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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Optimization of antigen dose for a receptor-binding domain-based subunit vaccine against MERS coronavirus</title><author><name sortKey="Tang, Jian" sort="Tang, Jian" uniqKey="Tang J" first="Jian" last="Tang">Jian Tang</name><affiliation><nlm:aff id="af0001"><institution>Xiang-Ya Medical College; Central South University;</institution>, Changsha,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="af0002"><institution>State Key Laboratory of Pathogen and Biosecurity; Beijing Institute of Microbiology and Epidemiology;</institution>, Beijing,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="af0003"><institution>Lindsley F Kimball Research Institute; New York Blood Center;</institution>, New York, NY,<country>USA</country></nlm:aff></affiliation></author><author><name sortKey="Zhang, Naru" sort="Zhang, Naru" uniqKey="Zhang N" first="Naru" last="Zhang">Naru Zhang</name><affiliation><nlm:aff id="af0003"><institution>Lindsley F Kimball Research Institute; New York Blood Center;</institution>, New York, NY,<country>USA</country></nlm:aff></affiliation></author><author><name sortKey="Tao, Xinrong" sort="Tao, Xinrong" uniqKey="Tao X" first="Xinrong" last="Tao">Xinrong Tao</name><affiliation><nlm:aff id="af0004"><institution>Department of Microbiology and Immunology and Center for Biodefense and Emerging Disease; University of Texas Medical Branch;</institution>, Galveston, TX,<country>USA</country></nlm:aff></affiliation></author><author><name sortKey="Zhao, Guangyu" sort="Zhao, Guangyu" uniqKey="Zhao G" first="Guangyu" last="Zhao">Guangyu Zhao</name><affiliation><nlm:aff id="af0002"><institution>State Key Laboratory of Pathogen and Biosecurity; Beijing Institute of Microbiology and Epidemiology;</institution>, Beijing,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Guo, Yan" sort="Guo, Yan" uniqKey="Guo Y" first="Yan" last="Guo">Yan Guo</name><affiliation><nlm:aff id="af0002"><institution>State Key Laboratory of Pathogen and Biosecurity; Beijing Institute of Microbiology and Epidemiology;</institution>, Beijing,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Tseng, Chien Te K" sort="Tseng, Chien Te K" uniqKey="Tseng C" first="Chien-Te K" last="Tseng">Chien-Te K. Tseng</name><affiliation><nlm:aff id="af0004"><institution>Department of Microbiology and Immunology and Center for Biodefense and Emerging Disease; University of Texas Medical Branch;</institution>, Galveston, TX,<country>USA</country></nlm:aff></affiliation></author><author><name sortKey="Jiang, Shibo" sort="Jiang, Shibo" uniqKey="Jiang S" first="Shibo" last="Jiang">Shibo Jiang</name><affiliation><nlm:aff id="af0003"><institution>Lindsley F Kimball Research Institute; New York Blood Center;</institution>, New York, NY,<country>USA</country></nlm:aff></affiliation><affiliation><nlm:aff id="af0005"><institution>Key Laboratory of Medical Molecular Virology of Ministries of Education and Health; Shanghai Medical College and Institute of Medical Microbiology; Fudan University;</institution>, Shanghai,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Du, Lanying" sort="Du, Lanying" uniqKey="Du L" first="Lanying" last="Du">Lanying Du</name><affiliation><nlm:aff id="af0003"><institution>Lindsley F Kimball Research Institute; New York Blood Center;</institution>, New York, NY,<country>USA</country></nlm:aff></affiliation></author><author><name sortKey="Zhou, Yusen" sort="Zhou, Yusen" uniqKey="Zhou Y" first="Yusen" last="Zhou">Yusen Zhou</name><affiliation><nlm:aff id="af0001"><institution>Xiang-Ya Medical College; Central South University;</institution>, Changsha,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="af0002"><institution>State Key Laboratory of Pathogen and Biosecurity; Beijing Institute of Microbiology and Epidemiology;</institution>, Beijing,<country>China</country></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">25874632</idno><idno type="pmc">4514392</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4514392</idno><idno type="RBID">PMC:4514392</idno><idno type="doi">10.1080/21645515.2015.1021527</idno><date when="2015">2015</date><idno type="wicri:Area/Pmc/Corpus">000D09</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000D09</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Optimization of antigen dose for a receptor-binding domain-based subunit vaccine against MERS coronavirus</title><author><name sortKey="Tang, Jian" sort="Tang, Jian" uniqKey="Tang J" first="Jian" last="Tang">Jian Tang</name><affiliation><nlm:aff id="af0001"><institution>Xiang-Ya Medical College; Central South University;</institution>, Changsha,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="af0002"><institution>State Key Laboratory of Pathogen and Biosecurity; Beijing Institute of Microbiology and Epidemiology;</institution>, Beijing,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="af0003"><institution>Lindsley F Kimball Research Institute; New York Blood Center;</institution>, New York, NY,<country>USA</country></nlm:aff></affiliation></author><author><name sortKey="Zhang, Naru" sort="Zhang, Naru" uniqKey="Zhang N" first="Naru" last="Zhang">Naru Zhang</name><affiliation><nlm:aff id="af0003"><institution>Lindsley F Kimball Research Institute; New York Blood Center;</institution>, New