-CoV IgG antibodies in the boosted mice using anti-mouse IgG<sub>1</sub>, IgG<sub>2a</sub>, IgG<sub>2b</sub> or IgG<sub>3</sub> second antibody by ELISA (<xref rid="FIG3">Fig. 3</xref>). Interestingly, the level of anti-SARS-CoV IgG<sub>2a</sub> in mice immunized with virion/alum was comparable to that in mice immunized with virion alone, whereas the level of anti-SARS-CoV IgG<sub>1</sub> was higher in mice with virion/alum than the mice with virion alone. In contrast, the levels of IgG<sub>2b</sub> and IgG<sub>3</sub> antibodies were fairly low in both groups. Therefore, our results indicated that vaccination with a combination of inactivated virion and alum induced a predominantly Th2-type immune response.</p><fig id="FIG3" orientation="portrait" position="float"><label>Fig. 3.</label><caption><p>IgG subclass of immunized serum. Mice were subcutaneously primed and boosted by injection in their footpads with 10 μg of UV-inactivated SARS-CoV virion (V), or virion with 2 mg of alum (VA). Serum was collected from individual mice at 1 week after boost and subjected to ELISA to detect SARS-specific IgG<sub>1</sub>, IgG<sub>2a</sub>, IgG<sub>2b</sub> and IgG<sub>3</sub> titer. The Y value is the reciprocal serum dilution number where the OD490nm ≥ 0.2 in each ELISA. Circles and bars represent the titer for each mouse and the mean, respectively; results are representative of two separate experiments.</p></caption><graphic xlink:href="intimmdxh143f03_lw"></graphic></fig><p>We also measured serum immunoglobulins other than IgG in the early and late phases of immunization. To avoid high IgG concentrations interfering with the detection of IgM and IgA antibodies, the serum IgG was absorbed with protein G-conjugated beads (>98%). The levels of anti-SARS-CoV IgM antibodies in the IgG-depleted sera, which were obtained 4 weeks after priming, were below our detection limit. Likewise, anti-SARS-CoV IgA antibody in the IgG-depleted sera, which were obtained 1 week after booster, was not detectable (data not shown).</p><p>Whether or not immune sera possess a neutralizing activity against SARS-CoV is a crucial aspect of vaccination. We estimated the neutralizing activity of sera obtained 1 week after boost inoculation (<xref rid="TBL1">Table 1</xref>). We observed that neutralizing activity against SARS-CoV was detected at a high level in sera of mice inoculated with virion/alum or virion alone. Taken together, these results indicate that subcutaneous vaccination with UV-inactivated SARS-CoV virion is able to elicit a sufficient amount of IgG antibodies with neutralizing activity.</p><table-wrap id="TBL1" orientation="portrait" position="float"><label>Table 1.</label><caption><p>Neutralizing activity in serum after vaccination</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" colspan="1" rowspan="1" valign="top"><hr></hr></th><th align="left" colspan="1" rowspan="1" valign="top"><hr></hr></th><th align="left" colspan="2" rowspan="1" valign="top"><hr></hr>Reciprocal endpoint titer<hr></hr></th><th align="left" colspan="1" rowspan="1" valign="top"></th></tr><tr><th align="left" colspan="1" rowspan="1" valign="top"><hr></hr></th><th align="left" colspan="1" rowspan="1" valign="top"><hr></hr></th><th align="left" colspan="1" rowspan="1" valign="top">Experiment 1<hr></hr></th><th align="left" colspan="1" rowspan="1" valign="top">Experiment 2<hr></hr></th></tr></thead><tbody><tr><td align="left" colspan="1" rowspan="1" valign="top">None/alum</td><td align="left" colspan="1" rowspan="1" valign="top"></td><td align="char" char="." colspan="1" rowspan="1" valign="top"><5<xref rid="TBLFN1"><sup>*</sup></xref></td><td align="char" char="." colspan="1" rowspan="1" valign="top"><5<xref rid="TBLFN1"><sup>*</sup></xref></td></tr><tr><td align="left" colspan="1" rowspan="1" valign="top">Virion</td><td align="char" char="." colspan="1" rowspan="1" valign="top">mouse 1</td><td align="char" char="." colspan="1" rowspan="1" valign="top">250</td><td align="char" char="." colspan="1" rowspan="1" valign="top">250</td></tr><tr><td align="left" colspan="1" rowspan="1" valign="top"></td><td align="char" char="." colspan="1" rowspan="1" valign="top">2</td><td align="char" char="." colspan="1" rowspan="1" valign="top">1250</td><td align="char" char="." colspan="1" rowspan="1" valign="top">250</td></tr><tr><td align="left" colspan="1" rowspan="1" valign="top"></td><td align="char" char="." colspan="1" rowspan="1" valign="top">3</td><td align="char" char="." colspan="1" rowspan="1" valign="top">1250</td><td align="char" char="." colspan="1" rowspan="1" valign="top">250</td></tr><tr><td align="left" colspan="1" rowspan="1" valign="top">Virion/alum</td><td align="char" char="." colspan="1" rowspan="1" valign="top">1</td><td align="char" char="." colspan="1" rowspan="1" valign="top">250</td><td align="char" char="." colspan="1" rowspan="1" valign="top">1250</td></tr><tr><td align="left" colspan="1" rowspan="1" valign="top"></td><td align="char" char="." colspan="1" rowspan="1" valign="top">2</td><td align="char" char="." colspan="1" rowspan="1" valign="top">1250</td><td align="char" char="." colspan="1" rowspan="1" valign="top">1250</td></tr><tr><td align="left" colspan="1" rowspan="1" valign="top"><hr></hr></td><td align="char" char="." colspan="1" rowspan="1" valign="top">3<hr></hr></td><td align="char" char="." colspan="1" rowspan="1" valign="top">1250<hr></hr></td><td align="char" char="." colspan="1" rowspan="1" valign="top">1250<hr></hr></td></tr></tbody></table><table-wrap-foot><fn id="TBLFN1"><label>*</label><p>All six mice examined did not have detectable neutralizing activity.