York, NY,<country>USA</country></nlm:aff></affiliation></author><author><name sortKey="Tao, Xinrong" sort="Tao, Xinrong" uniqKey="Tao X" first="Xinrong" last="Tao">Xinrong Tao</name><affiliation><nlm:aff id="af0004"><institution>Department of Microbiology and Immunology and Center for Biodefense and Emerging Disease; University of Texas Medical Branch;</institution>, Galveston, TX,<country>USA</country></nlm:aff></affiliation></author><author><name sortKey="Zhao, Guangyu" sort="Zhao, Guangyu" uniqKey="Zhao G" first="Guangyu" last="Zhao">Guangyu Zhao</name><affiliation><nlm:aff id="af0002"><institution>State Key Laboratory of Pathogen and Biosecurity; Beijing Institute of Microbiology and Epidemiology;</institution>, Beijing,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Guo, Yan" sort="Guo, Yan" uniqKey="Guo Y" first="Yan" last="Guo">Yan Guo</name><affiliation><nlm:aff id="af0002"><institution>State Key Laboratory of Pathogen and Biosecurity; Beijing Institute of Microbiology and Epidemiology;</institution>, Beijing,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Tseng, Chien Te K" sort="Tseng, Chien Te K" uniqKey="Tseng C" first="Chien-Te K" last="Tseng">Chien-Te K. Tseng</name><affiliation><nlm:aff id="af0004"><institution>Department of Microbiology and Immunology and Center for Biodefense and Emerging Disease; University of Texas Medical Branch;</institution>, Galveston, TX,<country>USA</country></nlm:aff></affiliation></author><author><name sortKey="Jiang, Shibo" sort="Jiang, Shibo" uniqKey="Jiang S" first="Shibo" last="Jiang">Shibo Jiang</name><affiliation><nlm:aff id="af0003"><institution>Lindsley F Kimball Research Institute; New York Blood Center;</institution>, New York, NY,<country>USA</country></nlm:aff></affiliation><affiliation><nlm:aff id="af0005"><institution>Key Laboratory of Medical Molecular Virology of Ministries of Education and Health; Shanghai Medical College and Institute of Medical Microbiology; Fudan University;</institution>, Shanghai,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Du, Lanying" sort="Du, Lanying" uniqKey="Du L" first="Lanying" last="Du">Lanying Du</name><affiliation><nlm:aff id="af0003"><institution>Lindsley F Kimball Research Institute; New York Blood Center;</institution>, New York, NY,<country>USA</country></nlm:aff></affiliation></author><author><name sortKey="Zhou, Yusen" sort="Zhou, Yusen" uniqKey="Zhou Y" first="Yusen" last="Zhou">Yusen Zhou</name><affiliation><nlm:aff id="af0001"><institution>Xiang-Ya Medical College; Central South University;</institution>, Changsha,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="af0002"><institution>State Key Laboratory of Pathogen and Biosecurity; Beijing Institute of Microbiology and Epidemiology;</institution>, Beijing,<country>China</country></nlm:aff></affiliation></author></analytic><series><title level="j">Human Vaccines & Immunotherapeutics</title><idno type="ISSN">2164-5515</idno><idno type="eISSN">2164-554X</idno><imprint><date when="2015">2015</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Middle East respiratory syndrome (MERS) is an emerging infectious disease caused by MERS coronavirus (MERS-CoV). The continuous increase of MERS cases has posed a serious threat to public health worldwide, calling for development of safe and effective MERS vaccines. We have previously shown that a recombinant protein containing residues 377–588 of MERS-CoV receptor-binding domain (RBD) fused with human Fc (S377-588-Fc) induced highly potent anti-MERS-CoV neutralizing antibodies in the presence of MF59 adjuvant. Here we optimized the doses of S377-588-Fc using MF59 as an adjuvant in order to elicit strong immune responses with minimal amount of antigen. Our results showed that S377-588-Fc at 1 μg was able to induce in the immunized mice potent humoral and cellular immune responses. Particularly, S377-588-Fc at 1 μg elicited strong neutralizing antibody responses against both pseudotyped and live MERS-CoV similar to those induced at 5 and 20 μg, respectively. These results suggest that this RBD-based subunit MERS vaccine candidate at the dose as low as one μg is sufficiently potent to induce strong humoral and cellular immune responses, including neutralizing antibodies, against MERS-CoV infection, thus providing guidance for determining the optimal dosage of RBD-based MERS vaccines in the future clinical trials and for applying the dose-sparing strategy in other subunit vaccine trials.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Drosten, C" uniqKey="Drosten C">C Drosten</name></author><author><name sortKey="Seilmaier, M" uniqKey="Seilmaier M">M Seilmaier</name></author><author><name sortKey="Corman, Vm" uniqKey="Corman V">VM Corman</name></author><author><name sortKey="Hartmann, W" uniqKey="Hartmann W">W Hartmann</name></author><author><name sortKey="Scheible, G" uniqKey="Scheible G">G Scheible</name></author><author><name sortKey="Sack, S" uniqKey="Sack S">S 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Hum Vaccin Immunother</journal-id><journal-id journal-id-type="iso-abbrev">Hum Vaccin Immunother</journal-id><journal-id journal-id-type="publisher-id">KHVI</journal-id><journal-id journal-id-type="publisher-id">khvi20</journal-id><journal-title-group><journal-title>Human Vaccines & Immunotherapeutics</journal-title></journal-title-group><issn pub-type="ppub">2164-5515</issn><issn