</p></fn><fn><p>Sera were obtained from mice 1 week after boost vaccination and subjected to SARS-CoV neutralizing activity assay as described in Methods. The titer is a reciprocal number of minimum serum dilution that inhibits the cytopathic effect.</p></fn></table-wrap-foot></table-wrap></sec><sec><title>UV-inactivated SARS-CoV induces serum IgG antibody specific for S and N proteins</title><p>Using the immune sera of mice boosted with virion/alum 1 week before, we analyzed the specificity of serum IgG by western blot analysis (see Methods). As shown in <xref rid="FIG4">Fig. 4(A)</xref>, the robust signal detected at 50 kDa corresponds to the N protein of SARS-CoV, as predicted by its genome size (<xref rid="BIB3">3</xref>,<xref rid="BIB4">4</xref>). A band near 200 kDa appears to correspond to S protein, analogous with the S protein of other human coronaviruses, HCV-229E and HCV-OC43, which are known to be heavily glycosylated and detected at 186 kDa and 190 kDa, respectively (<xref rid="BIB17">17</xref>). Our result is consistent with the data reported recently by Xiao <italic>et al.</italic> who expressed the full-length S glycoprotein of SARS-CoV Tor2 strain in 293 cells and showed that the protein ran ∼180–200 kDa in SDS gels (<xref rid="BIB18">18</xref>). The origins of the 120 kDa and the faint 37 kDa bands were unknown. However, similar bands were also detected on a fluorogram by using anti-N mAbs (Ohnishi, K., Sakaguchi, M., Takasuka, N. <italic>et al.</italic>, unpublished data), suggesting that it is related to N protein. The specificity of IgG in the immune sera was also determined by ELISA plates coated with lysates of cells infected with either S- or N-expressing recombinant vaccinia viruses (<xref rid="FIG4">Fig. 4B</xref>). The results indicated that anti-S as well as anti-N protein IgG antibodies were elicited by virion/alum vaccination.</p><fig id="FIG4" orientation="portrait" position="float"><label>Fig. 4.</label><caption><p>Specificity of the serum antibodies. (A) Purified UV-inactivated SARS-CoV virion (0.5 μg) was fractionated by SDS–PAGE and subjected to western blotting. Diluted pooled sera (1:1000) from mice primed and boosted with virion/alum were exploited to detect virus proteins. Upper and lower arrows indicate the predicted band of S (spike protein) and N (nucleocapside protein) of SARS-CoV, respectively. The size of molecular weight markers (kDa) is shown on the left. (B) S protein- or N protein-specific ELISA. ELISA plates were coated at the indicated dilution with 1% NP40 lysates of chick embryo fibroblasts that had been infected with S protein-expressing vaccinia virus (circle), N protein-expressing vaccinia virus (triangle) or uninfected (mock; square). Diluted serum (1:1000) from mice prime and boost immunized with virion/alum, was exploited for detection of virus proteins.</p></caption><graphic xlink:href="intimmdxh143f04_ht"></graphic></fig></sec><sec><title>UV-inactivated SARS-CoV whole virion induces T-cell response</title><p>To examine whether or not subcutaneously vaccinated mice gained an induced T-cell response against SARS-CoV, mice were immunized either with virion/alum, virion, or alum only via the footpad. T cells of these mice were enriched from the spleen and regional lymph nodes 1 week after a booster immunization and cultured with irradiated APCs in the presence or absence of UV-inactivated SARS-CoV virion at 1 or 10 μg/ml. As shown in <xref rid="FIG5">Fig. 5(A)</xref>, regional lymph node T cells proliferated <italic>in vitro</italic> in response to UV-inactivated virion in virion/alum-immunized mice and, to a lesser extent, in virion-immunized mice. Because mice inoculated with virion/alum showed a high basal level of proliferation of lymph node T cells in the absence of antigen, there is not much difference in the net proliferative response of these cells between the virion/alum group and the virion only group. On the other hand, in splenic T cells, a low level of proliferation was observed only in the virion/alum group of mice. The level of proliferation of these T cells, however, was virion-dose independent. Therefore, our results suggest that the subcutaneous injection of inactivated virion, even without alum, does induce T cell activation to some extent in the draining lymph node, a result which hardly occurs systemically.</p><fig id="FIG5" orientation="portrait" position="float"><label>Fig. 5.