pub-type="epub">2164-554X</issn><publisher><publisher-name>Taylor & Francis</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">25874632</article-id><article-id pub-id-type="pmc">4514392</article-id><article-id pub-id-type="publisher-id">1021527</article-id><article-id pub-id-type="doi">10.1080/21645515.2015.1021527</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Paper</subject></subj-group></article-categories><title-group><article-title>Optimization of antigen dose for a receptor-binding domain-based subunit vaccine against MERS coronavirus</article-title><alt-title alt-title-type="running-title">Antigen doses for MERS RBD subunit vaccines</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Tang</surname><given-names>Jian</given-names></name><xref ref-type="aff" rid="af0001"><sup>1</sup></xref><xref ref-type="aff" rid="af0002"><sup>2</sup></xref><xref ref-type="aff" rid="af0003"><sup>3</sup></xref><xref ref-type="author-notes" rid="afn0001"></xref></contrib><contrib contrib-type="author"><name><surname>Zhang</surname><given-names>Naru</given-names></name><xref ref-type="aff" rid="af0003"><sup>3</sup></xref><xref ref-type="author-notes" rid="afn0001"></xref></contrib><contrib contrib-type="author"><name><surname>Tao</surname><given-names>Xinrong</given-names></name><xref ref-type="aff" rid="af0004"><sup>4</sup></xref></contrib><contrib contrib-type="author"><name><surname>Zhao</surname><given-names>Guangyu</given-names></name><xref ref-type="aff" rid="af0002"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Guo</surname><given-names>Yan</given-names></name><xref ref-type="aff" rid="af0002"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Tseng</surname><given-names>Chien-Te K</given-names></name><xref ref-type="aff" rid="af0004"><sup>4</sup></xref></contrib><contrib contrib-type="author"><name><surname>Jiang</surname><given-names>Shibo</given-names></name><xref ref-type="aff" rid="af0003"><sup>3</sup></xref><xref ref-type="aff" rid="af0005"><sup>5</sup></xref><xref ref-type="corresp" rid="an0001"><sup>*</sup></xref></contrib><contrib contrib-type="author"><name><surname>Du</surname><given-names>Lanying</given-names></name><xref ref-type="aff" rid="af0003"><sup>3</sup></xref><xref ref-type="corresp" rid="an0001"><sup>*</sup></xref></contrib><contrib contrib-type="author"><name><surname>Zhou</surname><given-names>Yusen</given-names></name><xref ref-type="aff" rid="af0001"><sup>1</sup></xref><xref ref-type="aff" rid="af0002"><sup>2</sup></xref><xref ref-type="corresp" rid="an0001"><sup>*</sup></xref></contrib><aff id="af0001"><label>1</label><institution>Xiang-Ya Medical College; Central South University;</institution>, Changsha,<country>China</country></aff><aff id="af0002"><label>2</label><institution>State Key Laboratory of Pathogen and Biosecurity; Beijing Institute of Microbiology and Epidemiology;</institution>, Beijing,<country>China</country></aff><aff id="af0003"><label>3</label><institution>Lindsley F Kimball Research Institute; New York Blood Center;</institution>, New York, NY,<country>USA</country></aff><aff id="af0004"><label>4</label><institution>Department of Microbiology and Immunology and Center for Biodefense and Emerging Disease; University of Texas Medical Branch;</institution>, Galveston, TX,<country>USA</country></aff><aff id="af0005"><label>5</label><institution>Key Laboratory of Medical Molecular Virology of Ministries of Education and Health; Shanghai Medical College and Institute of Medical Microbiology; Fudan University;</institution>, Shanghai,<country>China</country></aff></contrib-group><author-notes><fn id="afn0001"><p><sup>#</sup>These authors equally contributed to this work.</p></fn><corresp id="an0001"><label>*</label>Correspondence to: Shibo Jiang; Email: <email xlink:href="sjiang@nybloodcenter.org">sjiang@nybloodcenter.org</email>; Lanying Du; Email: <email xlink:href="ldu@nybloodcenter.org">ldu@nybloodcenter.org</email>; Yusen Zhou; Email: <email xlink:href="yszhou@bmi.ac.cn">yszhou@bmi.ac.cn</email></corresp></author-notes><pub-date pub-type="collection"><year>2015</year></pub-date><pub-date pub-type="epub"><day>27</day><month>5</month><year>2015</year></pub-date><volume>11</volume><issue>5</issue><fpage seq="25">1244</fpage><lpage>1250</lpage><history><date date-type="received"><day>05</day><month>1</month><year>2015</year></date><date date-type="rev-recd"><day>04</day><month>2</month><year>2015</year></date><date date-type="accepted"><day>17</day><month>2</month><year>2015</year></date></history><permissions><copyright-statement>© 2015 Taylor & Francis Group, LLC</copyright-statement><copyright-year>2015</copyright-year><copyright-holder>Taylor & Francis Group, LLC</copyright-holder><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 content-type="pdf" xlink:href="KHVI_11_1021527.pdf"></self-uri><abstract><p>Middle East respiratory syndrome (MERS) is an emerging infectious disease caused by MERS coronavirus (MERS-CoV). The continuous increase of MERS cases has posed a serious threat to public health worldwide, calling for development of safe and effective MERS vaccines. We have previously shown that a recombinant protein containing residues 377–588 of MERS-CoV receptor-binding domain (RBD) fused with human Fc (S377-588-Fc) induced highly potent anti-MERS-CoV neutralizing antibodies in the presence of MF59 adjuvant. Here we optimized the doses of S377-588-Fc using MF59 as