</label><caption><p><italic>In vitro</italic> responses of SARS-CoV-specific T cells taken from mice vaccinated with inactivated SARS-CoV. Mice were subcutaneously primed with 10 μg of UV-inactivated SARS-CoV virion, or virion with 2 mg of alum, or none, and then boosted with the same dose in their footpads at 7 weeks after priming. Draining lymph nodes and spleens were isolated at 1 week after boost and stimulated with T-cell depleted splenocytes that had been pulsed with the indicated concentration of UV-inactivated SARS-CoV virion. These cells were cultured for 2–4 days and [<sup>3</sup>H]thymidine was added 8 h prior to the harvest. The peak response on day 4 after cultivation is shown in (A). (B) Culture supernatant was collected at day 2–4 post cultivation and the level of IL-2, IFN-γ, IL-4, IL-5 and TNF-α was determined by CBA kit. The maximum cytokine production at day 4 is shown. Results are representative of two separate experiments.</p></caption><graphic xlink:href="intimmdxh143f05_ht"></graphic></fig><p>We also measured the level of cytokine production in the supernatant of lymph node T cells stimulated with inactivated virion <italic>in vitro</italic> for 4 days. We found that the inactivated virion induced the production of all the cytokines (IL-2, IL-4, IL-5, IFN-γ and TNF-α) in T cells of virion/alum-immunized mice, in a dose-dependent manner (<xref rid="FIG5">Fig. 5B</xref>). Likewise, T cells of virion-immunized mice produced low, yet significant, levels of these cytokines in a dose-dependent manner, except IL-5. In contrast, lymph node T cells from normal mice did not produce any cytokines at all in response to virion, suggesting that the virion itself does not possess innate stimulating activity as bacterial products [such as lipopolysaccharide (LPS) and purified protein derivative of mycobacterium tuberculosis (PPD)] do. Taken together, these results suggest that subcutaneous vaccination with UV-inactivated SARS-CoV is able to activate CD4<sup>+</sup> T cells in regional lymph nodes, where T cells produce several immunoregulatory cytokines, including IFN-γ.</p></sec></sec><sec><title>Discussion</title><p>The present results demonstrated that even a single subcutaneous administration of UV-irradiated virion without alum adjuvant induced a high level of systemic anti-SARS-CoV antibody response in mice, probably followed by the generation of long-term antibody-secreting cells and memory cells in the bone marrow. Considering that polyvalent particulate structures such as hepatitis B virus surface antigen-based, HIV-1 Gag-based and Ty virus-like particles have been shown to elicit humoral as well as cellular immune responses (<xref rid="BIB19">19</xref>), these particulates probably have comparable dimensions and structures to the pathogens that are targeted for uptake by APCs to facilitate the induction of potent immune responses. The antibodies elicited in mice vaccinated by the current protocol with or without adjuvant recognized both the S and N proteins of SARS-CoV and were able to neutralize the infection of virus to Vero E6 cells. However, serum anti-SARS-CoV IgA antibody was not detectable, probably owing to the route of vaccination. In addition, the present vaccination protocol caused T cell response at the regional lymph nodes, although it did not allow for the induction of a sufficient cellular immune response systemically.</p><p>We show here the potentiality of subcutaneous injection of inactivated virion with alum, which is utilized for most of current human vaccinations. Alum has been used as an adjuvant for vaccines such as diphtheria, pertussis and tetanus, and these vaccines have a long safety record for human use (<xref rid="BIB20">20</xref>). We observed that the addition of alum to the vaccine formula resulted in a large augmentation of serum IgG<sub>1</sub> production, but not IgG<sub>2a</sub> production. The level of IgG<sub>1</sub> in alum-vaccinated mice reached a level similar to that found in hyper-immunized mice, which were subcutaneously injected with 5 μg of inactivated virion emulsified with a complete Freund adjuvant, followed by consecutive three-times intravenous boosters with 2 μg of virion. Alum is known to selectively stimulate an IgG<sub>1</sub> dominant, type 2 immune response [reviewed in (<xref rid="BIB21">21</xref>)]. Activation of complement by alum could contribute to the type 2-biased immune response partly via an inhibition of IL-12 production. Interestingly, a quite recent report demonstrated that an alum-induced Gr1<sup>+</sup> myeloid cell population produced IL-4 and activated B-cells (<xref rid="BIB22">22</xref>).