an adjuvant in order to elicit strong immune responses with minimal amount of antigen. Our results showed that S377-588-Fc at 1 μg was able to induce in the immunized mice potent humoral and cellular immune responses. Particularly, S377-588-Fc at 1 μg elicited strong neutralizing antibody responses against both pseudotyped and live MERS-CoV similar to those induced at 5 and 20 μg, respectively. These results suggest that this RBD-based subunit MERS vaccine candidate at the dose as low as one μg is sufficiently potent to induce strong humoral and cellular immune responses, including neutralizing antibodies, against MERS-CoV infection, thus providing guidance for determining the optimal dosage of RBD-based MERS vaccines in the future clinical trials and for applying the dose-sparing strategy in other subunit vaccine trials.</p></abstract><kwd-group kwd-group-type="author"><title>Keywords</title><kwd>antigen doses</kwd><kwd>MERS</kwd><kwd>MERS-CoV</kwd><kwd>receptor-binding domain</kwd><kwd>subunit vaccines</kwd></kwd-group><counts><fig-count count="4"></fig-count><table-count count="0"></table-count><ref-count count="43"></ref-count><page-count count="7"></page-count></counts></article-meta></front><body><sec sec-type="intro" id="s0001"><title>Introduction</title><p>Middle East respiratory syndrome (MERS) is a newly emerged infectious disease caused by MERS coronavirus (MERS-CoV).<sup><xref rid="cit0001" ref-type="bibr">1,2</xref></sup> First reported in Saudi Arabia in 2012,<sup><xref rid="cit0003" ref-type="bibr">3</xref></sup> the virus has now been identified in 20 other countries of the world and has led to the infection of 965 individuals with 357 deaths worldwide (a mortality rate ∼37%) (<uri xlink:href="http://www.who.int/csr/don/03-february-2015-mers/en/">http://www.who.int/csr/don/03-february-2015-mers/en/</uri>). Studies have indicated bats and camels as the natural reservoirs and intermediate transmission hosts of MERS-CoV, respectively, and they have, moreover, elucidated the bat-to-human transmission mechanism of MERS-CoV.<sup><xref rid="cit0004" ref-type="bibr">4-9</xref></sup> MERS-CoV has caused diseases in several family clusters and healthcare workers.<sup><xref rid="cit0010" ref-type="bibr">10-13</xref></sup> With continuous increase of human cases, MERS-CoV has posed a serious threat to public health worldwide, demonstrating the need to develop safe and effective vaccines against virus infection.</p><p>MERS-CoV spike (S) protein plays significant roles in mediating virus entry into target cells expressing viral receptor dipeptidyl peptidase 4 (DPP4) and subsequent fusion of virus and cell membranes.<sup><xref rid="cit0014" ref-type="bibr">14-16</xref></sup> To accomplish this, MERS-CoV depends on the receptor-binding domain (RBD) in the S1 subunit to bind host cellular receptors.<sup><xref rid="cit0017" ref-type="bibr">17-19</xref></sup> As such, RBD is an important target for the development of MERS vaccines.<sup><xref rid="cit0020" ref-type="bibr">20-24</xref></sup> Previous studies have mapped the RBD to the regions containing residues 358–588, 367–588, 377–588, and 367–606 of MERS-CoV S protein.<sup><xref rid="cit0017" ref-type="bibr">17–19,22,23,25</xref></sup></p><p>It is known that a fragment containing residues 377–588 of MERS-CoV RBD is a critical neutralizing domain.<sup><xref rid="cit0022" ref-type="bibr">22-23,26</xref></sup> After comparing 5 different RBD fragments respectively containing residues 350–588, 358–588, 367–588, 367–606, and 377–588 of MERS-CoV S protein fused with Fc of human IgG, namely S350-588-Fc, S358-588-Fc, S367-588-Fc, S367-606-Fc, S377-588-Fc, we found that S377-588-Fc induced the highest antibody responses and neutralizing antibodies in immunized animals.<sup><xref rid="cit0022" ref-type="bibr">22–23</xref></sup> We have further compared the effects of several commercially available adjuvants, such as Freund's, aluminum, Monophosphoryl lipid A, Montanide ISA51, and MF59, in the promotion of immunogenicity of the aforementioned S377-588-Fc, and demonstrated that MF59 is an ideal adjuvant for use with this protein.<sup><xref rid="cit0027" ref-type="bibr">27</xref></sup> However, the minimal dose of the RBD protein required to induce sufficient immune responses against MERS-CoV infection remains to be elucidated. This calls for further optimization of the antigen dose for MERS subunit vaccines.</p><p>In this study, we examined the immunization potential of different doses of S377-588-Fc and compared their ability to induce specific humoral and cellular immune responses, particularly neutralizing antibodies against infection of MERS-CoV, using S377-588-Fc as a model antigen and MF59 as a selected adjuvant.</p></sec><sec sec-type="results" id="s0002"><title>Results</title><sec id="s0002-0001"><title>S377-588-Fc at 1 μg was able to induce strong humoral immune responses</title><p>To optimize the dose of S377-588-Fc required to induce sufficient antibody responses, we immunized mice with S377-588-Fc at 1, 5, and 20 μg, respectively, and detected specific IgG antibody, as well as IgG1 and IgG2a subtypes, in immunized mouse sera.</p><p>As shown in <xref ref-type="fig" rid="f0001"><bold>Figure 1A</bold></xref>, S377-588-Fc at all 3 test doses was able to induce MERS-CoV S1-specific IgG antibody response, with the antibody rapidly reaching a high level after the 2nd immunization and maintaining similar levels thereafter, suggesting that 2 doses of S377-588-Fc formulated with MF59 adjuvant are sufficient to induce strong antibody responses. As expected, only a background level of IgG antibody response was induced in the adjuvant only group (S377-588-Fc at 0 μg).