</p><p>There are various diseases associated with animal coronavirus infection. The clinical manifestations of the disease and the correlates of protection with immunity have been studied extensively in these animal coronavirus infections [reviewed in (<xref rid="BIB7">7</xref>)]. Although antibodies and T cells may play a role in exacerbating the pathology in some animal coronavirus infections (<xref rid="BIB23">23</xref>,<xref rid="BIB24">24</xref>), both humoral and cellular immune responses are known to contribute to protection against coronavirus infection. In murine hepatitis virus, a Group 2 coronavirus, the mortality of susceptible mice was partially prevented by the transfer of immune serum containing neutralizing antibody prior to challenge (<xref rid="BIB25">25</xref>). Recently, Zhi-yong <italic>et al.</italic> reported in the murine acute infection model that the neutralizing antibody elicited by vaccination of DNA encoding S was protective, but cellular components of vaccinated mice were not required for the inhibition of viral replication (<xref rid="BIB26">26</xref>). Because a twice parenteral administration of inactivated virion with alum induced a high level of antibodies that are able to neutralize SARS-CoV, this vaccination protocol may have a certain effect on the protection of humans from SARS-CoV infection.</p><p>We observed that two successive inoculations with inactivated virus at 7 week intervals generated SARS-CoV-specific T cells. These cells were restimulated with the irradiated virus <italic>in vitro</italic>, but their response was low in terms of the level of proliferation and production of INF-γ and IL-2. However, irrespective of vaccination protocols with or without alum adjuvant, virus-primed T cells of vaccinated animals were capable of producing IL-4 at high levels upon <italic>in vitro</italic> stimulation, comparable to other reports for a variety of vaccination studies (<xref rid="BIB27">27</xref>,<xref rid="BIB28">28</xref>). This outlook seems compatible with the idea that the present vaccine protocol may tend to select T-cell subsets with Th2 phenotype. However, it remains to be elucidated whether such T cells may exhibit serological memory phenotype and persist in the immune system after vaccination as long as memory B cells, which may persist more than 180 days post vaccination. In addition, further analysis is needed to clarify whether T cell response is a crucial factor for long-term protection against SARS-CoV infections.</p><p>Efforts to develop a SARS-CoV vaccine have been carried out by many profitable or non-profitable organizations in various ways. For example, it has recently been reported that the combination of adenovirus vector expressing SARS-S, -M or -N protein elicited a neutralizing capacity in serum and N-specific T-cell response in rhesus macaques (<xref rid="BIB29">29</xref>). However, it is still uncertain whether or not the immunity against only these components of SARS-CoV is sufficient for virus protection. SARS-CoV tends to cause replication errors, which may allow the virus to escape the host-immune response and result in a seasonal outbreak. From this point of view, it resembles influenza virus. In influenza virus, inactivated HA vaccine showed incomplete protection but had a certain efficacy and safety record for a long period of time. Indeed, this approach has been used in the veterinary field, such as with the bovine coronavirus (<xref rid="BIB30">30</xref>) and canine coronavirus (<xref rid="BIB31">31</xref>). These advantages make a whole killed virion a prime candidate for a SARS vaccine, even if it may not have the best protective ability.</p><p>Unfortunately, no information is available so far on the immune correlates of protection against human coronaviruses, including SARS-CoV. In consideration that SARS-CoV transmission occurs by direct contact with droplets or by the fecal oral route, mucosal secretary IgA in both the lower respiratory tract and digestive tract seem to be crucially important. Failure to induce IgA-type antibodies in a current systemic vaccination method should be improved. Notably, IgA antibodies were detectable in the sera and bronchoalveolar lavage fluid obtained from mice hyper-immunized with UV-irradiated virus (data not shown). Therefore, if a non-toxic and more potent adjuvant becomes available for human use, the subcutaneous injection of inactivated virion would become an effective vaccination method to reduce the number of susceptible people.</p><p>In the future, it will be necessary to determine whether or not the inactivated whole virion vaccine possesses protective ability against SARS-CoV infection by the use of adequate animal models. Furthermore, whether the alum addition augmented the protection and the effective period of SARS-CoV virion vaccination should be addressed, because currently used inactivated influenza virus whole virion vaccine is significantly effective without any adjuvant. Meanwhile, we also need to develop a potent adjuvant for induction of a much stronger mucosal immunity, in addition to evaluating available methods of virion inactivation.</p></sec></body><back><fn-group><fn><p><italic>Transmitting editor</italic>: K. Sugamura</p></fn></fn-group><ack><p>We thank Ms R. 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