<fig id="f0001" orientation="portrait" position="float"><label>Figure 1.</label><caption><p><bold>Comparison of IgG antibodies induced by S377-588-Fc at different antigen doses</bold>. Mice were immunized without (0 µg) or with 1, 5, or 20 µg of S377-588-Fc, and sera were assessed for MERS-CoV S1-specific IgG antibody by ELISA. (<bold>A</bold>) IgG was tested in sera (1:3,200 dilution) of mice before (pre-immune) and 10 d post-each immunization, and the data are presented as mean A450 ± standard deviation (SD) from 5 mice in each group. (<bold>B</bold>) Sera from 10 d post-last immunization were tested for S1-specific IgG titers and expressed as the endpoint dilutions that remain positively detectable. The data are presented as mean titers ± SD from 5 mice in each group. Significant differences were noted between the groups immunized with 5 or 20 µg, respectively, and 1 µg of MERS-CoV RBD (*).</p></caption><graphic content-type="black-white" xlink:href="KHVI_A_1021527_F0001_B"></graphic></fig></p><p>We then calculated and compared the endpoint IgG titers from the 3rd sera of mice immunized with the 3 RBD doses. Results, as shown in <xref ref-type="fig" rid="f0001"><bold>Figure 1B</bold></xref>, revealed that S377-588-Fc at 1 μg induced high levels of IgG antibody response, but such response was significantly increased when the mice received 5 and 20 μg of the S377-588-Fc. Nevertheless, no significant difference was observed for IgG titers in the 5 and 20 μg immunization groups. As expected, mice receiving adjuvant only (S377-588-Fc at 0 μg) failed to induce MERS-CoV S1-specific IgG antibody response (<xref ref-type="fig" rid="f0001"><bold>Fig. 1B</bold></xref>).</p><p>To elucidate the IgG subtypes induced by different doses of S377-588-Fc, we detected IgG1 and IgG2a production using mouse sera from the 3rd immunization. As shown in <xref ref-type="fig" rid="f0002"><bold>Figure 2</bold></xref>, high titers of IgG1 (Th2) and IgG2a (Th1) antibodies were induced by 1, 5, and 20 μg of S377-588-Fc. However, S377-588-Fc at 5 and 20 μg induced a significantly higher level of IgG1 antibody than that at 1 μg (<xref ref-type="fig" rid="f0002"><bold>Fig. 2A</bold></xref>). Notably, while no significant difference was revealed between titers of IgG1 and IgG2a antibodies induced by 5 and 20 μg of S377-588-Fc, 5 μg of S377-588-Fc appears to elicit stronger IgG2a antibodies than either 1 or 20 μg (<xref ref-type="fig" rid="f0002"><bold>Fig. 2B</bold></xref>). As expected, no IgG subtypes were induced in the adjuvant control group (<xref ref-type="fig" rid="f0002"><bold>Fig. 2</bold></xref>).
<fig id="f0002" orientation="portrait" position="float"><label>Figure 2.</label><caption><p><bold>Comparison of antibody subtypes induced by S377-588-Fc at different antigen doses</bold>. Mice were immunized without (0 µg) or with 1, 5, or 20 µg of S377-588-Fc, and sera from 10 d post-last immunization were tested for MERS-CoV S1-specific IgG1 and IgG2a subtypes by ELISA. The titers were expressed as the endpoint dilutions that remained positively detectable, and the values are presented as mean ± SD from 5 mice in each group. Significant differences were noted in the IgG1 responses between the groups immunized with 5 or 20 µg, respectively, and 1 µg of MERS-CoV RBD (*).</p></caption><graphic content-type="black-white" xlink:href="KHVI_A_1021527_F0002_B"></graphic></fig></p><p>The above results suggest that 1 μg of S377-588-Fc is sufficient to induce high titers of RBD-specific antibody responses. Although S377-588-Fc at 5 and 20 µg could induce higher titers of total IgG and IgG1 subtype than those at 1 µg, the increased level of IgG and IgG1 may not necessarily provide stronger neutralizing antibody response that is required for protecting animals from MERS-CoV infection.</p></sec><sec id="s0002-0002"><title>S377-588-Fc at 1 μg induced high levels of neutralizing antibody responses, similar to those induced by 5 and 20 μg in immunized mice</title><p>To elucidate the neutralizing potential induced by different doses of S377-588-Fc and determine the minimal dose of such an antigen required to elicit strong neutralization against MERS-CoV infection, we investigated neutralizing antibodies in mouse sera from the 3rd immunization based on MERS pseudovirus and live MERS-CoV neutralization assays. Results, as shown in <xref ref-type="fig" rid="f0003"><bold>Figure 3</bold></xref>, demonstrated that S377-588-Fc at all 3 doses tested was able to induce potent neutralizing antibody titers against infections of MERS pseudovirus in Huh-7 cells (<xref ref-type="fig" rid="f0003"><bold>Fig. 3A</bold></xref>) and live MERS-CoV in Vero E6 cells (<xref ref-type="fig" rid="f0003"><bold>Fig. 3B</bold></xref>). There were no significant differences in the neutralizing activity among the sera of mice immunized with S377-588-Fc at 1, 5, and 20 µg, respectively, in the presence of MF59 adjuvant, while the sera from the adjuvant control mice (S377-588-Fc at 0 μg) showed no neutralizing activity (<xref ref-type="fig" rid="f0003"><bold>Fig. 3</bold></xref>). These data suggest that S377-588-Fc at 1 μg concentration is able to induce sufficient MERS-CoV neutralizing antibodies in the immunized mice.
<fig id="f0003" orientation="portrait" position="float"><label>Figure 3.</label><caption><p><bold>Comparison of neutralization induced by S377-588-Fc at different antigen doses</bold>. Mice were immunized without (0 µg) or with 1, 5, or 20 µg of S377-588-Fc, and sera from 10 d post-last immunization were tested for neutralization against MERS pseudovirus (<bold>A</bold>) and live MERS-CoV infection (<bold>B</bold>) in DPP4-expressing Huh-7 and Vero E6 cells, respectively. MERS pseudovirus neutralization data were expressed as 50% neutralizing antibody titers (NT<sub>50</sub>), while live MERS-CoV-based neutralizing antibody titers were presented as the reciprocal of the highest dilution of sera that resulted in a complete inhibition of virus-induced CPE in at least 50% of the wells (NT<sub>50</sub>). The data are presented as mean ± SD from 5 mice in each group.</p></caption><graphic content-type="black-white" xlink:href="KHVI_A_1021527_F0003_B"></graphic></fig></p></sec><sec id="s0002-0003"><title>S377-588-Fc at 1 μg induced high levels of IFN-γ-expressing T cell responses in immunized mice</title><p>To compare the cellular immune responses induced by different doses of S377-588-Fc, we immunized mice with the protein at 1, 5, and 20 μg, respectively, and detected MERS-CoV S1-specific IL-2-and IFN-γ-expressing T cells in mouse splenocytes collected from the 3rd immunization. As shown in <xref ref-type="fig" rid="f0004"><bold>Figure 4</bold></xref>, S377-588-Fc at 1 μg was capable of inducing strong IFN-γ-expressing T cells in both CD4<sup>+</sup> (<xref ref-type="fig" rid="f0004"><bold>Fig. 4A</bold></xref>) and CD8<sup>+</sup> (<xref ref-type="fig" rid="f0004"><bold>Fig. 4B</bold></xref>) populations, while S377-588-Fc at the increased doses (5 or 20 μg) did not induce higher T cell responses. In addition, S377-588-Fc at 1, 5, or 20 μg failed to elicit strong IL-2-expressing CD4<sup>+</sup> and CD8<sup>+</sup> T cells. As expected, the adjuvant control group (S377-588-Fc at 0 μg) induced a background level of specific T cell responses. These results suggest that 1 μg of S377-588-Fc is sufficient to induce potent IFN-γ-expressing T cell responses in immunized mice.
<fig id="f0004" orientation="portrait" position="float"><label>Figure 4.</label><caption><p><bold>Comparison of cellular immune responses induced by S377-588-Fc at different antigen doses</bold>. Mice were immunized without (0 µg) or with 1, 5, or 20 µg of S377-588-Fc, and splenocytes from 10 d post-last immunization were tested for MERS-CoV S1-specific T cell responses by flow cytometric analysis. The frequencies of IL-2-(upper left corner) and IFN-γ-(bottom right corner) producing cells were expressed as percentages of CD4<sup>+</sup> (<bold>A</bold>) or CD8<sup>+</sup> (<bold>B</bold>) T cells. The samples were tested in triplicate and presented as mean ± SD from 5 mice in each group.</p></caption><graphic content-type="color" xlink:href="KHVI_A_1021527_F0004_C"></graphic></fig></p></sec></sec><sec sec-type="discussion" id="s0003"><title>Discussion</title><p>Development of effective vaccines is urgently needed to prevent continuous epidemic of MERS. Currently, several MERS vaccine candidates have been tested in preclinical settings, some of which show immunogenicity.<sup><xref rid="cit0021" ref-type="bibr">21,23</xref>,<xref rid="cit0024" ref-type="bibr">24,28–30</xref></sup> It was reported that a modified vaccinia virus Ankara (MVA) expressing full-length S protein and adenoviruses encoding full-length S protein or S1 subunit induced S-specific antibody responses that neutralized MERS-CoV infection <italic>in vitro</italic>,<sup><xref rid="cit0028" ref-type="bibr">28,29</xref></sup> indicating the potential of developing viral vector-based MERS vaccines. The nanoparticle-conjugated full-length S protein elicited neutralizing antibodies in mice, bringing some hopes for developing nanoparticle-based MERS vaccine.<sup><xref rid="cit0031" ref-type="bibr">31</xref></sup> An engineered replication-competent, propagation-defective MERS-CoV provides a platform to develop attenuated viruses as MERS vaccine candidates.<sup><xref rid="cit0030" ref-type="bibr">30</xref></sup> We and others have identified several protein fragments, including residues 350-588, 358-588, 367-588, 367-606, 377-588 and 377-622, of the RBD of MERS-CoV S protein that induced neutralizing antibodies in mice or rabbits,<sup><xref rid="cit0021" ref-type="bibr">21-23,25</xref></sup> suggesting the potential to develop subunit vaccines against MERS-CoV.</p><p>Among various vaccine types, such as those based on inactivated and live-attenuated viruses and viral vectors, recombinant protein-based subunit vaccines are considered to be the safest type of vaccines against virus infection.<sup><xref rid="cit0020" ref-type="bibr">20,26,32</xref></sup> However, the efficacy of subunit vaccines largely depends on the identification of suitable antigens and selection of appropriate adjuvants.<sup><xref rid="cit0020" ref-type="bibr">20,32</xref></sup> We have shown that a recombinant protein containing residues 377-588 of MERS-CoV RBD elicited the highest neutralizing antibodies among the 5 RBD fragments tested against MERS-CoV infection.<sup><xref rid="cit0022" ref-type="bibr">22,23,27</xref></sup>In addition, we have demonstrated that MF59 is most potent among the 5 adjuvants, including Freund's, aluminum, Monophosphoryl lipid A, MF59, and Montanide ISA51, to augment the immunogenicity of S377-588-Fc to induce strong antibody responses, neutralizing antibodies, and protection against MERS-CoV infection, suggesting it an ideal adjuvant for MERS-CoV RBD-based subunit vaccines.<sup><xref rid="cit0027" ref-type="bibr">27</xref></sup> Apart from antigens and adjuvants, an equally crucial, but often neglected, aspect of immunization is the optimization of antigen dosage to find the minimal antigen dose able to induce strong immune responses.<sup><xref rid="cit0033" ref-type="bibr">33,34</xref></sup></p><p>In this study, we compared the levels of immune responses in mice immunized with S377-588-Fc at 1, 5, and 20 μg, respectively, formulated with MF59 adjuvant. This range of doses was selected based on our previous studies showing that 10 μg of this protein induced strong immune responses with neutralizing activity that protected all of the vaccinated mice from challenge of MERS-CoV.<sup><xref rid="cit0022" ref-type="bibr">22,23</xref></sup> Here, we found that S377-588-Fc at 1 μg was able to elicit strong humoral and cellular immune responses in the immunized mice. Particularly, this protein at 1 μg induced high levels of neutralizing antibodies against infections of both MERS pseudovirus and live MERS-CoV, similar to those induced at 5 and 20 μg, respectively, suggesting that 1 μg of S377-588-Fc formulated with MF59 is sufficient to induce strong neutralizing antibody response capable of protecting mice from MERS-CoV challenge, as that was induced by 10 μg of S377-588-Fc formulated with MF59 in our previous study.<sup><xref rid="cit0027" ref-type="bibr">27</xref></sup> Therefore, only about 10% of S377-588-Fc that we tested before is actually needed to achieve the efficacy for prevention of MERS-CoV infection in vaccinated mice, based on the results from the present study.</p><p>Application of the lowest possible amount of the antigen and fewer injections is an important dose sparing strategy for a vaccine with low productivity (e.g., a subunit vaccine), especially during a pandemic or epidemic of an emerging infectious disease, like MERS. This study provides important information for rational design of optimal dosages of vaccines against MERS and other emerging infectious diseases for their future clinical trials.</p></sec><sec sec-type="materials|methods" id="s0004"><title>Materials and Methods</title><sec id="s0004-0001"><title>Ethics statement</title><p>Four-to six-week-old female BALB/c mice were used in the study. The animal studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal protocol was approved by the Committee on the Ethics of Animal Experiments of the New York Blood Center (Permit Number: 194.15).</p></sec><sec id="s0004-0002"><title>Recombinant MERS-CoV RBD and S1 proteins</title><p>The recombinant S377-588-Fc containing residues 377-588 of MERS-CoV spike plus a C-terminal Fc tag (S377-588-Fc, hereinafter named RBD) was used as the antigen to optimize antigen doses for RBD-based subunit vaccines. The construction, expression and purification of the S377-588-Fc were described previously by fusing the RBD gene with human IgG Fc (InvivoGen, San Diego, CA), expressing the S377-588-Fc protein in 293T cell culture supernatant, and purifying it by Protein A affinity chromatography (GE Healthcare, Piscataway, NJ).<sup><xref rid="cit0022" ref-type="bibr">22,23</xref></sup> MERS-CoV S1 protein (residues 18–725) plus a C-terminal His<sub>6</sub> (S1-His) was constructed using the pJW4303 expression vector (Jiangsu Taizhou Haiyuan Protein Biotech, Co., Ltd., China), expressed in 293T cell culture supernatant, and purified using Ni-NTA Superflow (Qiagen, Valencia, CA).<sup><xref rid="cit0023" ref-type="bibr">23,24</xref></sup></p></sec><sec id="s0004-0003"><title>Animal immunization and sample collection</title><p>Mice were immunized with S377-588-Fc as previously described with some modifications.<sup><xref rid="cit0021" ref-type="bibr">21,23,35</xref></sup> Briefly, mice were subcutaneously prime-immunized with S377-588-Fc at 1, 5, and 20 μg, respectively, in the presence of MF59 adjuvant,<sup><xref rid="cit0036" ref-type="bibr">36</xref></sup> and boosted twice with the same immunogen and adjuvant at 3-week intervals. Adjuvant only without antigen (0 μg) was included as the negative control. Sera were collected before immunization and 10 d post-each vaccination to assess MERS-CoV S1-specific antibody responses and neutralizing antibodies. Immunized mice were sacrificed at 10 d after the last immunization, and splenocytes were collected to detect MERS-CoV S1-specific T cell responses.</p></sec><sec id="s0004-0004"><title>ELISA</title><p>ELISA was used to test MERS-CoV S1-specific antibody responses in mouse sera based on our previously described protocols.<sup><xref rid="cit0023" ref-type="bibr">23,37</xref></sup> Briefly, ELISA plates were precoated with MERS-CoV S1-His protein overnight at 4°C, followed by addition of serially diluted sera and incubation at 37°C for 1 h. After four washes with PBST, the plates were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (1:3000, GE Healthcare), IgG1 (1:2000), or IgG2a (1:5000) (Invitrogen, Carlsbad, CA), respectively, at 37°C for 1 h, and washed 4 times. The reaction was visualized by substrate 3,3′,5,5′-tetramethylbenzidine (TMB) (Invitrogen, Carlsbad, CA) and stopped with 1 N H<sub>2</sub>SO<sub>4</sub>. The absorbance at 450 nm (A450) was measured by ELISA plate reader (Tecan, San Jose, CA).</p></sec><sec id="s0004-0005"><title>MERS pseudovirus neutralization assay</title><p>This was done as previously described with some modifications.<sup><xref rid="cit0038" ref-type="bibr">38,39</xref></sup> Briefly, 293T cells were co-transfected with a plasmid encoding Env-defective, luciferase-expressing HIV-1 genome (pNL4-3.luc.RE) and a plasmid encoding MERS-CoV (EMC-2012 strain) S protein using the calcium phosphate method. Cells were changed into fresh DMEM 8 h later, and pseudovirus-containing supernatants were harvested 72 h post-transfection for single-cycle infection of Huh-7 cells. The pseudovirus was incubated with serially diluted mouse sera at 37°C for 1 h before adding to the cells preplated in 96-well culture plates. Twenty-four hours later, cells were refed with fresh medium, which was followed by lysing cells 72 h later using cell lysis buffer (Promega, Madison, WI) and transferring the lysates into 96-well luminometer plates. Luciferase substrate (Promega) was added to the plates, and relative luciferase activity was determined in an Infinite 200 PRO Luminator (Tecan). MERS pseudovirus neutralization was calculated and expressed as 50% neutralizing antibody titer, NT<sub>50</sub>.<sup><xref rid="cit0040" ref-type="bibr">40</xref></sup></p></sec><sec id="s0004-0006"><title>Live MERS-CoV neutralization assay</title><p>A standard micro-neutralization assay was used to confirm the anti-MERS-CoV neutralizing antibodies as previously described.<sup><xref rid="cit0023" ref-type="bibr">23,41</xref></sup> Briefly, mouse sera were diluted at serial 2-fold in 96-well tissue culture plates and incubated for 1 h at room temperature with ∼100 infectious MERS-CoV (EMC-2012) before transfer to Vero E6 cells. Seventy-two hours later, the neutralizing capacity of serum samples was assessed by determining the presence or absence of virus-induced cytopathic effect (CPE). Neutralizing antibody titers were expressed as the reciprocal of the highest dilution of sera that completely inhibited virus-induced CPE in at least 50% of the wells (NT<sub>50</sub>).</p></sec><sec id="s0004-0007"><title>Intracellular cytokine staining and flow cytometry analysis</title><p>MERS-CoV S1-specific cellular immune responses were evaluated in immunized mice by intracellular cytokine staining followed by flow cytometric analysis as previously described.<sup><xref rid="cit0021" ref-type="bibr">21,42,43</xref></sup> Briefly, mouse splenocytes (2 × 10<sup>6</sup>) were stimulated with MERS-CoV S1-His protein for 5 h at 37°C with 5% CO<sub>2</sub> in the presence of GolgiPlug™ containing brefeldin A (1 μl/ml; BD Biosciences, San Jose, CA). The cells were stained with conjugated anti-mouse-CD4 (APC) and -CD8 (P-Cy5-5) antibodies for 30 min at 4°C. After washes, the cells were fixed using the Cytofix/Cytoperm™ Kit (BD Biosciences) and stained with anti-mouse-IL-2 (FITC) and -IFN-γ (PE) (BD Biosciences) antibodies for 30 min at 4°C. The stained cells were analyzed using a FACSCalibur (BD Biosciences) and FACSDiva software v.6.1.2 (BD Biosciences).</p></sec><sec id="s0004-0008"><title>Statistical analysis</title><p>Values are presented as mean and standard deviation (SD). Statistical significance among different vaccination groups was calculated by Student's <italic>t</italic>-test using GraphPad Prism statistical software. <italic>P</italic> values less than 0.05 were considered statistically significant.</p></sec></sec></body><back><sec id="s0005" sec-type="other"><title>Disclosure of Potential Conflicts of Interest</title><p>No potential conflicts of interest were disclosed.</p></sec><sec><title>Funding</title><p>This study was supported by China National Project of Infectious Diseases grant (2014ZX10004001004), NIH grant (R21AI109094), and an intramural fund of New York Blood Center (NYB000068).</p></sec><ref-list><title>References</title><ref id="cit0001"><label>1.</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Drosten</surname><given-names>C</given-names></name>, <name name-style="western"><surname>Seilmaier</surname><given-names>M</given-names></name>, <name name-style="western"><surname>Corman</surname><given-names>VM</given-names></name>, <name name-style="western"><surname>Hartmann</surname><given-names>W</given-names></name>, <name name-style="western"><surname>Scheible</surname><given-names>G</given-names></name>, <name name-style="western"><surname>Sack</surname><given-names>S</given-names></name>, <name name-style="western"><surname>Guggemos</surname><given-names>W</given-names></name>, <name name-style="western"><surname>Kallies</surname><given-names>R</given-names></name>, <name name-style="western"><surname>Muth</surname><given-names>D</given-names></name>, <name name-style="western"><surname>Junglen</surname><given-names>S</given-names></name>, et al.</person-group><article-title>Clinical features and virological analysis of a case of Middle East respiratory syndrome coronavirus infection</article-title>. <source>Lancet Infect Dis</source><year>2013</year>; 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